中国流行株HIV-1B/C重组亚型包膜蛋白真核表达载体的构建及稳定表达细胞株的筛选

Construction of Eukaryotic Expression Vector and Screening of Cell Strain for Stable Expression of HIV-1 Subtype B/C Env

目的获得高质量的中国流行株HIV-1B/C重组亚型包膜蛋白抗原。方法改造表达载体,以构建中国流行株HIV-1B/C重组亚型包膜蛋白基因带有筛选标记的真核细胞表达质粒,将所构建质粒转染真核细胞,用含有抗性的培养基筛选出能够高效、持续表达包膜蛋白的稳定表达细胞株。结果所构建的表达载体pVRPEnv转染细胞后可表达目的蛋白,并建立了稳定表达细胞系。结论获得了可以高效、持续稳定表达中国流行株HIV-1B/C重组亚型包膜蛋白的细胞株。

Objective To obtain the envelope protein（Env） of HIV-1 subtype B/C epidemic in China. Methods Insert puromycin gene into plnsmid pVR to construct a modified eukaryotic expression vector pVRP. Insert the Env gene of HIV-1 subtype B/C into the modified vector pVRP,then transfect 293T cells with the constructed recombinant plasmid pVRPEnv in the mediation of lipofectamine. Screen the cell strain for stable and high expression of Env with the medium containing puromycin. Identify the expressed protein by Western blot. Results Sequencing result proved that the eukaryotic expression vector pVR was successfully modified. Both restriction analysis and PCR amplification showed that an eukaryotic expression vector pVRPEnv was successfully constructed. Western blot proved the expression of gp160 in the screened positive cell clones integrated with Env gene. Conclusion A cell strain for high and stable expression of HIV-1 subtype B/C Env was obtained.