含结核分枝杆菌Ag85A基因的2型重组腺相关病毒的构建和免疫原性

Construction of Recombinant Adeno-Associated Virus Type 2 Containing Ag85A Gene of Mycobacterium tuberculosis

目的构建含结核分枝杆菌Ag85A基因的2型重组腺相关病毒并初步研究其免疫原性。方法采用PCR法从结核杆菌H37Rv株扩增Ag85A基因,将PCR扩增产物克隆于2型腺相关病毒(AAV-2)表达质粒pSNAV中,构建重组质粒pSNAV-Ag85A;用脂质体转染的方法将重组质粒转入BHK-21细胞中,G418筛选得到能表达目的基因混合细胞系BHK-Ag85A;用具有rAAV-2包装功能的辅助病毒感染BHK-Ag85A,纯化后得到rAAV-2-Ag85A;Western blotting检测重组病毒Ag85A基因在BHK-21细胞中的表达;rAAV-2-Ag85A免疫Balb/c小鼠,ELISA法检测血清中抗-Ag85A抗体,^(51)Cr释放分析检测细胞毒性T淋巴细胞(CTL)活性。结果PCR扩增的Ag85A基因序列与GenBank公布的序列一致,纯化后得到的rAAV-2-Ag85A滴度为1×10^(12)virus particles/ml;Western blotting检测到rAAV-2-Ag85A在BHK-21细胞中能够表达出一相对分子质量为32000的多肽;rAAV-2-Ag85A免疫的Balb/c小鼠,抗-Ag85A抗体的滴度可达1∶1024,同时还可激发Ag85A特异性的CTL产生。结论rAAV-2-Ag85A构建成功,rAAV-2-Ag85A免疫小鼠可以同时诱导体液和细胞免疫反应的出现。rAAV-2-Ag85A对于防止结核分枝杆菌感染,尤其是作为结核病的治疗性疫苗可能具有潜在的应用价值,值得进一步深入研究。

Objective To construct a recombinant adeno-associated virus type 2 （ rAAV-2 ） containing Ag85A gene of Mycobacterium tuberculosis, and to investigate the immunogenicity of the recombinant protein. Methods Ag85A gene was amplified by polymerase chain reaction （PCR） from the genome of Mycobacterium tuberculosis H37Rv strain. The amplified PCR product was cloned into the adeno-associated virus expressing vector pSNAV. The recombinant pSNAV-Ag85A was transfected into BHK-21 cell by using LipofectamineTM 2000. After transfection, the cells were screened with G418. The cells expressing Ag85A （BHK-Ag85A） were then infected with the helper virus for the packaging of the rAAV-2. After purification, rAAV-2-Ag85A was obtained. Expression of rAAV-Ag85A in BHK-21 was detected by Western blotting. Immune sera from rAAV-2-Ag85A immunized Balb/c mice were tested for anti-Ag85A antibodies by the ELISA method. Cytotoxicity of the T-lymphocyte （CTL） was determined using the ^51Cr release assay. Results The sequence of the amplified gene segment was identical to the Ag85A sequence reported in GenBank. The titer of the recombinant virus was 1 ×10^12 yirus particles/ml. The expressed Ag85A protein had a molecular mass of 32 kD. After immunization of Balb/c mice with rAAV-2-Ag85A, the titer of anti-Ag85A antibody was 1：1 024. Specific CTL activity was also detected. Conclusion Recombinant virus expressing Ag85A was successfully constructed, rAAV- 2-Ag85A can induce both humoral and cellular immune response. The results indicated that the rAAV-2-Ag85A may be developed as a vaccine for the prevention infection by Mycobacterium tuberculosis.