猪繁殖与呼吸综合征病毒Hn-1/06株M基因的原核表达与重组蛋白的复性研究

摘要

The gene segments tM deleting hydrophobic region sequence were successfully amplified from recombinant plasmids pTG19-T-M by PCR. The fragment was inserted into the prokaryotic expression vector pET-32a to construct the recombinant plasmid pET-tM,and expressed in E. Coli BL21. The result indicated that the fusion protein pET-tM was successfully expressed. SDS-PAGE analysis demonstrated the molecular weight of reorganization protein existing in cytorrhyctes type was 29 kD and amounted to 31% of the mass of bacterial proteins in cvtorrhvctes. The renaturation of reorganization protein was carried on by dilution and the dialysis after treatment with the 8 mol ·L~(-1) urea. The renaturation reorganization protein was analyzed by Western-blot,the result demonstrated that the renaturation reorganization protein was recognized by the PRRSV masculine blood serum.%采用PCR方法从重组质柱pTG19-T-M扩增得到缺失N端疏水区序列的基因片段tM.将tM与原核表达载体pET-32a进行连接并转化,重组质粒经鉴定并测序.结果表明,目的基因在大肠杆菌BL21细胞中成功地表达了醉繁殖与呼吸病毒重组蛋白pET-tM.表达量为31%.表达产物经SDS-PAGE分析,得到分子量约为29 kD的重组蛋白且以包涵体形式存在.8 mol·L~(-1)尿素变性溶解包涵体,再用稀释与透析相结合的方法对重组蛋白进行复性.复性蛋白经Western-blot检测,结果证明复性蛋白可被PRRSV阳性血清识别用.