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

摘要:

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，并在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。

关键词:关键词：抗原Ag85B;牛结核分枝杆菌;重组腺病毒;γ干扰素;CD69

Abstract:

Abstract: [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 lipofectamineTM2000. 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 108 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.

Key words:Keywords: Ag85B; Mycobacterium bovis; recombinant adenovirus; IFN-γ; CD69