表面展示副结核分枝杆菌重组蛋白rMAP62-54c的大肠杆菌载体疫苗的构建及其免疫效果评价  

Construction of Escherichia coli based vaccine with surface display of Mycobacterium avium subsp.Paratuberculosis recombinant protein rMAP62-54c and evaluation of its immune efficacy

作　　者：丁仕豪 周薇[1] 许秋 张卓[1] 陆瑶 刘思国[1] 于申业[1] DING Shi-hao;ZHOU Wei;XU Qiu;ZHANG Zhuo;LU Yao;LIU Si-guo;YU Shen-ye(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2020年第10期1032-1038,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划(2016YFD0500905,2016YFD0501608);国家自然科学基金项目(31470893)。

摘　　要：为开发高效表面展示副结核分枝杆菌(Mycobacterium avium subsp.Paratuberculosis,MAP)抗原的大肠杆菌活载体疫苗,本研究将融合的目的基因map0862-2154c克隆于pET-INP载体,构建了重组质粒pET-INP-6254c。将该重组质粒转入大肠杆菌BL21(DE3)中,IPTG诱导表达后经western blot及流式细胞术等技术检测目的蛋白的表达及定位,western blot分析结果显示,诱导的全菌以及超声后的上清和沉淀在75 ku处均出现目的条带;流式细胞术结果显示,重组蛋白rMAP62-54c能够表达并展示到细胞表面。将110只BABL/c小鼠分为4组:INP-28a/BL21对照组(n=30)和INP-rMAP62-54c/BL21疫苗组(n=30)(均以5×10^5cfu/只腹腔注射免疫小鼠3次),PBS对照组(n=30)(注射等体积PBS),三免后以5×10^5cfu/只MAP标准株K-10感染上述3组小鼠;空白组(n=20)(不做处理)。然后通过ELISA、流式细胞术等方法定期检测各组小鼠血清IgG抗体水平、脾脏CD4+T/CD8+T细胞含量及比值、细胞因子IFN-γ、IL-17A及IL-10水平和小鼠增重率及其肝脏、脾脏和结肠病理损伤评价免疫效果。结果显示,重组菌INP-rMAP62-54c/BL21免疫组小鼠脾脏CD4+T/CD8+T细胞比值自免疫2周起即显著高于两个对照组(p<0.01);抗体检测结果显示,自小鼠免疫4周起,重组菌INP-rMAP62-54c/BL21诱导小鼠产生了高水平的特异性血清IgG抗体,与两个对照组相比差异极显著(p<0.001);细胞因子检测结果显示,攻毒后疫苗组小鼠血清中IFN-γ、IL-17与两个对照组相比差异极显著(p<0.01);攻毒14周疫苗组小鼠血清中IL-10分泌量明显下降,与两个对照组相比差异显著(p<0.05);随着攻毒时间的延长INP-rMAP62-54c/BL21疫苗组小鼠体质量保持增加趋势,而两个对照组小鼠体质量一直下降;病理组织学结果显示,疫苗组小鼠与空白组小鼠相比脏器未出现明显病变。本研究首次构建了INP-rMAP62-54c/BL21表面展示大肠杆菌,其能显著增强小鼠对MAP的抵抗能力,并且能刺激小鼠To develop an efficient Escherichia coli(E.coli)based vaccine with surface display of Mycobacterium avium subsp.Paratuberculosis(MAP)antigen,the fused gene map0862-2154c was inserted into p ET-INP vector,and the resulting recombinant plasmid was transferred into E.coli BL21(DE3).Western blot and flow cytometry were used to verify the expression and location of recombinant protein rMAP62-54c in E.coli after IPTG induction.One hundred and ten BABL/c female mice were divided into four groups.INP-28a control group(n=30)and INP-rMAP62-54 vaccine group(n=30)were intraperi-toneally injected with 5×10^5 cfu/mouse for three times,respectively;PBS group(n=30)was injected with equal volume PBS.Mice in the above three groups were challenged by 5×10^5 cfu MAP standard strain of K-10 after the last injection,and blank group(n=20)was accepted no treatment.To evaluate the immune effect,the serum IgG antibody level and cytokines(eg,IFN-γ,IL-17 A and IL-10)were detected regularly by ELISA,and the percentage and ratio of splenic CD4^+T/CD8^+T cells were measured by flow cytometry,and the weight gain rate and the pathological damage(liver,spleen,and ileum)were also examined.The flow cytometry results showed that the recombinant protein rMAP62-54c was successfully expressed and displayed on the cell surface.Compared with the two control groups,the ratio of splenic CD4^+T/CD8^+T cells became significant(p<0.01)since 2 weeks after immunization.The antibody detection results showed that,since 4 weeks after immunization,specific serum IgG antibodies were induced by INPrMAP62-54c/BL21 in mice,significantly higher than the two control groups(p<0.001).The results of cytokine detection showed that the serum levels of IFN-γand IL-17 in the vaccine group were significantly higher than those of the control groups(p<0.01).The IL-10 secretion in serum of vaccine group decreased significantly(p<0.05)compared with the two control groups on 14-week post challenge.As the infection went on,the weight of mice in INP-rMAP62-54c/BL21 vaccine group ke

关 键 词：副结核分枝杆菌 重组蛋白rMAP62-54c 表面展示 免疫效果 

分 类 号：S855.1[农业科学—临床兽医学]