在大肠杆菌中高效表达人Ⅰ型免疫缺陷病毒p24蛋白

目的表达人Ⅰ型免疫缺陷病毒（HIV—1）衣壳蛋白p24，为制备抗p24单克隆抗体及其诊断杭原奠定基础。方法将编码HIV—1p24蛋白的P24(gag)基因片段，克隆到原核表达载体PET—17b的T7噬菌体启动子下游，构建重组表达质粒;转化大肠杆菌BL2（DE3），IPTG诱导表达，表达产物以SDS-PAGE、Westernblotting及点免疫印迹分析。结果构建成功重组表达质粒pET24，经IPG诱导，p24(gag)基因片段在大肠杆菌BL21（DE3）中高效表达，产物为30kDa的810—p24融合蛋白，表达量占菌体总蛋白的38.4％;重组p24蛋白均与抗p24单克隆抗体及HIV—1阳性血清发生特异性反应。结论重组P24蛋白具有较好的免疫活性，是制备抗P24单克隆抗体及诊断试剂的理想抗原。

HIGH0 LEVEL EXPRESSION OF P24 PROTEIN OF HUMAN IMMUNODEFICIENCY VIRUS TYPE IN E. COLI

Aim Expressing the capsid protein (p24) of human immunodeficiency virus type 1 (HIV-1 ) for preparing monoclonal antibody against HIV. Methods The p24 gene fragment encoding the p24 protein was recombined and inserted into expression vector PET─ 17b under the control of the bacteriophage T7 promoter. The recombinant vector was transferred into Escherchia coli (E. coli) BL21 (DE3), and induced by IPTG.The expressed protein was analyzed by SDS-PAGE, Western blotting, and immunodot blotting. Results A recombiant plasmid pET24 expressing the p24 protein of HIV─ 1 was successfully constructed. Induced by IPTG,the pET24 plasmid highly expressed a 30kDa protein of s10-p24 spanning 284 amino acid residues. The total amount of the fusion protein was approximately 38. 4% of the celluar protein in E. coli. Western blotting and immunodot blotting indicated that the recombinant protein could be recognized by anti-p24 monoclonal antibody and HIV-1 positive sera respectively. Conclusions The recombinant protein is useful for preparing specific antibody and diagnostic usage.