人巨细胞病毒结构蛋白p150、p38的原核表达纯化及免疫性鉴定

应用聚合酶链式反应(PCR)扩增HCMV结构蛋白p150aa595~aa614/aa1006~aa1048和p38aa117~aa373基因片段,将其分别克隆于表达载体pGEX-4T-1和pET30 a-SetB中,转化大肠杆菌BL21(DE3),IPTG诱导表达,重组质粒经酶切鉴定及DNA测序证实pGEX-4T-1/p150aa595~aa614/aa1006~aa1048和pET30 a-SetB/p38aa117~aa373构建正确;通过优化表达和纯化,分别获得分子量约为35kD和43kD纯化的表达产物。经亲和层析纯化以免疫印迹实验对纯化的重组蛋白进行免疫反应性检测,证实重组蛋白p150aa595~aa614/aa1006~aa1048、p38aa117~aa373能够与抗HCMV IgM阳性血清反应,说明表达的重组蛋白p150aa595~aa614/aa1006~aa1048、p38aa117~aa373具有良好的免疫性,对HCMV的原发感染具有潜在的应用价值。

Expression, purification and identification of recombinant viral structural proteins p150 and p38 of human cytomegalovirus

Both gene fragments of P150(amino acids aa 595 to 614 and 1006 to 1048 which were fused together) and p38(aa 117 to 373) were amplified by PCR from template DNA of human cytomegalovirus.The two target DNA fragments were cloned into expression vector pGEX-4T-1 and pET30a-SetB separately.After transformed into competent E.coli BL21(DE3) cell and induced by IPTG,the recombinant fusion proteins p150_(aa595~aa614/aa1006~aa1048)and p38_(aa117~aa373) were expressed and then purified by an affinity chromatography.Restriction enzymes cleavage and DNA sequencing confirmed that the plasmid p150_(aa595~aa614/aa1006~aa1048) and pET30a-SetB/ p38_(aa117~aa373) were correctly constructed.Two purified recombinant_()proteins with molucular weight about 35kD and 43kD were obtained by optimizing the conditions for both expression and purification.Western blot showed that these two recombinant proteins could be recognized by positive IgM serum of human cytomegalovirus.These two recombinant proteins showed good immunity and had a potential value for diagnosis of primary HCMV infection.