人/猴免疫缺陷病毒SHIV衣壳蛋白p27的表达纯化及活性鉴定

Expression, purification and bioactivity test of SHIV capsid p27

利用PCR技术从质粒SHIV89.6P中扩增p27gag基因,并克隆入pMD18-T载体,测序鉴定;将p27gag基因插入表达载体pET32aT7启动子下游,构成重组质粒pET32a-p27并使其在大肠杆菌BL21中高效表达,最后利用固定化金属离子(Ni2+)配体亲和层析技术从表达蛋白中纯化目的蛋白,Western Blot检测活性。结果显示,融合蛋白p27经SDS-PAGE电泳观察可见45ku的明显条带,与预期分子质量大小一致且多以可溶形式表达,目的蛋白占菌体总蛋白的36.8%;经镍柱纯化,获得目的蛋白p27纯度可达98%;经Western Blot试验检测,证明此融合蛋白p27能与SHIV阳性血清发生特异性反应,具有较好的抗原活性。该研究为进一步开发早期诊断试剂盒和基因工程疫苗奠定了初步基础。

According to the published SHIV sequences, p27 gag gene was amplified from the plasmid SHIV89.6P by PCR method, cloned into the pMD18-T vector and sequenced. Under the control of the bacteriophage T7 promoter, the gag gene fragment of p27 was inserted into the pET32a vector in order to construct the pET32a-p27 recombined vector. After highly expresson in E.coli, the purified p27 protein was prepared by metal-ligand affinity chromatograpby（IMAC）. The accuracy of the inserted gene and activity of the SHIV p27 protein were detected by SDS-PAGE and Western Blot. The result indicated that the fused protein p27 had a single expected band about 45 ku in SDS-PAGE and accumulated up to 36.8% of the total bacteria protein. The final p27 protein purification could reach 98%. Western Blot indicated that the recombinant protein could be recognized by SHIV positive sera. This study lays a foudation for developing the early confirmation test kit and genetic engineering vaccine.