sHLA-G1重链分子、β_2m的体外表达及纯化研究

In vitro expression and purification of heavy chain and β_2m of soluble HLA-G1

目的获得sHLA-G1重链分子及轻链β2微球蛋白(β2m)基因体外表达并纯化的相关蛋白质。方法RT-PCR扩增可溶性HLA-G1重链分子及人β2m轻链的cDNA序列,构建表达载体pET28a(+)/sHLA-G1及pET28a(+)/β2m,导入大肠杆菌BL21(DE3),IPTG诱导sHLA-G1及β2m蛋白表达,并以Ni-NTA亲和层析和CM-FF弱阳离子柱分别纯化,透析后浓缩保存。SDS-PAGE,Western-Blot鉴定目的蛋白的表达和纯化。结果成功克隆了sHLA-G1及2βm基因并构建了pET28a(+)-sHLA-G1、pET28a(+)-β2m原核高效表达载体;表达产物以可溶性形式存在,表达量>30%,纯化后产物纯度达到95%。结论成功表达并纯化出的sHLA-G1重链分子及轻链β2m有助于阐明可溶性HLA-G1的功能。

Objective To obtain purified heavy chain molecule and β-2microglobulin （β2m） light chain of soluble HLA-G1 protein. Methods RT-PCR was used to amplify sHLA-G heavy chain and β2m light chain genes from total RNA obtained from human placenta tissue by TRIzol. After DNA sequencing and restrictive enzyme digestion, the DNA fragments of sHLA-G heavy chain were purified from the cloning plasmids digested by BamHⅠand EcoR Ⅰ , while β2 m were digested by Nde Ⅰ and Hind Ⅲ. Then the fragments were inserted into plasmid pET28a （＋）, digested by the same enzyme, to construct recombinant expression. Expressions of sHLA-G1 heavy chain and β2m light chain by plasmids pET28a（＋ ）/sHLA-G1 and pET28a（＋ ）/β22m, were induced by ITPG in E. coli BL21. The sHLA- G1 heavy chain was purified by Co-affinity chromatography, and β2 m purified by CM-FF weak cationic column. The purification and expression of the protein was identified by SDS-PAGE and Westernblot. Results The sHLA-G1 and β2m gene were homologous with the published gene sequences of sHLA-G1. The expression rate of sHLA-G1 heavy chain molecule and β2m was 30% and 25 %, respectively, and their molecular weights were about 37KD and 12KD, respectively. The purity of sHLA-G1 heavy chain and β2m protein could also reach about 95 %. Conclusion The prokaryotic expression vectors for heavy chain molecule and β2 m of sHLA-G1 have been established successfully, providing firm basis for the clarification of the functiofi of sHLA-G1.