可溶性HLA-G1重链分子的构建、表达及纯化

Expression and its purification of a heavy chain molecule of soluble HLA-G in E.coli

目的:获取原核表达的可溶性HLA- G1重链分子(heavychainofsolubleHLA G1,sHLA -G1 hc) ,为进一步构建可溶性HLA- G1单体分子奠定基础。方法:应用引物点突变技术,RT -PCR扩增去信号肽的可溶性HLA -G1重链分子的cDNA序列,构建表达载体pET2 2b sHLA -G1 -hc,导入大肠杆菌BL2 1(DE3)Plys,IPTG诱导sHLA-G1- hc- 6 (his融合蛋白表达,提取包涵体蛋白,溶解后经Ni- NTA亲和层析纯化,透析后浓缩保存。SDS PAGE、Westernblot鉴定目的蛋白的表达和纯化。结果:克隆基因序列符合重链基因特征;表达产物以包涵体形式为主,表达量占菌体总蛋白的2 0 % ;纯化后证明表达产物具有较高纯度达到95 %。结论:成功构建可溶性HLA -G1重链分子原核表达载体,为阐明可溶性HLA- G1的功能奠定基础。

Objective:To obtain purified heavey chain molecule of soluble HLA-G1 protein produced by prokaryotic expression system,and to provide a firm basis on construction of soluble HLA-G1 protein.Methods:Through point mutation of primer,sHLA- G1-hc cDNA sequences without encoding signal peptide were amplified by RT-PCR followed by sequencing. After restrictive enzyme digestion,the subclonal fragments were inserted into expression vectors to construct the recombinant expression vectors pET22b(+)/sHLA-G1-hc,which were then expressed as G1-hc-6×his fusion proteins induced by IPTG.The fusion proteins was expressed mostly in the form of inclusion bodies.After inclusion bodies dissolving in 8 mol/L urea,then flowing through Ni 2+ affinity chromatography and finally dialysis,the purification effects of this fusion proteins were identified by SDS-PAGE and westernblot.Results:The G1-hc gene was homologous with the published gene sequences of sHLA-G1. The target proteins (MW 33 kD ) expressed by E.coli took account of 20% total cell proteins, the purified recombinant proteins with purity up to 95%.Conclusion:The prokaryotic expression vector for heavey chain molecule of sHLA-G1 have been established successfully, providing firm basis on clarification the function of sHLA-G1.