HIV-1Gag蛋白在大肠杆菌和重组杆状病毒系统中的高效表达及通用型温控原核表达载体的构建

将中国株HIV－1B亚型的gag全基因序列，克隆到杆状病毒表达载体pfastbacI中，构建了重组质粒pfastGag，利用细菌／杆状病毒表达系统筛选重组杆状病毒，在昆虫细胞中高效表达了HIV－1Gag蛋白。通过改造原核表达载体pBV220和pET28，构建了一种新的通用型温控原核表达载体质粒pVV5，该载体携带PrPl串联温控启功子及His—Tag纯化标签，利于目的蛋白表达与纯化。将HIV－1gag基因的1148一1857编码序列，分别插入到pVV5b、pET28b的相应位点，构建了重组表达质粒pEG1b、pEG7b，二者在不同受体菌中，表达重组蛋白的量分别占全菌体蛋白总量的42％和28％。利用IMAC金属螯合层析柱，对包涵体中的重组p24蛋白进行纯化，纯度超过80％；纯化后的重组蛋白可与HIV－1型标准阳性血清发生较强的免疫学反应。

EXPRESSION OF HIV-1 GAG PROTEIN IN E.coli AND INSECT CELLS WITHI THE CONSTRUCTION OF CURRENT NOVEL EXPRESSION VECTORI

The full length of subtype B HIV - 1 gag gene were cloned into baculoviruses transfer vector pfastbacl and the recombinant plasmid pfastGag was constructed. After transformation and con - transfecting the recombinant Bacimid with S19 cells, the recombinant baculoviruses expressed HIV - 1 Gag protein were obtained. New kinds of novel fusion expression vector pCC5 were designed and consructed these Vectors containing the phage promoter pR and pL as well as 5' - terminal Sequence coding the peptide of 6xhis-tag that fit for the effective expression and purification of foreign protein. HIV-1 P24 gag gene was cloned into plasmid PVV5 and PE128b respectively and the target products could be expressed successfully.