人巨细胞病毒截短被膜磷蛋白pp65的原核表达及抗原性分析

摘要

Objective To express the fragment of tegument protein pp65 of human cytomegalovirus ( HCMV) in E. co-li, and identify its antigenicity initially. Methods The target gene was amplified by PCR and cloned into expression vector pET30a ( + ). After transformated the recombinant plasmid into E. coli, BL21 ( DE3 ) , the expression was analyzed initially by SDS-PAGE, and the antigenicity of recombinant protein was detected by ELISA. Results The fusion protein about 30 kD was obtained, the P/N value of pp65 antigen and virus antigen were 2. 737,2. 535 respectively. Conclusions This study express the fragment of HCMV pp65 successfully, and prove that its antigenicity is superior than virus antigen; This may provide foundation for the vaccinal research and manufacture of the HCMV detection kit.%目的 利用大肠杆菌表达系统表达截短人巨细胞病毒(HCMV) pp65蛋白,并对其抗原性进行初步鉴定.方法 采用PCR扩增HCMV pp65基因961～1 686 bp(321aa-562aa)片段,并将其插入表达载体pET30a(+),转化BL21(DE3)菌株,十二烷基硫酸钠—聚丙稀酰胺凝胶(SDS-PAGE)分析蛋白表达,常规镍柱法纯化目的蛋白,以ELISA法初步检测其抗原性.结果 获得相对分子质量约为30 kD的融合蛋白,pp65抗原和全病毒抗原的P/N值分别为2.737、2.535.结论 本研究成功表达截短HCMV pp65抗原,并证实其蛋白抗原性优于全病毒抗原;此为HCMV检测试剂盒的研制和疫苗的研究提供了依据.