【摘要】 目的　构建编码HIV 1外膜糖蛋白的gp12 0和gp4 1基因杆状病毒转移载体 ,并在昆虫细胞中表达gp12 0和gp4 1重组蛋白。方法　从HIV 1基因克隆PNL4 3(NY5 /LAV)中应用聚合链反应扩增目的基因gp12 0和gp4 1,克隆到PGEM T载体中,限制性内切酶酶切、DNA序列分析鉴定目的基因 ,再经EcoRⅠ和BamHⅠ双酶切后定向克隆到杆状病毒转移载体PACSecG2T中 ,再次测序鉴定。通过在sf9昆虫细胞中同源重组、空斑筛选、病毒鉴定、SDS PAGE、Western blot对重组病毒和重组蛋白进行分析。结果　限制性内切酶酶切和DNA序列分析表明 ,gp12 0和gp4 1正确克隆到杆状病毒转移载体PACSecG2T中 ;SDS PAGE和Western blot结果表明在昆虫细胞中成功表达了HIV外膜糖蛋白gp12 0和gp4 1。结论　成功构建了PACSecG2T gp12 0和PAC SecG2T gp4 1杆状病毒转移载体 ,并在杆状病毒 昆虫细胞表达系统中表达了HIV 1gp12 0和gp4 1重组蛋白 ,Wes tern blot证明具有较好的免疫原性更多还原

【Abstract】 Objective The cDNA coding HIV gp120 and gp41 envelope (en v) proteins were cloned from PNL4 3 containing HIV genome and inserted into bac ulovirus transfer vector. The recombinant protein of HIV gp120 and gp41 were exp ressed in sf9 insect cells.Me thods HIV gp120 and gp41 wer e ampl ified by PCR from PNL4 3 plasmid. The PCR product was cloned into PGEM T vecto r by TA cloning strategy and the recombinant plasmid was screened by restriction analysis and direct sequencing. PGEM gp120 and PGEM gp41 digested by restri ction endonuclease was cloned to the baculovirus transfer vector PACSecG2T and con firmed by sequencing. The recombinant baculovirus and recombinant protein were i dentified and analysed by homologously recombination in sf9 insect cells, plaque assay, SDS PAGE and Western blot.Results gp120 and gp41 that were cloned to the baculovirus transfer vector PACSecG2T were assessed by restri ction analysis and direct sequencing. The recombinant proteins of HIV gp120 and HIV gp41 were expressed in sf9 insect cells.Conclusion The PACS ecG2T gp120 and PACSecG2T gp41 baculovirus transfer vector were constructed su ccessfully, the recombinant proteins of HIV gp120 and HIV gp41 were expressed in baculovirus insect cell systems. The activity of immunity of HIV gp120 and HIV gp41 were confirmed by Western blot. [更多还原