论文部分内容阅读
目的:应用序列特异性PCR技术(PCR-SSP)分析不同细胞系中MICA*008等位基因特异性位点,筛选出人MICA*008基因型,并构建其真核表达载体。方法:PCR-SSP技术分析本实验室保存的多种人源性细胞系中多态性基因MICA的特异性位点,预筛选出包含MICA*008等位基因的细胞系,应用RT-PCR扩增MICA*008基因片段,随后克隆到pcDNA3(+)真核表达载体,用经过测序、鉴定的pcDNA3(+)/MICA*008质粒转染结肠癌细胞系sw480,通过流式细胞术检测MICA膜蛋白表达水平。结果:预筛选出包含MICA*008基因型的细胞系,成功构建了pcDNA3(+)/MICA*008质粒,pcDNA3(+)/MICA*008转染sw480细胞系后,其MICA膜蛋白表达水平明显提高。结论:验证了应用PCR-SSP技术对细胞系中对特定MICA等位基因预筛选的可行性,成功构建了多态性MICA基因中MICA*008等位基因的真核表达载体pcDNA3(+)/MICA*008并转染结肠癌细胞系sw480,为后续体外研究MICA*008在结肠癌及其他肿瘤的作用奠定了基础。
OBJECTIVE: To analyze the MICA * 008 allele-specific loci in different cell lines by PCR-SSP and to screen the human MICA * 008 genotype and construct its eukaryotic expression vector. Methods: PCR-SSP was used to analyze the specific sites of MICA in many human cell lines preserved in our laboratory. The cell lines containing the MICA * 008 alleles were pre-screened and amplified by RT-PCR The MICA * 008 gene fragment was amplified and cloned into the eukaryotic expression vector pcDNA3 (+). The transfected colon cancer cell line sw480 was transfected with the pcDNA3 (+) / MICA * 008 plasmid. The MICA membrane was detected by flow cytometry Protein expression levels. Results: The cell line containing MICA * 008 genotype was pre-screened and the pcDNA3 (+) / MICA * 008 plasmid was successfully constructed. The expression of MICA membrane protein in pcDNA3 (+) / MICA * 008 transfected sw480 cell line was significantly improve. Conclusion: The feasibility of pre-screening specific MICA alleles in cell lines by PCR-SSP was verified. The eukaryotic expression vector pcDNA3 (+) / MICA * 008 of the MICA * MICA * 008 and transfected into colon cancer cell line sw480, which laid the foundation for the subsequent study on the role of MICA * 008 in colon cancer and other tumors.