幽门螺杆菌ureB基因在乳球菌中食品级表达及免疫反应性
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摘要
幽门螺杆菌(Helicobacter pylori,H. pylori)感染是胃炎、胃癌和消化道溃疡的重要病因。目前临床主要用抗生素和抑酸剂联合疗法,虽然取得了一定疗效,但抗生素经济负担高、耐药株出现和药物的副作用等使得抗生素治疗受限。在此情况下,疫苗就成了最有效的方法,具有很好的应用前景。
尿素酶(Urease,Ure)是H. pylori的主要致病因子,UreB亚基已经通过基因工程在大肠杆菌中表达,并在粘膜免疫佐剂配合下,能引起免疫应答。所有的经口免疫模型必须用霍乱毒(Cholera toxin,CT)或大肠杆菌不耐热肠毒素(E.coli labile toxin,LT)作为免疫佐剂。但是CT和LT都有一定的毒性而不能用于人体,加上目前疫苗大多采用鼠伤寒减毒沙门氏菌为抗原提呈载体,对免疫力低下的人群危害大,限制了疫苗的使用。
但用乳酸乳球菌(Lactococcus lactis,L. lactis)做为活菌疫苗的宿主菌,就可以避免上述危害。但传统的基因改良菌往往以抗生素抗性基因作为筛选标记,这就存在潜在的因生物转基因污染而引起的抗生素耐药的危害。因此完全的食品级表达系统的研发一直是该领域的热点。
材料与方法
1.从重组质粒pMD19-T-ureB双酶切得到ureB基因；双酶切胶回收乳酸菌载体pNZ8149(该载体以lacF基因为筛选标记),连接后电转化入lacF基因缺失的乳酸乳球菌NZ3900。挑阳性克隆进行酶切、PCR和测序鉴定。
2.用正交试验,对影响目的蛋白诱导产量的因素,如诱导剂浓度、诱导时机和诱导持续时间进行实验,用Brandford法测定目的蛋白浓度,通过方差分析得出最适宜表达条件。
3.纯化重组大肠杆菌TB1/pMAL-C2X-UreB的诱导产物MBP-UreB,混合弗氏免疫佐剂免疫小鼠,制备抗UreB免疫血清。用Western-blot免疫印迹法研究重组乳球菌NZ3900/pNZ8149-ureB表达的UreB蛋白的免疫反应性。
结果
1.PCR扩增、酶切鉴定,食品级表达载体pNZ8149-ureB构建成功,转化得到重组工程菌NZ3900/pNZ8149-ureB。重组菌用乳联菌肤(Nisin)诱导后,SDS-PAGE电泳可见64.1KD的目的蛋白UreB。
2.正交实验表明,各因素对实验结果影响大小次序是：诱导时机、诱导剂浓度、诱导时间,只有诱导时机对实验结果的影响有统计学意义;表达条件的最优组合：在菌体培养到OD600为0.4时加入终浓度为25ng/mL的Nisin,诱导表达5h。
3.制备的小鼠抗UreB血清,经ELISA鉴定效价为1：800。将乳酸菌诱导表达的目的蛋白UreB转移到硝酸纤维膜上,用制备的小鼠血清进行Western blot分析,可在相应位置显示杂交条带,说明乳酸菌诱导表达的UreB具有良好的免疫反应原性。
结论
1.成功构建了表达UreB的不含任何抗生素抗性筛选标记的乳酸乳球菌食品级原核表达系统。
2.利用正交实验获得了重组工程菌NZ3900/pNZ8149-ureB的最优表达条件。
3.乳酸乳球菌食品级表达系统诱导表达的UreB蛋白有良好的免疫原性,为研制H. pylori的直接口服疫苗奠定基础。
Helicobacter pylori (H. pylori) is the principal cause of chronic gastritis, pepti ulcers and stomach cancer. Current therapies are mainly based on antibiotics together with antiacid. Although the antibiotic-based triple treatments have recently acquired good therapeutic effect, it is not practical for global control for the high cost of antibiotic, problems with patient's compliance and the increasing drug resistant strains. So, under these circumstances vaccination against H. pylori infection has been considered the best way to control H. pylori infection all over the word.
Urease is acid stable, appears to be essential for stomach colonization by H. pylori. The subunit B(UreB) which has been cloned in E. coli.UreB combination with immunogenic adjuvant can stimulate the mouse to producing immunoresponse against challenge of H. pylori. In all the oral immunization experiments, without immunogenic adjuvant, UreB can not protect the animals gainst challenge of H. pylori. The Cholera toxin (CT) and E. coli heat-labile toxin (LT) used to mucosal adjuvant. Because of the toxicity in volunteers, CT or LT can not be given to people.
By contrast, using Lactococcus lactis (L.lactis) to deliver protective antigens to the mucosal surface may be a way to solve these problems. L. lactis is nonpathogenic food-grade bacterium that is generally recognized as safe and widely used in food industry. But these traditional genetically modified organism usually used antibiotics resistance gene screening method. So it has potential risk of the antibiotic-resistant. A number of reaserch groups have reported that L. lactis can be widespread used for the expression of heterogeneous genes.
Method
1.ureB gene was obtained from the recombinant vector of pMD19-T-ureB by NcoⅠand XbaⅠenzyme digestion. The ureB gene was inserted into pNZ8149 food-grade expression vector which lacF is the food-grade selection.Then the recombinant plasmid pNZ8149-ureB was electrotransformed into L. lactis NZ3900.Due to the lacF deletion, strain NZ3900 is unable to grow on lactose.
2. L25 (53) orthogonal design was used to optimization the expression conditions of UreB.Different dosage of inducer, the OD6oo of culture, incubating time after iuduced were chosen. Five levels had set for each factor. Then Brandford method was used to work out the expression quantity of the target protein. ANOVA method was used to analyze the results.
3.This study acquired fusion protein MBP-UreB from E. coli TB1/pMAL-C2X-UreB. The MBP-UreB fusion protein immunized mice to produce anti-UreB immune serum. Western-blot was performed to check the immunological activity of UreB expressed by food-gread gene expression system.
Results
1.The food-grade expression vector was successfully constructed through PCR and enzyme digestion.The recombinant plasmid was electrotransformed into L. lactis NZ3900.
2.Nisin 25ng/mL was added to the medium when the recombinant grew to OD600≈0.4 then incubate 5h.As a result, the maximum yield of UreB was estimated to be 7% of total soluble cellular proteins.
3.The titre of the antiserum was determined to be 1:800 by enzyme lined immunosorbent assay (ELISA).Western blot analysis showed that the UreB protein expressed by L. lactis transformant had favorable immunoreactivity.
Conclusions
1.Because all the fragments used to construct the expression vector system L. lactis NZ3900/pNZ8149-ureB were obtained from food-grade bacteriat, the expression vector system can be regarded as a food-grade expression vector system.
2.The optimal expression conditions for targeted protein expressed by NZ3900/pNZ8149-ureB have been obtained by orthogonal design. 3.The recombinant protein UreB expressed by L. lactis was detected by Western-blot. All the results make an appealing case for construction of the food-grade vaccine for H. pylori.
尿素酶(Urease,Ure)是H. pylori的主要致病因子,UreB亚基已经通过基因工程在大肠杆菌中表达,并在粘膜免疫佐剂配合下,能引起免疫应答。所有的经口免疫模型必须用霍乱毒(Cholera toxin,CT)或大肠杆菌不耐热肠毒素(E.coli labile toxin,LT)作为免疫佐剂。但是CT和LT都有一定的毒性而不能用于人体,加上目前疫苗大多采用鼠伤寒减毒沙门氏菌为抗原提呈载体,对免疫力低下的人群危害大,限制了疫苗的使用。
但用乳酸乳球菌(Lactococcus lactis,L. lactis)做为活菌疫苗的宿主菌,就可以避免上述危害。但传统的基因改良菌往往以抗生素抗性基因作为筛选标记,这就存在潜在的因生物转基因污染而引起的抗生素耐药的危害。因此完全的食品级表达系统的研发一直是该领域的热点。
材料与方法
1.从重组质粒pMD19-T-ureB双酶切得到ureB基因；双酶切胶回收乳酸菌载体pNZ8149(该载体以lacF基因为筛选标记),连接后电转化入lacF基因缺失的乳酸乳球菌NZ3900。挑阳性克隆进行酶切、PCR和测序鉴定。
2.用正交试验,对影响目的蛋白诱导产量的因素,如诱导剂浓度、诱导时机和诱导持续时间进行实验,用Brandford法测定目的蛋白浓度,通过方差分析得出最适宜表达条件。
3.纯化重组大肠杆菌TB1/pMAL-C2X-UreB的诱导产物MBP-UreB,混合弗氏免疫佐剂免疫小鼠,制备抗UreB免疫血清。用Western-blot免疫印迹法研究重组乳球菌NZ3900/pNZ8149-ureB表达的UreB蛋白的免疫反应性。
结果
1.PCR扩增、酶切鉴定,食品级表达载体pNZ8149-ureB构建成功,转化得到重组工程菌NZ3900/pNZ8149-ureB。重组菌用乳联菌肤(Nisin)诱导后,SDS-PAGE电泳可见64.1KD的目的蛋白UreB。
2.正交实验表明,各因素对实验结果影响大小次序是：诱导时机、诱导剂浓度、诱导时间,只有诱导时机对实验结果的影响有统计学意义;表达条件的最优组合：在菌体培养到OD600为0.4时加入终浓度为25ng/mL的Nisin,诱导表达5h。
3.制备的小鼠抗UreB血清,经ELISA鉴定效价为1：800。将乳酸菌诱导表达的目的蛋白UreB转移到硝酸纤维膜上,用制备的小鼠血清进行Western blot分析,可在相应位置显示杂交条带,说明乳酸菌诱导表达的UreB具有良好的免疫反应原性。
结论
1.成功构建了表达UreB的不含任何抗生素抗性筛选标记的乳酸乳球菌食品级原核表达系统。
2.利用正交实验获得了重组工程菌NZ3900/pNZ8149-ureB的最优表达条件。
3.乳酸乳球菌食品级表达系统诱导表达的UreB蛋白有良好的免疫原性,为研制H. pylori的直接口服疫苗奠定基础。
Helicobacter pylori (H. pylori) is the principal cause of chronic gastritis, pepti ulcers and stomach cancer. Current therapies are mainly based on antibiotics together with antiacid. Although the antibiotic-based triple treatments have recently acquired good therapeutic effect, it is not practical for global control for the high cost of antibiotic, problems with patient's compliance and the increasing drug resistant strains. So, under these circumstances vaccination against H. pylori infection has been considered the best way to control H. pylori infection all over the word.
Urease is acid stable, appears to be essential for stomach colonization by H. pylori. The subunit B(UreB) which has been cloned in E. coli.UreB combination with immunogenic adjuvant can stimulate the mouse to producing immunoresponse against challenge of H. pylori. In all the oral immunization experiments, without immunogenic adjuvant, UreB can not protect the animals gainst challenge of H. pylori. The Cholera toxin (CT) and E. coli heat-labile toxin (LT) used to mucosal adjuvant. Because of the toxicity in volunteers, CT or LT can not be given to people.
By contrast, using Lactococcus lactis (L.lactis) to deliver protective antigens to the mucosal surface may be a way to solve these problems. L. lactis is nonpathogenic food-grade bacterium that is generally recognized as safe and widely used in food industry. But these traditional genetically modified organism usually used antibiotics resistance gene screening method. So it has potential risk of the antibiotic-resistant. A number of reaserch groups have reported that L. lactis can be widespread used for the expression of heterogeneous genes.
Method
1.ureB gene was obtained from the recombinant vector of pMD19-T-ureB by NcoⅠand XbaⅠenzyme digestion. The ureB gene was inserted into pNZ8149 food-grade expression vector which lacF is the food-grade selection.Then the recombinant plasmid pNZ8149-ureB was electrotransformed into L. lactis NZ3900.Due to the lacF deletion, strain NZ3900 is unable to grow on lactose.
2. L25 (53) orthogonal design was used to optimization the expression conditions of UreB.Different dosage of inducer, the OD6oo of culture, incubating time after iuduced were chosen. Five levels had set for each factor. Then Brandford method was used to work out the expression quantity of the target protein. ANOVA method was used to analyze the results.
3.This study acquired fusion protein MBP-UreB from E. coli TB1/pMAL-C2X-UreB. The MBP-UreB fusion protein immunized mice to produce anti-UreB immune serum. Western-blot was performed to check the immunological activity of UreB expressed by food-gread gene expression system.
Results
1.The food-grade expression vector was successfully constructed through PCR and enzyme digestion.The recombinant plasmid was electrotransformed into L. lactis NZ3900.
2.Nisin 25ng/mL was added to the medium when the recombinant grew to OD600≈0.4 then incubate 5h.As a result, the maximum yield of UreB was estimated to be 7% of total soluble cellular proteins.
3.The titre of the antiserum was determined to be 1:800 by enzyme lined immunosorbent assay (ELISA).Western blot analysis showed that the UreB protein expressed by L. lactis transformant had favorable immunoreactivity.
Conclusions
1.Because all the fragments used to construct the expression vector system L. lactis NZ3900/pNZ8149-ureB were obtained from food-grade bacteriat, the expression vector system can be regarded as a food-grade expression vector system.
2.The optimal expression conditions for targeted protein expressed by NZ3900/pNZ8149-ureB have been obtained by orthogonal design. 3.The recombinant protein UreB expressed by L. lactis was detected by Western-blot. All the results make an appealing case for construction of the food-grade vaccine for H. pylori.
引文
[1]Marshall BJ,Warren JR.Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration[J].Lancet,1984,1:1311-1315
[2]Yasui W,Sentani K,Molecular pathobiology of gastric cancer.Scand j surg [J].2006, 22(4):225-231
[3]王凯娟,王润田.中国幽门螺杆菌感染流行病学Meta分析[J].中华流行病学杂志,2003,24(6)：443-445
[4]Wang KX,Chen L.Helicobacter pylori L-form and patients with chronic gastritis [J].Gastroenterol,2004,10(9):1306-1309
[5]Trajkov D,Stardelova K,Dimitrova M,et al.Helicobacter pylori and gastric carcinoma [J].Prilozi,2007,28(2):39-46
[6]Peter S,Beglinger C.Helicobacter pylori and gastric cancer:the causal relationship [J].Digestion,2007,75(1):25-35
[7]陈洁,陈旻湖.幽门螺杆菌感染的免疫致病机制及疫苗的保护作用机制[J].国外医学,内科学分册,1999,26(12)：524-526
[8]Corthesy-Theulaz I,Davin C,Michetti P,et al.Helicobacter pylori urease elicits protection against Helicobacter felis infection in mice [J].Gastroenterology,1993,56(S):64-70
[9]郭鹰,邹全明,朱永红,等.BALB/C小鼠口服幽门螺杆菌疫苗rltB-hspA的免疫应答[J].免疫学杂志,2004,20(3)：201-204
[10]张荣光,段广才,郗园林.幽门螺杆菌omp11基因的克隆及序列分析[J].中华微生物学和免疫学杂志,2003,23(12)：951-954
[11]Marchetti M,Rossi M,Giannelli V,et al.Protection against Helicobacter pylori infection in mice by intragastric vaccination with H.pyliri antigents in achieved using a non-toxic mutant of E.coli heat-labile enterotoxin (LT) as adjuvant [J].Vaccine,1998,16(1):33-37
[12]Labigne A.J Bacteriol,1991,173(6):1920-1931
[13]Kreiss C, Buclin T,Cosma M,et al.Safety of oral immunization with recombinant urease in patients with Helicobacter pylori infection[J].Lancet,1996,347(9015):1630-1631
[14]Michetti P,Kreiss C,Kotloff KL,et al.Oral immunization with urease and Eschechia Coli heat-labile enterotoxin is safe and immunogenic in Helicobacter pylori-infected adults [J].Gastroenterology,1999,116(4):804-812
[15]Gomea-Duarte OG,Lucas B,Yan ZX,et al.Protection of mice against gastric colonization by Helicobacter pylori by single oral dose immunization with attenuated Salmonella typhimurium producing urease subunits A and B [J].Vaccine,1998,16(5):460-471
[16]Corthesy-Theulaz IE,Hopkins S,Bachmann D,et al.Mice are protected from Helicobacter pylori infection by nasal immunization with attenuated Salmonella typhimurium phopc expressing urease A and B subunits [J].Infect Immun,1998,66(2):581-586
[17]刘晓峰,胡家露,张霞,等.减毒鼠伤寒杆菌为载体的幽门螺杆菌尿素酶口服疫苗的 制备[J].解放军医学杂志,2003,28(7)：633-635
[18]朱森林,陈旻湖,陈洁,等.表达幽门螺杆菌HpaA基因的减毒鼠伤寒沙门氏菌对小鼠的免疫保护作用[J].中国人兽共患病杂志,2002,18(2)：32-36
[19]郭本恒主编.益生菌[M].第1版,北京,化学工业出版社.2004
[20]Olivera M L.Areas A P,Campos I B,et al.Induction of systemic and mucosal immune response and decrease in Streptococcus pneumoniae colonization by nasal inoculation of mice with recombinant lactic acid bacteria expressing pneumococcal surface antigen A [J].Microbes and infection,2006,8(4):1016-1024
[21]Sheppler L,Vogel M,Marti P,et al.Intranasal immunisation using recombinant Lactobacillus johnsonii as a new strategy to prevent allergic disease [J].Vaccine,2005, 23(9):1126-1134
[22]Yao X Y,Yuan M M,Li D J.Mucosal inculation of Lactobacillus expressing hCGbeta induces anti-hCGbeta antibody response in mice of different strains [J].Methods,2006, 38(2):124-132
[23]薛迎迎,杨汝德.乳酸菌基因组学与基因工程的研究新进展[J].现代食品科技,2008,24(6)： 617-620
[24]Xiaojuan Zhang,Guangcai Duan,Rongguang Zhang,et al.Optimized expression of Helicobacter pylori ureB gene in the Lactococcus lactis Nisin-controlled gene expression (NICE) system and experimental study of its immunoreactivity [J].Curr Microbiol,2009 58:308-314
[25]Joseph Sambrook,David W. Russell.Molecular Cloning:A Laboratory Manual,3rd ed [M].Cold Spring Harbor Laboratory Press,2001
[26]Lembo A,caladonna L,Magron T,et al.Helicobacter pylori infection, immune response and vaccination [J].Curr Drug Targets Immune Endocr Metabol Desord,2001,1(3): 119～208
[27]Sougioltzis S,Lee CK,Alsabli M.Safely and efficacy of E coli enterooxin adjuvant for urease based rectal immunization against Helicobacter pylori [J].Vaccine,2002,21(3): 194-201
[28]朱森林,廖文俊.表达幽门螺杆菌Ure-B减毒鼠伤寒沙门氏菌对小鼠的免疫保护作用[J].中山医科大学学报,2001,22(6)：418-422
[29]Martin M C,Alonso J C,Suarez J E,et al.Genertion of food-grade recombinamt Lactic acid bacterium strains by site-specific recombination [J].Applied Environmental Microbiology,2000,66(6):2599-2604
[30]史达,宋岩,李一经.乳酸乳球菌作为黏膜免疫活载体疫苗传递抗原的研究进展[J].微生物学报,2006,46(4)：680-683
[31]郭兴华,益生乳酸细菌：分子生物学及生物技术[M].北京科学技术出版社,2008：129-130
[32]Bermudez-Humaran L G,Langella P,Miyoshi A,et al.Production of human papillomavirus type 16 E7 protein in Lactococcus lactis [J].Applied and Enviromental Microbiology,2002,68(2):917-922
[33]Wells JM,Wilson PW,NortonPM,et al.Lactoeoceus lactis:high-level expression of tetanus toxin fragment C and protection against lethal challenge [J].Mol Microbiol,1993, 8(6):1155-1162
[34]Lee MH,Roussel Y,Wilks M,et al.Expression of Helicobacter pylori urease subunit B gene in Lactococcus lactis MG1363 and its use as a vaccine delivery system against H.pylori infection in mice [J].Vaeeine,2001,19(28-29):3927-3933
[35]Kim SJ,Jun do Y,Yong CH,et al.Expression of Helicobacter pylori cag12 gene in Lactococcus lactis MG1363 and its oral administration to induce systemic anti-Cag12 immune response in mice [J].Appl Microbiol Biotechnol,2006,72(3):462-470
[36]李文妹,陆惠民,张惠琴,等.弓形虫P30乳酸球菌口服疫苗诱导小鼠的体液免疫反应及保护性观察,中国寄生虫病防治杂志,2004,17(2)：90-92
[37]孙强正,徐建国.乳酸乳球菌食品级表达载体的研究进展[J].中国微生态学杂志,2006,18(3)：260-261
[38]FU X,XUJG.Development of a chromosome-plasmid balanced lethal system for Lactobacillus acidophilus with thyA gene as selective marker [J].Microbiol Immunol, 2000,44:551-556
[39]熊衍文.乳酸乳球菌食品级载体系统的构建及外源基因的克隆和表达[D].[博十学位论文],2004,中国疾病控制中心
[40]Platteeuw C,Alen-Boerriger I,van Schalkwijk S,et al.Food-grade cloning and expression system for Lactococcus lactis [J].Appl Environ Microbiol,1996,15:839-847
[41]卫文仲,向华,谭华荣.人铜锌超氧化物歧化酶基因在乳酸乳球菌中食品级表达[J].微生物学报,2003,3(2)：377-354
[42]贾兴元,陈星,苏畅,等.人工合成苯丙氨酸脱氨酶基因在乳酸乳球菌中的食品级表达[J].中华微生物学和免疫学杂志,2006,26(1)：23-26
[43]Frosethb R,Herman RE E,Mckayl L.Cloning of nisin resistance determinant and replication origin on 7-kilobase EcoR I fragment of pNP400 from Sstrep to coccus lactis subsp diacetylactis DRC3[J].Appl Environ Microbiol 1988,54:2136-2139
[44]Igor Mierau,Kees Olieman,James Mond,et al.Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications [J].Microbial Cell Factories,2005,4(1):4-16
[45]陈星,高斌,贾兴元,等.使用偏爱密码子大幅度提高苯丙氨酸脱氨酶在食品级乳酸乳球菌中的表达[J].生物工程学报,2006,22(2)：187-190
[46]方积乾.卫生统计学[M].(第五版),北京人民卫生出版社,2005
[47]白雪飞.幽门螺杆菌抗原rUreB、rOmp11经3种黏膜途径接种BALB/c小鼠的免疫效果研究[D].[硕士学位论文],2006,郑州大学
[48]孙强正,李振军,李娟,等.霍乱毒素B亚单位在乳酸乳球菌食品级表达系统中的分泌性表达[J].中华流行病学杂志,2009,30(12)：1288-1291
[1]薛迎迎,杨汝德.乳酸菌基因组学与基因工程的研究新进展[J].现代食品科技.2008,24(6)：617-620
[2]Ahmed,F.E.Genetically modified probiotics in foods.Trends Biotechnol [J].2003, 21(11):491-497
[3]Kleerebezem M,Hugenholtz J.Metabolic pathway engineering in Lactic acid bacteria.Current Opinion in Biotechnology [J].2003,14(2):232-237
[4]周正任.医学微生物学[M].北京：人民卫生出版社,2003
[5]Matsuzaki T.Immunomodulation by treatment with Lactobacillus casei strain Shirota.International Journal of Food Microbiology [J].1998,41(2):133-140
[6]Hosoda M,Hashimoto H,Morita H,et al.Antimutagenicity of Milk Cultured with Lactic Acid Bacteria against N-Methyl-N'-Nitro-N-Nitrosoguanidine [J].J Dairy Sci,1992, 75(4):976-981
[7]贾士芳,郭兴华.活菌制剂的现状和未来.生物工程进展[J].1996,16(2)：16-20
[8]Shroff K E,Meslin K,Cebra JJ.Commensal enteric bacteria engender a self-limiting humoral mucosal immune response while permanently colonizing the gut [J].Infection and Immunity,1995,63(10):3904-3913
[9]Gronlund M M,Arvilommi H,Kero P,et al.Importance of intestinal colonisation in the maturation of humoral immunity in early infancy:a prospective follow up study of healthy infants aged 0-6 months [J].Archieves of Disease in Child,2000,83(3):186～192
[10]Ivory K,Chambers SJ,Pin C,et al.Oral delivery of Lactobacillus casei Shirota modifies allergen-induced immune responses in allergic rhinitis[J].Clinical & Experimental Allergy,2008,38(8):1282-1289
[11]王跃,罗强,陈淑惠,等.双歧杆菌脂磷壁酸对结肠癌细胞Toll样受体表达的影响[J].中华微生物学免疫学杂志,2005,25(10)：803-805
[12]Ouwehand A,Isolauri E,Salminen S,et al.The role of the intestinal microflora for the development of the immune system in early childhood [J].European Journal of Nutrition, 2002,41(11):32-37
[13]焦凤超,李迎晓,刘纪成,等.减毒沙门氏菌运送的口服DNA疫苗研究进展[J].中国兽医学杂志,2008,44(4)：50-51
[14]Neirynck S,Steidler L.Delivery of therapeutic proteins through Lactococcus lactis [J].Biotechnol Genet Eng Rev,2006,22:253-266
[15]Lother Steidler,Pieter Rottiers.Therapeutic drug delivery by genetically modified Lactococcus lactis [J].Annals of the New York Academy of Sciences,2006,1072(1): 176-186
[16]Olivera ML,Areas AP,Campos IB,et al.Induction of systemic and mucosal immune response and decrease in Streptococcus pneumoniae colonization by nasal inoculation of mice with recombinant lactic acid bacteria expressing pneumococcal surface antigen A[J].Microbes and infection,2006,8(4):1016-1024
[17]Seegers JF.Lactobacillus as live vaccine delivery vectors:progress and prospects [J].Trends in biotechnology,2002,20(12):508-515
[18]Norton PM.,Wells JM.,Brown HW,et al.Protection against tetanus toxin in mice nasally immunized with recombinant Lactococcus lactis expressing tetanus toxin fragment C[J].Vaccine,1997,15(6-7):616-619
[19]Ming HL,Yvonne R,Mark Wilks,et al.Expression of Helicobacterpylori urease subunit B gene in Lactococcus lactis MG1363 and its use as a vaccine delivery system against H. pylori infection in mice [J].Vaccine,2001,19(28-29):3927-3935
[20]Li YG,Tian FL,Gao FS,et al.Immune responses generated by Lactobacillus as a carrier in DNA immunization against foot-and-mouth disease virus[J].Vaccine,2007,25(5): 902-911
[21]Poo H,Pyo H M,Lee T Y,et al.Oral administration of an human papillomavirus type 16 E7 displayed on Lactobacillus casei induces E7-specific antitumor effects in C57/BL6 mice[J].Int J Cancer,2006,119(7):1702-1709
[22]李桂伟,乔薪瑗,刘伯臣,等.表达猪轮状病毒VP4基因重组乳酸球菌表达载体构建及免疫原性分析[J].中国农业科学,2009,42(10)：3672-3678
[23]许云华.表达GPV VP3和CPV VP2结构蛋白重组乳酸乳球菌的构建[D].[硕士学位论文],2008,扬州大学
[24]Vinay S,Jean PA, Maarten L,et al.Intranasal delivery of influenza subunit vaccine formulated with GEM particles as an adjuvant [J].The AAPS Journal,2010,12(2): 109-116
[25]Buccato S,Maione D,Rinaudo C D,et al.Use of Lactococcus lactis Expressing Pili from Group B Streptococcus as a Broad-Coverage Vaccine against Streptococcal Disease[J].The Journal of Infectious Diseases,2006,194(1):331-340
[26]Kim SJ,Jun DY,Yang CH,et al.Expression of Helicobacter pylori cag12 gene in Lactococcus lactis MG1363 and its oral administration to induce systemic anti-Cagl2 immune response in mice[J].Applied Microbiology and Biotechnology,2006,72(3): 462-470
[27]Sheppler L,Vogel M,Marti P,et al.Intranasal immunisation using recombinant Lactobacillus johnsonii as a new strategy to prevent allergic disease[J].Vaccine,2005, 23(9):1126-1134
[28]Yao XY,Yuan MM,Li DJ.Mucosal inculation of Lactobacillus expressing hCGbeta induces anti-hCGbeta antibody response in mice of different strains [J].Methods,2006, 38(2):124-132
[29]Ramasamy R,Yasawardena S,Zomer A,et al.Immunogenicity of a malaria parasite antigen displayed by Lactococcus lactis in oral immunisations[J].Vaccine,2006,24(18): 3900-3908
[30]孙强正,徐建国.乳酸乳球菌食品级表达载体的研究进展[J].中国微生态学杂志,2006,18(3)：260-261
[31]FU X,XU J G.Development of a chromosome-plasmid balanced lethal system for Lactobacillus acidophilus with thyA gene as selective marker [J].Microbiol Immunol, 2000,44:551-556
[32]孙强正,熊衍文,李振军,等.乳酸乳球菌食品级载体的构建及Mn-SOD基因的克隆表达[J].中国人兽共患病学报,2006,22(6)：498-501
[33]Bron PA,Benchimol MG,Lambert J,et al.Use of the alr Gene as a Food-Grade Selection Marker in Lactic Acid Bacteria [J].Appl Environ Microbiol.2002,68(11): 5663-5670
[34]Glenting J,Madsen S M,Vrang A,et al.A plasmid selection system in Lactococcus lactis and its use for gene expression in L.lactis and human kidney fibroblasts [J].Appl Environ Microbiol,2002,68(10):5051-5056
[35]卫文仲,向华,谭华荣.人铜锌超氧化物歧化酶基因在乳酸乳球菌中食品级表达[J].微生物学报,2003,3(2)：377-354
[36]贾兴元,陈星,苏畅,等.人工合成苯丙氨酸脱氨酶基因在乳酸乳球菌中的食品级表达[J].中华微生物学和免疫学杂志,2006,26(1)：23-26
[37]Frosethb R,Herman RE E,Mckayl L.Cloning of nisin resistance determinant and replication origin on 7-kilobase EcoR I fragment of pNP400 from Sstrep to coccus lactis subsp diacetylactis DRC3[J].Appl Environ Microbiol 1988,54(8):2136-2139
[38]Igor Mierau,Kees Olieman,James Mond,et al.Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications [J].Microbial Cell Factories,2005,4(1):16-28
[39]Mierau I,Kleerebezem,M.10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis [J].Applied Microbiology and Biotechnology,2005,68(6):705-717
[40]Madsen SM,Arnau J,Vrang A,et al.Molecular characterization of the pH inducible and growth phase dependent promoter P170 of Lactococcus lactis [J].Molmicrobiol,1999, 32(1):75-87
[41]Sanders JW,Leenhouts K,Burghoorn J,et al.A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation [J].Molmicrobiol,1999,32(1):75-87
[42]Sanders JW,Venema G,Kok J.A chloride-inducible gene expression cassette and its used in induce lysis of Lactococcus lactis [J].Appl Environ Microbiol,1997,63(12):4877-4882
[43]孙强正,熊衍文,叶长芸,等.食品级分泌表达载体的构建及报告蛋白在乳酸乳球菌中表达[J].微生物学报,2008,48(3)：293-298
[44]Sandrine AL,Maarten L,Jolanda N,et al.Lactococcus lactis GEM particles displaying pneumococcal antigens induce local and systemic immune responses following intranasal immunization [J].Vaccine,2006,24(26):434-5441
[45]Shida K,Makino K,Morishita A,et al.Lactobacillus casei Inhibits Antigen-Induced IgE Secretion through Regulation of Cytokine Production in Murine Splenocyte Cultures [J].Int Arch Allergy Immunol,1998,115(4):278-287
[46]Miettinen M,Vuopio-Varkila J,Varkila K.Production of human tumor necrosis factor alpha, interleukin-6,and interleukin-10 is induced by lactic acid bacteria [J].Infect. Immun.,1996,64(12):5403-5405
[47]Schiffrin EJ,Brassart D,Servin AL,et al.Immune modulationof blood leukocytes in humans by lactic acid bacteria:criteria for strain selection [J].Am J Clin Nutr,1997, 66(2):515S-520S
[48]Tejada-Simon MV,J stunol ZL,Pestka J J.Effects of lactic acid bacteria ingestion of basal cytokine mRNA and immunoglobulin levels in the mouse [J].Food Prot,1999,62 (3):287-291
[49]Haller D,Blum S,Bode C,et al.Activation of human peripheral blood mornouclear cells by nonpathogenic bacteria in vitro:evidence of NK cells as proiary targets [J].Infect Immun,2000,68(2):752-759
[50]Aattouri N,Lemonnier D.Production of interferon thermophilus:role of CD4+ and CD8 + lymphocytes induced by Streptococcus[J].Nutr Biochem,1997,8(1):25-31
[51]Steidler L,Hans W,Schotte L,et al.Treatment of murine colitis by Lactococcus lactis secreting interleukin-10[J].Science,2000,289(5483):1311-1312
[52]Vandenbroucke K.,Hans W.,Vanhuysse J.,et al.Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice [J].Gastroenterology,2004,127(2):502-513
[53]Steidler,L.,Neirynck S.,Huyghebaert N.,et al.Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10[J].Nature Biotechnol,2003,21(7):785-789
[2]Yasui W,Sentani K,Molecular pathobiology of gastric cancer.Scand j surg [J].2006, 22(4):225-231
[3]王凯娟,王润田.中国幽门螺杆菌感染流行病学Meta分析[J].中华流行病学杂志,2003,24(6)：443-445
[4]Wang KX,Chen L.Helicobacter pylori L-form and patients with chronic gastritis [J].Gastroenterol,2004,10(9):1306-1309
[5]Trajkov D,Stardelova K,Dimitrova M,et al.Helicobacter pylori and gastric carcinoma [J].Prilozi,2007,28(2):39-46
[6]Peter S,Beglinger C.Helicobacter pylori and gastric cancer:the causal relationship [J].Digestion,2007,75(1):25-35
[7]陈洁,陈旻湖.幽门螺杆菌感染的免疫致病机制及疫苗的保护作用机制[J].国外医学,内科学分册,1999,26(12)：524-526
[8]Corthesy-Theulaz I,Davin C,Michetti P,et al.Helicobacter pylori urease elicits protection against Helicobacter felis infection in mice [J].Gastroenterology,1993,56(S):64-70
[9]郭鹰,邹全明,朱永红,等.BALB/C小鼠口服幽门螺杆菌疫苗rltB-hspA的免疫应答[J].免疫学杂志,2004,20(3)：201-204
[10]张荣光,段广才,郗园林.幽门螺杆菌omp11基因的克隆及序列分析[J].中华微生物学和免疫学杂志,2003,23(12)：951-954
[11]Marchetti M,Rossi M,Giannelli V,et al.Protection against Helicobacter pylori infection in mice by intragastric vaccination with H.pyliri antigents in achieved using a non-toxic mutant of E.coli heat-labile enterotoxin (LT) as adjuvant [J].Vaccine,1998,16(1):33-37
[12]Labigne A.J Bacteriol,1991,173(6):1920-1931
[13]Kreiss C, Buclin T,Cosma M,et al.Safety of oral immunization with recombinant urease in patients with Helicobacter pylori infection[J].Lancet,1996,347(9015):1630-1631
[14]Michetti P,Kreiss C,Kotloff KL,et al.Oral immunization with urease and Eschechia Coli heat-labile enterotoxin is safe and immunogenic in Helicobacter pylori-infected adults [J].Gastroenterology,1999,116(4):804-812
[15]Gomea-Duarte OG,Lucas B,Yan ZX,et al.Protection of mice against gastric colonization by Helicobacter pylori by single oral dose immunization with attenuated Salmonella typhimurium producing urease subunits A and B [J].Vaccine,1998,16(5):460-471
[16]Corthesy-Theulaz IE,Hopkins S,Bachmann D,et al.Mice are protected from Helicobacter pylori infection by nasal immunization with attenuated Salmonella typhimurium phopc expressing urease A and B subunits [J].Infect Immun,1998,66(2):581-586
[17]刘晓峰,胡家露,张霞,等.减毒鼠伤寒杆菌为载体的幽门螺杆菌尿素酶口服疫苗的 制备[J].解放军医学杂志,2003,28(7)：633-635
[18]朱森林,陈旻湖,陈洁,等.表达幽门螺杆菌HpaA基因的减毒鼠伤寒沙门氏菌对小鼠的免疫保护作用[J].中国人兽共患病杂志,2002,18(2)：32-36
[19]郭本恒主编.益生菌[M].第1版,北京,化学工业出版社.2004
[20]Olivera M L.Areas A P,Campos I B,et al.Induction of systemic and mucosal immune response and decrease in Streptococcus pneumoniae colonization by nasal inoculation of mice with recombinant lactic acid bacteria expressing pneumococcal surface antigen A [J].Microbes and infection,2006,8(4):1016-1024
[21]Sheppler L,Vogel M,Marti P,et al.Intranasal immunisation using recombinant Lactobacillus johnsonii as a new strategy to prevent allergic disease [J].Vaccine,2005, 23(9):1126-1134
[22]Yao X Y,Yuan M M,Li D J.Mucosal inculation of Lactobacillus expressing hCGbeta induces anti-hCGbeta antibody response in mice of different strains [J].Methods,2006, 38(2):124-132
[23]薛迎迎,杨汝德.乳酸菌基因组学与基因工程的研究新进展[J].现代食品科技,2008,24(6)： 617-620
[24]Xiaojuan Zhang,Guangcai Duan,Rongguang Zhang,et al.Optimized expression of Helicobacter pylori ureB gene in the Lactococcus lactis Nisin-controlled gene expression (NICE) system and experimental study of its immunoreactivity [J].Curr Microbiol,2009 58:308-314
[25]Joseph Sambrook,David W. Russell.Molecular Cloning:A Laboratory Manual,3rd ed [M].Cold Spring Harbor Laboratory Press,2001
[26]Lembo A,caladonna L,Magron T,et al.Helicobacter pylori infection, immune response and vaccination [J].Curr Drug Targets Immune Endocr Metabol Desord,2001,1(3): 119～208
[27]Sougioltzis S,Lee CK,Alsabli M.Safely and efficacy of E coli enterooxin adjuvant for urease based rectal immunization against Helicobacter pylori [J].Vaccine,2002,21(3): 194-201
[28]朱森林,廖文俊.表达幽门螺杆菌Ure-B减毒鼠伤寒沙门氏菌对小鼠的免疫保护作用[J].中山医科大学学报,2001,22(6)：418-422
[29]Martin M C,Alonso J C,Suarez J E,et al.Genertion of food-grade recombinamt Lactic acid bacterium strains by site-specific recombination [J].Applied Environmental Microbiology,2000,66(6):2599-2604
[30]史达,宋岩,李一经.乳酸乳球菌作为黏膜免疫活载体疫苗传递抗原的研究进展[J].微生物学报,2006,46(4)：680-683
[31]郭兴华,益生乳酸细菌：分子生物学及生物技术[M].北京科学技术出版社,2008：129-130
[32]Bermudez-Humaran L G,Langella P,Miyoshi A,et al.Production of human papillomavirus type 16 E7 protein in Lactococcus lactis [J].Applied and Enviromental Microbiology,2002,68(2):917-922
[33]Wells JM,Wilson PW,NortonPM,et al.Lactoeoceus lactis:high-level expression of tetanus toxin fragment C and protection against lethal challenge [J].Mol Microbiol,1993, 8(6):1155-1162
[34]Lee MH,Roussel Y,Wilks M,et al.Expression of Helicobacter pylori urease subunit B gene in Lactococcus lactis MG1363 and its use as a vaccine delivery system against H.pylori infection in mice [J].Vaeeine,2001,19(28-29):3927-3933
[35]Kim SJ,Jun do Y,Yong CH,et al.Expression of Helicobacter pylori cag12 gene in Lactococcus lactis MG1363 and its oral administration to induce systemic anti-Cag12 immune response in mice [J].Appl Microbiol Biotechnol,2006,72(3):462-470
[36]李文妹,陆惠民,张惠琴,等.弓形虫P30乳酸球菌口服疫苗诱导小鼠的体液免疫反应及保护性观察,中国寄生虫病防治杂志,2004,17(2)：90-92
[37]孙强正,徐建国.乳酸乳球菌食品级表达载体的研究进展[J].中国微生态学杂志,2006,18(3)：260-261
[38]FU X,XUJG.Development of a chromosome-plasmid balanced lethal system for Lactobacillus acidophilus with thyA gene as selective marker [J].Microbiol Immunol, 2000,44:551-556
[39]熊衍文.乳酸乳球菌食品级载体系统的构建及外源基因的克隆和表达[D].[博十学位论文],2004,中国疾病控制中心
[40]Platteeuw C,Alen-Boerriger I,van Schalkwijk S,et al.Food-grade cloning and expression system for Lactococcus lactis [J].Appl Environ Microbiol,1996,15:839-847
[41]卫文仲,向华,谭华荣.人铜锌超氧化物歧化酶基因在乳酸乳球菌中食品级表达[J].微生物学报,2003,3(2)：377-354
[42]贾兴元,陈星,苏畅,等.人工合成苯丙氨酸脱氨酶基因在乳酸乳球菌中的食品级表达[J].中华微生物学和免疫学杂志,2006,26(1)：23-26
[43]Frosethb R,Herman RE E,Mckayl L.Cloning of nisin resistance determinant and replication origin on 7-kilobase EcoR I fragment of pNP400 from Sstrep to coccus lactis subsp diacetylactis DRC3[J].Appl Environ Microbiol 1988,54:2136-2139
[44]Igor Mierau,Kees Olieman,James Mond,et al.Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications [J].Microbial Cell Factories,2005,4(1):4-16
[45]陈星,高斌,贾兴元,等.使用偏爱密码子大幅度提高苯丙氨酸脱氨酶在食品级乳酸乳球菌中的表达[J].生物工程学报,2006,22(2)：187-190
[46]方积乾.卫生统计学[M].(第五版),北京人民卫生出版社,2005
[47]白雪飞.幽门螺杆菌抗原rUreB、rOmp11经3种黏膜途径接种BALB/c小鼠的免疫效果研究[D].[硕士学位论文],2006,郑州大学
[48]孙强正,李振军,李娟,等.霍乱毒素B亚单位在乳酸乳球菌食品级表达系统中的分泌性表达[J].中华流行病学杂志,2009,30(12)：1288-1291
[1]薛迎迎,杨汝德.乳酸菌基因组学与基因工程的研究新进展[J].现代食品科技.2008,24(6)：617-620
[2]Ahmed,F.E.Genetically modified probiotics in foods.Trends Biotechnol [J].2003, 21(11):491-497
[3]Kleerebezem M,Hugenholtz J.Metabolic pathway engineering in Lactic acid bacteria.Current Opinion in Biotechnology [J].2003,14(2):232-237
[4]周正任.医学微生物学[M].北京：人民卫生出版社,2003
[5]Matsuzaki T.Immunomodulation by treatment with Lactobacillus casei strain Shirota.International Journal of Food Microbiology [J].1998,41(2):133-140
[6]Hosoda M,Hashimoto H,Morita H,et al.Antimutagenicity of Milk Cultured with Lactic Acid Bacteria against N-Methyl-N'-Nitro-N-Nitrosoguanidine [J].J Dairy Sci,1992, 75(4):976-981
[7]贾士芳,郭兴华.活菌制剂的现状和未来.生物工程进展[J].1996,16(2)：16-20
[8]Shroff K E,Meslin K,Cebra JJ.Commensal enteric bacteria engender a self-limiting humoral mucosal immune response while permanently colonizing the gut [J].Infection and Immunity,1995,63(10):3904-3913
[9]Gronlund M M,Arvilommi H,Kero P,et al.Importance of intestinal colonisation in the maturation of humoral immunity in early infancy:a prospective follow up study of healthy infants aged 0-6 months [J].Archieves of Disease in Child,2000,83(3):186～192
[10]Ivory K,Chambers SJ,Pin C,et al.Oral delivery of Lactobacillus casei Shirota modifies allergen-induced immune responses in allergic rhinitis[J].Clinical & Experimental Allergy,2008,38(8):1282-1289
[11]王跃,罗强,陈淑惠,等.双歧杆菌脂磷壁酸对结肠癌细胞Toll样受体表达的影响[J].中华微生物学免疫学杂志,2005,25(10)：803-805
[12]Ouwehand A,Isolauri E,Salminen S,et al.The role of the intestinal microflora for the development of the immune system in early childhood [J].European Journal of Nutrition, 2002,41(11):32-37
[13]焦凤超,李迎晓,刘纪成,等.减毒沙门氏菌运送的口服DNA疫苗研究进展[J].中国兽医学杂志,2008,44(4)：50-51
[14]Neirynck S,Steidler L.Delivery of therapeutic proteins through Lactococcus lactis [J].Biotechnol Genet Eng Rev,2006,22:253-266
[15]Lother Steidler,Pieter Rottiers.Therapeutic drug delivery by genetically modified Lactococcus lactis [J].Annals of the New York Academy of Sciences,2006,1072(1): 176-186
[16]Olivera ML,Areas AP,Campos IB,et al.Induction of systemic and mucosal immune response and decrease in Streptococcus pneumoniae colonization by nasal inoculation of mice with recombinant lactic acid bacteria expressing pneumococcal surface antigen A[J].Microbes and infection,2006,8(4):1016-1024
[17]Seegers JF.Lactobacillus as live vaccine delivery vectors:progress and prospects [J].Trends in biotechnology,2002,20(12):508-515
[18]Norton PM.,Wells JM.,Brown HW,et al.Protection against tetanus toxin in mice nasally immunized with recombinant Lactococcus lactis expressing tetanus toxin fragment C[J].Vaccine,1997,15(6-7):616-619
[19]Ming HL,Yvonne R,Mark Wilks,et al.Expression of Helicobacterpylori urease subunit B gene in Lactococcus lactis MG1363 and its use as a vaccine delivery system against H. pylori infection in mice [J].Vaccine,2001,19(28-29):3927-3935
[20]Li YG,Tian FL,Gao FS,et al.Immune responses generated by Lactobacillus as a carrier in DNA immunization against foot-and-mouth disease virus[J].Vaccine,2007,25(5): 902-911
[21]Poo H,Pyo H M,Lee T Y,et al.Oral administration of an human papillomavirus type 16 E7 displayed on Lactobacillus casei induces E7-specific antitumor effects in C57/BL6 mice[J].Int J Cancer,2006,119(7):1702-1709
[22]李桂伟,乔薪瑗,刘伯臣,等.表达猪轮状病毒VP4基因重组乳酸球菌表达载体构建及免疫原性分析[J].中国农业科学,2009,42(10)：3672-3678
[23]许云华.表达GPV VP3和CPV VP2结构蛋白重组乳酸乳球菌的构建[D].[硕士学位论文],2008,扬州大学
[24]Vinay S,Jean PA, Maarten L,et al.Intranasal delivery of influenza subunit vaccine formulated with GEM particles as an adjuvant [J].The AAPS Journal,2010,12(2): 109-116
[25]Buccato S,Maione D,Rinaudo C D,et al.Use of Lactococcus lactis Expressing Pili from Group B Streptococcus as a Broad-Coverage Vaccine against Streptococcal Disease[J].The Journal of Infectious Diseases,2006,194(1):331-340
[26]Kim SJ,Jun DY,Yang CH,et al.Expression of Helicobacter pylori cag12 gene in Lactococcus lactis MG1363 and its oral administration to induce systemic anti-Cagl2 immune response in mice[J].Applied Microbiology and Biotechnology,2006,72(3): 462-470
[27]Sheppler L,Vogel M,Marti P,et al.Intranasal immunisation using recombinant Lactobacillus johnsonii as a new strategy to prevent allergic disease[J].Vaccine,2005, 23(9):1126-1134
[28]Yao XY,Yuan MM,Li DJ.Mucosal inculation of Lactobacillus expressing hCGbeta induces anti-hCGbeta antibody response in mice of different strains [J].Methods,2006, 38(2):124-132
[29]Ramasamy R,Yasawardena S,Zomer A,et al.Immunogenicity of a malaria parasite antigen displayed by Lactococcus lactis in oral immunisations[J].Vaccine,2006,24(18): 3900-3908
[30]孙强正,徐建国.乳酸乳球菌食品级表达载体的研究进展[J].中国微生态学杂志,2006,18(3)：260-261
[31]FU X,XU J G.Development of a chromosome-plasmid balanced lethal system for Lactobacillus acidophilus with thyA gene as selective marker [J].Microbiol Immunol, 2000,44:551-556
[32]孙强正,熊衍文,李振军,等.乳酸乳球菌食品级载体的构建及Mn-SOD基因的克隆表达[J].中国人兽共患病学报,2006,22(6)：498-501
[33]Bron PA,Benchimol MG,Lambert J,et al.Use of the alr Gene as a Food-Grade Selection Marker in Lactic Acid Bacteria [J].Appl Environ Microbiol.2002,68(11): 5663-5670
[34]Glenting J,Madsen S M,Vrang A,et al.A plasmid selection system in Lactococcus lactis and its use for gene expression in L.lactis and human kidney fibroblasts [J].Appl Environ Microbiol,2002,68(10):5051-5056
[35]卫文仲,向华,谭华荣.人铜锌超氧化物歧化酶基因在乳酸乳球菌中食品级表达[J].微生物学报,2003,3(2)：377-354
[36]贾兴元,陈星,苏畅,等.人工合成苯丙氨酸脱氨酶基因在乳酸乳球菌中的食品级表达[J].中华微生物学和免疫学杂志,2006,26(1)：23-26
[37]Frosethb R,Herman RE E,Mckayl L.Cloning of nisin resistance determinant and replication origin on 7-kilobase EcoR I fragment of pNP400 from Sstrep to coccus lactis subsp diacetylactis DRC3[J].Appl Environ Microbiol 1988,54(8):2136-2139
[38]Igor Mierau,Kees Olieman,James Mond,et al.Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications [J].Microbial Cell Factories,2005,4(1):16-28
[39]Mierau I,Kleerebezem,M.10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis [J].Applied Microbiology and Biotechnology,2005,68(6):705-717
[40]Madsen SM,Arnau J,Vrang A,et al.Molecular characterization of the pH inducible and growth phase dependent promoter P170 of Lactococcus lactis [J].Molmicrobiol,1999, 32(1):75-87
[41]Sanders JW,Leenhouts K,Burghoorn J,et al.A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation [J].Molmicrobiol,1999,32(1):75-87
[42]Sanders JW,Venema G,Kok J.A chloride-inducible gene expression cassette and its used in induce lysis of Lactococcus lactis [J].Appl Environ Microbiol,1997,63(12):4877-4882
[43]孙强正,熊衍文,叶长芸,等.食品级分泌表达载体的构建及报告蛋白在乳酸乳球菌中表达[J].微生物学报,2008,48(3)：293-298
[44]Sandrine AL,Maarten L,Jolanda N,et al.Lactococcus lactis GEM particles displaying pneumococcal antigens induce local and systemic immune responses following intranasal immunization [J].Vaccine,2006,24(26):434-5441
[45]Shida K,Makino K,Morishita A,et al.Lactobacillus casei Inhibits Antigen-Induced IgE Secretion through Regulation of Cytokine Production in Murine Splenocyte Cultures [J].Int Arch Allergy Immunol,1998,115(4):278-287
[46]Miettinen M,Vuopio-Varkila J,Varkila K.Production of human tumor necrosis factor alpha, interleukin-6,and interleukin-10 is induced by lactic acid bacteria [J].Infect. Immun.,1996,64(12):5403-5405
[47]Schiffrin EJ,Brassart D,Servin AL,et al.Immune modulationof blood leukocytes in humans by lactic acid bacteria:criteria for strain selection [J].Am J Clin Nutr,1997, 66(2):515S-520S
[48]Tejada-Simon MV,J stunol ZL,Pestka J J.Effects of lactic acid bacteria ingestion of basal cytokine mRNA and immunoglobulin levels in the mouse [J].Food Prot,1999,62 (3):287-291
[49]Haller D,Blum S,Bode C,et al.Activation of human peripheral blood mornouclear cells by nonpathogenic bacteria in vitro:evidence of NK cells as proiary targets [J].Infect Immun,2000,68(2):752-759
[50]Aattouri N,Lemonnier D.Production of interferon thermophilus:role of CD4+ and CD8 + lymphocytes induced by Streptococcus[J].Nutr Biochem,1997,8(1):25-31
[51]Steidler L,Hans W,Schotte L,et al.Treatment of murine colitis by Lactococcus lactis secreting interleukin-10[J].Science,2000,289(5483):1311-1312
[52]Vandenbroucke K.,Hans W.,Vanhuysse J.,et al.Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice [J].Gastroenterology,2004,127(2):502-513
[53]Steidler,L.,Neirynck S.,Huyghebaert N.,et al.Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10[J].Nature Biotechnol,2003,21(7):785-789