结核分支杆菌MPT83在真核、原核表达载体中的克隆、表达与鉴定

为了克隆、鉴定和表达结核分支杆菌抗原蛋白 MPT83 ,为结核病的诊断以及亚单位疫苗、核酸疫苗的制备和应用奠定基础。以结核分支杆菌标准菌株 H3 7RV基因组 DNA为模板 ,PCR扩增目的基因 mpt83 ,扩增产物酶切后分别克隆到真核、原核表达载体 p JW43 0 3和p ET2 2 b( )中 ,构成重组真、原核表达载体 83eu和 MPT83。经酶切和序列鉴定为正确的重组真核表达载体 83 eu和重组原核表达载体MPT83 ,分别转染 COS7细胞和转化 BL2 1( DE3 ) plys S。结果表明 ,经 SDS-PAGE、Westernblotting检测显示 ,IPTG诱导的原核表达载体MPT83在 2 .6万处有正确而特异的蛋白条带 ;转染 COS7细胞的真核表达载体 83 eu,Westernblotting检测表明也有与结核分支杆菌抗血清特异反应的蛋白条带

Cloning,Expression and Identification of Mycobacterium Tuberculosis Protein MPT83 in Eukaryotic Expression Vector and Prokaryotic Expression Vector

Cloning,identification and expression of antigen MPT83 from Mycobacterium Tuberculosis play a role in diagnosis of tuberculosis,preparation of subunit vaccine and DNA vaccine and application of those vaccines. We amplified gene mpt83 based on the genome DNA sequence of H37RV by PCR and then cloned them into eukaryotic expression vector pJW4303 and prokaryotic expression vector pET22b( ) after digested with restriction endonuclease corresponding to the enzyme sites on the vectors. We named those constructions 83eu and MPT83. After identified correctly by digestion with restriction endonuclease and sequencing,the vectors 83eu transferred COS-7 cells and MPT83 transformed E.coli BL21(DE3)plysS respectively. Result:SDS-PAGE and Western blotting analysis of the protein induced by IPTG after the prokaryotic expression vector MPT83 transformed BL21(DE3)plysS demonstrated a specific protein about 26KD;western blotting also showed a specific protein reacting actively with anti-tuberculosis serum after the eukaryotic expression vector 83eu transferred COS-7 cells.