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目的克隆人红细胞血型糖蛋白C(GYPC)基因,构建GYPC重组逆转录病毒载体,以获得稳定表达GYPC的L929细胞。方法RT-PCR扩增人GYPC基因,克隆入T载体后,将GYPC双酶切亚克隆入逆转录病毒载体pEGZ,用此重组载体转染L929细胞,Zeocin筛选得到阳性克隆;用RT-PCR及流式细胞术等方法对重组载体进行鉴定。结果克隆了人GYPC基因,并获得稳定表达GYPC的L929细胞系。结论应用基因工程技术,成功构建了含人GYPC基因重组逆转录病毒载体和稳定表达人GYPC的细胞株,建立了红细胞血型抗原的异源表达系统,为血清中抗GYPC特异性抗体的检测及GYPC单克隆抗体的制备和研究奠定了基础。
Objective To clone human erythrocyte glycoprotein C (GYPC) gene and construct GYPC recombinant retroviral vector to obtain stable GYPC-expressing L929 cells. Methods The human GYPC gene was amplified by RT-PCR, cloned into T vector, subcloned into retroviral vector pEGZ by double digestion with GYPC, and then transfected into L929 cells with Zeocin. The positive clones were screened by RT-PCR and Flow cytometry and other methods to identify the recombinant vector. Results The human GYPC gene was cloned and the L929 cell line stably expressing GYPC was obtained. Conclusion Recombinant retroviral vector containing human GYPC gene and cell line stably expressing human GYPC were successfully constructed by genetic engineering technique. The heterologous expression system of erythrocyte blood group antigen was established to detect the anti-GYPC specific antibody in serum and GYPC Monoclonal antibody preparation and research laid the foundation.