用小鼠模型分析表达牛结核分枝杆菌Ag85B重组腺病毒的细胞免疫特性  

Construction of recombinant adenovirus expressing Ag85B of Mycobacterium bovis and its cellular immunoproperties in mice

作　　者：周海霞[1] 陈祥[1] 季琰[1] 周卫东 胡茂志 黄金林[1] 潘志明[1] 焦新安[1] 

机构地区：[1]扬州大学江苏省人兽共患病学重点实验室,扬州225009 [2]扬州大学测试中心,扬州225009

出　　处：《微生物学报》2010年第6期811-816,共6页Acta Microbiologica Sinica

基　　金：国家"973项目"(2006CB504404);国家科技重大专项(2008ZX10003-010);公益性行业科研专项(200903027);江苏省科技攻关计划(BE2007340)~~

摘　　要：【目的】构建表达牛结核分枝杆菌抗原Ag85B重组腺病毒rAd-Ag85B,并用小鼠模型分析其细胞免疫特点。【方法】采用PCR方法,扩增结核分枝杆菌Ag85B的编码基因fbpB,测序后构建pDC516-Ag85B重组质粒。利用脂质体将pDC516-Ag85B与pBHGfrtΔE1,3FLP共转染Ad293细胞包装成重组病毒rAd-Ag85B。空斑纯化后用电镜负染、目的基因转录和蛋白表达进行rAd-Ag85B验证。同时将rAd-Ag85B和rAd(wtAd)分别经皮下注射免疫BALB/c小鼠,二免二周后取小鼠脾脏细胞,进行CD69表面分子表达、淋巴细胞增殖和ELISPOT实验分析。【结果】在电镜下能观察到包装的重组病毒粒子,且在转录和蛋白水平上验证了rAd-Ag85B构建成功。免疫试验结果显示,rAd-Ag85B能激活CD4+T和CD8+T细胞表面分子CD69的表达,并引起淋巴细胞增殖。ELISPOT表明rAd-Ag85B呈现Th1免疫特点。【结论】成功构建的rAd-Ag85B能引起机体针对PPD蛋白或Ag85B多肽的Th1免疫应答。[Objective]We constructed recombinant adenovirus rAd-Ag85B expressing antigen 85B of Mycobacterium bovis and analyzed cellular immune responses after mice immunization. [Methods]We amplified the fbpB gene from genomic DNA of BCG by PCR and cloned into pMD18-T vector for sequence analysis. Then we constructed pDC516-Ag85B and co-transfected with plasmid pBHGfrtΔE1,3FLP into Ad293 cells by lipofectamine^TM 2000. Then we identified the recombinant Ad-Ag85B virus by RT-PCR,Western blot assay and TEM observation after negative staining. BALB /c mice were subcutaneously immunized with the dose of 10^8 TCID50 rAd-Ag85B or rAd. Two weeks after immunization,CD69 expression,T lymphocyte proliferation and ELISPOT were detected in spleen cells of immunized mice. [Results]We obtained the correct recombinant adenovirus rAd-Ag85B expressed Ag85B antigen. The level of both splenic CD69 ^＋ CD4^ ＋ T and CD69^ ＋ CD8 ^＋ T cells of rAd-Ag85B group were significantly higher than those of rAd control. The SI index of rAd-Ag85B was much higher than rAd. In ELISPOT assay more IFN-γ specific secreting cell spots other than IL-4 were detected in rAd-Ag85B group. [Conclusion] The recombinant adenovirus rAd-Ag85B can induce specific Th1 cell immune responses in mice.

关 键 词：抗原Ag85B 牛结核分枝杆菌 重组腺病毒 Γ干扰素 CD69 

分 类 号：R392[医药卫生—免疫学]