摘要
合成引物扩增HIV 1p2 4基因 ,并将其克隆到 pQE 30质粒中 ,使其在大肠杆菌E .coliM 15中以IPTG诱导高效表达 ,经SDS PAGE分析 ,该表达产物约占菌体总蛋白 2 0 % ,并且以可溶蛋白的形式存在于细菌裂解液上清之中。经镍离子柱亲和层析一步纯化 ,洗脱产物中 p2 4蛋白纯度达95 %。ELISA分析表明 ,该蛋白可与HIV感染者血清发生特异性免疫反应。以此蛋白交联Sepharose 4B ,亲和层析纯化HIV感染者血清中的抗体 ,用所得抗体与HIV确认试剂反应 ,发现该纯化抗体仅与确认试剂中的 p2 4蛋白反应。上述结果表明在大肠杆菌中已经高效表达了可溶性HIV 1p2 4蛋白 。
The HIV 1 p24 gene (gag gene) was amplified by polymerase chain reaction (PCR) and cloned into an E.coli expression vehicle pQE30 between BamH I and Kpn I sites. After induction with IPTG, the transformed E.coli strain M15 expressed the HIV 1 p24 gene efficiently. The recombinant p24 protein was expressed as a soluble protein, composing 20% of the total protein. After onestep purification with Ni NTA + affinity chromatography, the protein was purified to almost homogeneity. When applied to ELISA, the p24 protein exhibited good specific reactivity with sera of HIV infected individuals. The antibodies against recombinant p24 protein from HIV infected plasma were purified and confirmed with HIV Western blot kit that the purified antibodies can only react with HIV 1 p24 antigen. It suggests that the recombinant HIV 1 p24 protein is suitable for assembling the HIV diagnostic kits.
出处
《中国病毒学》
CSCD
2000年第2期116-121,共6页
Virologica Sinica
