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目的将含周期型马来丝虫半胱氨酸蛋白酶抑制剂(Bm-CPI)基因的重组质粒pc DNA3.1(+)-Bm-CPI转染人宫颈癌细胞(Hela细胞)获得重组质粒稳定转染细胞株,为重组蛋白的获得提供基础。方法将成功构建的重组质粒pc DNA3.1(+)-Bm-CPI转染Hela细胞,以G418筛选转染细胞,通过RT-PCR和SDS-PAGE鉴定G418筛选后的单克隆抗性细胞株。结果重组真核表达质粒pc DNA3.1(+)-Bm-CPI转染Hela细胞后,d 14可见G418抗性细胞株开始形成。G418抗性Hela细胞株筛选、扩大培养后RT-PCR扩增出621bp左右的目的条带;SDS-PAGE检测获得了明显的目的条带。结论实验证实pc DNA3.1(+)-Bm-CPI重组质粒被成功转入Hela细胞,并获得稳定表达;为进一步研究Bm-CPI表达、蛋白纯化和测定其生物学活性奠定了基础。
OBJECTIVE: To construct recombinant plasmid pcDNA3.1 (+) - Bm-CPI containing the Bm-CPI gene and to transfer the recombinant plasmid to Hela cells Transfection of cell lines, to provide a basis for the recombinant protein. Methods The recombinant plasmid pcDNA3.1 (+) - Bm-CPI was successfully transfected into Hela cells. The transfected cells were screened by G418. The monoclonal cell line G418 was screened by RT-PCR and SDS-PAGE. Results After transfection of Hela cells with recombinant plasmid pcDNA3.1 (+) - Bm-CPI, G418 resistant cell lines began to form. G418-resistant Hela cell lines were screened. After amplification, the target band of about 621bp was amplified by RT-PCR. Obvious bands of interest were obtained by SDS-PAGE. Conclusions The recombinant plasmid pcDNA3.1 (+) - Bm-CPI was successfully transfected into Hela cells and the stable expression was confirmed. It is the basis for further study of Bm-CPI expression, protein purification and determination of its biological activity.