细粒棘球绦虫EgM123重组耻垢分枝杆菌疫苗在小鼠体内的表达及其免疫应答  

Expression and immunogenicity of Echinococcus granulosus EgM123 expressed in Mycobacterium smegmatis in mice

作　　者：何黎 齐文静 郑雪婷 郭刚 李军 张文宝 HE Li;QI Wen-jing;ZHEN Xue-ting;GUO Gang;LI Jun;ZHANG Wen-bao(College ofBasic Medicine,Xinjiang Medical University,Urumqi,China 830054;State Key Laboratory of Pathogenesis,Prevention and Treatment of High Incidence Diseases in Central Asia,Clinical Medicine Institute,The First Hospital Affiliated with Xinjiang Medical Universityy)

机构地区：[1]新疆医科大学基础医学院,新疆乌鲁木齐830054 [2]中亚高发病成因与防治国家重点实验室,临床医学研究院,新疆医科大学第一附属医院

出　　处：《中国病原生物学杂志》2018年第12期1314-1318,1323,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.U1303203).

摘　　要：目的构建表达细粒棘球绦虫成虫特异性基因EgM123的耻垢分枝杆菌疫苗株,接种BALB/c小鼠,观察其免疫原性。方法 PCR扩增EgM123片段,定向克隆至大肠埃希菌-分枝杆菌穿梭质粒pMV261中,构建重组质粒pMV261-EgM123。连接产物转化至大肠埃希菌DH5α中,培养后提取质粒,测序确定阅读框架正确,后电穿孔转入耻垢分枝杆菌,构建表达EgM123的重组耻垢分枝杆菌疫苗(rMS-EgM123),经热诱导后采用SDS-PAGE和Western blot检测目的蛋白表达。在培养过程中连续检测菌液A600值,绘制重组菌生长曲线。取重组菌接种小鼠,检测基础接种和加强免疫接种后血清特异抗体水平。结果 PCR扩增出大小为594bp的EgM123片段,EgM123基因正确插入到穿梭质粒pMV261中,测序鉴定阅读框架正确。重组耻垢分枝杆菌连续传10代培养,重组质粒DNA序列未发生变异。Western blot检测表达产物能被抗EgM123鼠血清识别。用重组耻垢分枝杆菌疫苗加强免疫后的小鼠血清特异性抗体呈持续上升趋势,单次加强免疫8周后抗体水平(A405值为2.504±0.659)显著高于亚单位疫苗组(P<0.05)。结论重组耻垢杆菌疫苗菌能表达细粒棘球绦虫成虫EgM123蛋白,且该重组疫苗可通过加强免疫增强基础免疫诱导小鼠产生的抗体水平。Objectives To construct and express Echinococcus granulosus EgM123 in a Mycobacterium smegmatis strain as a vaccine to determine its immunogenicity in mice. Methods EgM123 was amplified using PCR and inserted into the shuttle plasmid pMV261.The recombinant pMV261-EgM123 was transformed into E.coli DH5α for cloning.After the open-reading-frame(ORF)was verified as correct with sequencing,the vector was transformed into M.smegmatis via electroporation as a live Mycobacterium vaccine(rMS-EgM123).The stability of the recombinant plasmid expressed in M.smegmatis was determined in 10 continuous passages with no mutation in the ORF.SDS-PAGE and Western blot analysis were used to identify and detect the expression of EgM123 in M.smegmatis.Mice were primarily vaccinated with recombinant His-tagged protein(His-EgM123).The mice were then given a booster with rMS-EgM123.Levels of antibodies against His-EgM123 in the sera of mice were determined using ELISA. Results Amplification of recombinant pMV261 yielded a fragment 594 bp in size,and sequencing indicated that the ORF of EgM123 was correct.Expression of the recombinant protein was induced in M.smegmatis cultured at 42°C for 5hours.Western blot analysis indicated that a band 29 kDa was recognized by the specific anti-EgM123 serum obtained from mice.Analysis of the serum antibody indicated that the specific antibody level was boosted(A405up to 2.504±0.659)after vaccination with rMSEgM123,and it continued to increase for 8weeks until the end of the experiment.The serum titer was significantly higher than that in mice given a booster with subunit EgM123. Conclusion Recombinant EgM123 expressed in M.smeg-matis as a live bacterium vaccine boosted production of specific antibodies against EgM123 in mice.The vaccine can be used to vaccinate dogs against E.granulosus infection.

关 键 词：包虫病 EgM123 重组耻垢分枝杆菌 疫苗 

分 类 号：R383.33[医药卫生—医学寄生虫学]