结核分支杆菌HSP65 DNA疫苗的制备和重组蛋白的原核表达

结核分支杆菌分子量为65ku的热应激蛋白(HSP65)是一种非常重要的抗原，为了研制结核病核酸疫苗，构建编码HSP65DNA，并将其分别克隆到原核和真核载体中进行了表达。以标准结核分支杆菌H37Rv基因组DNA为模板，用PCR法扩增出HSP65基因，经限制性内切酶消化后，插入真核表达栽体pJW4303中，获得重组质粒pJW-HSP65。同时将HSP65基因插入原核表达栽体pET-22b( )，获得重组质粒pET22b-HSP65。将pET22b-HSP65重组质粒转化大肠杆菌蛋白酶缺陷型菌株BL21(DE3)／PolysS，用IPTG诱导，进行蛋白表达。结果表明，经酶切鉴定和序列测定证实插入片断为目的基因HSP65，构建成功了真核重组质粒pJW-HSP65即可作为结核病DNA疫苗。经SDS-PAGE检验证明可以在大肠杆菌细胞中高效表达，将表达蛋白进行纯化，作为保护性结核杆菌抗原以便检测HSP65DNA疫苗的免疫效果。

The Preparation of Mycobacterium tuberculosis HSP65 DNA Vaccine and the Prokaryotic Expression of the Recombinant HSP65 Protein

The purpose of this study is to construct DNA Vaccine of the Mycobacterium tuberculosis 65 ku Heat shock protein and clone it into prokaryotic expression vector to express, because it is a very important antigen against tuberculosis infection. The method: The HSP65 gene is amplified from Mycobacterium tuberculosis H37Rv genome DNA using PCR method. The PCR product is digested by the restriction endonucleases and then ligated to the eukaryotic expression vector pJW4303 which is digested by the same restriction endonucleases. The recombinant plasmid is called pJW-HSP65. Meantime, the HSP65 gene is inserted into the prokaryotic expression vector pET-22b( ) to product the recombinant prokaryotic expression vector pET22b-HSP65. Plasmids containing the right insertion were sequenced to confirm their identity. Transform the BL21(DE3)/PolysS strain with this recombinant prokaryotic expression vector,and induce it with IPTG. The HSP65 protein is highly expressed. After purification, the expressed protein can be used as protective Mycobacterium Tuberculosis antigen to detect the immunity efficiency of the HSP65 DNA Vaccine.In conclusion, we have successfully constructed HSP65 DNA vaccine and its protein has been expressed.