摘要
为研究人免疫缺陷病毒（HIV－1）的分子生物学，发展艾滋病的诊断试剂，克隆了含HIV－1env和gag基因cDNA的质粒pUCF12。用PvuⅡ和HincⅡ降解pUCF12质粒，得到含部分p17、全部p24和全部p15基因的cDNA片段，用BglⅡ降解pUCF12质粒得到含有部分gp120和全部gp41的cDNA片段。将该两个片段分别亚克隆到表达载体PGex上，在pTac启动子的调控下，部分gag基因和env基因在大肠杆菌中表达．在SDS－PAGE中，GST＋gag融合蛋白呈现一条约70kD的染色带，其表达水平约占菌体总蛋白的18％．而GST－env蛋白则呈现一条约65kD的蛋白带，该表达蛋白约占菌体总蛋白的15％。经免疫印迹分析，该两种表达产物可分别与抗-P24和抗-gP41的单克隆抗体反应，用Abbott试剂也证明为HIV－1抗原蛋白。
In order to study the molecular biology of HIV-1 and to develop the diagnostic reagent for AIDS, the plasmid pUCF12 containing HIV-1 env and gag gene cDNA was cloned.The cDNA fragment containing P24, P15 and partial P17 was obtained by degrading pUCF12with Pvu Ⅱand Hine Ⅱ. However,after pUCF12 was degraded with Bg1Ⅱ, the cDNA fragment containing gp41 and partial gp120 was obtained. Each of the 2 fagments was subcloned into pGex (a carrier), then partial gag and env genes were expressed in E. coli under the control of pTac promotor. SDS-PAGE proved that GST+gag fusion protein showed a single band with a molecular weight of 70KDa, and the expressive level reached 18 % of its total bacterial protein i however,GST-env showed a single band with a molecular weight of 65KDa, and 15% of its total bacterial protein was expressed. Western blotting proved that the 2 expressed products could react with anti-p24 and anti-gp41 McAbs respectively. Both of the 2 products were proved to be HIV-1 antigentic protein detected by Abbott kit.
出处
《中国生物制品学杂志》
CAS
CSCD
1995年第2期49-52,共4页
Chinese Journal of Biologicals
