结核杆菌pcHSP65真核表达载体的构建及其DNA免疫实验

Construction of eukaryotic expression vector and DNA immune experiment pcHSP65 of mycobacterium tuberculosis

目的 :构建以结核分枝杆菌热休克蛋白 65kD基因为基础的核酸疫苗。方法 :采用聚合酶链反应从结核杆菌H37Rv株基因中 ,扩增出HSP65的编码基因 ,经限制性核酸内切酶消化后 ,插入真核表达载体pcDNA3 .1 (- )的相应酶切位点 ,并将此重组质粒免疫动物。结果 :重组质粒的插入基因经序列测定证实为结核分枝杆菌HSP65。DNA免疫小鼠体内产生特异性抗体 ,能抵抗结核杆菌的感染。结论 :以HSP65编码基因为基础的真核表达载体构建成功 ,并能引起特异性动物免疫反应 。

Objective:To construct nucleotide vaccine based on HSP65 gene of mycobacterium tuberculosis.Methods:The gene encoding heat shock protein 65 kilodalton was amplified from genome of mycobacterium tuberculosis H37Rv strain by polymerase chain reaction(PCR) and correctly inserted into corresponding sites of eukaryotic expression vector pcDNA3.1(-) after restriction endonuclease digestion.The experimental mice was vaccined by this recombinant plasmid DNA.Results:The gene fragment inserted into the vector pcDNA3.1(-) was the HSP65 gene of mycobacterium tuberculosis H37Rv strain,which was confirmed by partial nucleotide sequencing.There was specific antibody produce in experimental mice vaccined by the recombinant plasmid DNA and this could protect mice against infection of mycobacterium tuberculosis.Conclusion:The successful construction of recombinant eukaryotic expression vector based on HSP65 gene of mycobacterium tuberculosis,such recombinant plasmid DNA induced specific antibody production in experimental mice,lays the foundation for further researching in prevention and treatment of tuberculosis.