表达HIV-1 gp160膜蛋白靶细胞的制备及鉴定

Preparation and Identification of Target Cells Expressing HTV-1 gp160 Membrane Protein

目的获得表达INV-1 gp160膜蛋白的靶细胞,用于HIV-1特异性细胞毒性淋巴细胞(CTL)和抗HIV-1重组毒素细胞杀伤活性检测。方法采用PCR方法,从质粒pJen 10中扩增HIV-1 gp160基因,插入pGEM-T载体,测序后克隆入pIRESl neo载体中构建成重组质粒pIRE160,酶切鉴定正确后,转染人肝癌细胞(SMMG-7721),CA18加压筛选至细胞不再死亡为止,并采用Western blot和间接免疫荧光法对制备的靶细胞进行检测。结果HIV-1 gp160在SMMC7721表面表达,并裂解为gp120和gp41蛋白。结论已成功得到稳定表达HIV-1 gp160的靶细胞,且表达的蛋白具有良好的特异性。

Objective To obtain the target cells expressing HIV-1 gp160 membrane protein used for the determination of killing activities of HTV-1 specific cytotoxic T lymphocytes and anti-HTV-1 recombinant toxin cells.Methods Amplify HTV-1 gp!60 gene from plasmid pJen 10 by PCR, insert into pGEM-T vector, sequence and clone into pIRESl neo vector to construct a recombinant plasmid pIRE 160. Identify the recombinant plasmid by digestion with restriction endonuclease, transform to human hepatoma cell SMMC-7721 and subject to pressure screening with G418 until no death of cell occurs. Detect the prepared target cells by West-em blot and indirect immunofluorescence method. Results HTV-1 gp160 was expressed on the surface of SMMC7721 cells and lyzed into gp120 and gp41. Conclusion The target cells for stable expression of HIV-1 gp160 was successfully prepared, and the expressed protein showed good specificity.