鸭疫里默氏杆菌致病相关因子研究
|
摘要
鸭疫里默氏杆菌是一个多宿主的病原菌,除主要引起家鸭的渗出性炎症以外,还感染鹅、鸡、火鸡、鸽子、野鸭、鹦鹉等其它鸟类,甚至已从猪、青鱼体内分离鉴定出该细菌。目前鸭疫里默氏杆菌已成为危害养鸭业的主要病原之一,呈世界性流行趋势,发病率和死亡率一般为10-30%,但部分鸭场达95%以上。该细菌的危害除引起动物发病、死亡外,还因感染耐过动物饲料转化率低、生长发育迟缓造成严重的经济损失,甚至由于抗菌药物的大量使用而引起食品安全问题。自1982年以来,尤其是在东亚和东南亚地区,鸭疫里默氏杆菌感染严重制约了肉鸭的集约化养殖。
鸭疫里默氏杆菌血清型复杂,各血清型菌株间缺乏交叉免疫保护;极易产生耐药性,流行菌株多表现为多重耐药。关于其致病相关因子尚不明确,而对于致病机制的研究处于起步阶段。作者在研究鸭疫里默氏杆菌液化明胶菌株的流行病学特征的过程中发现,鸭疫里默氏杆菌液化明胶特性与致病性有关。基于此,本文以液化明胶活性成分(明胶酶)为对象,研究鸭疫里默氏杆菌的致病相关因子,为进一步研究细菌的致病机制奠定基础。
1.鸭疫里默氏杆菌流行菌株的分离鉴定及液化明胶特性测定
利用巧克力营养琼脂平板从2004-2008年收集于重庆、四川等25个地区的661份疑似鸭疫里默氏杆菌感染的自然病例中分离、鉴定出548株鸭疫里默氏杆菌,其中512株对10日龄鸭具有毒力,25株细菌经鸭体内传1-3代后获得毒力,11株在鸭体内传7代仍不具有毒力。利用明胶-营养琼脂平板和明胶-琼脂平板分别测定细菌及培养滤液的液化明胶特性,有毒力的537株细菌及培养滤液都能液化明胶,占分离菌株的98%;不具有毒力的11株细菌及培养滤液不能液化明胶,占分离菌株的2%。鸭疫里默氏杆菌液化明胶特性稳定,液化明胶和不液化明胶菌株在巧克力培养基和鸭体内分别传30和10代后未发现明显的变化。液化明胶的鸭疫里默氏杆菌培养滤液的明胶酶谱呈现6条活性带,不同菌株各谱带的活性存在差异,且各谱带活性能被EDTA抑制。
2.鸭疫里默氏杆菌产明胶酶的条件优化及酶的分离纯化
本研究在建立定量测定鸭疫里默氏杆菌明胶酶活性的基础上,对鸭疫里默氏杆菌产酶条件进行了优化。研究结果表明,产酶培养基的基本组成为2%蛋白胨、0.5%酵母粉和0.5%氯化钠,初始pH值为7.4;培养基含终浓度5mMCa~(2+)有利于酶的产生;最适培养温度为37℃,在振荡条件下的最适培养时间为24h。
在最佳条件下培养AF株细菌,培养滤液经分子截流量为10kDa超滤膜浓缩、最终饱和度为70%硫酸铵盐析、DEAE-Sepharose Fast Flow阴离子交换层析、phenyl-sepharose CL-4B疏水柱层析及Superdex-75凝胶柱层析分离纯化,获得具有液化明胶活性的蛋白质组分(明胶酶),活性回收率为28.4%。分离纯化的明胶酶的比活力为17076.0U/mg;经SDS-PAGE检测为分子量59kDa单带;明胶酶谱与培养滤液的第5活性条带一致。
3.鸭疫里默氏杆菌明胶酶的生物学活性研究
本研究利用鸭胚成纤维细胞和10日龄雏鸭模型对分离纯化的鸭疫里默氏杆菌明胶酶的生物学活性进行了研究。明胶酶对鸭胚成纤维细胞具有细胞毒性,半数致死浓度(LC_(50))和半数生长抑制浓度(GI_(50))分别为1.45U/ml和0.16U/ml;导致细胞形态发生异常,主要表现为细胞膜降解、细胞质丧失与细胞核浓缩。对30株鸭疫里默氏杆菌临床分离株的培养滤液检测发现,具有毒力及能液化明胶的27株细菌均能引起与明胶酶一致的细胞形态变化,而无毒力及不液化明胶的3株细菌的培养滤液不表现细胞毒性。
明胶酶对雏鸭具有毒性,能增强鸭疫里默氏杆菌对动物的侵袭力,增加菌血症的严重程度,缩短感染潜伏期和致死动物的时间,显著提高动物的死亡率。颈静脉注射5000U/ml能致死10日龄雏鸭,2000U/ml能引起发病,而500U/ml的作用不明显。发病动物主要表现嗜睡懒动、腿软无力、甩头、张口呼吸及头颈歪斜等症状;死亡动物有肝脏轻微肿大、硬脑膜有出血点、大脑水肿等大体病理变化。在组织学上,2000U/ml作用8h鸭的心肌纤维肿胀变粗、横纹不明显,纤维间质水肿、可见微细颗粒和空泡;肝细胞索不规则,肝血窦变小,肝细胞肿胀、胞浆中出现大小不等的空泡;脾中央动脉内皮细胞肿胀、脱落,脾小体局灶性出血及小淋巴管扩张,淋巴鞘数量增多、管径扩大,淋巴细胞不同程度变性、坏死。在超微结构上,心肌细胞线粒体肿大,毛细血管的内皮细胞中多空泡、核膜不均一;肝细胞核浓缩、边缘有皱褶,毛细血管内皮细胞间连接不紧密、红细胞形态不规则;脾淋巴细胞核膜不规则、染色质核周边浓集。
明胶酶经静脉注射后对雏鸭外周血血液指标的影响明显。雏鸭外周血中红细胞(RBC)的数量与C3b花环率显著下降,在9h最低,分别为(1.58±0.05)×10~(12)·L~(-1)和(18.5±1.32)%。白细胞(WBC)的数量减少;对绵羊红细胞的吞噬率(BP)与吞噬指数(IP)降低,在6h时最低,分别为(27.7±2.0)%和1.053±0.015,与对照差异极显著(P＜0.01);对金黄色葡萄球菌的杀菌指数下降,在9h时最低为0.4562±0.0205,与对照差异极显著(P＜0.01)。外周血中淋巴细胞(LT)数量减少;a-醋酸萘酯酶阳性T淋巴细胞百分率降低。血清谷草/谷丙转氨酶活性、碱性磷酸酶活性及总胆固醇和血清葡萄糖水平显著升高。血清总补体活性(CH_(50))显著下降。
明胶酶的作用对雏鸭脾脏损伤严重,导致脾淋巴细胞的增殖活性和生成IgG的能力下降,9h最低,分别为0.185±0.022和0.237±0.038,与对照差异极显著(p＜0.01);可引起雏鸭血-脑屏障机能障碍,表现为脑脊液中的白蛋白含量升高,血-脑屏障的通透性增大,基底膜粗糙、部分节段的连续性中断,脑血管周围水肿、管腔面不光滑、内皮细胞肿胀、连接处开放。
4.鸭疫里默氏杆菌明胶酶基因克隆及分析
从AF株培养滤液中分离纯化的明胶酶N-氨基酸残基序列为:天冬酰胺-苏氨酸-甘氨酸-丙氨酸-丙氨酸-谷氨酸-甘氨酸-苏氨酸-组氨酸-甘氨酸(NTGAAEGTHG),在NCBI中未能找到同源序列,仅第3-10位氨基酸残基与类鼻疽伯克霍尔德菌406e株的一个假设保守蛋白的第555-562一致。在此基础上,以N端氨基酸序列设计引物,用单特性引物PCR(SSP-PCR)从细菌基因组DNA文库中克隆出197bp的DNA片段,然后利用热不对称交错PCR(Tail-PCR)进行基因组步移,获得2150bp核苷酸序列。该序列中有一个1872bp的ORF,编码624aa,其中包含NTGAAEGTHG序列;1-24aa为信号肽;成熟蛋白存在3个跨膜区,分别在25-45aa、211-232aa和416-435aa;在蛋白质数据库中未能找到同源性序列。
5.鸭疫里默氏杆菌明胶酶的理化特性测定
鸭疫里默氏杆菌明胶酶不降解Ⅰ型胶原蛋白维、干酪素、牛血清白蛋白V及Azocoll试剂;降解明胶的最适作用温度和pH值分别为37℃和7.8,在4℃24h及37℃2h内稳定、65℃0.1h内完全失活;Ca~(2+)、Mg~(2+)、Na~+和Zn~(2+)对酶有一定激活作用,Cu~(2+)、Cd~(2+)、Mn~(2+)、Hg~(2+)及金属离子络合物EDTA和EGTA对酶活性有强烈的抑制作用,但络合物的抑制作用可以被Ca~(2+)逆转;蛋白质化学修饰剂2-ME和DEPC不影响酶活,而PMSF、ADD和NBS导致酶活显著下降。
本文在研究明胶酶生物学活性过程中,与鸭疫里默氏杆菌感染模型进行比较,结果表明二者在引起雏鸭血液、肝、脾及血-脑屏障等的生物化学与组织学变化及机能障碍方面相似。这进一步表明,明胶酶是鸭疫里默氏杆菌的致病相关因子之一,在其致病过程中起着重要的作用。
Riemerella anatipestifer infection characterized by exudative septicaemia is primarily a disease of domestic ducks.This bacterium is also isolated from other domestic poultry,such as geese,chickens,turkeys,pigeons,wild ducks and parrots and was also reported from snail craps and domesticated pigs.Now,this bacterium is a major bacterial pathogen of ducks in all over the world.Mortality varies from 10 to 30%,but it can be as high as 95%and is influenced by predisposing viral and bacterial infections.R.anatipestifer not only accounts for major economic losses in industrialized duck production due to high mortality,reduced growth rate,poor feed conversion,increased condemnations,and high treatment costs,but also causes food safety for antibacterial.In east and south-east Asia,R.anatipestifer infection has been a problem in intensive production of meat ducks since 1982.
The occurrence of different serotypes of R.anatipestifer in field cases has been reported.Unfortunately,no cross-protection has been observed among different serotypes of R.anatipestifer.Drug resistant strains of R.anatipestifer are very popular and some strains are multidrug resistant.In contrast,there has been little work on the virulence factor and pathogenesis of this organism.We have studied the epidemiological characteristic of R.anatipestifer strains which liquefacted gelatin,and it has been found that the pathogenicity of R.anatipestifer related to gelatin liquefaction.To facilitate study of the pathogenesis,gelatinase was identified and verified as a potential virulence factor of R.anatipestifer.
1.Isolation of R.anatipestifer from ducks and determination of gelatin liquefaction
In the study,chocolate nutrient agar was used and 548 R.anatipestifer field strains were isolated from representing 661outbreaks of Riemerellosis in China between 2004 and 2008.537 strains had pathogenicity to 10-day-old ducks and 11 strains were avirulence.The nutrient gelatin agar plate and gelatin agar plate were used to evaluate gelatin liquefaction of R.anatipestifer and it's culture filtrate,respectively.The results showed that the virulence R.anatipestifer and its culture filtrate liquefied gelatin and avirulence strains did not.When this bacterium was cultured 10 passages in vivo or 30 passages in vitro,its activity of gelatin liquefaction did not changed obviously. Zymography showed gelatinase activity in culture filtrate of gelatin liquefaction strains and there were 6 gelatinolytic bands on a darkly stained gel.The activity of gelatinase was estimated by using densitometry and there were different among R.anatipestifer strains.
2.Optimal enzyme production condition for R.anatipestifer AF strain and purification of gelatinase
A quantitative assay method of gelatinase has been established and the fermentation was studied.The results showed that the suitable condition of R. anatipestifer AF strain producing gelatinase were 2%Peptone,0.5%yeast powder, 0.5%sodium chloride,initial pH 7.4,37℃and shaking culture 24 hours.At the same time,5mmol/L Ca~(2+) promoted the producing level of gelatinase.
Purification protocol of active compound was designed according to typical protein purification strategy and R.anatipestifer AF strain active compound was purified with the following program.The purification of active compound was started by concentrating the supernatantfluid in an Amicon(M_t cut-off 10,000).Second, concentrate was precipitated with ammonium sulfate at 15%saturation,and then at 70%saturation.The third step was DEAE-Sepharose anion-exchange chromatography. Fourth,phenyl-sepharose CL-4B was used in hydrophobic interaction chromatography. Finally,Superdex-75 column was used in gel permeation chromatography.The active compound obtained after steps 5 was SDS-PAGE-pure.The molecular weight was about 59kDa which was determined with SDS-PAGE.From now on,the active compound was named gelatinase.The specific activity of gelatinase was 17076.0 unit per milligram protein and it was accorded to the fifth bands of culture filtrate in zymography.
3.Study on biological activity of R.anatipestifer gelatinase
In this study,duck embryo fibroblast(DEF) and 10-day-old ducklings were used to research the biological activity of R.anatipestifer gelatinase.
Cytotoxicity of gelatinase was observed using the DEF.The median lethal concentration(LC_(50)) and the median growth inhibiting(GI_(50)) were 1.45U/ml and 0.16U/ml,respectively.DEF treated with gelatinase resulted in striking morphologic changes including losses of cell membrane and cytoplasm,and nucleus being condensed. Most of the clinical strains 90%(27/30) had the capability of hydrolyzing gelatin, causing disease and altering the morphology of DEF,While the rest 10%(3/30) are on the contrary.
Moreover,the damage after ducklings intrajugular injection of gelatinase was studied.The results showed gelatinase not only enhanced the invasion of R. anatipestifer,increased bacteremia,shortened stage of latency and course of disease, but also raised mortality obviously.10-days old duckling was died after intrajugular injection of 5000U/ml.Injection 2000U/ml,the duckling was fall ill,but clinical symptom did not been discovered injected 500U/ml each ducklings.Ducklings treated with gelatinase resulted in drowsiness,debility,throwing head,buccal respiration, neurological symptom,liver intumesce,bleeding point on cerebral dura mater,Cerebral edema,and so on.Though light microscope and electron microscope,the pathological changes and ultrastructural changes of main organs in the duckling which was treated with 2000U/ml by intrajugular injection were observed.After 8h,there were cardiac muscle fibers coarsened,transverse striation in nubibus,interstitial substance oedema and many vacuo-luses.Liver cell cords were irregularity and liver sinusoid shrinked. Hepatocyte was swollen and there were many vacuoluses in endochylema.Spleen resulted in endotheliocyte of central arterys swelling and ablate,acinus lienalis focal bleeding and angioleukectasia,leukomonocyte apomorphosis and necro-sis.Under electron microscope,parenchymal cells such as cadiocyte,hepatocyte,splenic lymphocyte and endothelial cell of micrangium were damaged.The structure of organelles were changed and the main manifestations were that mitochondria of cadiocyte was Swollen,cell nucleus of hepatocyte was condensed and crenulated, caryotheca of splenic lymphocyte was irregularity and caryotin was enriched at ambitus.
Ducklings after intrajugular injection of gelatinase at 10 days of age were studied to determine the dynamic changes of some blood indices.The results showed that the akaryocyte and its chaplet rate of C3b decreased,the 9~(th) hour was the ravine and they were(1.58±0.05)×10~(12)·L~(-1) and(18.5±1.32)%,respectively.The numbers of white blood cell of peripheral blood decreased,the rate of phagocytosis and index of phagocytosis to sheep red blood cell were lowest at 9h,and they were(27.7±2.0)%and 1.053±0.01, respectively.Gelatinase decreased bactericidal index of WBC significantly when compared with controls,maximal damage was seen at 9h.Leukomonocyte and the percentage of ANAE~+-T of peripheral blood were decreased,too.The results of serum biochemical indices showed that GOT,GPT,ALP,CHOI and GLU were all obviously enhanced,but serum total complement activity(CH_(50)) was declined.
Gelatinase damaged spleen of ducklings and the worst damage was discovered at 9h.At that time,the generation activity and production IgG of splenic lymphocyte were 0.185±0.022 and 0.237±0.038,respectively.Blood brain barrier dysfunction occur in ducklings injected with gelatinase,and the damages showed at higher concentration of albumin in CSF and higher permeability in blood brain barrier,Cerebral vasogenic edema,lumens rough,junction break,basement membrane rough and break.
4.Gene cloning and analysis
N-terminal first ten amino acids of gelatinase protein were sequenced as NTGAAEGTHG.In NCBI,Sequence alignments indicated that the same sequence was not founded.According to the analysis of gelatinase N-terminal first ten amino acid sequence,the primer encoding sequence was designed and 197bp DNA fragment was cloned by single specific primer polymerase chain reaction(SSP-PCR) from genome DNA library of R.anatipestifer AF strain.And then,Genome walking by thermal asymmetric interlaced PCR(Tail-PCR) was used and 2150bp DNA fragment was cloned.One ORF was found out and it encoded 624 amino acids.According to bioinformation,N-terminal first twenty-four amino acids were signal peptide,and mature protein has three transmembrane domain.Unfortunately,homophylic sequence was not found in NCBI.The results showed that the gelatinase gene was a new gene probably.
5.Study on physico-chemical property of R.anatipestifer gelatinase
The activities of gelatinase were tested byⅠcollagen fibers,casein,bovine serum albumin V and Azocoll.As the results showed they were not substrate of gelatinase.The optimum pH and temperature of the gelatinase were found to be 7.8 and 37℃.The activity of gelatinase was retained after storing at 4℃for 24h and at 37℃for 2h,but activity was not detected after storing at 65℃for 0.1h.Enzyme testing for inhibition and activation of the gelatinase indicated that EDTA,EGTA,Cu~(2+),Cd~(2+),Mn~(2+),Hg~(2+), PMSF,ADD and NBS inhibited enzymatic activity,2-ME and DEPC did not affected activity,but Ca~(2+),Mg~(2+),Na~+ and Zn~(2+) activated the activity.Moreover,Ca~(2+) counteracted the inhibition of EDTA and EGTA.
Animal model of R.anatipestifer infection was studied in order to showing the relationship of R.anatipestifer and gelatinase.The results showed the effects of gelatinase to ducklings were similar to R.anatipestifer infection at dysfunction, pathohistology,biochemistry,and so on.All results indicated that the gelatinase was a virulence factor and it was important to the infection of R.anatipestifer.
鸭疫里默氏杆菌血清型复杂,各血清型菌株间缺乏交叉免疫保护;极易产生耐药性,流行菌株多表现为多重耐药。关于其致病相关因子尚不明确,而对于致病机制的研究处于起步阶段。作者在研究鸭疫里默氏杆菌液化明胶菌株的流行病学特征的过程中发现,鸭疫里默氏杆菌液化明胶特性与致病性有关。基于此,本文以液化明胶活性成分(明胶酶)为对象,研究鸭疫里默氏杆菌的致病相关因子,为进一步研究细菌的致病机制奠定基础。
1.鸭疫里默氏杆菌流行菌株的分离鉴定及液化明胶特性测定
利用巧克力营养琼脂平板从2004-2008年收集于重庆、四川等25个地区的661份疑似鸭疫里默氏杆菌感染的自然病例中分离、鉴定出548株鸭疫里默氏杆菌,其中512株对10日龄鸭具有毒力,25株细菌经鸭体内传1-3代后获得毒力,11株在鸭体内传7代仍不具有毒力。利用明胶-营养琼脂平板和明胶-琼脂平板分别测定细菌及培养滤液的液化明胶特性,有毒力的537株细菌及培养滤液都能液化明胶,占分离菌株的98%;不具有毒力的11株细菌及培养滤液不能液化明胶,占分离菌株的2%。鸭疫里默氏杆菌液化明胶特性稳定,液化明胶和不液化明胶菌株在巧克力培养基和鸭体内分别传30和10代后未发现明显的变化。液化明胶的鸭疫里默氏杆菌培养滤液的明胶酶谱呈现6条活性带,不同菌株各谱带的活性存在差异,且各谱带活性能被EDTA抑制。
2.鸭疫里默氏杆菌产明胶酶的条件优化及酶的分离纯化
本研究在建立定量测定鸭疫里默氏杆菌明胶酶活性的基础上,对鸭疫里默氏杆菌产酶条件进行了优化。研究结果表明,产酶培养基的基本组成为2%蛋白胨、0.5%酵母粉和0.5%氯化钠,初始pH值为7.4;培养基含终浓度5mMCa~(2+)有利于酶的产生;最适培养温度为37℃,在振荡条件下的最适培养时间为24h。
在最佳条件下培养AF株细菌,培养滤液经分子截流量为10kDa超滤膜浓缩、最终饱和度为70%硫酸铵盐析、DEAE-Sepharose Fast Flow阴离子交换层析、phenyl-sepharose CL-4B疏水柱层析及Superdex-75凝胶柱层析分离纯化,获得具有液化明胶活性的蛋白质组分(明胶酶),活性回收率为28.4%。分离纯化的明胶酶的比活力为17076.0U/mg;经SDS-PAGE检测为分子量59kDa单带;明胶酶谱与培养滤液的第5活性条带一致。
3.鸭疫里默氏杆菌明胶酶的生物学活性研究
本研究利用鸭胚成纤维细胞和10日龄雏鸭模型对分离纯化的鸭疫里默氏杆菌明胶酶的生物学活性进行了研究。明胶酶对鸭胚成纤维细胞具有细胞毒性,半数致死浓度(LC_(50))和半数生长抑制浓度(GI_(50))分别为1.45U/ml和0.16U/ml;导致细胞形态发生异常,主要表现为细胞膜降解、细胞质丧失与细胞核浓缩。对30株鸭疫里默氏杆菌临床分离株的培养滤液检测发现,具有毒力及能液化明胶的27株细菌均能引起与明胶酶一致的细胞形态变化,而无毒力及不液化明胶的3株细菌的培养滤液不表现细胞毒性。
明胶酶对雏鸭具有毒性,能增强鸭疫里默氏杆菌对动物的侵袭力,增加菌血症的严重程度,缩短感染潜伏期和致死动物的时间,显著提高动物的死亡率。颈静脉注射5000U/ml能致死10日龄雏鸭,2000U/ml能引起发病,而500U/ml的作用不明显。发病动物主要表现嗜睡懒动、腿软无力、甩头、张口呼吸及头颈歪斜等症状;死亡动物有肝脏轻微肿大、硬脑膜有出血点、大脑水肿等大体病理变化。在组织学上,2000U/ml作用8h鸭的心肌纤维肿胀变粗、横纹不明显,纤维间质水肿、可见微细颗粒和空泡;肝细胞索不规则,肝血窦变小,肝细胞肿胀、胞浆中出现大小不等的空泡;脾中央动脉内皮细胞肿胀、脱落,脾小体局灶性出血及小淋巴管扩张,淋巴鞘数量增多、管径扩大,淋巴细胞不同程度变性、坏死。在超微结构上,心肌细胞线粒体肿大,毛细血管的内皮细胞中多空泡、核膜不均一;肝细胞核浓缩、边缘有皱褶,毛细血管内皮细胞间连接不紧密、红细胞形态不规则;脾淋巴细胞核膜不规则、染色质核周边浓集。
明胶酶经静脉注射后对雏鸭外周血血液指标的影响明显。雏鸭外周血中红细胞(RBC)的数量与C3b花环率显著下降,在9h最低,分别为(1.58±0.05)×10~(12)·L~(-1)和(18.5±1.32)%。白细胞(WBC)的数量减少;对绵羊红细胞的吞噬率(BP)与吞噬指数(IP)降低,在6h时最低,分别为(27.7±2.0)%和1.053±0.015,与对照差异极显著(P＜0.01);对金黄色葡萄球菌的杀菌指数下降,在9h时最低为0.4562±0.0205,与对照差异极显著(P＜0.01)。外周血中淋巴细胞(LT)数量减少;a-醋酸萘酯酶阳性T淋巴细胞百分率降低。血清谷草/谷丙转氨酶活性、碱性磷酸酶活性及总胆固醇和血清葡萄糖水平显著升高。血清总补体活性(CH_(50))显著下降。
明胶酶的作用对雏鸭脾脏损伤严重,导致脾淋巴细胞的增殖活性和生成IgG的能力下降,9h最低,分别为0.185±0.022和0.237±0.038,与对照差异极显著(p＜0.01);可引起雏鸭血-脑屏障机能障碍,表现为脑脊液中的白蛋白含量升高,血-脑屏障的通透性增大,基底膜粗糙、部分节段的连续性中断,脑血管周围水肿、管腔面不光滑、内皮细胞肿胀、连接处开放。
4.鸭疫里默氏杆菌明胶酶基因克隆及分析
从AF株培养滤液中分离纯化的明胶酶N-氨基酸残基序列为:天冬酰胺-苏氨酸-甘氨酸-丙氨酸-丙氨酸-谷氨酸-甘氨酸-苏氨酸-组氨酸-甘氨酸(NTGAAEGTHG),在NCBI中未能找到同源序列,仅第3-10位氨基酸残基与类鼻疽伯克霍尔德菌406e株的一个假设保守蛋白的第555-562一致。在此基础上,以N端氨基酸序列设计引物,用单特性引物PCR(SSP-PCR)从细菌基因组DNA文库中克隆出197bp的DNA片段,然后利用热不对称交错PCR(Tail-PCR)进行基因组步移,获得2150bp核苷酸序列。该序列中有一个1872bp的ORF,编码624aa,其中包含NTGAAEGTHG序列;1-24aa为信号肽;成熟蛋白存在3个跨膜区,分别在25-45aa、211-232aa和416-435aa;在蛋白质数据库中未能找到同源性序列。
5.鸭疫里默氏杆菌明胶酶的理化特性测定
鸭疫里默氏杆菌明胶酶不降解Ⅰ型胶原蛋白维、干酪素、牛血清白蛋白V及Azocoll试剂;降解明胶的最适作用温度和pH值分别为37℃和7.8,在4℃24h及37℃2h内稳定、65℃0.1h内完全失活;Ca~(2+)、Mg~(2+)、Na~+和Zn~(2+)对酶有一定激活作用,Cu~(2+)、Cd~(2+)、Mn~(2+)、Hg~(2+)及金属离子络合物EDTA和EGTA对酶活性有强烈的抑制作用,但络合物的抑制作用可以被Ca~(2+)逆转;蛋白质化学修饰剂2-ME和DEPC不影响酶活,而PMSF、ADD和NBS导致酶活显著下降。
本文在研究明胶酶生物学活性过程中,与鸭疫里默氏杆菌感染模型进行比较,结果表明二者在引起雏鸭血液、肝、脾及血-脑屏障等的生物化学与组织学变化及机能障碍方面相似。这进一步表明,明胶酶是鸭疫里默氏杆菌的致病相关因子之一,在其致病过程中起着重要的作用。
Riemerella anatipestifer infection characterized by exudative septicaemia is primarily a disease of domestic ducks.This bacterium is also isolated from other domestic poultry,such as geese,chickens,turkeys,pigeons,wild ducks and parrots and was also reported from snail craps and domesticated pigs.Now,this bacterium is a major bacterial pathogen of ducks in all over the world.Mortality varies from 10 to 30%,but it can be as high as 95%and is influenced by predisposing viral and bacterial infections.R.anatipestifer not only accounts for major economic losses in industrialized duck production due to high mortality,reduced growth rate,poor feed conversion,increased condemnations,and high treatment costs,but also causes food safety for antibacterial.In east and south-east Asia,R.anatipestifer infection has been a problem in intensive production of meat ducks since 1982.
The occurrence of different serotypes of R.anatipestifer in field cases has been reported.Unfortunately,no cross-protection has been observed among different serotypes of R.anatipestifer.Drug resistant strains of R.anatipestifer are very popular and some strains are multidrug resistant.In contrast,there has been little work on the virulence factor and pathogenesis of this organism.We have studied the epidemiological characteristic of R.anatipestifer strains which liquefacted gelatin,and it has been found that the pathogenicity of R.anatipestifer related to gelatin liquefaction.To facilitate study of the pathogenesis,gelatinase was identified and verified as a potential virulence factor of R.anatipestifer.
1.Isolation of R.anatipestifer from ducks and determination of gelatin liquefaction
In the study,chocolate nutrient agar was used and 548 R.anatipestifer field strains were isolated from representing 661outbreaks of Riemerellosis in China between 2004 and 2008.537 strains had pathogenicity to 10-day-old ducks and 11 strains were avirulence.The nutrient gelatin agar plate and gelatin agar plate were used to evaluate gelatin liquefaction of R.anatipestifer and it's culture filtrate,respectively.The results showed that the virulence R.anatipestifer and its culture filtrate liquefied gelatin and avirulence strains did not.When this bacterium was cultured 10 passages in vivo or 30 passages in vitro,its activity of gelatin liquefaction did not changed obviously. Zymography showed gelatinase activity in culture filtrate of gelatin liquefaction strains and there were 6 gelatinolytic bands on a darkly stained gel.The activity of gelatinase was estimated by using densitometry and there were different among R.anatipestifer strains.
2.Optimal enzyme production condition for R.anatipestifer AF strain and purification of gelatinase
A quantitative assay method of gelatinase has been established and the fermentation was studied.The results showed that the suitable condition of R. anatipestifer AF strain producing gelatinase were 2%Peptone,0.5%yeast powder, 0.5%sodium chloride,initial pH 7.4,37℃and shaking culture 24 hours.At the same time,5mmol/L Ca~(2+) promoted the producing level of gelatinase.
Purification protocol of active compound was designed according to typical protein purification strategy and R.anatipestifer AF strain active compound was purified with the following program.The purification of active compound was started by concentrating the supernatantfluid in an Amicon(M_t cut-off 10,000).Second, concentrate was precipitated with ammonium sulfate at 15%saturation,and then at 70%saturation.The third step was DEAE-Sepharose anion-exchange chromatography. Fourth,phenyl-sepharose CL-4B was used in hydrophobic interaction chromatography. Finally,Superdex-75 column was used in gel permeation chromatography.The active compound obtained after steps 5 was SDS-PAGE-pure.The molecular weight was about 59kDa which was determined with SDS-PAGE.From now on,the active compound was named gelatinase.The specific activity of gelatinase was 17076.0 unit per milligram protein and it was accorded to the fifth bands of culture filtrate in zymography.
3.Study on biological activity of R.anatipestifer gelatinase
In this study,duck embryo fibroblast(DEF) and 10-day-old ducklings were used to research the biological activity of R.anatipestifer gelatinase.
Cytotoxicity of gelatinase was observed using the DEF.The median lethal concentration(LC_(50)) and the median growth inhibiting(GI_(50)) were 1.45U/ml and 0.16U/ml,respectively.DEF treated with gelatinase resulted in striking morphologic changes including losses of cell membrane and cytoplasm,and nucleus being condensed. Most of the clinical strains 90%(27/30) had the capability of hydrolyzing gelatin, causing disease and altering the morphology of DEF,While the rest 10%(3/30) are on the contrary.
Moreover,the damage after ducklings intrajugular injection of gelatinase was studied.The results showed gelatinase not only enhanced the invasion of R. anatipestifer,increased bacteremia,shortened stage of latency and course of disease, but also raised mortality obviously.10-days old duckling was died after intrajugular injection of 5000U/ml.Injection 2000U/ml,the duckling was fall ill,but clinical symptom did not been discovered injected 500U/ml each ducklings.Ducklings treated with gelatinase resulted in drowsiness,debility,throwing head,buccal respiration, neurological symptom,liver intumesce,bleeding point on cerebral dura mater,Cerebral edema,and so on.Though light microscope and electron microscope,the pathological changes and ultrastructural changes of main organs in the duckling which was treated with 2000U/ml by intrajugular injection were observed.After 8h,there were cardiac muscle fibers coarsened,transverse striation in nubibus,interstitial substance oedema and many vacuo-luses.Liver cell cords were irregularity and liver sinusoid shrinked. Hepatocyte was swollen and there were many vacuoluses in endochylema.Spleen resulted in endotheliocyte of central arterys swelling and ablate,acinus lienalis focal bleeding and angioleukectasia,leukomonocyte apomorphosis and necro-sis.Under electron microscope,parenchymal cells such as cadiocyte,hepatocyte,splenic lymphocyte and endothelial cell of micrangium were damaged.The structure of organelles were changed and the main manifestations were that mitochondria of cadiocyte was Swollen,cell nucleus of hepatocyte was condensed and crenulated, caryotheca of splenic lymphocyte was irregularity and caryotin was enriched at ambitus.
Ducklings after intrajugular injection of gelatinase at 10 days of age were studied to determine the dynamic changes of some blood indices.The results showed that the akaryocyte and its chaplet rate of C3b decreased,the 9~(th) hour was the ravine and they were(1.58±0.05)×10~(12)·L~(-1) and(18.5±1.32)%,respectively.The numbers of white blood cell of peripheral blood decreased,the rate of phagocytosis and index of phagocytosis to sheep red blood cell were lowest at 9h,and they were(27.7±2.0)%and 1.053±0.01, respectively.Gelatinase decreased bactericidal index of WBC significantly when compared with controls,maximal damage was seen at 9h.Leukomonocyte and the percentage of ANAE~+-T of peripheral blood were decreased,too.The results of serum biochemical indices showed that GOT,GPT,ALP,CHOI and GLU were all obviously enhanced,but serum total complement activity(CH_(50)) was declined.
Gelatinase damaged spleen of ducklings and the worst damage was discovered at 9h.At that time,the generation activity and production IgG of splenic lymphocyte were 0.185±0.022 and 0.237±0.038,respectively.Blood brain barrier dysfunction occur in ducklings injected with gelatinase,and the damages showed at higher concentration of albumin in CSF and higher permeability in blood brain barrier,Cerebral vasogenic edema,lumens rough,junction break,basement membrane rough and break.
4.Gene cloning and analysis
N-terminal first ten amino acids of gelatinase protein were sequenced as NTGAAEGTHG.In NCBI,Sequence alignments indicated that the same sequence was not founded.According to the analysis of gelatinase N-terminal first ten amino acid sequence,the primer encoding sequence was designed and 197bp DNA fragment was cloned by single specific primer polymerase chain reaction(SSP-PCR) from genome DNA library of R.anatipestifer AF strain.And then,Genome walking by thermal asymmetric interlaced PCR(Tail-PCR) was used and 2150bp DNA fragment was cloned.One ORF was found out and it encoded 624 amino acids.According to bioinformation,N-terminal first twenty-four amino acids were signal peptide,and mature protein has three transmembrane domain.Unfortunately,homophylic sequence was not found in NCBI.The results showed that the gelatinase gene was a new gene probably.
5.Study on physico-chemical property of R.anatipestifer gelatinase
The activities of gelatinase were tested byⅠcollagen fibers,casein,bovine serum albumin V and Azocoll.As the results showed they were not substrate of gelatinase.The optimum pH and temperature of the gelatinase were found to be 7.8 and 37℃.The activity of gelatinase was retained after storing at 4℃for 24h and at 37℃for 2h,but activity was not detected after storing at 65℃for 0.1h.Enzyme testing for inhibition and activation of the gelatinase indicated that EDTA,EGTA,Cu~(2+),Cd~(2+),Mn~(2+),Hg~(2+), PMSF,ADD and NBS inhibited enzymatic activity,2-ME and DEPC did not affected activity,but Ca~(2+),Mg~(2+),Na~+ and Zn~(2+) activated the activity.Moreover,Ca~(2+) counteracted the inhibition of EDTA and EGTA.
Animal model of R.anatipestifer infection was studied in order to showing the relationship of R.anatipestifer and gelatinase.The results showed the effects of gelatinase to ducklings were similar to R.anatipestifer infection at dysfunction, pathohistology,biochemistry,and so on.All results indicated that the gelatinase was a virulence factor and it was important to the infection of R.anatipestifer.
引文
[1]B.W.卡尔尼克 主编.禽病学[M].第10版.高福,苏敬良 主译.北京:中国农业出版社,1999.10:195-202
[2]程安春,汪铭书,陈孝跃,等.我国鸭疫里默氏杆菌血清型调查及新血清型的发现和病原特性[J].中国兽医学报,2003,23(4):320-323
[3]郭玉璞.北京鸭小鸭传染性浆膜炎的调查研究[J].畜牧兽医学报,1982,13(2):107-114
[4]吴札洁,陈泽祥.鸭疫里氏杆菌不同血清型广西株的分离鉴定[J].中国兽医科,2001,31(3):13-14
[5]王春霞,王林川.广东省肇庆地区鸭疫里氏杆菌病的调查[J].中国兽医科技,2001,21(7):11-12
[6]陈兴生,李布勇.海南省鸭疫里氏杆菌病流行病学调查[J].中国预防兽医学报,1999,21(3):231-232
[7]鲍国连,佟承刚,韦强,等.鸭传染性浆膜炎流行病学与病原特性研究[J].浙江农业学报,1997,9(3):161-164
[8]汪铭书,程安春,陈孝跃,等.血清4型鸭疫里氏杆菌在我国的发现极其病原特性研究[J].中国兽医杂志,2002,38(12):5-8
[9]张大丙,郭玉璞.北京地区鸭传染性浆膜炎的流行病学调查[J].中国预防兽医学报,1999,21(4):260-263
[10]张大丙,郭玉璞.我国鸭疫里氏杆菌血清型的鉴定[J].畜牧兽医学报,1999,30(6):536-542
[11]李继祥,徐刚,王孝友,等.重庆地区鸭传染性浆膜炎病原分离鉴定[J].中国预防兽医学报,2000,22(6):420-422
[12]Hendrickson J M,Hilbert K F.A new and seroious septicaemic disease of young ducks with a description of the causative organisms,N.S.cornell Vet.1932,22:239-252
[13]Bangun A,Tripathy D,Hanson LE,et al.Studies of pasteurella anatipestifer and approach to its classification[J].Avian disease1981,25:326-337.
[14]Piechulla K,Pohl S,Mannheim W,et al.Phenotypic and genetic relationships of so-called Moraxlla(pasteurella anatipestifer)to the Flavobacterium /cytophagagroup[J].Veterinary Microbiology,1986,11(3):261-270
[15]Ryll M,Hinz KH.Exclusion of strain 670/89 as type strain for serovar 20 of Riemerella anatipestifer[J].Berliner and Munchener Tierarztliche Wochenschrift,2000,113(2):65-66
[16]Segers p w,Mannheim M,Vancanneyt K,et al.Riemerella anatipestifer gen.nov,comb.tne causative agent of septicemia anserum exsudativa,and its phylogenetic affiliation within the flavobacterium-cytophaga rRNA homology group[J].Int J.Syst Bacteriol,1993,43:768-776
[17]Subramaniam S,Chua K L,Tan H M,et al.Phylogenetic position of Riemerella anatipestifer based on 16s rRNA gene sequences[J].International Journal of systematic Bacteriology 1997,47:562-565
[18]苏敬良,吕艳丽,郭玉璞.鸭疫里氏杆菌免疫原性研究Ⅰ.荚膜的形态学观察及荚膜物质的提取[J].中国兽医杂志.1998,24(1):8-10
[19]韦强,鲍国连,季权安,等.鸭疫里氏杆菌形态特征的电镜观察[J].畜牧兽医学报,2004,35(3),314-317
[20]胡清海,陈鸿军,刘晓文,等.鸭疫里氏菌生长曲线的测定[J].畜牧与兽医.2002,34(8):8-9
[21]刘颖,苏敬良,刘文华,等.鸭疫里默氏菌的药物敏感性检测[J].中国兽医杂志.2005,41(5):10-12
[22]Hinz K H,Ryll M,Kohler B.Detection of acid production from carbohydrates by Riemerella anatipestifer and related organisms using the buffered single substrate test[J].Veterinary Microbiology,1998,60:277-284
[23]胡青海,李刚,郑明球.鸭疫里氏杆菌江苏分离株G+C含量测定DNA同源性研究[J].南京农业大学学报.1998,21(4):87-91
[24]苏敬良,郭玉璞,吕艳丽.鸭疫里默氏杆菌免疫原性研究Ⅱ.荚膜提取物免疫原性测定[J].中国兽医杂志,1998,24(8):3-5
[25]黄承洪.鸭疫里默氏杆菌分离鉴定及荚膜多糖特性研究[硕士学位论文].重庆:西南大学,2006
[26]Tan H C,Loh H,Yap E H.The immunogenic proteins of Pasteurella anatipestifer[J].Singapore Journal of Primary Industries,1991,19(1):1-7
[27]Subramaniam S,Huang B,Loh H,et,al.Characterization of a predominant immunogenic outer membrane protein of Riemerella anatipestifer[J].Clin Diagn Lab Immuno,2000,7(2):168-174
[28]Tsai HJ,LIU Y T,Tseng C S,et al.Genetic variation of the ompA and 16S rRNA genes of Riemerella anatipestife[J].Avian Pathology,2005,34(4):354-359
[29]苏敬良,郭玉璞,吕艳丽等.鸭疫里氏杆菌外膜蛋白免疫原性研究[J].畜牧兽医学报,1999,30(5):444-448
[30]程安春,汪铭书,刘兆宇,等.1,2,4和5型鸭疫里默氏杆菌外膜蛋白多肽的组成及免疫原性研究[C].中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集,四川成都,2002
[31]程安春,汪铭书,刘兆宇,等.血清2型鸭疫里默氏杆菌外膜蛋白多肽的组成及免疫原性[J].中国兽医学报,2004,24(4):329-332
[32]程龙飞,施少华,李文杨,等.1、2型鸭疫里默氏菌外膜蛋白分析[J].福建农林大学学报(自然科学版),2005,34(2):260-262
[33]林文华,郑秀莲,苏杰夫,等.Riemerella anatipestifer 外膜蛋白质的电泳图谱[J].家畜卫生试验所研究报告,2000,36(1):53-58
[34]林文华.Riemerella anafipestifer外膜蛋白质的生物学特性[博士论文].台北:台湾大学,2003
[35]刘燕,韦强,鲍国连,等.鸭疫里默氏杆菌外膜蛋白生物学特性研究[J].生物工程学报,2008,24(4):586-591
[36]Harry E G.Pasteurella anatipestifer sero types isolated from cases of anatipestifer septicaemia in ducks[J].Veterinary Record,1969,84:673
[37]Brogden KA,Rhoades KR,Rimler RB.Serologic types and physiologic characteristics of 46 avian Pasteurella anatipestifer cultures[J].Avian Dis.1982,26(4):891-896
[38]Bisgaard M.Antigenic studies on Pasteurella anatipestifer species increate sedis,using slide and tube agglutination[J].Avian Pathology,1982,11(2):341- 350
[39]Sandhu T S,Leister M L.Serotypes of Pasteurella anatipestifer isolates from poultry in different countries[J].Avian Pathology,1991,20(2):233-239
[40]Loh H,Teot P,Tan H C.Serotypes of Pasteurella anatiestifer isolates from duck s in Singapore:aproposal of new serotypes[J].Avian Pathology,1992,21(4):453-459
[41]Pathanasophon P,Sawada T,Tanticharoenyos T.New serotypes of Riemerella anatipestifer isolated from ducks in Thailand[J].Avian Pathology,1995,24(1):195-199
[42]Pathanasophon P,Phuektes P,Tanticharoenyos T,et al.A potential new serotype of Riemerella anatipestifer isolated from ducks in Thailan[J].Avian Pathology,2002,31(3):267-270
[43]张大丙,郭玉璞.15型鸭疫里氏杆菌分离株的鉴定[J].中国兽医杂志,2002,38(3):23-24
[44]胡清海,张知良,苗晋锋,等.江苏安徽两省鸭疫里默氏杆菌病的流行病学调查研究[J].中国兽医科技,2001,31(8):30-31
[45]李文杨,程龙飞,彭春香,等.福建省鸭疫里默氏菌血清流行病学调查[J].中国预防兽医学报,2002,24(2):138-139
[46]韦强,鲍国连,季权安,等.浙江省鸭疫里氏杆茵的优势血清型[J].浙江农业学报,2003,15(4):255-256
[47]张大丙,曲丰发,郑献进.鸭疫里默氏杆菌血清型的研究概况[J].中国兽医杂志,2006,42(11):38-40
[48]黄承洪,李继祥,黄伟,等.重庆和四川地区鸭疫里默氏杆菌流行动态研究[J].中国预防兽医学报,2007,29(1):67-70
[49]Huang B,Subramaniam S,Chua KL,et al.Molecular fingerprinting of Riemerella anatipestifer by repetitive sequence PCR[J].Veterinary Microbiology,1999,67(3):213-219
[50]Chang C F,Hung P E,Chang Y F.Molecular characterization of a plasmid isolated from Riemerella anatipestifer[J].Avian Pathology,1998,27(4):339-345
[51]Weng S C,Lin W H,Chang Y F,et al.Identification of a virulence-associated protein homolog gene and ISRal in a plasmid of Riemerella anatipestifer[J].FEMS Microbiology Letters,1999,179(1):11-19
[52]Karen C C,Chua K L,Subramaniam S,et al.Identification and Characterization of CAMP Cohemolysin as a Potential Virulence Factor of Riemerella anatipestifer[J].Journal of Bacteriology,2002,184(7):1932-1939
[53]方饮.鸭疫里默氏杆菌抗体间接ELISA检测方法的建立及应用研究[硕士学位论文].重庆:西南大学,2007
[54]刘燕,韦强,鲍国连,等.鸭疫里默氏杆菌强、弱菌株外膜蛋白的比较蛋白质组学研究[J].生物化学与生物物理进展,2008,35(6):691-694
[55]邱振豪.鸭疫里莫氏杆菌菌株间之基因差异分析(硕士学位论文).台湾:台湾大学,2006
[56]郑娟.利用SCOTS技术筛选鸭疫华氏杆菌体内外差异表达的基因(硕士学位论文).武汉:华中农业大学,2008
[57]陈芬芬.鸭疫里默氏杆菌体内诱导抗原基因的筛选与应用研究(硕士学位论文).武汉:华中农业大学,2008
[58]Asplin WJ.A septicaemic disease of ducklings[J].Vet.Rec,1955,67:854-858
[59]Sandhu T S.Immunization of White Pekin ducklings against Pasteurlla anatipestifer infection[J].Avian Dis,1979,23(3):662-669
[60]高福,郭玉璞.小鸭传染胜浆膜炎疫苗的研究:Ⅱ型鸭疫里氏杆菌的灭活苗初步研究[J].中国兽医杂志,1987,13(6):51-53
[61]刘永德,吴祖立,叶陈梁.鸭疫巴氏杆菌与大肠杆菌二联苗研究[J].中国畜禽传染病,1997,93(2):4-6
[62]胡青海,刘晓文.鸭疫里氏杆菌病三价油乳剂灭活疫苗的研究[J].中国兽医科技,2002,32(7):7-8
[63]程安春,汪铭书,郭宇飞,等.鸭疫里默氏菌1,2,4和5型铝胶复合佐剂四价灭活疫苗的研究[J].黑龙江畜牧兽医,2004,(12):9-13
[64]黄承锋,吕敏娜,张玉团,等.鸭疫里默氏杆菌和大肠杆菌双联灭活蜂胶苗研制及应用[J].广东畜牧兽医科技,2006,22(2):34-35
[65]Sandhu T S.Immunogenicity and safety of alive Pasteurella anatipestifer vaccine in White Pekin ducklings:Laboratory and field trials[J].Avian Pathology,1991,20(3):423-432
[66]Higgins D A,Henry R.R,Kounev ZV.Duck immune responses to R.anatipestifer vaccines[J].Developmental and Comparative Immunology,2000,24(2-3):153-167
[67]赵洁.鸭疫里默氏杆菌自然弱毒菌株的分离鉴定及特性研究[硕士学位论文].重庆:西南大学,2008
[68]黄国安.鸭疫里氏杆菌毒力相关蛋白基因VapD1之次单位疫苗及DNA疫苗建构及对鸭免疫保护效果[D].台湾大学兽医学研究所,2002
[69]李继祥,徐刚,杜华茂,等.抗鸭传染性浆膜炎兽药及其制备方法.中华人民共和国国家知识产权局发明专利公报,2001,17(14):13
[70]Balebona MC,Mori(?)go MA,Borrego JJ.Role of extracellular products in the pathogenicity of Vibrio strainson cultured gilt-head seabream(Sparus aurata)[J].Microbiologia,1995,11(4):439-446
[71]Coque TM,Patterson JE,Steckelberg JM,ET AL.Incidence of hemolysin,gelatinase,and aggregation substance among enterococci isolated from patients with endocarditis and other infections and from feces of hospitalized and community-based persons[J].Infect Dis,1995,171(5):1223-1229
[72]Kanemitsu K,Nishino T,Kunishima H,et al.Quantitative determination of gelatinase activity among enterococci[J].Journal of Microbiological Methods,2001,47(1):11-16
[73]Eisner H A,Sobottka I,Mack D,et al.Virulence Factors of Enterococcus faecalis and Enterococcus faecium Blood Culture Isolates[J].Eurooean Journal of Clinical Microbiology & Infectious Diseases,2000,19(1):39-42
[74]Roberta C,Monica I,Lucia B,et al.Survey for virulence determinants among Enterococcus faecalis isolated from different sources[J].Journal of Medical Microbiology,2004,53(1):13-20
[75]Kavindra V S,Xiang Q,George M W,et al.Generation and Testing of Mutants of Enterococcus faecalis in a Mouse[J].The Journal of Infectious Diseases 1998,178(11):1416-1420
[76]Qin X,Kavindra V S,George M W,et al.Characterization of fsr,a Regulator Controlling Expression of Gelatinase and Serine Protease in Enterococcus faecalis OG1RF[J].Journary of Bacteriology,2001,183(11):3372-3382
[77]Costi DS,Eleftherios M,Kavindra V S,et al.Virulence Effect of Enterococcus faecalis Protease Genes and the Quorum-Sensing Locus fsr in Caenorhabditis elegans and Mice[J].Infection and Immunity,2002,70(10):5647-5650
[78]Michael E,Eleftherios M,Frederick M A,et al.Contribution of Gelatinase,Serine Protease,and fsr to the Pathogenesis of Enterococcus faecalis Endophthalmitis[J].Infection and Immunity,2004,72(6):3628-3633
[79]Zeng J,Teng F,George M.W,et al.Translocation of Enterococcus faecalis Strains across a Monolayer of Polarized Human Enterocyte-Like T84 Cells[J].Journal of Clinical Microbiology,2004,42(3):1149-1154
[80]Zeng J,Teng F,Barbara E M.Gelatinase Is Important for Translocation of Enterococcus faecalis across Polarized Human Enterocyte-Like T84 Cells[J].Infection and Immunity,2005,73(3):1606-1612
[81]Hoiby N,Krogh J H,Moser C,et al.Pseudomonas aeruginosa and the in vitroand in vivo biofilm mode of growth[J].Microbes and Infection,2001,3(1):23-35
[82]Costerton JW,Stewart PS,Greenberg EP,et al.Bacterial biofilms:A common cause of persistent infections[J].Science,1999,284(5418):1318-1322.
[83]李新,王金良.细菌耐药性机制的研究进展[J].国外医学临床生物化学与检验学分册,2001,22(5):261-262
[84]Lynn E H,Marta.The Enterococcus faecalis fsr two-Component system controls biofilm development through production of gelatinase[J].Journal of Bacteriology,2004,186(17):5629-5639
[85]Blanco A R,Simona L T M,Babini G,et al.Epigallocatechin-3-gallate inhibits gelatinase activity of some bacterial isolates from ocular infection,and limits their invasion through gelatin[J].Biochimica and Biophysica Acta,2003,1620:273-281
[86]Pirkko L M,Don B.C,Florence A,et al.Purification and Substrate Specificity of a Strongly Hydrophobic Extracellular Metalloendopeptidase("Gelatinase") from Streptococcus fuecalis (Strain OGI-IO)[J].The Journal of Biological Chemistry,1989,264(6):3325-3334
[87]Yan AS,Mark CS,Ping HE,et al.Nucleotide Sequence of the Gelatinase Gene(gelE) fromEnterococcus faecalis subsp.Liquefaciens [J].Infection and Immunity, 1991, 59(1):415-420
[88]Qin X, Kavindra VS, George MW, et al.Effects of Enterococcus faecalis fsr Genes on Productionof Gelatinase and a Serine Protease and Virulence [J].Infection and Immunity, 2000,68(5): 2579-2586
[89]Nakayama J, Cao Y, Horii T, et al.Gelatinase biosynthesis-activating pheromone: apeptide lactone that mediates a quorum sensing in Enterococcus faecalis [J].Molecular Microbiology,2001, 41(1):145-154
[90]Dupre I, Zanetti S, Schito A M, et al.Incidence of virulence determinants in clinical Enterococcus faecium and Enterococcus faecalis isolates collected in Sardinia (Italy) [J].Journal of Medical Microbiology, 2003, 52(2):491-498
[91]Nakayama J, Kariyama R , Kumon H.Description of a 23.9-Kilobase chromosomal deletion containing a region encoding fsr genes which mainly determines the gelatinase negative phenotype of clinical isolates of enterococcus faecalis in urine [J].Applied and Environmental Microbiology, 2002, 68(6): 3152-3155
[92]Simon GL, Gorbach SL.Intestinal flora in health and disease [J].Gastroenterology 1984;86(1):174-193
[93]Gibson SA, Macfarlane GT.Studies on the proteolytic activity of Bacteroides fragilis [J].J Gen Microbiol, 1988,134(1): 19-27
[94]Weikel C S, Grieco F D, Reuben J, et al.Human colonic epithelial cells, HT29/C1, treated with crude Bacteroides fragilis enterotoxin dramatically alter their morphology[J].Infection and Immunity, 1992, 60(2):321-327
[95]Tassell LV, Lyerly DM, Wilkins TD.Purification and characterization of an enterotoxin from Bacteroides fragilis [J].Infection and Immunity, 1992, 60(4): 1343-1350
[96]Tassell LV, Lyerly D M, Wilkins T D.Characterization of Enterotoxigenic Bacteroides fragilis by a Toxin-Specific Enzyme-Linked Immunosorbent Assay [J].Clinical and Diagnostic Laboratory Immunology, 1994, l(5):578-584
[97]Eaton TJ, Gasson MJ.Molecular screening of enterococcus virulence determinants and potential for genetic exchange between food and medical isolates [J].Applied and Environmental Microbiology, 2001, 67(4): 1628-1635
[98]Richard J, David M, Roger L, et al.Proteolytic activity of the bacteroides fragilis enterotoxin causes fluid secretion and intestinal damage in vivo [J].Infection and immunity, 1995, 63(10):3820-3826
[99]Moncrief J S, Richard J R, Barroso L A, et al.The enterotoxin of bacteroides fragilis is a metalloprotease [J].Infection and immunity, 1995, 63(1):175—181
[100]Augusto A F, Simy L B, Jai W S, et al.Mutation of the zinc-binding metalloprotease motif affects bacteroides fragilis toxin activity but does not affect propeptide processing[J].Infection and immunity, 2005, 73(8): 5273-5277
[101]Hatheway C L.Toxigenic clostridia[J].Clin Microbiol Rev.1990,3(1):66-98
[102]Kuniko Y,Osamu M,Junzaburo M,et al.Cloning and nucleotide sequence analysis of the colh gene from clostnidium histolyticum encoding a collagenase and a gelatinase[J].Journal of Bacteriology,1994,176(21):6489-6496
[103]Matsushita O,Chang M J,Katayama S,et al.Gene duplication and multiplicity of collagenases in clostridium histolyticum[j].Journal of Bacteriology,1999,181(3):923-933
[104]Matsushita O K,Yoshihara S I,Katayama J M,et al.Purification and character- ization of a Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the correspondinggene[J].J Bacteriol1994,176(1):149-156
[105]Rada E,Zambrano E A,Rodr(?)guez I Y,et al.Gelatinase activity in Mycobacterium bovis protein extract[J],Int J Lepr Other Mycobact Dis.2002,70(4):260-268
[106]Papageorgakopoulou N,Plakoutsi F,Stephanopoulou A,et al.Study of the gelatinolytic activities Escherichia coli cells before and after starvation in seawater by substrate gel electrophoresis[J].Microbiol Res.1996,151(3):329-335
[107]Conlan JW,Baskerville A,Ashworthl AE.Separation of Legionella pneumophila proteases and purification of a protease which produces lesions like those of Legionnaires's disease in guinea pig lung[J].Journal of general microbiology,1986,32(6):1565-1574
[108]Dowling JN,Saha AK,Glew RH.Virulence factors of the family Legionellaceae[J].Microbiol Rev,1992,56(1):32-60
[109]柯娜,肖昌松,应启峰,等.一个可降解直链烷基苯磺酸盐的新种[J].微生物学报,2003;42(1):1-7
[110]J.萨姆布鲁克.D.W.拉塞尔著.分子克隆实验指南(上下册)[M].北京:科学出版社.2003
[111]C.W.迪芬巴赫.G.S.德维克斯勒.PCR技术实验指南[M].北京:科学出版社.2000
[112]罗玉峰,钱心元,房燕丽.猪丹毒QL 251弱毒菌株的稳定性试验[J].中国兽药杂志,1997,31(1):19-21
[113]Roche SM,Gracieux P,Albert I,et al.Experimental validation of low virulence in field strains of listeria monocytogenes[J].Infection and Immunity,2003,7 1(6):3429-3436
[114]Pickett M J,Greenwood J R,Harvey S M.Tests for detecting degradation of gelatin:comparison of five methods[J].J Clin Microbiol,1991,29(10):2322-2325
[115]Frazier W C.A method for the deteetion of changes in gelatin due to bacteria[J].Journary infection Diseases,1926,39(2):302-309
[116]Amanda E S,Tonima D,Kanwal M,et al.Potential involvement of gelatinases and their inhibitors in mannheimia haemolytica pneumonia in cattle[J].Infection and immunity,2004,72(8):4393-4400
[117]Ratnikov BI,Deryugina EI,Strongin AY.Gelatin zymography and substrate cleavage assays of matrix metalloproteinase-2 in breast carcinoma cells overexpressing membrane type-1matrix metalloproteinase[J].Lab Invest.2002,82(11):1583-1590
[118]Marta T,Rafael F.Assessment of Gelatinases(MMP-2 and MMP-9 by Gelatin Zymography [J].Methods in Molecular Medicine,2001,57(1):163-174
[119]Shona R E,Nell H,Alan J.K,et al.Matrix metalloproteinase expression and activity in human airwaysmooth muscle cells[J].British Journal of Pharmacology,2004,142(7):1318-1324
[120]徐宁.发光杆菌产生胶原酶及其应用于制备胶原蛋白的研究(硕士学位论文).四川成都:四川大学,2004
[121]张龙翔,张庭芳,李令暖,等.生化实验方法和技术[M].北京:高等教育出版社,1997,163-165
[122]Rosen H.A modified ninhydrin colorimetric analysis for amino acids[J].Archives of Biochemistry and Biophysics,1957,67(1):10-15
[123]Wang HT,Hsu JT.Optimal protease production condition for Prevotella ruminicola 23 and characterization of its extracellular crude protease[J].Anaerobe,2005,11(3):155-62
[124]Ust(?)ri z F J,Laca A,Garc(?)a L A,et al.Fermentation conditions increasing protease production by Serratia marcescens in fresh whey[J].Rev.T(?)c.Ing.Univ.Zulia,2008,31(1):79 - 89
[125]孟喜龙,刘永杰,陆承平.嗜水气单胞菌产弹性蛋白酶条件的优化[J].南京农业大学学报,2007,30(2):98-101
[126]Takac s,Elmas S,Calik P,et al.Separation of the protease enzymes of Bacillus licheniformis from the fermentation medium by crossflow ultrafiltration[J].Journal of Chemical Technology and Biotechnology,2000,75(2):491-499
[127]Flaschel E,Wandrey C,Kula M R.Ultrafltration for the separation of biocatalysts[J].Advances in Biochemical Engineering/Biotechnology,1983,26(1):73-82
[128]Kalashnikova EE,Chemyshova MP,Ignatov VV.The Extracellular Proteases of the Phytopatho- genic Bacterium Xanthomonas campestris[J].Microbiology,2003,72(4):443-447
[129]Coseteng M Y,Lee C Y.Changes in apple polyphendoxidase and polyphenol concentrations in relation to degree of browing[J].Journal of Food Science,1987,52(1):985-989
[130]汪家政编著.蛋白质技术手册[M].北京:科学出版社.2000
[131]Gold O G,Jordan H V,van Houte J.The prevalence of enterococci in the human mouth and their pathogenicity in animal models[J].Arch.Oral BWl.1975,20(7):473-477
[132]殷震,刘景华.动物病毒学[M].第2版.北京:中国农业出版社,1997:210-231
[133]Parody J P,Alvarez M D,Quiroga A,et al.Hepatocytes isolated from preneoplastic rat livers are resistant to ethacrynic acid cytotoxicity[J].Arch Toxicol,2007,81(8):565-573
[134]Cullen K J,Newkirk K A,Schumaker L M,et al.Glutathione S-transferase pi amplification is associated with cisplatin resistance in head and neck squamous cell carcinoma cell lines and primary tumors[J].Cancer Res,2003,63(23):97-102
[135]储卫华,陆承平.嗜水气单胞菌胞外蛋白酶对鲫鱼的致病性[M].南京农业大学学报,2000,23(2):80-84
[136]黎晓敏,郭丽敏,唐发书,等.鸭传染性浆膜炎病鸭免疫器官的光镜及电镜观察[J].畜牧兽医学报,2004,35(6):702-704
[137]Sachar L A,Winter K K,Sicher N,et al.Photometric method for estimation of elastase activity[J].Proc Soc Exp Biol Med,1955,90(2):323-326
[138]刘玉斌,苟仕金.动物免疫学实验技术[M].长春:吉林科学技术出版社,1989,234-236
[139]方静,崔恒敏,彭西,何敏.雏鸭胸腺生长指标的动态观测及组织学观察[J].中国兽医科学,2006,36(5):401-405
[140]陈龙,庞训胜,高勤学等.感染IBDV雏鸡血液激素水平和ANAE阳生淋巳细胞动态变化[J].中国兽医学报,1999,19(1):22-25
[141]聂其灼,陈小慧.牛白血病病毒感染牛群外周血内酸性-醋酸萘酯酶阳性淋巴细胞百分率变化的研究[J].南京农业大学学报,1985,(1):60-67
[142]朱立平,陈学清主编.免疫学常用实验方法[M].北京:人民军医出版社,2003,120-122
[143]郭峰,虞紫茜,赵中平,等.红细胞免疫功能的初步研究[J].中华医学杂志,1982,62(12):715-716
[144]饶家荣,王旭东,钱光云.樱桃谷鸭红细胞免疫功能研究及其意义[J].畜牧兽医杂志,1999,18(3):20-22
[145]黎德兵,崔恒敏,赵翠燕.鸭疫里默扦菌病发病讥理的研究Ⅳ-试验感染雏鸭红细胞免疫功能的变化[J].黑龙江畜牧兽医,2007,(9):61-62
[146]林祥梅,赵增连.鸭源流感病毒感染对鸡和家鸭红细胞免疫功能的影响[J].中国兽医学报,2000,20(2):135-137
[147]张秋胜,陈昌福.异育银鲫对嗜水气单胞茵灭活菌苗的免疫应答[J].华中农业大学学报,2001,20(3):271-274
[148]王德云,胡元亮,孔祥峰,等.中药成分对雏鸡外周血T淋巴细胞转化的影响[J].中国兽医学报,2004,24(6):578-580
[149]王德云,胡元亮,孔祥峰等.中药成分对培养的鸡脾脏淋巴细胞增殖的影响[J].畜牧兽医学报,2005,36(11),1210-1214
[150]储岳峰,李祥瑞,胡元亮.9种中药成分对小鼠免疫细胞活性的影响[J].南京农业大学学报,2004,27(1):97-100
[151]姚根宏,侯亚义,沈苏南,等.睾酮在体外对脾淋巴细胞增殖反应和分泌IgG的影响[J].卫生研究,2002,31(5):333-335
[152]Lee J D,Tsail Y,Chen C H,et al.Blood brain barrier dysfunction occurring in mice infected with Angiostrongyluscantonensis[J].Acta Tropica,2006,97(2):204-211
[153]Seyfert S,Becher A,Ohring R,et al.The permeability of the blood CSF barrier in hydrocephalus,polyradiculitis and meningitis[J].J Neurol,2004,251:355-356
[154]Siegel I,Liu TL,Gleicher N.The red-cell immune system[J].Lancet,1981,2(8246):556-559
[155]郭峰,张励力.用 C3b致敏感酵母菌测定血清免疫复合物[J].上海免疫学杂志,1982,2(1):32-37
[156]Robert C,Monica I,Lucia B,et al.Survey for virulence determinants among Enterococcus faecalis isolated from different sources[J].Journal of Medical microbiology,2004, 25(53):13-20
[157]Gumey K J,Estrada E Y,Rosenberg G A,et al.Blood-brain barrier disruption bystromelysin-1facilitates neutrophil infiltration in neuroinflammation[J].Neurobiology of Disease,2006,23(10):87-96
[158]傅童生,傅运生.实验性鸭疫里默氏菌病临床病理学动态变化[J].中国兽医学报,2002,22(6):563-564
[159]黎德兵,崔恒敏,章蕾.鸭疫里默氏杆菌病发病机理的研究Ⅰ:人工感染雏鸭部分血液指标的动态变化[J].黑龙江畜牧兽医,2004,(1):8-10
[160]ShinYP,Yong PS,Chong HK,et al.Immune evasion of enterococcus faecalis by an extracellular gelatinase that cleaves C3 and iC3[J].The Journal of Immunology,2008,181:6328-6336
[161]李影林.中华医学检验全书[M].北京:人民卫生出版社,1996
[162]Anderas W,Gregor Z,Joachim G,et al.Decreased virulence of a pneumolysin deficient strain of streptococcus pneumoniae in murine meningitis[J].Infect Irnrnun,2002,20(11):6504-6508
[163]Catarina R,Gabriei AM,Gustavo H,et al.Acute blood brain barrier permeabili- zation in rats after systemic phoneutria nigriventer venom[J].Brain Res,2007,(1149):18-29
[164]Weller R O.Pathology of cerebrospinal fluid and interstitial fluid of the CNS:significance for alzheimer disease,prion disorders and multiple sclerosisb[J].J Neuropathol Exp Neurol,1998,(57):885-894
[165]Jortner B S,Porro R,Leibovitz L,et al.Centralnervous system lesions of spon- taneous Pasteurella anatipestifer infection in ducklings[J].AvianDis,1969,13(1):27-35
[166]Vorbrodt A W,Dobrogowska D H,Ueno M,et al.lmmunocytochemical studies of protam ine-induced blood brain barrier opening to endogenous albumin[J].Acta Neuropathol,1995,(89):491-499
[167]Diane A,Mario K,Gary A,et al.Neutral proteases and disruption of the blood brain barrier in rat[J].Brain Res,1997,767(2):259-264
[168]Matsudaira P.Sequence from picomole quantities of proteins electroblotted onto polyvinyl-lidene difluoride membranes[J].J Biol Chem,1987,262(21):10035-10038
[169]周建,王小行,高小平.转移缓冲液对蛋白质印迹的影响[J].生物化学与生物物理进展,2000,27(3):331-332
[170]Moncrief JS,Richard JR,Lisa AB,et al.The Enterotoxin of Bacteroides fragilis is a Metalloprotease[J].Infection and immunity,1995,63(1):175-181
[171]Shamala V,Ames G F L.Genome walking by single specific primer-polymerase chain reaction:SSP-PCR[J].Gene,1989,84(1):1-8
[172]颜子颖,王海林译.精编分子生物学实验指南[M].北京:科学出版社,1999
[173]Liu YG,Whittier RF.Thermal asymmetric interlaced PCR:automatable ampli- fication and sequencing of insert end fragments from P1 and YAC clones for chromo- some walking[J].Genomics,1995,25(3):674-681
[174]Li ZT,Gray DJ.Isolation by improved thermal asymmetric interlaced PCR and characterization of a seedspecific 2S albumin gene and its promoter from grape(Vitis vinifera L)[J].Genome,2005,48(2):312-320
[175]孔俊,刘文轩.Kas基因启动子区的克隆及功能初步分析[J].上海大学学报(自然科学版),2005,11(6):652-656
[176]仇艳光,田景汉,葛荣朝,等.TAIL-PCR的改良及其在分离小麦基因启动子中的应用[J].生物工程学报,2008,24(4):695-699
[177]Wang L,Gao Q,Wang YZ,et al.Isolation and sequence analysis of two CYC-like genes,SiCYC1A and SiCYC1B,from zygomorphic and actinomorphic cultivars of Saintpaulia ionantha(Gesneriaceae)[J].Acta Phytotaxonomica Sinica,2006,44(4):353-361
[178]杨英军,周鹏.番木瓜proteinase omega基因启动子的克隆及功能初步研究[J].云南植物研究2005,27(5):545-551
[179]薛晓晶,徐福洲,史爱华,等.副猪嗜血杆菌aroA基因鉴定及遗传进化分析[J].微生物学报,2008,48(8):1100-1103
[180]Patricia M,Larry B.Rapid identification of gelatin and casein hydrolysis using TCA[J].Journal of Microbiological Methods,2007,69(2) 391-393
[181]谢莉萍,林静瑜,肖锐,等.合浦珠母贝碱性磷酸酶的分离纯化与性质研究.海洋科学,2000,24(10):37-40
[182]Mayerhofer HJ,Marshall RT,White CH,et al.Characterization of a Heat-Stable Protease of Pseudomonas fluorescens P26[J].Applied microbiology,1973,25(1):44-48
[183]Hoshino T,Ishizaki K,Sakamoto T,et al.Isolation of a Pseudomonas species from fish intestine that produces a protease active at low temperature[J].Letters in Applied Microbiology,1997,25(1):70-72
[184]朱俭,曹凯鸣,周润琦,等.生物化学实验[M].上海:上海科学技术出版社,1981
[185]Basma G,Alya SK,Moncef N.Stability studies of protease from Bacillus cereus BG1[J].Enzyme and Microbial Technology,2003,32(3):513-518
[186]姜军,杨晓达,王夔.汞和金属离子及多硫化物对木瓜蛋白酶活性的抑制作用[J].中国药学,2007,16(1):13-18
[187]Amtul J S,Amtul N S,Noreen K.Comparison in effect of different metal ions,pH and reducing agent on the protease activity in human hyper mature and mature cataract[J].Journal Zhejiang Univ Sci B.2007,8(8):599-603
[188]John WR,Keith NJ.Proteolytic activity of a rumen anaerobic fungus[J].FEMS Microbiology Letters,2006,29(1-2):19-25
[189]周海梦,王洪睿.蛋白质化学修饰[M].北京:清华大学出版社,1998,19-43
[190]张树政,孟广震,何忠效.酶学研究技术(上册)[M].北京:科学出版社,1987,289-311
[191]储卫华,陆承平.嗜水气单胞菌胞外蛋白酶的化学修饰[J].中国生物化学与分子生物学报,2001,17(3):372-375
[192]潘继承,汪劲松,向显智,等.粘质赛氏菌胞外蛋白酶的化学修饰[J].生物化学杂志,1997,13(4):483-486
[2]程安春,汪铭书,陈孝跃,等.我国鸭疫里默氏杆菌血清型调查及新血清型的发现和病原特性[J].中国兽医学报,2003,23(4):320-323
[3]郭玉璞.北京鸭小鸭传染性浆膜炎的调查研究[J].畜牧兽医学报,1982,13(2):107-114
[4]吴札洁,陈泽祥.鸭疫里氏杆菌不同血清型广西株的分离鉴定[J].中国兽医科,2001,31(3):13-14
[5]王春霞,王林川.广东省肇庆地区鸭疫里氏杆菌病的调查[J].中国兽医科技,2001,21(7):11-12
[6]陈兴生,李布勇.海南省鸭疫里氏杆菌病流行病学调查[J].中国预防兽医学报,1999,21(3):231-232
[7]鲍国连,佟承刚,韦强,等.鸭传染性浆膜炎流行病学与病原特性研究[J].浙江农业学报,1997,9(3):161-164
[8]汪铭书,程安春,陈孝跃,等.血清4型鸭疫里氏杆菌在我国的发现极其病原特性研究[J].中国兽医杂志,2002,38(12):5-8
[9]张大丙,郭玉璞.北京地区鸭传染性浆膜炎的流行病学调查[J].中国预防兽医学报,1999,21(4):260-263
[10]张大丙,郭玉璞.我国鸭疫里氏杆菌血清型的鉴定[J].畜牧兽医学报,1999,30(6):536-542
[11]李继祥,徐刚,王孝友,等.重庆地区鸭传染性浆膜炎病原分离鉴定[J].中国预防兽医学报,2000,22(6):420-422
[12]Hendrickson J M,Hilbert K F.A new and seroious septicaemic disease of young ducks with a description of the causative organisms,N.S.cornell Vet.1932,22:239-252
[13]Bangun A,Tripathy D,Hanson LE,et al.Studies of pasteurella anatipestifer and approach to its classification[J].Avian disease1981,25:326-337.
[14]Piechulla K,Pohl S,Mannheim W,et al.Phenotypic and genetic relationships of so-called Moraxlla(pasteurella anatipestifer)to the Flavobacterium /cytophagagroup[J].Veterinary Microbiology,1986,11(3):261-270
[15]Ryll M,Hinz KH.Exclusion of strain 670/89 as type strain for serovar 20 of Riemerella anatipestifer[J].Berliner and Munchener Tierarztliche Wochenschrift,2000,113(2):65-66
[16]Segers p w,Mannheim M,Vancanneyt K,et al.Riemerella anatipestifer gen.nov,comb.tne causative agent of septicemia anserum exsudativa,and its phylogenetic affiliation within the flavobacterium-cytophaga rRNA homology group[J].Int J.Syst Bacteriol,1993,43:768-776
[17]Subramaniam S,Chua K L,Tan H M,et al.Phylogenetic position of Riemerella anatipestifer based on 16s rRNA gene sequences[J].International Journal of systematic Bacteriology 1997,47:562-565
[18]苏敬良,吕艳丽,郭玉璞.鸭疫里氏杆菌免疫原性研究Ⅰ.荚膜的形态学观察及荚膜物质的提取[J].中国兽医杂志.1998,24(1):8-10
[19]韦强,鲍国连,季权安,等.鸭疫里氏杆菌形态特征的电镜观察[J].畜牧兽医学报,2004,35(3),314-317
[20]胡清海,陈鸿军,刘晓文,等.鸭疫里氏菌生长曲线的测定[J].畜牧与兽医.2002,34(8):8-9
[21]刘颖,苏敬良,刘文华,等.鸭疫里默氏菌的药物敏感性检测[J].中国兽医杂志.2005,41(5):10-12
[22]Hinz K H,Ryll M,Kohler B.Detection of acid production from carbohydrates by Riemerella anatipestifer and related organisms using the buffered single substrate test[J].Veterinary Microbiology,1998,60:277-284
[23]胡青海,李刚,郑明球.鸭疫里氏杆菌江苏分离株G+C含量测定DNA同源性研究[J].南京农业大学学报.1998,21(4):87-91
[24]苏敬良,郭玉璞,吕艳丽.鸭疫里默氏杆菌免疫原性研究Ⅱ.荚膜提取物免疫原性测定[J].中国兽医杂志,1998,24(8):3-5
[25]黄承洪.鸭疫里默氏杆菌分离鉴定及荚膜多糖特性研究[硕士学位论文].重庆:西南大学,2006
[26]Tan H C,Loh H,Yap E H.The immunogenic proteins of Pasteurella anatipestifer[J].Singapore Journal of Primary Industries,1991,19(1):1-7
[27]Subramaniam S,Huang B,Loh H,et,al.Characterization of a predominant immunogenic outer membrane protein of Riemerella anatipestifer[J].Clin Diagn Lab Immuno,2000,7(2):168-174
[28]Tsai HJ,LIU Y T,Tseng C S,et al.Genetic variation of the ompA and 16S rRNA genes of Riemerella anatipestife[J].Avian Pathology,2005,34(4):354-359
[29]苏敬良,郭玉璞,吕艳丽等.鸭疫里氏杆菌外膜蛋白免疫原性研究[J].畜牧兽医学报,1999,30(5):444-448
[30]程安春,汪铭书,刘兆宇,等.1,2,4和5型鸭疫里默氏杆菌外膜蛋白多肽的组成及免疫原性研究[C].中国畜牧兽医学会禽病学分会第十一次学术研讨会论文集,四川成都,2002
[31]程安春,汪铭书,刘兆宇,等.血清2型鸭疫里默氏杆菌外膜蛋白多肽的组成及免疫原性[J].中国兽医学报,2004,24(4):329-332
[32]程龙飞,施少华,李文杨,等.1、2型鸭疫里默氏菌外膜蛋白分析[J].福建农林大学学报(自然科学版),2005,34(2):260-262
[33]林文华,郑秀莲,苏杰夫,等.Riemerella anatipestifer 外膜蛋白质的电泳图谱[J].家畜卫生试验所研究报告,2000,36(1):53-58
[34]林文华.Riemerella anafipestifer外膜蛋白质的生物学特性[博士论文].台北:台湾大学,2003
[35]刘燕,韦强,鲍国连,等.鸭疫里默氏杆菌外膜蛋白生物学特性研究[J].生物工程学报,2008,24(4):586-591
[36]Harry E G.Pasteurella anatipestifer sero types isolated from cases of anatipestifer septicaemia in ducks[J].Veterinary Record,1969,84:673
[37]Brogden KA,Rhoades KR,Rimler RB.Serologic types and physiologic characteristics of 46 avian Pasteurella anatipestifer cultures[J].Avian Dis.1982,26(4):891-896
[38]Bisgaard M.Antigenic studies on Pasteurella anatipestifer species increate sedis,using slide and tube agglutination[J].Avian Pathology,1982,11(2):341- 350
[39]Sandhu T S,Leister M L.Serotypes of Pasteurella anatipestifer isolates from poultry in different countries[J].Avian Pathology,1991,20(2):233-239
[40]Loh H,Teot P,Tan H C.Serotypes of Pasteurella anatiestifer isolates from duck s in Singapore:aproposal of new serotypes[J].Avian Pathology,1992,21(4):453-459
[41]Pathanasophon P,Sawada T,Tanticharoenyos T.New serotypes of Riemerella anatipestifer isolated from ducks in Thailand[J].Avian Pathology,1995,24(1):195-199
[42]Pathanasophon P,Phuektes P,Tanticharoenyos T,et al.A potential new serotype of Riemerella anatipestifer isolated from ducks in Thailan[J].Avian Pathology,2002,31(3):267-270
[43]张大丙,郭玉璞.15型鸭疫里氏杆菌分离株的鉴定[J].中国兽医杂志,2002,38(3):23-24
[44]胡清海,张知良,苗晋锋,等.江苏安徽两省鸭疫里默氏杆菌病的流行病学调查研究[J].中国兽医科技,2001,31(8):30-31
[45]李文杨,程龙飞,彭春香,等.福建省鸭疫里默氏菌血清流行病学调查[J].中国预防兽医学报,2002,24(2):138-139
[46]韦强,鲍国连,季权安,等.浙江省鸭疫里氏杆茵的优势血清型[J].浙江农业学报,2003,15(4):255-256
[47]张大丙,曲丰发,郑献进.鸭疫里默氏杆菌血清型的研究概况[J].中国兽医杂志,2006,42(11):38-40
[48]黄承洪,李继祥,黄伟,等.重庆和四川地区鸭疫里默氏杆菌流行动态研究[J].中国预防兽医学报,2007,29(1):67-70
[49]Huang B,Subramaniam S,Chua KL,et al.Molecular fingerprinting of Riemerella anatipestifer by repetitive sequence PCR[J].Veterinary Microbiology,1999,67(3):213-219
[50]Chang C F,Hung P E,Chang Y F.Molecular characterization of a plasmid isolated from Riemerella anatipestifer[J].Avian Pathology,1998,27(4):339-345
[51]Weng S C,Lin W H,Chang Y F,et al.Identification of a virulence-associated protein homolog gene and ISRal in a plasmid of Riemerella anatipestifer[J].FEMS Microbiology Letters,1999,179(1):11-19
[52]Karen C C,Chua K L,Subramaniam S,et al.Identification and Characterization of CAMP Cohemolysin as a Potential Virulence Factor of Riemerella anatipestifer[J].Journal of Bacteriology,2002,184(7):1932-1939
[53]方饮.鸭疫里默氏杆菌抗体间接ELISA检测方法的建立及应用研究[硕士学位论文].重庆:西南大学,2007
[54]刘燕,韦强,鲍国连,等.鸭疫里默氏杆菌强、弱菌株外膜蛋白的比较蛋白质组学研究[J].生物化学与生物物理进展,2008,35(6):691-694
[55]邱振豪.鸭疫里莫氏杆菌菌株间之基因差异分析(硕士学位论文).台湾:台湾大学,2006
[56]郑娟.利用SCOTS技术筛选鸭疫华氏杆菌体内外差异表达的基因(硕士学位论文).武汉:华中农业大学,2008
[57]陈芬芬.鸭疫里默氏杆菌体内诱导抗原基因的筛选与应用研究(硕士学位论文).武汉:华中农业大学,2008
[58]Asplin WJ.A septicaemic disease of ducklings[J].Vet.Rec,1955,67:854-858
[59]Sandhu T S.Immunization of White Pekin ducklings against Pasteurlla anatipestifer infection[J].Avian Dis,1979,23(3):662-669
[60]高福,郭玉璞.小鸭传染胜浆膜炎疫苗的研究:Ⅱ型鸭疫里氏杆菌的灭活苗初步研究[J].中国兽医杂志,1987,13(6):51-53
[61]刘永德,吴祖立,叶陈梁.鸭疫巴氏杆菌与大肠杆菌二联苗研究[J].中国畜禽传染病,1997,93(2):4-6
[62]胡青海,刘晓文.鸭疫里氏杆菌病三价油乳剂灭活疫苗的研究[J].中国兽医科技,2002,32(7):7-8
[63]程安春,汪铭书,郭宇飞,等.鸭疫里默氏菌1,2,4和5型铝胶复合佐剂四价灭活疫苗的研究[J].黑龙江畜牧兽医,2004,(12):9-13
[64]黄承锋,吕敏娜,张玉团,等.鸭疫里默氏杆菌和大肠杆菌双联灭活蜂胶苗研制及应用[J].广东畜牧兽医科技,2006,22(2):34-35
[65]Sandhu T S.Immunogenicity and safety of alive Pasteurella anatipestifer vaccine in White Pekin ducklings:Laboratory and field trials[J].Avian Pathology,1991,20(3):423-432
[66]Higgins D A,Henry R.R,Kounev ZV.Duck immune responses to R.anatipestifer vaccines[J].Developmental and Comparative Immunology,2000,24(2-3):153-167
[67]赵洁.鸭疫里默氏杆菌自然弱毒菌株的分离鉴定及特性研究[硕士学位论文].重庆:西南大学,2008
[68]黄国安.鸭疫里氏杆菌毒力相关蛋白基因VapD1之次单位疫苗及DNA疫苗建构及对鸭免疫保护效果[D].台湾大学兽医学研究所,2002
[69]李继祥,徐刚,杜华茂,等.抗鸭传染性浆膜炎兽药及其制备方法.中华人民共和国国家知识产权局发明专利公报,2001,17(14):13
[70]Balebona MC,Mori(?)go MA,Borrego JJ.Role of extracellular products in the pathogenicity of Vibrio strainson cultured gilt-head seabream(Sparus aurata)[J].Microbiologia,1995,11(4):439-446
[71]Coque TM,Patterson JE,Steckelberg JM,ET AL.Incidence of hemolysin,gelatinase,and aggregation substance among enterococci isolated from patients with endocarditis and other infections and from feces of hospitalized and community-based persons[J].Infect Dis,1995,171(5):1223-1229
[72]Kanemitsu K,Nishino T,Kunishima H,et al.Quantitative determination of gelatinase activity among enterococci[J].Journal of Microbiological Methods,2001,47(1):11-16
[73]Eisner H A,Sobottka I,Mack D,et al.Virulence Factors of Enterococcus faecalis and Enterococcus faecium Blood Culture Isolates[J].Eurooean Journal of Clinical Microbiology & Infectious Diseases,2000,19(1):39-42
[74]Roberta C,Monica I,Lucia B,et al.Survey for virulence determinants among Enterococcus faecalis isolated from different sources[J].Journal of Medical Microbiology,2004,53(1):13-20
[75]Kavindra V S,Xiang Q,George M W,et al.Generation and Testing of Mutants of Enterococcus faecalis in a Mouse[J].The Journal of Infectious Diseases 1998,178(11):1416-1420
[76]Qin X,Kavindra V S,George M W,et al.Characterization of fsr,a Regulator Controlling Expression of Gelatinase and Serine Protease in Enterococcus faecalis OG1RF[J].Journary of Bacteriology,2001,183(11):3372-3382
[77]Costi DS,Eleftherios M,Kavindra V S,et al.Virulence Effect of Enterococcus faecalis Protease Genes and the Quorum-Sensing Locus fsr in Caenorhabditis elegans and Mice[J].Infection and Immunity,2002,70(10):5647-5650
[78]Michael E,Eleftherios M,Frederick M A,et al.Contribution of Gelatinase,Serine Protease,and fsr to the Pathogenesis of Enterococcus faecalis Endophthalmitis[J].Infection and Immunity,2004,72(6):3628-3633
[79]Zeng J,Teng F,George M.W,et al.Translocation of Enterococcus faecalis Strains across a Monolayer of Polarized Human Enterocyte-Like T84 Cells[J].Journal of Clinical Microbiology,2004,42(3):1149-1154
[80]Zeng J,Teng F,Barbara E M.Gelatinase Is Important for Translocation of Enterococcus faecalis across Polarized Human Enterocyte-Like T84 Cells[J].Infection and Immunity,2005,73(3):1606-1612
[81]Hoiby N,Krogh J H,Moser C,et al.Pseudomonas aeruginosa and the in vitroand in vivo biofilm mode of growth[J].Microbes and Infection,2001,3(1):23-35
[82]Costerton JW,Stewart PS,Greenberg EP,et al.Bacterial biofilms:A common cause of persistent infections[J].Science,1999,284(5418):1318-1322.
[83]李新,王金良.细菌耐药性机制的研究进展[J].国外医学临床生物化学与检验学分册,2001,22(5):261-262
[84]Lynn E H,Marta.The Enterococcus faecalis fsr two-Component system controls biofilm development through production of gelatinase[J].Journal of Bacteriology,2004,186(17):5629-5639
[85]Blanco A R,Simona L T M,Babini G,et al.Epigallocatechin-3-gallate inhibits gelatinase activity of some bacterial isolates from ocular infection,and limits their invasion through gelatin[J].Biochimica and Biophysica Acta,2003,1620:273-281
[86]Pirkko L M,Don B.C,Florence A,et al.Purification and Substrate Specificity of a Strongly Hydrophobic Extracellular Metalloendopeptidase("Gelatinase") from Streptococcus fuecalis (Strain OGI-IO)[J].The Journal of Biological Chemistry,1989,264(6):3325-3334
[87]Yan AS,Mark CS,Ping HE,et al.Nucleotide Sequence of the Gelatinase Gene(gelE) fromEnterococcus faecalis subsp.Liquefaciens [J].Infection and Immunity, 1991, 59(1):415-420
[88]Qin X, Kavindra VS, George MW, et al.Effects of Enterococcus faecalis fsr Genes on Productionof Gelatinase and a Serine Protease and Virulence [J].Infection and Immunity, 2000,68(5): 2579-2586
[89]Nakayama J, Cao Y, Horii T, et al.Gelatinase biosynthesis-activating pheromone: apeptide lactone that mediates a quorum sensing in Enterococcus faecalis [J].Molecular Microbiology,2001, 41(1):145-154
[90]Dupre I, Zanetti S, Schito A M, et al.Incidence of virulence determinants in clinical Enterococcus faecium and Enterococcus faecalis isolates collected in Sardinia (Italy) [J].Journal of Medical Microbiology, 2003, 52(2):491-498
[91]Nakayama J, Kariyama R , Kumon H.Description of a 23.9-Kilobase chromosomal deletion containing a region encoding fsr genes which mainly determines the gelatinase negative phenotype of clinical isolates of enterococcus faecalis in urine [J].Applied and Environmental Microbiology, 2002, 68(6): 3152-3155
[92]Simon GL, Gorbach SL.Intestinal flora in health and disease [J].Gastroenterology 1984;86(1):174-193
[93]Gibson SA, Macfarlane GT.Studies on the proteolytic activity of Bacteroides fragilis [J].J Gen Microbiol, 1988,134(1): 19-27
[94]Weikel C S, Grieco F D, Reuben J, et al.Human colonic epithelial cells, HT29/C1, treated with crude Bacteroides fragilis enterotoxin dramatically alter their morphology[J].Infection and Immunity, 1992, 60(2):321-327
[95]Tassell LV, Lyerly DM, Wilkins TD.Purification and characterization of an enterotoxin from Bacteroides fragilis [J].Infection and Immunity, 1992, 60(4): 1343-1350
[96]Tassell LV, Lyerly D M, Wilkins T D.Characterization of Enterotoxigenic Bacteroides fragilis by a Toxin-Specific Enzyme-Linked Immunosorbent Assay [J].Clinical and Diagnostic Laboratory Immunology, 1994, l(5):578-584
[97]Eaton TJ, Gasson MJ.Molecular screening of enterococcus virulence determinants and potential for genetic exchange between food and medical isolates [J].Applied and Environmental Microbiology, 2001, 67(4): 1628-1635
[98]Richard J, David M, Roger L, et al.Proteolytic activity of the bacteroides fragilis enterotoxin causes fluid secretion and intestinal damage in vivo [J].Infection and immunity, 1995, 63(10):3820-3826
[99]Moncrief J S, Richard J R, Barroso L A, et al.The enterotoxin of bacteroides fragilis is a metalloprotease [J].Infection and immunity, 1995, 63(1):175—181
[100]Augusto A F, Simy L B, Jai W S, et al.Mutation of the zinc-binding metalloprotease motif affects bacteroides fragilis toxin activity but does not affect propeptide processing[J].Infection and immunity, 2005, 73(8): 5273-5277
[101]Hatheway C L.Toxigenic clostridia[J].Clin Microbiol Rev.1990,3(1):66-98
[102]Kuniko Y,Osamu M,Junzaburo M,et al.Cloning and nucleotide sequence analysis of the colh gene from clostnidium histolyticum encoding a collagenase and a gelatinase[J].Journal of Bacteriology,1994,176(21):6489-6496
[103]Matsushita O,Chang M J,Katayama S,et al.Gene duplication and multiplicity of collagenases in clostridium histolyticum[j].Journal of Bacteriology,1999,181(3):923-933
[104]Matsushita O K,Yoshihara S I,Katayama J M,et al.Purification and character- ization of a Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the correspondinggene[J].J Bacteriol1994,176(1):149-156
[105]Rada E,Zambrano E A,Rodr(?)guez I Y,et al.Gelatinase activity in Mycobacterium bovis protein extract[J],Int J Lepr Other Mycobact Dis.2002,70(4):260-268
[106]Papageorgakopoulou N,Plakoutsi F,Stephanopoulou A,et al.Study of the gelatinolytic activities Escherichia coli cells before and after starvation in seawater by substrate gel electrophoresis[J].Microbiol Res.1996,151(3):329-335
[107]Conlan JW,Baskerville A,Ashworthl AE.Separation of Legionella pneumophila proteases and purification of a protease which produces lesions like those of Legionnaires's disease in guinea pig lung[J].Journal of general microbiology,1986,32(6):1565-1574
[108]Dowling JN,Saha AK,Glew RH.Virulence factors of the family Legionellaceae[J].Microbiol Rev,1992,56(1):32-60
[109]柯娜,肖昌松,应启峰,等.一个可降解直链烷基苯磺酸盐的新种[J].微生物学报,2003;42(1):1-7
[110]J.萨姆布鲁克.D.W.拉塞尔著.分子克隆实验指南(上下册)[M].北京:科学出版社.2003
[111]C.W.迪芬巴赫.G.S.德维克斯勒.PCR技术实验指南[M].北京:科学出版社.2000
[112]罗玉峰,钱心元,房燕丽.猪丹毒QL 251弱毒菌株的稳定性试验[J].中国兽药杂志,1997,31(1):19-21
[113]Roche SM,Gracieux P,Albert I,et al.Experimental validation of low virulence in field strains of listeria monocytogenes[J].Infection and Immunity,2003,7 1(6):3429-3436
[114]Pickett M J,Greenwood J R,Harvey S M.Tests for detecting degradation of gelatin:comparison of five methods[J].J Clin Microbiol,1991,29(10):2322-2325
[115]Frazier W C.A method for the deteetion of changes in gelatin due to bacteria[J].Journary infection Diseases,1926,39(2):302-309
[116]Amanda E S,Tonima D,Kanwal M,et al.Potential involvement of gelatinases and their inhibitors in mannheimia haemolytica pneumonia in cattle[J].Infection and immunity,2004,72(8):4393-4400
[117]Ratnikov BI,Deryugina EI,Strongin AY.Gelatin zymography and substrate cleavage assays of matrix metalloproteinase-2 in breast carcinoma cells overexpressing membrane type-1matrix metalloproteinase[J].Lab Invest.2002,82(11):1583-1590
[118]Marta T,Rafael F.Assessment of Gelatinases(MMP-2 and MMP-9 by Gelatin Zymography [J].Methods in Molecular Medicine,2001,57(1):163-174
[119]Shona R E,Nell H,Alan J.K,et al.Matrix metalloproteinase expression and activity in human airwaysmooth muscle cells[J].British Journal of Pharmacology,2004,142(7):1318-1324
[120]徐宁.发光杆菌产生胶原酶及其应用于制备胶原蛋白的研究(硕士学位论文).四川成都:四川大学,2004
[121]张龙翔,张庭芳,李令暖,等.生化实验方法和技术[M].北京:高等教育出版社,1997,163-165
[122]Rosen H.A modified ninhydrin colorimetric analysis for amino acids[J].Archives of Biochemistry and Biophysics,1957,67(1):10-15
[123]Wang HT,Hsu JT.Optimal protease production condition for Prevotella ruminicola 23 and characterization of its extracellular crude protease[J].Anaerobe,2005,11(3):155-62
[124]Ust(?)ri z F J,Laca A,Garc(?)a L A,et al.Fermentation conditions increasing protease production by Serratia marcescens in fresh whey[J].Rev.T(?)c.Ing.Univ.Zulia,2008,31(1):79 - 89
[125]孟喜龙,刘永杰,陆承平.嗜水气单胞菌产弹性蛋白酶条件的优化[J].南京农业大学学报,2007,30(2):98-101
[126]Takac s,Elmas S,Calik P,et al.Separation of the protease enzymes of Bacillus licheniformis from the fermentation medium by crossflow ultrafiltration[J].Journal of Chemical Technology and Biotechnology,2000,75(2):491-499
[127]Flaschel E,Wandrey C,Kula M R.Ultrafltration for the separation of biocatalysts[J].Advances in Biochemical Engineering/Biotechnology,1983,26(1):73-82
[128]Kalashnikova EE,Chemyshova MP,Ignatov VV.The Extracellular Proteases of the Phytopatho- genic Bacterium Xanthomonas campestris[J].Microbiology,2003,72(4):443-447
[129]Coseteng M Y,Lee C Y.Changes in apple polyphendoxidase and polyphenol concentrations in relation to degree of browing[J].Journal of Food Science,1987,52(1):985-989
[130]汪家政编著.蛋白质技术手册[M].北京:科学出版社.2000
[131]Gold O G,Jordan H V,van Houte J.The prevalence of enterococci in the human mouth and their pathogenicity in animal models[J].Arch.Oral BWl.1975,20(7):473-477
[132]殷震,刘景华.动物病毒学[M].第2版.北京:中国农业出版社,1997:210-231
[133]Parody J P,Alvarez M D,Quiroga A,et al.Hepatocytes isolated from preneoplastic rat livers are resistant to ethacrynic acid cytotoxicity[J].Arch Toxicol,2007,81(8):565-573
[134]Cullen K J,Newkirk K A,Schumaker L M,et al.Glutathione S-transferase pi amplification is associated with cisplatin resistance in head and neck squamous cell carcinoma cell lines and primary tumors[J].Cancer Res,2003,63(23):97-102
[135]储卫华,陆承平.嗜水气单胞菌胞外蛋白酶对鲫鱼的致病性[M].南京农业大学学报,2000,23(2):80-84
[136]黎晓敏,郭丽敏,唐发书,等.鸭传染性浆膜炎病鸭免疫器官的光镜及电镜观察[J].畜牧兽医学报,2004,35(6):702-704
[137]Sachar L A,Winter K K,Sicher N,et al.Photometric method for estimation of elastase activity[J].Proc Soc Exp Biol Med,1955,90(2):323-326
[138]刘玉斌,苟仕金.动物免疫学实验技术[M].长春:吉林科学技术出版社,1989,234-236
[139]方静,崔恒敏,彭西,何敏.雏鸭胸腺生长指标的动态观测及组织学观察[J].中国兽医科学,2006,36(5):401-405
[140]陈龙,庞训胜,高勤学等.感染IBDV雏鸡血液激素水平和ANAE阳生淋巳细胞动态变化[J].中国兽医学报,1999,19(1):22-25
[141]聂其灼,陈小慧.牛白血病病毒感染牛群外周血内酸性-醋酸萘酯酶阳性淋巴细胞百分率变化的研究[J].南京农业大学学报,1985,(1):60-67
[142]朱立平,陈学清主编.免疫学常用实验方法[M].北京:人民军医出版社,2003,120-122
[143]郭峰,虞紫茜,赵中平,等.红细胞免疫功能的初步研究[J].中华医学杂志,1982,62(12):715-716
[144]饶家荣,王旭东,钱光云.樱桃谷鸭红细胞免疫功能研究及其意义[J].畜牧兽医杂志,1999,18(3):20-22
[145]黎德兵,崔恒敏,赵翠燕.鸭疫里默扦菌病发病讥理的研究Ⅳ-试验感染雏鸭红细胞免疫功能的变化[J].黑龙江畜牧兽医,2007,(9):61-62
[146]林祥梅,赵增连.鸭源流感病毒感染对鸡和家鸭红细胞免疫功能的影响[J].中国兽医学报,2000,20(2):135-137
[147]张秋胜,陈昌福.异育银鲫对嗜水气单胞茵灭活菌苗的免疫应答[J].华中农业大学学报,2001,20(3):271-274
[148]王德云,胡元亮,孔祥峰,等.中药成分对雏鸡外周血T淋巴细胞转化的影响[J].中国兽医学报,2004,24(6):578-580
[149]王德云,胡元亮,孔祥峰等.中药成分对培养的鸡脾脏淋巴细胞增殖的影响[J].畜牧兽医学报,2005,36(11),1210-1214
[150]储岳峰,李祥瑞,胡元亮.9种中药成分对小鼠免疫细胞活性的影响[J].南京农业大学学报,2004,27(1):97-100
[151]姚根宏,侯亚义,沈苏南,等.睾酮在体外对脾淋巴细胞增殖反应和分泌IgG的影响[J].卫生研究,2002,31(5):333-335
[152]Lee J D,Tsail Y,Chen C H,et al.Blood brain barrier dysfunction occurring in mice infected with Angiostrongyluscantonensis[J].Acta Tropica,2006,97(2):204-211
[153]Seyfert S,Becher A,Ohring R,et al.The permeability of the blood CSF barrier in hydrocephalus,polyradiculitis and meningitis[J].J Neurol,2004,251:355-356
[154]Siegel I,Liu TL,Gleicher N.The red-cell immune system[J].Lancet,1981,2(8246):556-559
[155]郭峰,张励力.用 C3b致敏感酵母菌测定血清免疫复合物[J].上海免疫学杂志,1982,2(1):32-37
[156]Robert C,Monica I,Lucia B,et al.Survey for virulence determinants among Enterococcus faecalis isolated from different sources[J].Journal of Medical microbiology,2004, 25(53):13-20
[157]Gumey K J,Estrada E Y,Rosenberg G A,et al.Blood-brain barrier disruption bystromelysin-1facilitates neutrophil infiltration in neuroinflammation[J].Neurobiology of Disease,2006,23(10):87-96
[158]傅童生,傅运生.实验性鸭疫里默氏菌病临床病理学动态变化[J].中国兽医学报,2002,22(6):563-564
[159]黎德兵,崔恒敏,章蕾.鸭疫里默氏杆菌病发病机理的研究Ⅰ:人工感染雏鸭部分血液指标的动态变化[J].黑龙江畜牧兽医,2004,(1):8-10
[160]ShinYP,Yong PS,Chong HK,et al.Immune evasion of enterococcus faecalis by an extracellular gelatinase that cleaves C3 and iC3[J].The Journal of Immunology,2008,181:6328-6336
[161]李影林.中华医学检验全书[M].北京:人民卫生出版社,1996
[162]Anderas W,Gregor Z,Joachim G,et al.Decreased virulence of a pneumolysin deficient strain of streptococcus pneumoniae in murine meningitis[J].Infect Irnrnun,2002,20(11):6504-6508
[163]Catarina R,Gabriei AM,Gustavo H,et al.Acute blood brain barrier permeabili- zation in rats after systemic phoneutria nigriventer venom[J].Brain Res,2007,(1149):18-29
[164]Weller R O.Pathology of cerebrospinal fluid and interstitial fluid of the CNS:significance for alzheimer disease,prion disorders and multiple sclerosisb[J].J Neuropathol Exp Neurol,1998,(57):885-894
[165]Jortner B S,Porro R,Leibovitz L,et al.Centralnervous system lesions of spon- taneous Pasteurella anatipestifer infection in ducklings[J].AvianDis,1969,13(1):27-35
[166]Vorbrodt A W,Dobrogowska D H,Ueno M,et al.lmmunocytochemical studies of protam ine-induced blood brain barrier opening to endogenous albumin[J].Acta Neuropathol,1995,(89):491-499
[167]Diane A,Mario K,Gary A,et al.Neutral proteases and disruption of the blood brain barrier in rat[J].Brain Res,1997,767(2):259-264
[168]Matsudaira P.Sequence from picomole quantities of proteins electroblotted onto polyvinyl-lidene difluoride membranes[J].J Biol Chem,1987,262(21):10035-10038
[169]周建,王小行,高小平.转移缓冲液对蛋白质印迹的影响[J].生物化学与生物物理进展,2000,27(3):331-332
[170]Moncrief JS,Richard JR,Lisa AB,et al.The Enterotoxin of Bacteroides fragilis is a Metalloprotease[J].Infection and immunity,1995,63(1):175-181
[171]Shamala V,Ames G F L.Genome walking by single specific primer-polymerase chain reaction:SSP-PCR[J].Gene,1989,84(1):1-8
[172]颜子颖,王海林译.精编分子生物学实验指南[M].北京:科学出版社,1999
[173]Liu YG,Whittier RF.Thermal asymmetric interlaced PCR:automatable ampli- fication and sequencing of insert end fragments from P1 and YAC clones for chromo- some walking[J].Genomics,1995,25(3):674-681
[174]Li ZT,Gray DJ.Isolation by improved thermal asymmetric interlaced PCR and characterization of a seedspecific 2S albumin gene and its promoter from grape(Vitis vinifera L)[J].Genome,2005,48(2):312-320
[175]孔俊,刘文轩.Kas基因启动子区的克隆及功能初步分析[J].上海大学学报(自然科学版),2005,11(6):652-656
[176]仇艳光,田景汉,葛荣朝,等.TAIL-PCR的改良及其在分离小麦基因启动子中的应用[J].生物工程学报,2008,24(4):695-699
[177]Wang L,Gao Q,Wang YZ,et al.Isolation and sequence analysis of two CYC-like genes,SiCYC1A and SiCYC1B,from zygomorphic and actinomorphic cultivars of Saintpaulia ionantha(Gesneriaceae)[J].Acta Phytotaxonomica Sinica,2006,44(4):353-361
[178]杨英军,周鹏.番木瓜proteinase omega基因启动子的克隆及功能初步研究[J].云南植物研究2005,27(5):545-551
[179]薛晓晶,徐福洲,史爱华,等.副猪嗜血杆菌aroA基因鉴定及遗传进化分析[J].微生物学报,2008,48(8):1100-1103
[180]Patricia M,Larry B.Rapid identification of gelatin and casein hydrolysis using TCA[J].Journal of Microbiological Methods,2007,69(2) 391-393
[181]谢莉萍,林静瑜,肖锐,等.合浦珠母贝碱性磷酸酶的分离纯化与性质研究.海洋科学,2000,24(10):37-40
[182]Mayerhofer HJ,Marshall RT,White CH,et al.Characterization of a Heat-Stable Protease of Pseudomonas fluorescens P26[J].Applied microbiology,1973,25(1):44-48
[183]Hoshino T,Ishizaki K,Sakamoto T,et al.Isolation of a Pseudomonas species from fish intestine that produces a protease active at low temperature[J].Letters in Applied Microbiology,1997,25(1):70-72
[184]朱俭,曹凯鸣,周润琦,等.生物化学实验[M].上海:上海科学技术出版社,1981
[185]Basma G,Alya SK,Moncef N.Stability studies of protease from Bacillus cereus BG1[J].Enzyme and Microbial Technology,2003,32(3):513-518
[186]姜军,杨晓达,王夔.汞和金属离子及多硫化物对木瓜蛋白酶活性的抑制作用[J].中国药学,2007,16(1):13-18
[187]Amtul J S,Amtul N S,Noreen K.Comparison in effect of different metal ions,pH and reducing agent on the protease activity in human hyper mature and mature cataract[J].Journal Zhejiang Univ Sci B.2007,8(8):599-603
[188]John WR,Keith NJ.Proteolytic activity of a rumen anaerobic fungus[J].FEMS Microbiology Letters,2006,29(1-2):19-25
[189]周海梦,王洪睿.蛋白质化学修饰[M].北京:清华大学出版社,1998,19-43
[190]张树政,孟广震,何忠效.酶学研究技术(上册)[M].北京:科学出版社,1987,289-311
[191]储卫华,陆承平.嗜水气单胞菌胞外蛋白酶的化学修饰[J].中国生物化学与分子生物学报,2001,17(3):372-375
[192]潘继承,汪劲松,向显智,等.粘质赛氏菌胞外蛋白酶的化学修饰[J].生物化学杂志,1997,13(4):483-486