几种布鲁菌抗原的免疫学特性研究
|
摘要
布鲁菌(Brucella)是布鲁菌病(Brucellosis)的病原体。现已发现布鲁菌有7个生物种:羊布鲁菌(B.melitensis)、牛布鲁菌(B.abortus)、猪布鲁菌(B. suis)、犬布鲁菌(B. canis)、绵羊布鲁菌(B. ovis)、鼠布鲁菌(B.neotomae)和海洋哺乳动物布鲁菌(B.maris)。各种间具有高度的同源性,但在毒力、生物学性状以及感染人类或畜类后的临床表现和流行特征等方面存在差异。其中既可以感染畜类也可以感染人类的生物种有羊、牛、猪和犬布鲁菌。在我国流行的主要有羊、牛和猪布鲁菌三种,且以羊布鲁菌最为常见,其致病力也最强。
布鲁菌是一种细胞内寄生的革兰阴性菌,感染后可以诱导机体产生保护性体液免疫和细胞免疫反应。细胞免疫可清除细胞内布鲁菌,在抗布鲁菌感染免疫中占主要地位。现有的布鲁菌疫苗为灭活疫苗和减毒活疫苗,在一定程度上对于疫情的预防和控制起到积极作用,但是也存在一些问题:(1)灭活疫苗虽然可以起到一定的保护作用,但是免疫效果比较有限,不能引起足够的细胞免疫应答;( 2)减毒活疫苗由于毒力恢复反弹而感染动物或人类,其应用受到限制;(3)在疫区筛查检测时,常用的血清学方法如试管凝集试验等无法区分传统疫苗免疫的动物和受到布鲁菌感染的动物,这对于隔离病畜,进而防治疫情扩散起到制约作用。因此,国内外均在进行布鲁菌新型疫苗包括DNA疫苗的研究。目前,已经发现了一些具有保护作用的布鲁菌抗原基因,如编码L7/L12、BCSP31和OMP31等抗原的基因。单独的保护抗原基因虽然可以诱导一定的免疫反应,起到一定的保护作用,但是存在保护效果较差,保护时间较短等缺陷。因此,研制联合多个保护性抗原基因的DNA疫苗是布鲁菌疫苗研究的热点之一。
本课题在前期研究的基础上,对布鲁菌rp1L、omp31、bcsp31、omp22、omp2b等基因进行免疫学分析研究:制备了相应的单克隆抗体(monoclonalantibody,mAb);对多个抗原基因的各种组合进行了免疫学分析,以期筛选出最佳的组合方式。
1、几种布鲁菌外膜蛋白的表达、纯化及其单克隆抗体的制备
构建了几种布鲁菌外膜蛋白基因的原核表达载体,并将其分别命名为pGEX-4T-1-omp31、pGEX-4T-1-omp22、pGEX-4T-1-omp25d、pGEX-4T-1-omp2b。将这些质粒分别转化至大肠埃希菌并诱导表达,采用GST亲和层析纯化的方法,分别获得纯化的布鲁菌OMP31、OMP22、OMP25d和OMP2b蛋白。用纯化的蛋白检测布鲁菌全菌免疫的BALB/c小鼠血清,取高效价的小鼠脾细胞与骨髓瘤Sp2/0细胞融合,ELISA间接法筛选阳性克隆,3次克隆化后建立杂交瘤细胞系。采用小鼠腹腔接种杂交瘤细胞制备腹水,正辛酸-硫酸铵法纯化mAb。采用Western blot和ELISA等方法鉴定mAb特性。
结果获得了2株抗布鲁菌OMP22蛋白的mAb,2株抗布鲁菌OMP25d蛋白的mAb,2株抗布鲁菌OMP31蛋白的mAb以及1株抗布鲁菌OMP2b蛋白的mAb。这7株单克隆抗体特异性强、敏感性较好,与大肠埃希菌、金黄色葡萄球菌、枯草芽孢杆菌、结核分枝杆菌和绿脓假单胞菌等无交叉反应。
2、重组DNA的制备和动物免疫
分别构建了pcDNA3.1-rp1L、pcDNA3.1-omp31、pcDNA3.1-bcsp31、pcDNA3.1-omp22和pcDNA3.1-omp2b真核表达载体。分别用这些质粒转染CHO细胞,其所表达的蛋白可被兔抗布鲁菌血清识别。再将上述质粒大量提取和纯化,并按照如下方式进行组合后分别免疫C57BL/6小鼠,并检测各项免疫学指标,以减毒活疫苗M5株和生理盐水为对照。
(1)将pcDNA3.1-rp1L、pcDNA3.1-omp31和pcDNA3.1-bcsp31三组重组DNA等量混合免疫C57BL/6小鼠,并将这种组合命名为ROB。
(2)将pcDNA3.1-omp22和pcDNA3.1-omp2b分别免疫C57BL/6小鼠。
(3)将ROB和pcDNA3.1-omp22(命名为Ⅰ4);ROB和pcDNA3.1-omp2b(Ⅱ4);ROB和pcDNA3.1-omp22、pcDNA3.1-omp2b(Ⅲ5)三组分别免疫C57BL/6小鼠。
3、重组DNA免疫效果的评价
ROB组重组DNA免疫小鼠在末次免疫后4周的抗体水平最高,平均抗体滴度为1 : 1280(P<0.01);所产生的Th1型细胞因子如TNF-α和IFN-γ等均比M5株对照组高出2倍以上(P<0.05);所诱导的脾淋巴细胞增殖能力明显强于M5株对照组(P<0.05);并具有更强的细胞杀伤能力(P<0.05),提示获得的ROB重组DNA在细胞免疫水平方面要强于M5疫苗株。
布鲁菌omp22组和omp2b组均刺激了较高的体液免疫水平(均大于1:1000,P<0.05);在检测IgG2a/IgG1比值方面,omp22组和omp2b组均优于M5组( P<0.05); omp2b组诱导的TNF-α和IFN-γ水平高于M5组( P<0.05); pcDNA3.1-omp2b免疫动物的脾细胞经特异性抗原刺激后,所产生的IFN-γ的细胞数目约是M5组的4倍(P<0.05); CTL杀伤实验表明,在效靶比为100时,omp2b组的杀伤能力明显强于M5组(P<0.05)。omp22组的各项指标也明显好于M5组,且具有统计学意义(P<0.05)。
Ⅱ4组所诱导的特异性IgG为1:1280(P<0.05);IgG2a/IgG1比值是2.8(P<0.05)。在细胞免疫方面,Ⅱ4组诱导的TNF-α和IFN-γ高于M5组(P<0.05);Ⅱ4组免疫小鼠的脾细胞经特异性抗原刺激后能产生IFN-γ的细胞数目为64±3.4,高于M5组(P<0.05); CTL杀伤实验表明,在效靶比为100时,Ⅱ4组的杀伤效果优于M5组(P<0.05)。
将Ⅱ4组和ROB组数据进行比对,其免疫效果优于原先ROB组,也优于M5疫苗组,我们将这四种抗原基因组成的组合命名为ROBO。综上所述,重组DNA所引起的细胞免疫应答水平要高于传统疫苗,本实验结果为进一步研究布鲁菌新型DNA疫苗奠定了较好的理论基础和物质基础。
Brucella is the pathogen of brucellosis. There have been found sevenBrucellaspecies:B.melitensis,B.abortus,B.suis,B.canis,B.ovis,B.neotomaeandB.maris.Thespeciesarehigh in homology, buttherearedifferent fromtoxicity,biologicalcharacteristics,clinicalmanifestationsandepidemicfeaturesinhumansandanimals.Inallthespecies,onlyB.melitensis,B.abortus,B.suisandB.caniscaninfectbothanimalsandhumans.InChina,themainbrucellaspeciesareB.melitensis,B.abortusandB.suis,andthemostprevalentisB.melitensiswhichisalsoasthemostvirulent.
Brucella is an intracellular parasitic Gram-negative bacterium. InfectionwithBrucellacan induceprotective humoral immunityandcellular immunity.Cellular immunity can clear brucella in cells, and plays an important role inanti-infection immunity of Brucella. Brucella vaccines are available for theinactivatedvaccinesandliveattenuatedvaccines.Toacertainextent,theyareuseful,buttherearemanyproblems:(1)inactivatedvaccinesaresoineffectivetocauseadequatecellularimmuneresponses(2)liveattenuatedvaccinesarelikelytocauseanimaldiseaseandinfecthumans(3)theycannotdistinguishbetweenimmunized animals and infected animals, which are important for epidemiccontrolandeconomy.Therefore,theresearcherslooktoinvestinnewvaccinessuchasDNA vaccines.Sofar,some antigens withprotectiveeffecthas beenfound,suchastheL7/L12,BCSP31andOMP31,whichcaninduceaprotectiveimmune response, but the protection of individual antigens is not effectiveenough cause the protection time is short and other defects. Therefore, thedevelopmentofmultivalentvaccine,highlevelsofexpressionofDNAvaccineshasbecomeimperative.
On the basis of the previous studies, this item was focused on theimmunological analysis ofBrucella rp1L, omp31, bcsp31, omp22 and omp2bgenes:preparationofmonoclonalantibodiesaccordinglytofilteroutthebestcombination, various combinations of multivalent recombinant DNAs wereanalyzed.
1 Cloning, expression ans purification of several Brucellaantigen genesandpreparationofthemonoclonalantibodies
TheplasmidspGEX-4T-1-omp31,pGEX-4T-1-omp22,pGEX-4T-1-omp25d,pGEX-4T-1-omp2b were transformed into E. coli and induced by IPTG. ThecorrespondingproteinswerepurifiedbyGSTaffinitychromatographymethod.
After using whole bacteria to immune mice, antibody was screened by purified proteins. The immunized mouse spleen cells were infused with myeloma Sp2/0 cell, some steps as followed: hybridoma selection and cloning preparation of hybridoma ascites purification of mAb identification of mAb characteristics.
There were 2 anti-OMP31 mAbs, 2 anti-OMP22 mAbs, 2 anti-OMP25d mAbs and 1 anti-OMP2b mAb. These seven mAbs were with high specificity and sensitivity. They did not cross-react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis and Bacillus aeruginosus.
2 Preparation of recombinant DNAs and immunizing animals
pcDNA3.1-rp1L, pcDNA3.1-omp31, pcDNA3.1-bcsp31, pcDNA3.1-omp22 and pcDNA3.1-omp2b vectors were constructed and measured their transient expression in CHO cells by serum from Brucella infected rabbit. Then a large number of the plasmids were extracted and purified. Immunization was performed in several ways as followed. Some immunological tests were performed. M5 and saline were as controls
(1) pcDNA3.1-rp1L, pcDNA3.1-omp31 and pcDNA3.1-bcsp31 equally were combined as trivalent DNAs to immunize C57BL/6 mice. This group was named as ROB.
(2) We immunized C57BL/6 mice by pcDNA3.1-omp22 or pcDNA3.1-omp2b separately.
(3) Three groups as ROB and pcDNA3.1-omp22 (Ⅰ4 ) ROB and pcDNA3.1-omp2b (Ⅱ4 ) ROB and pcDNA3.1-omp22, pcDNA3.1-omp2b (Ⅲ5) were performed to immunize C57BL / 6.
3 Evaluation of recombinant DNAs
4 weeks after the last immunization, ROB group were on the highest specific antibody level (1: 1280, P <0.01) Th1-type cytokines such as TNF-αand IFN-γ of ROB were more than 2 times higher than M5 group (P <0.05) ROB had higher ability in CTL test. The level of cellular immunity that ROB obtained was stronger than the vaccine strain M5.
Brucella omp22 group and omp2b group were at a high level of humoral immunity (greater than 1:1000, P <0.05) the ratio of IgG2a/IgG1 were better than the M5 group (P <0.05) TNF-αand IFN-γlevels of omp2b group were higher than M5 group (P <0.05) IFN-γproduced by the the number of cells of omp2b group was about 4 times more than M5 group (P <0.05) CTL test showed that at e/t ratio of 100, omp2b group were strongler than M5 group (P <0.05). omp22 group was significantly better than M5 group (P <0.05).
pecific IgG ofⅡ4 group was 1:1280 (P <0.05) IgG2a/IgG1 ratio was 2.8 (P <0.05). In terms of cellular immunity,Ⅱ4 group induced higher levels of TNF-αand IFN-γthan M5 group (P <0.05) Cells ofⅡ4 group mice spleen cells stimulated by specific antigen to produce IFN-γwere 64±3.4, higher than M5 group (P <0.05) CTL test showed that when e/t ratio was at 100,Ⅱ4 group was better than M5 group (P <0.05).
Ⅱ4 ROB group was better than all the other groups including M5, ROB and others. we calledⅡ4 as ROBO.
In summary, the levels of cell immune response of recombinant DNA were higher than the traditional vaccine. The results of this study are a good theoretical foundation and physical infrastructure for researching in performing a new DNA vaccine against brucellosis.
布鲁菌是一种细胞内寄生的革兰阴性菌,感染后可以诱导机体产生保护性体液免疫和细胞免疫反应。细胞免疫可清除细胞内布鲁菌,在抗布鲁菌感染免疫中占主要地位。现有的布鲁菌疫苗为灭活疫苗和减毒活疫苗,在一定程度上对于疫情的预防和控制起到积极作用,但是也存在一些问题:(1)灭活疫苗虽然可以起到一定的保护作用,但是免疫效果比较有限,不能引起足够的细胞免疫应答;( 2)减毒活疫苗由于毒力恢复反弹而感染动物或人类,其应用受到限制;(3)在疫区筛查检测时,常用的血清学方法如试管凝集试验等无法区分传统疫苗免疫的动物和受到布鲁菌感染的动物,这对于隔离病畜,进而防治疫情扩散起到制约作用。因此,国内外均在进行布鲁菌新型疫苗包括DNA疫苗的研究。目前,已经发现了一些具有保护作用的布鲁菌抗原基因,如编码L7/L12、BCSP31和OMP31等抗原的基因。单独的保护抗原基因虽然可以诱导一定的免疫反应,起到一定的保护作用,但是存在保护效果较差,保护时间较短等缺陷。因此,研制联合多个保护性抗原基因的DNA疫苗是布鲁菌疫苗研究的热点之一。
本课题在前期研究的基础上,对布鲁菌rp1L、omp31、bcsp31、omp22、omp2b等基因进行免疫学分析研究:制备了相应的单克隆抗体(monoclonalantibody,mAb);对多个抗原基因的各种组合进行了免疫学分析,以期筛选出最佳的组合方式。
1、几种布鲁菌外膜蛋白的表达、纯化及其单克隆抗体的制备
构建了几种布鲁菌外膜蛋白基因的原核表达载体,并将其分别命名为pGEX-4T-1-omp31、pGEX-4T-1-omp22、pGEX-4T-1-omp25d、pGEX-4T-1-omp2b。将这些质粒分别转化至大肠埃希菌并诱导表达,采用GST亲和层析纯化的方法,分别获得纯化的布鲁菌OMP31、OMP22、OMP25d和OMP2b蛋白。用纯化的蛋白检测布鲁菌全菌免疫的BALB/c小鼠血清,取高效价的小鼠脾细胞与骨髓瘤Sp2/0细胞融合,ELISA间接法筛选阳性克隆,3次克隆化后建立杂交瘤细胞系。采用小鼠腹腔接种杂交瘤细胞制备腹水,正辛酸-硫酸铵法纯化mAb。采用Western blot和ELISA等方法鉴定mAb特性。
结果获得了2株抗布鲁菌OMP22蛋白的mAb,2株抗布鲁菌OMP25d蛋白的mAb,2株抗布鲁菌OMP31蛋白的mAb以及1株抗布鲁菌OMP2b蛋白的mAb。这7株单克隆抗体特异性强、敏感性较好,与大肠埃希菌、金黄色葡萄球菌、枯草芽孢杆菌、结核分枝杆菌和绿脓假单胞菌等无交叉反应。
2、重组DNA的制备和动物免疫
分别构建了pcDNA3.1-rp1L、pcDNA3.1-omp31、pcDNA3.1-bcsp31、pcDNA3.1-omp22和pcDNA3.1-omp2b真核表达载体。分别用这些质粒转染CHO细胞,其所表达的蛋白可被兔抗布鲁菌血清识别。再将上述质粒大量提取和纯化,并按照如下方式进行组合后分别免疫C57BL/6小鼠,并检测各项免疫学指标,以减毒活疫苗M5株和生理盐水为对照。
(1)将pcDNA3.1-rp1L、pcDNA3.1-omp31和pcDNA3.1-bcsp31三组重组DNA等量混合免疫C57BL/6小鼠,并将这种组合命名为ROB。
(2)将pcDNA3.1-omp22和pcDNA3.1-omp2b分别免疫C57BL/6小鼠。
(3)将ROB和pcDNA3.1-omp22(命名为Ⅰ4);ROB和pcDNA3.1-omp2b(Ⅱ4);ROB和pcDNA3.1-omp22、pcDNA3.1-omp2b(Ⅲ5)三组分别免疫C57BL/6小鼠。
3、重组DNA免疫效果的评价
ROB组重组DNA免疫小鼠在末次免疫后4周的抗体水平最高,平均抗体滴度为1 : 1280(P<0.01);所产生的Th1型细胞因子如TNF-α和IFN-γ等均比M5株对照组高出2倍以上(P<0.05);所诱导的脾淋巴细胞增殖能力明显强于M5株对照组(P<0.05);并具有更强的细胞杀伤能力(P<0.05),提示获得的ROB重组DNA在细胞免疫水平方面要强于M5疫苗株。
布鲁菌omp22组和omp2b组均刺激了较高的体液免疫水平(均大于1:1000,P<0.05);在检测IgG2a/IgG1比值方面,omp22组和omp2b组均优于M5组( P<0.05); omp2b组诱导的TNF-α和IFN-γ水平高于M5组( P<0.05); pcDNA3.1-omp2b免疫动物的脾细胞经特异性抗原刺激后,所产生的IFN-γ的细胞数目约是M5组的4倍(P<0.05); CTL杀伤实验表明,在效靶比为100时,omp2b组的杀伤能力明显强于M5组(P<0.05)。omp22组的各项指标也明显好于M5组,且具有统计学意义(P<0.05)。
Ⅱ4组所诱导的特异性IgG为1:1280(P<0.05);IgG2a/IgG1比值是2.8(P<0.05)。在细胞免疫方面,Ⅱ4组诱导的TNF-α和IFN-γ高于M5组(P<0.05);Ⅱ4组免疫小鼠的脾细胞经特异性抗原刺激后能产生IFN-γ的细胞数目为64±3.4,高于M5组(P<0.05); CTL杀伤实验表明,在效靶比为100时,Ⅱ4组的杀伤效果优于M5组(P<0.05)。
将Ⅱ4组和ROB组数据进行比对,其免疫效果优于原先ROB组,也优于M5疫苗组,我们将这四种抗原基因组成的组合命名为ROBO。综上所述,重组DNA所引起的细胞免疫应答水平要高于传统疫苗,本实验结果为进一步研究布鲁菌新型DNA疫苗奠定了较好的理论基础和物质基础。
Brucella is the pathogen of brucellosis. There have been found sevenBrucellaspecies:B.melitensis,B.abortus,B.suis,B.canis,B.ovis,B.neotomaeandB.maris.Thespeciesarehigh in homology, buttherearedifferent fromtoxicity,biologicalcharacteristics,clinicalmanifestationsandepidemicfeaturesinhumansandanimals.Inallthespecies,onlyB.melitensis,B.abortus,B.suisandB.caniscaninfectbothanimalsandhumans.InChina,themainbrucellaspeciesareB.melitensis,B.abortusandB.suis,andthemostprevalentisB.melitensiswhichisalsoasthemostvirulent.
Brucella is an intracellular parasitic Gram-negative bacterium. InfectionwithBrucellacan induceprotective humoral immunityandcellular immunity.Cellular immunity can clear brucella in cells, and plays an important role inanti-infection immunity of Brucella. Brucella vaccines are available for theinactivatedvaccinesandliveattenuatedvaccines.Toacertainextent,theyareuseful,buttherearemanyproblems:(1)inactivatedvaccinesaresoineffectivetocauseadequatecellularimmuneresponses(2)liveattenuatedvaccinesarelikelytocauseanimaldiseaseandinfecthumans(3)theycannotdistinguishbetweenimmunized animals and infected animals, which are important for epidemiccontrolandeconomy.Therefore,theresearcherslooktoinvestinnewvaccinessuchasDNA vaccines.Sofar,some antigens withprotectiveeffecthas beenfound,suchastheL7/L12,BCSP31andOMP31,whichcaninduceaprotectiveimmune response, but the protection of individual antigens is not effectiveenough cause the protection time is short and other defects. Therefore, thedevelopmentofmultivalentvaccine,highlevelsofexpressionofDNAvaccineshasbecomeimperative.
On the basis of the previous studies, this item was focused on theimmunological analysis ofBrucella rp1L, omp31, bcsp31, omp22 and omp2bgenes:preparationofmonoclonalantibodiesaccordinglytofilteroutthebestcombination, various combinations of multivalent recombinant DNAs wereanalyzed.
1 Cloning, expression ans purification of several Brucellaantigen genesandpreparationofthemonoclonalantibodies
TheplasmidspGEX-4T-1-omp31,pGEX-4T-1-omp22,pGEX-4T-1-omp25d,pGEX-4T-1-omp2b were transformed into E. coli and induced by IPTG. ThecorrespondingproteinswerepurifiedbyGSTaffinitychromatographymethod.
After using whole bacteria to immune mice, antibody was screened by purified proteins. The immunized mouse spleen cells were infused with myeloma Sp2/0 cell, some steps as followed: hybridoma selection and cloning preparation of hybridoma ascites purification of mAb identification of mAb characteristics.
There were 2 anti-OMP31 mAbs, 2 anti-OMP22 mAbs, 2 anti-OMP25d mAbs and 1 anti-OMP2b mAb. These seven mAbs were with high specificity and sensitivity. They did not cross-react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis and Bacillus aeruginosus.
2 Preparation of recombinant DNAs and immunizing animals
pcDNA3.1-rp1L, pcDNA3.1-omp31, pcDNA3.1-bcsp31, pcDNA3.1-omp22 and pcDNA3.1-omp2b vectors were constructed and measured their transient expression in CHO cells by serum from Brucella infected rabbit. Then a large number of the plasmids were extracted and purified. Immunization was performed in several ways as followed. Some immunological tests were performed. M5 and saline were as controls
(1) pcDNA3.1-rp1L, pcDNA3.1-omp31 and pcDNA3.1-bcsp31 equally were combined as trivalent DNAs to immunize C57BL/6 mice. This group was named as ROB.
(2) We immunized C57BL/6 mice by pcDNA3.1-omp22 or pcDNA3.1-omp2b separately.
(3) Three groups as ROB and pcDNA3.1-omp22 (Ⅰ4 ) ROB and pcDNA3.1-omp2b (Ⅱ4 ) ROB and pcDNA3.1-omp22, pcDNA3.1-omp2b (Ⅲ5) were performed to immunize C57BL / 6.
3 Evaluation of recombinant DNAs
4 weeks after the last immunization, ROB group were on the highest specific antibody level (1: 1280, P <0.01) Th1-type cytokines such as TNF-αand IFN-γ of ROB were more than 2 times higher than M5 group (P <0.05) ROB had higher ability in CTL test. The level of cellular immunity that ROB obtained was stronger than the vaccine strain M5.
Brucella omp22 group and omp2b group were at a high level of humoral immunity (greater than 1:1000, P <0.05) the ratio of IgG2a/IgG1 were better than the M5 group (P <0.05) TNF-αand IFN-γlevels of omp2b group were higher than M5 group (P <0.05) IFN-γproduced by the the number of cells of omp2b group was about 4 times more than M5 group (P <0.05) CTL test showed that at e/t ratio of 100, omp2b group were strongler than M5 group (P <0.05). omp22 group was significantly better than M5 group (P <0.05).
pecific IgG ofⅡ4 group was 1:1280 (P <0.05) IgG2a/IgG1 ratio was 2.8 (P <0.05). In terms of cellular immunity,Ⅱ4 group induced higher levels of TNF-αand IFN-γthan M5 group (P <0.05) Cells ofⅡ4 group mice spleen cells stimulated by specific antigen to produce IFN-γwere 64±3.4, higher than M5 group (P <0.05) CTL test showed that when e/t ratio was at 100,Ⅱ4 group was better than M5 group (P <0.05).
Ⅱ4 ROB group was better than all the other groups including M5, ROB and others. we calledⅡ4 as ROBO.
In summary, the levels of cell immune response of recombinant DNA were higher than the traditional vaccine. The results of this study are a good theoretical foundation and physical infrastructure for researching in performing a new DNA vaccine against brucellosis.
引文
1. Jahans KL, Foster G, Broughton ES. The characterization of Brucella strains isolated from marine mammals. Vet Microbiol. 1997, 57(1):373-382.
2. Mayfield JE, Betsy JB. The cloning, expression, and nucleotide sequence of a gene coding for an immunogenic Brucella abortus protein.Gene. 1988, 63 (1):1-8.
3. Mehmet D, Bilgehan A. Human brucellosis: an overview. Infect Dis. 2003, 7: 173-182.
4. Jinkyung K, Gary A. Molecular host-pathogen interaction in brucellosis: current understanding and future approaches to vaccine development for mice and humans. Clin Microbio Review. 2003, 16:65-78.
5. Zhang FL, Wu XA, Luo W, Bai WT, Liu Y, Yan Y, Wang HT, Xu ZK. The expression and genetic immunization of chimeric fragment of Hantaan virus M and S segments. BBRC. 2007, 354(4): 858-863.
6. Akdeniz H, Irmak H, Anlar O et al. Central nervous system brucellosis: presentation, diagnosis and treatment. J Infect. 1998, 36: 297–301.
7. Shang DQ. Research on Brucella spp. Chin J Ctrl Endem Dis. 2004, 19: 204–213.
8. Franco MP, Mulder M, Gilman RH et al. Human brucellosis. Lancet Infect Dis. 2007, 7: 775–786.
9. Hoover D, Friedlander A. Brucellosis. In Zajtchuk R, ed. Textbook of Military Medicine: Medical Aspects of Chemical and Biological Warfare. Washington, DC: US Department of the Army, Surgeon General, and the Borden Institute 1997: 513-521.
10. Amélie G, Philippe BP, Fran ois B. Brucellose par bioterrorisme. La Presse Médicale, 2004, 33( 2): 119-122.
11. World Health Organization: Fact Sheet N173. World Health Organization, Geneva, Switzerland July 1997.
12. Pappas G., Papadimitriou P, Akritidis N, Christou L and Tsianos E.V. The new global map of human brucellosis. Lancet Infect Dis. 2006, 6: 91–99.
13.赵忠鹏,王希良.布鲁菌毒力因子研究进展.微生物学免疫学进展. 2007, 35 (1):50-53.
14.徐志凯.临床微生物学.北京:高等教育出版社, 2007. 244-245.
15. Gross A, Terraza A, Ouahrani-Bettache S, Liautard JP, Dornand J In vitro Brucella suis infection prevents the programmed cell death of human monocytic cells. Infect Immun. 2000, 68, 342-351.
16. Pappas G, Akritidis N, Bosilkovski M and Tsianos E. Brucellosis. N Engl J Med. 2005, 352: 2325–2336.
17. Farshad S, Javad M, Banpour M. Prodution and characterization of monoclonal antibodies against Brucella abortus S (99) surface antigens. Iran Biomed. 2002, 6 (1): 7-12.
18.吐尔洪努尔,谷文喜,何倩倪,热合木江,布合丽切木依干巴地,刘丽娅.布鲁菌病研究进展.动物医学进展. 2007, 28(7): 82-87.
19. Bowden RA, Cloeckaert A, Zygmunt M S. Evaluation of immunogenicity and protective activity in BALB/c mice of the 25kDa major outer membrane protein of Brucella melitensis (OMP25) expressed in Escherichia coli. Jmed Micobiol. 1998, 47: 39-48.
20. Gerhardt G, Schurig NA, Michael J. Brucellosis vaccine: past, present and future. Vet Microbiol. 1990, 20(20): 479-496.
21. Bocharova EV, Gudkov AT, Arsenieva AS.Topology of the secondarystructure elements of ribosomal protein L7/L12 from E. coli in solution. FEBS Letters. 1996, 379:291-294.
22. Simge B, Boyle SM, Vemulapalli R, Sriranganathan N, Schurig GG, Toth TE. Immune responses of mice to vaccinia virus recombinants expressing either Listeria monocytogenes partial listeriolysin or Brucella abortus ribosomal L7/L12 protein. Vet Microbiol, 2005, 109(1-2): 11-17.
23. Zhongpeng Z, Min L, Deyan L, Li X, Shuo W, Yueqiang D, Penghui Y, Xiliang W. Protection of mice from Brucella infection by immunization with attenuated Salmonella enterica serovar typhimurium expressing A L7/L12 and BLS fusion antigen of Brucella. Vaccine. 2009, 27(38): 5214-5219.
24. Victoria DM, Juliana Cassataro, Carlos A. Brucella outer membrane protein Omp31 is a haemin-binding protein. Microbes Infec.2006, 8(5):1203-1208.
25. Silvia M. Estein, Pablo et al. Immunogenicity of recombinant Omp31 from Brucella melitensis in rams and serum bactericidal activity against B. ovis. Vet Microbiol. 2004, 102:203-213.
26. Bricker BJ, Tabatabai LB, Deyoe BL et al. Conservation of antigenicity in a 31-kDa Brucella protein. Vet Microbio. 1988, 18:313–325.
27. Van MD, Kennedy GA, Olsen SC, et al. Brucellosis induced by RB51 vaccine in a pregnant heifer.J Am Vet Med Assoc. 1999, 215: 1491-1493.
28. Pugh GW Jr, Tabatabai LB. Variation of Brucella abortus 2308 infection in BALB/c mice induced by prior vaccination with salt-extractable periplasmic proteins from Brucella abortus 19. Infect Immun. 1996, 64(2): 548-556.
29. Halling SM, Detilleux PG, Tatum FM, et al. Deletion of the BCSP31 gene of B.Abortus by replacement. Infect. Immun.1991, 59:3863-3868.
30. Thomas JS, Mayfield JE, Louisa B. et al. Oral Immunization of Micewith Attenuated Salmonella typhimurium Containing a Recombinant PlasmidWhich Codes for Production of a 31-Kilodalton Protein of Brucella abortus. Infect onand immunity.1990, 58(7): 2048-2055.
31. Cloeckaert A, Verger M, Grayon M, et al. Classification of Brucella spp. Isolated from marine manuals by DNA polymorphism at the Omp2 locus. Microbes Infect. 2001, 3: 729-738.
32. Cassataro J, Pasquevich K, Bruno L, et a1. Antibody reactivity to Omp3l from Brucella melitensis in human and animal infections by smooth and rough Brucellae. Clin Diagn Lab Immunol, 2004, 11(1):111-114.
33. Delvecchio G, Kapat V , RedKat J, et al . The genome of Brucella melitensis. Vet Microbiol. 2002, 90 (1): 587 -592.
34. Mart′n-Mart′n AI, Caro-Herna′ndez P, Orduna A, et al. Importance of the Omp25/Omp31 family in the internalization and intracellular replication of virulent B. ovis in murine macrophages and HeLa cells. Microbes Infec. 2008, 10: 706-710.
35. Martín-Martín AI, Caro-Hernández P, Sancho P, Tejedor C, Cloeckaert A, Fernández-Lago L, Vizcaíno N. Analysis of the occurrence and distribution of the Omp25/Omp31 family of surface proteins in the six classical Brucella species. Vet. Microbiol. 2009, 137(1-2): 74-82.
36. Edmonds D, Cloeckaert A, Hagius D, et al. Pathogenicity and protective activity in pregnant goats of a Brucella melitensis Delta Omp25 deletion mutant. Res Vet Sci. 2002, 72: 235–239.
37. Manterola L, Guzman-Verri C, Chaves-Olarte E, et al. BvrR/BvrS-controlled outer membrane proteins Omp3a and Omp3b are not essential for Brucella abortus virulence. Infect Immun. 2007, 75: 4867-4874.
38. Vizcaino N, Caro-Hernandez P, Cloeckaert A, et a1. DNA polymorphism in the Omp25/Omp31 family of Brucella spp: identification of a 1.7-kbinversion in Brucella cetaceae and of a 15.1-kb genomie island, absent from Brucella ovis, related to the synthesis of smooth lipopolysaccharide. Microbes Infect. 2004, 6 (9):821-834.
39.尚德秋.布鲁氏菌病流行病学研究近况.中华流行病学杂志,.1998, 19(2): 107-110.
40. Bagchi TD. Immune mechanisms in murine brucellosis: studies with strain RB51, a rough mutant of Brucella abortus. USA, VA, 1990: 24-61.
41. Donnelly JJ, Wahren B, Liu MA. DNA vaccines: progress and challenges. J Immunol. 2005, 175(2):633–639.
42. Pasquali P, Rosanna A, Pistoia C. Brucella abortus RB51 induces protection in mice orally infected with the virulent strain B. abortus 2308. Infect Immun. 2003, 71(5): 2326-2330.
43. Ashfrd DA, Pietra J, Lingappa J. Adverse events in humans associated with accidental exposure to the livestock brucellosis vaccine RB51. Vaccine. 2004, 22(25-26): 3435-3439.
44. Davis DS, Elizer PH. Brucella vaccines in wildlife. Vet Microbiol. 2002, 90(11):533-544.
45. Munoz-Montesino C, Andrews E, Rivers R, Gonzalez-Smith A, Moraga-Cid G, Folch H, et al. Intraspleen delivery of a DNA vaccine coding for superoxide dismutase (SOD) of Brucella abortus induces SOD-specific CD4+ and CD8+ T cells. Infect Immun. 2004, 72(4): 2081-2087.
46. Elzer PH, Enright FM. Evaluation of a rough mutant of Brucella melitensis in pregnant goats. Res Vet Sci. 1998, 64: 259-260.
47. Murphy EA, Sathiyaseelan J, Parent MA. Interferon-gamma is crucial for surviving a Brucella abortus infection in both resistant C57BL/6 and susceptible BALB/c mice.Immunology. 2001, 103:511-518.
48.罗德炎,韩玉霞,王希良.布氏杆菌病新型疫苗的研究进展.免疫学杂志. 2004, 20(3): 43-45.
49. Klinman DM., Klaschik S, Tross D, Shirota H, Steinhagen F. FDA guidance on prophylactic DNA vaccines: Analysis and recommendations. Vaccine. 2010, 28(16): 2801-2865.
50. Mallick AI, Singha H, Chaudhuri P, Nadeemc A, Khand SA, Dard KA, Owaisa M. Liposomised recombinant ribosomal L7/L12 protein protects BALB/c mice against Brucella abortus 544 infection. Vaccine. 2007, 25: 3692-3704.
51. Gupta VK, Rout PK, Vihan VS. Induction of immune response in mice with a DNA vaccine encoding outer membrane protein (omp31) of Brucella melitensis 16M. Res Vet Sci. 2007, 82(3): 305-313.
52. Kurar E, Splitter GA. Nucleic acid vaccinationof Brucella abortus ribosomal L7/L12 gene elicitsimmune response. Vacine. 1997, 15(17-18): 1851-1857.
53.曾政,邓小红.布鲁氏菌DNA疫苗的发展:过去、现在和未来.国外医学免疫学分册. 2005, 28(3): 143-146
54. Kaushik P, Singh DK, Kumar SV, Tiwari Ak, Shukla G. Protection of mice against Brucella abortus 544 challenge by vaccination with recombinant OMP28 adjuvanted with CpG. Vet Res Com.2010, 34( 2):119-132.
55. Park JH , Song MH, Lee CH, Lee MK Park YM.Expression of the human cancer/testis antigen NY-SAR-35 is activated by CpG island hypomethylation. Biotechnology Letters. 2011.[Equ before print]
56.司瑞,白文涛,徐志凯.羊布鲁菌BCSP31、OMP31、L7/L12基因的原核表达载体构建及表达.中国人兽共患病杂志.2005, 21(11):961-964
57. Kumar G, Rathore G, Sengupta U et al. Production of monoclonal antibodies specific to major outer membrane protein of Edwardsiella tarda. CompImmunol Microbiol Infect Dis. 2010, 33: 133–144.
58.徐志凯.实用单克隆抗体技术.陕西科技出版社.1992:31-41.
59. Hoffmann F, Heuvel J, Zidek N. Minimizing inclusion body formation during recombinant protein production in Escherichia coli at bench and pilot plant scale. Enzyme Microb Tech. 2004, 34: 235–241.
60. Hayashi K, Kojima C. pCold-GST vector: A novel cold-shock vector containing GST tag for soluble protein production. Protein Expres Purif. 2008, 62: 120–127.
61. Vernet E, Kotzsch A, Voldborg B, Sundstr m M. Screening of genetic parameters for soluble protein expression in Escherichia coli . Protein Expres Purif. 2011, 77, (1): 104-111.
62. Cai X, Wang JF, Wang YY et al. Expression, purification and characterization of recombinant human interleukin-22 in Pichia pastoris. Mol Biol Rep. 2010, 37: 2609-2613.
63. Song JP, Chen WT, Lu ZS et al Soluble expression, purification, and characterization of recombinant human flotillin-2 (reggie-1) in Escherichia coli. Mol Biol Rep. 2011, DOI: 10.1007/s11033-010-0335-4
64. Liu SX, Fu ZP, Mu RM et al. Expression and characterization of Momordica Chanrantia anti-hyperglycaemic peptide in Escherichia coli. Mol Biol Rep. 2010, 37: 1781-1786.
65. Cabrita LD, Bottomley SP. Protein expression and refolding - A practical guide to getting the most out of inclusion bodies. Biotech Annu Rev. 2004, 10: 31–50.
66.蛋白质纯化与鉴定实验指南,朱厚础等译.北京,科学出版社, 1999, 7:179-213.
67. Cloeckaert A, Verger JM, Grayon M, Vizcaíno N. Molecular andimmunological characterization of the major outer membrane proteins of Brucella . FEMS Microbiol Letters. 1996, 145(1): 1-8.
68. GSTrap FF, Hitrap affinity columns. Instruction 71-5016-96 AK. GE Healthcare. p6-7
69.胡刚,徐志凯,张芳琳,吴兴安,阎岩,白文涛,于澜,王海涛.抗汉滩病毒双链抗体重组杆状病毒在昆虫细胞中的表达.免疫学杂志. 2005, 21(2): 84-87.
70. Karmali PP, Majeti BK, Sreedhar B. In vitro gene transfer efficacies and serum compatibility profiles of novel mono-, di-, and tri-histidinylated cationic transfection lipids: a structure-activity investigation. Bioconjug Chem. 2006, 17(1): 159-171.
71. Malecki M, Swoboda P, Pachecka J. Recombinant adenoassociated virus derived vectors (rAAV2) efficiently transducer ovarian and hepatocellular carcinoma cells-implication for cancer gene therapy. Acta Pol Pharm. 2009, 66(1): 93-99.
72. Plasmid DNA purification user manual, Macherey-Nagel, Germany, 2008:13-14.
73. Kurar E. Nucleic acid vaccination of Brucella abortus ribosomal L7/L12 gene elicits immune response. Vaccine. 1997, 15(17-18): 1851-1857.
74. Estein M, Cassataro J, Vizcaino N. The recombinant Omp31 from Brucella melitensis alone or associated with rough lipopolysaccharide induce protection against Brucella ovis infection in Balb/c mice. Microbes Infec. 2003, 5(2): 85-93.
75. Boschiroli ML, Foulongne V, O'Callaghan D. Brucellosis: a worldwide zoonosis. Curr Opin Microbiol. 2001, 4(1): 58-64.
76. Yu DH, Hu XD, Cai H, Li M. A combined DNA vaccine encoding BCSP31,SOD, and L7/L12 confers high protection against Brucella abortus 2308 by inducing specific CTL responses. DNA Cell Biol. 2007, 26: 435-443.
77.陈慰峰.医学免疫学.第3版.北京.人民卫生出版社, 2001, 9: 89-96.
78.王旭东,国外医学免疫学分册. 1993, 3: 154-155.
79. Mosmann T. and Sad S. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today. 1996, 17: 138-146.
80. Algood HM, Lin PL, Flynn JL Tumor necrosis factor and chemokine interactions in the formation and maintenance of granulomas in tuberculosis. Clin Infect Dis. 2005, 41: 189-193.
81. Salgame P. Host innate and Th1 responses and the bacterial factors that control Mycobacterium tuberculosis infection. Curr Opin Immunol. 2005, 17: 374-380.
82. Scheffold A, Huhn J, and Hofer T. Regulation of CD4+CD25+ regulatory T cell activity: it takes (IL-) two to tango. Eur J Immunol. 2005, 35: 1336-1341.
83. Serbina NV, Lazarevic V, and Flynn JL. CD4(+) T cells are required for the development of cytotoxic CD8(+) T cells during Mycobacterium tuberculosis infection. J Immunol. 2001, 167: 6991-7000.
84. Vizcaíno N, Caro-Hernández P, Cloeckaert A, Fernández-Lago L. DNA polymorphism in the omp25/omp31 family of Brucella spp.: identification of a 1.7-kb inversion in Brucella cetaceae and of a 15.1-kb genomic island, absent from Brucella ovis, related to the synthesis of smooth lipopolysaccharide. Microbes Infec. 2004, 6(9): 821-834.
2. Mayfield JE, Betsy JB. The cloning, expression, and nucleotide sequence of a gene coding for an immunogenic Brucella abortus protein.Gene. 1988, 63 (1):1-8.
3. Mehmet D, Bilgehan A. Human brucellosis: an overview. Infect Dis. 2003, 7: 173-182.
4. Jinkyung K, Gary A. Molecular host-pathogen interaction in brucellosis: current understanding and future approaches to vaccine development for mice and humans. Clin Microbio Review. 2003, 16:65-78.
5. Zhang FL, Wu XA, Luo W, Bai WT, Liu Y, Yan Y, Wang HT, Xu ZK. The expression and genetic immunization of chimeric fragment of Hantaan virus M and S segments. BBRC. 2007, 354(4): 858-863.
6. Akdeniz H, Irmak H, Anlar O et al. Central nervous system brucellosis: presentation, diagnosis and treatment. J Infect. 1998, 36: 297–301.
7. Shang DQ. Research on Brucella spp. Chin J Ctrl Endem Dis. 2004, 19: 204–213.
8. Franco MP, Mulder M, Gilman RH et al. Human brucellosis. Lancet Infect Dis. 2007, 7: 775–786.
9. Hoover D, Friedlander A. Brucellosis. In Zajtchuk R, ed. Textbook of Military Medicine: Medical Aspects of Chemical and Biological Warfare. Washington, DC: US Department of the Army, Surgeon General, and the Borden Institute 1997: 513-521.
10. Amélie G, Philippe BP, Fran ois B. Brucellose par bioterrorisme. La Presse Médicale, 2004, 33( 2): 119-122.
11. World Health Organization: Fact Sheet N173. World Health Organization, Geneva, Switzerland July 1997.
12. Pappas G., Papadimitriou P, Akritidis N, Christou L and Tsianos E.V. The new global map of human brucellosis. Lancet Infect Dis. 2006, 6: 91–99.
13.赵忠鹏,王希良.布鲁菌毒力因子研究进展.微生物学免疫学进展. 2007, 35 (1):50-53.
14.徐志凯.临床微生物学.北京:高等教育出版社, 2007. 244-245.
15. Gross A, Terraza A, Ouahrani-Bettache S, Liautard JP, Dornand J In vitro Brucella suis infection prevents the programmed cell death of human monocytic cells. Infect Immun. 2000, 68, 342-351.
16. Pappas G, Akritidis N, Bosilkovski M and Tsianos E. Brucellosis. N Engl J Med. 2005, 352: 2325–2336.
17. Farshad S, Javad M, Banpour M. Prodution and characterization of monoclonal antibodies against Brucella abortus S (99) surface antigens. Iran Biomed. 2002, 6 (1): 7-12.
18.吐尔洪努尔,谷文喜,何倩倪,热合木江,布合丽切木依干巴地,刘丽娅.布鲁菌病研究进展.动物医学进展. 2007, 28(7): 82-87.
19. Bowden RA, Cloeckaert A, Zygmunt M S. Evaluation of immunogenicity and protective activity in BALB/c mice of the 25kDa major outer membrane protein of Brucella melitensis (OMP25) expressed in Escherichia coli. Jmed Micobiol. 1998, 47: 39-48.
20. Gerhardt G, Schurig NA, Michael J. Brucellosis vaccine: past, present and future. Vet Microbiol. 1990, 20(20): 479-496.
21. Bocharova EV, Gudkov AT, Arsenieva AS.Topology of the secondarystructure elements of ribosomal protein L7/L12 from E. coli in solution. FEBS Letters. 1996, 379:291-294.
22. Simge B, Boyle SM, Vemulapalli R, Sriranganathan N, Schurig GG, Toth TE. Immune responses of mice to vaccinia virus recombinants expressing either Listeria monocytogenes partial listeriolysin or Brucella abortus ribosomal L7/L12 protein. Vet Microbiol, 2005, 109(1-2): 11-17.
23. Zhongpeng Z, Min L, Deyan L, Li X, Shuo W, Yueqiang D, Penghui Y, Xiliang W. Protection of mice from Brucella infection by immunization with attenuated Salmonella enterica serovar typhimurium expressing A L7/L12 and BLS fusion antigen of Brucella. Vaccine. 2009, 27(38): 5214-5219.
24. Victoria DM, Juliana Cassataro, Carlos A. Brucella outer membrane protein Omp31 is a haemin-binding protein. Microbes Infec.2006, 8(5):1203-1208.
25. Silvia M. Estein, Pablo et al. Immunogenicity of recombinant Omp31 from Brucella melitensis in rams and serum bactericidal activity against B. ovis. Vet Microbiol. 2004, 102:203-213.
26. Bricker BJ, Tabatabai LB, Deyoe BL et al. Conservation of antigenicity in a 31-kDa Brucella protein. Vet Microbio. 1988, 18:313–325.
27. Van MD, Kennedy GA, Olsen SC, et al. Brucellosis induced by RB51 vaccine in a pregnant heifer.J Am Vet Med Assoc. 1999, 215: 1491-1493.
28. Pugh GW Jr, Tabatabai LB. Variation of Brucella abortus 2308 infection in BALB/c mice induced by prior vaccination with salt-extractable periplasmic proteins from Brucella abortus 19. Infect Immun. 1996, 64(2): 548-556.
29. Halling SM, Detilleux PG, Tatum FM, et al. Deletion of the BCSP31 gene of B.Abortus by replacement. Infect. Immun.1991, 59:3863-3868.
30. Thomas JS, Mayfield JE, Louisa B. et al. Oral Immunization of Micewith Attenuated Salmonella typhimurium Containing a Recombinant PlasmidWhich Codes for Production of a 31-Kilodalton Protein of Brucella abortus. Infect onand immunity.1990, 58(7): 2048-2055.
31. Cloeckaert A, Verger M, Grayon M, et al. Classification of Brucella spp. Isolated from marine manuals by DNA polymorphism at the Omp2 locus. Microbes Infect. 2001, 3: 729-738.
32. Cassataro J, Pasquevich K, Bruno L, et a1. Antibody reactivity to Omp3l from Brucella melitensis in human and animal infections by smooth and rough Brucellae. Clin Diagn Lab Immunol, 2004, 11(1):111-114.
33. Delvecchio G, Kapat V , RedKat J, et al . The genome of Brucella melitensis. Vet Microbiol. 2002, 90 (1): 587 -592.
34. Mart′n-Mart′n AI, Caro-Herna′ndez P, Orduna A, et al. Importance of the Omp25/Omp31 family in the internalization and intracellular replication of virulent B. ovis in murine macrophages and HeLa cells. Microbes Infec. 2008, 10: 706-710.
35. Martín-Martín AI, Caro-Hernández P, Sancho P, Tejedor C, Cloeckaert A, Fernández-Lago L, Vizcaíno N. Analysis of the occurrence and distribution of the Omp25/Omp31 family of surface proteins in the six classical Brucella species. Vet. Microbiol. 2009, 137(1-2): 74-82.
36. Edmonds D, Cloeckaert A, Hagius D, et al. Pathogenicity and protective activity in pregnant goats of a Brucella melitensis Delta Omp25 deletion mutant. Res Vet Sci. 2002, 72: 235–239.
37. Manterola L, Guzman-Verri C, Chaves-Olarte E, et al. BvrR/BvrS-controlled outer membrane proteins Omp3a and Omp3b are not essential for Brucella abortus virulence. Infect Immun. 2007, 75: 4867-4874.
38. Vizcaino N, Caro-Hernandez P, Cloeckaert A, et a1. DNA polymorphism in the Omp25/Omp31 family of Brucella spp: identification of a 1.7-kbinversion in Brucella cetaceae and of a 15.1-kb genomie island, absent from Brucella ovis, related to the synthesis of smooth lipopolysaccharide. Microbes Infect. 2004, 6 (9):821-834.
39.尚德秋.布鲁氏菌病流行病学研究近况.中华流行病学杂志,.1998, 19(2): 107-110.
40. Bagchi TD. Immune mechanisms in murine brucellosis: studies with strain RB51, a rough mutant of Brucella abortus. USA, VA, 1990: 24-61.
41. Donnelly JJ, Wahren B, Liu MA. DNA vaccines: progress and challenges. J Immunol. 2005, 175(2):633–639.
42. Pasquali P, Rosanna A, Pistoia C. Brucella abortus RB51 induces protection in mice orally infected with the virulent strain B. abortus 2308. Infect Immun. 2003, 71(5): 2326-2330.
43. Ashfrd DA, Pietra J, Lingappa J. Adverse events in humans associated with accidental exposure to the livestock brucellosis vaccine RB51. Vaccine. 2004, 22(25-26): 3435-3439.
44. Davis DS, Elizer PH. Brucella vaccines in wildlife. Vet Microbiol. 2002, 90(11):533-544.
45. Munoz-Montesino C, Andrews E, Rivers R, Gonzalez-Smith A, Moraga-Cid G, Folch H, et al. Intraspleen delivery of a DNA vaccine coding for superoxide dismutase (SOD) of Brucella abortus induces SOD-specific CD4+ and CD8+ T cells. Infect Immun. 2004, 72(4): 2081-2087.
46. Elzer PH, Enright FM. Evaluation of a rough mutant of Brucella melitensis in pregnant goats. Res Vet Sci. 1998, 64: 259-260.
47. Murphy EA, Sathiyaseelan J, Parent MA. Interferon-gamma is crucial for surviving a Brucella abortus infection in both resistant C57BL/6 and susceptible BALB/c mice.Immunology. 2001, 103:511-518.
48.罗德炎,韩玉霞,王希良.布氏杆菌病新型疫苗的研究进展.免疫学杂志. 2004, 20(3): 43-45.
49. Klinman DM., Klaschik S, Tross D, Shirota H, Steinhagen F. FDA guidance on prophylactic DNA vaccines: Analysis and recommendations. Vaccine. 2010, 28(16): 2801-2865.
50. Mallick AI, Singha H, Chaudhuri P, Nadeemc A, Khand SA, Dard KA, Owaisa M. Liposomised recombinant ribosomal L7/L12 protein protects BALB/c mice against Brucella abortus 544 infection. Vaccine. 2007, 25: 3692-3704.
51. Gupta VK, Rout PK, Vihan VS. Induction of immune response in mice with a DNA vaccine encoding outer membrane protein (omp31) of Brucella melitensis 16M. Res Vet Sci. 2007, 82(3): 305-313.
52. Kurar E, Splitter GA. Nucleic acid vaccinationof Brucella abortus ribosomal L7/L12 gene elicitsimmune response. Vacine. 1997, 15(17-18): 1851-1857.
53.曾政,邓小红.布鲁氏菌DNA疫苗的发展:过去、现在和未来.国外医学免疫学分册. 2005, 28(3): 143-146
54. Kaushik P, Singh DK, Kumar SV, Tiwari Ak, Shukla G. Protection of mice against Brucella abortus 544 challenge by vaccination with recombinant OMP28 adjuvanted with CpG. Vet Res Com.2010, 34( 2):119-132.
55. Park JH , Song MH, Lee CH, Lee MK Park YM.Expression of the human cancer/testis antigen NY-SAR-35 is activated by CpG island hypomethylation. Biotechnology Letters. 2011.[Equ before print]
56.司瑞,白文涛,徐志凯.羊布鲁菌BCSP31、OMP31、L7/L12基因的原核表达载体构建及表达.中国人兽共患病杂志.2005, 21(11):961-964
57. Kumar G, Rathore G, Sengupta U et al. Production of monoclonal antibodies specific to major outer membrane protein of Edwardsiella tarda. CompImmunol Microbiol Infect Dis. 2010, 33: 133–144.
58.徐志凯.实用单克隆抗体技术.陕西科技出版社.1992:31-41.
59. Hoffmann F, Heuvel J, Zidek N. Minimizing inclusion body formation during recombinant protein production in Escherichia coli at bench and pilot plant scale. Enzyme Microb Tech. 2004, 34: 235–241.
60. Hayashi K, Kojima C. pCold-GST vector: A novel cold-shock vector containing GST tag for soluble protein production. Protein Expres Purif. 2008, 62: 120–127.
61. Vernet E, Kotzsch A, Voldborg B, Sundstr m M. Screening of genetic parameters for soluble protein expression in Escherichia coli . Protein Expres Purif. 2011, 77, (1): 104-111.
62. Cai X, Wang JF, Wang YY et al. Expression, purification and characterization of recombinant human interleukin-22 in Pichia pastoris. Mol Biol Rep. 2010, 37: 2609-2613.
63. Song JP, Chen WT, Lu ZS et al Soluble expression, purification, and characterization of recombinant human flotillin-2 (reggie-1) in Escherichia coli. Mol Biol Rep. 2011, DOI: 10.1007/s11033-010-0335-4
64. Liu SX, Fu ZP, Mu RM et al. Expression and characterization of Momordica Chanrantia anti-hyperglycaemic peptide in Escherichia coli. Mol Biol Rep. 2010, 37: 1781-1786.
65. Cabrita LD, Bottomley SP. Protein expression and refolding - A practical guide to getting the most out of inclusion bodies. Biotech Annu Rev. 2004, 10: 31–50.
66.蛋白质纯化与鉴定实验指南,朱厚础等译.北京,科学出版社, 1999, 7:179-213.
67. Cloeckaert A, Verger JM, Grayon M, Vizcaíno N. Molecular andimmunological characterization of the major outer membrane proteins of Brucella . FEMS Microbiol Letters. 1996, 145(1): 1-8.
68. GSTrap FF, Hitrap affinity columns. Instruction 71-5016-96 AK. GE Healthcare. p6-7
69.胡刚,徐志凯,张芳琳,吴兴安,阎岩,白文涛,于澜,王海涛.抗汉滩病毒双链抗体重组杆状病毒在昆虫细胞中的表达.免疫学杂志. 2005, 21(2): 84-87.
70. Karmali PP, Majeti BK, Sreedhar B. In vitro gene transfer efficacies and serum compatibility profiles of novel mono-, di-, and tri-histidinylated cationic transfection lipids: a structure-activity investigation. Bioconjug Chem. 2006, 17(1): 159-171.
71. Malecki M, Swoboda P, Pachecka J. Recombinant adenoassociated virus derived vectors (rAAV2) efficiently transducer ovarian and hepatocellular carcinoma cells-implication for cancer gene therapy. Acta Pol Pharm. 2009, 66(1): 93-99.
72. Plasmid DNA purification user manual, Macherey-Nagel, Germany, 2008:13-14.
73. Kurar E. Nucleic acid vaccination of Brucella abortus ribosomal L7/L12 gene elicits immune response. Vaccine. 1997, 15(17-18): 1851-1857.
74. Estein M, Cassataro J, Vizcaino N. The recombinant Omp31 from Brucella melitensis alone or associated with rough lipopolysaccharide induce protection against Brucella ovis infection in Balb/c mice. Microbes Infec. 2003, 5(2): 85-93.
75. Boschiroli ML, Foulongne V, O'Callaghan D. Brucellosis: a worldwide zoonosis. Curr Opin Microbiol. 2001, 4(1): 58-64.
76. Yu DH, Hu XD, Cai H, Li M. A combined DNA vaccine encoding BCSP31,SOD, and L7/L12 confers high protection against Brucella abortus 2308 by inducing specific CTL responses. DNA Cell Biol. 2007, 26: 435-443.
77.陈慰峰.医学免疫学.第3版.北京.人民卫生出版社, 2001, 9: 89-96.
78.王旭东,国外医学免疫学分册. 1993, 3: 154-155.
79. Mosmann T. and Sad S. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today. 1996, 17: 138-146.
80. Algood HM, Lin PL, Flynn JL Tumor necrosis factor and chemokine interactions in the formation and maintenance of granulomas in tuberculosis. Clin Infect Dis. 2005, 41: 189-193.
81. Salgame P. Host innate and Th1 responses and the bacterial factors that control Mycobacterium tuberculosis infection. Curr Opin Immunol. 2005, 17: 374-380.
82. Scheffold A, Huhn J, and Hofer T. Regulation of CD4+CD25+ regulatory T cell activity: it takes (IL-) two to tango. Eur J Immunol. 2005, 35: 1336-1341.
83. Serbina NV, Lazarevic V, and Flynn JL. CD4(+) T cells are required for the development of cytotoxic CD8(+) T cells during Mycobacterium tuberculosis infection. J Immunol. 2001, 167: 6991-7000.
84. Vizcaíno N, Caro-Hernández P, Cloeckaert A, Fernández-Lago L. DNA polymorphism in the omp25/omp31 family of Brucella spp.: identification of a 1.7-kb inversion in Brucella cetaceae and of a 15.1-kb genomic island, absent from Brucella ovis, related to the synthesis of smooth lipopolysaccharide. Microbes Infec. 2004, 6(9): 821-834.