结核分枝杆菌Ag85A基因DNA疫苗的构建和免疫原性研究

目的　构建编码结核分枝杆菌蛋白Ag85A基因为基础的DNA疫苗。 方法　采用聚合酶链反应从结核杆菌H37Rv株基因组中 ,扩增出分泌蛋白Ag85A成熟蛋白的编码基因 ,用限制性内切酶消化后 ,插入克隆载体 pGEM TEasy中。经酶切鉴定与序列测定证实后 ,以亚克隆法构建于真核表达载体 pcDNA3.1的相应酶切位点。重组质粒肌注免疫小鼠 ,4周后用ELISA法检测抗体滴度。结果　结核分枝杆菌H37Rv株Ag85A成熟蛋白的编码基因经序列测定证实无突变 ;经BamHⅠ和EcoRⅠ双酶切鉴定证实克隆基因正确插入载体 pcDNA3.1。ELISA法检测几何平均滴度为 1∶10 0 0。结论　以Ag85A成熟蛋白的编码基因为基础的DNA疫苗的成功构建和表达 ,为进一步研究其在结核病与肿瘤防治方面的作用奠定了基础

Construction and immunogenicity of DNA vaccine of Ag85A gene of mycobacterium tuberculosis

Objective To construct DNA vaccine based on Ag85A gene of mycobacterium tuberculosis. Methods The gene encoding Ag85A mature protein was amplified by polymerase chain reaction (PCR) from genome of mycobacterium tuberculosis H37Rv strain, and was inserted into cloning vector pGEM-T Easy after restriction endonuclease digestion. Gene fragment encoding Ag85A mature protein was correctly inserted into the vector, which was confirmed by partial nucleotide sequencing and restriction endonuclease digestion, and then was subcloned to the same sites of eukaryotic expressing vector pcDNA3.1. When the mice were vaccinated with recombinant eukaryotic expressing vector 4 weeks later, titers of serum antibody against Ag85A were detected by ELISA.Results The nucleotide sequencing of the gene encoding Ag85A mature form of mycobacterium tuberculosis H37Rv strain was identical and there were no undesired mutations, the cloning gene was correctly inserted into the vector pcDNA3.1 as confirmed by restriction endonuclease digestion of BamHI and EcoRI. Mean geometric antibody titer of Ag85A was 1∶1 000 by ELISA. Conclusion DNA vaccine constructed and expressed successfully based on the gene encoding Ag85A mature protein of mycobacterium tuberculosis lays a foundation for further research into the prevention and treatment of tuberculosis and carcinoma.