摘要
目的克隆人B-CAM/Lu基因,构建其逆转录病毒表达载体,获得稳定高表达B-CAM/Lu的L929细胞株。方法RT-PCR技术克隆人B-CAM/Lu基因,并插入逆转录病毒载体pEGZ中,该重组逆转录病毒载体与pH456、pH460两个辅助病毒载体一起,共转染包装细胞293T,并感染L929细胞,经Zeocin筛选获得的细胞株通过RT-PCR、Western blot及间接免疫荧光鉴定细胞中B-CAM/Lu基因mRNA的转录和蛋白表达。结果成功获得人B-CAM/Lu基因,测序正确,亚克隆至逆转录病毒载体pEGZ后,经转染筛选,获得的抗性L929细胞株通过RT-PCR和Western blot分析,检测到B-CAM/Lu mRNA的转录和目的蛋白的表达,通过间接免疫荧光确定该蛋白定位表达在L929转基因细胞膜上。结论成功克隆并构建了人B-CAM/Lu基因的重组逆转录病毒表达载体,获得稳定高表达B-CAM/Lu蛋白的L929细胞株,为将其作为免疫源,制备B-CAM/Lu单抗用于镰刀型红细胞病及一些肿瘤疾病的检测诊断打下了基础。
Objective To construct the retrovirus expression vector of human B-CAM/Lu gene, and express it in L929 cells. Methods Human B-CAM/Lu gene amplified by RT-PCR was inserted into retrovirus vector pEGZ by genetic recombination technique. The recombinant plasmid together with its two helper virus vectors was cotransfected into the package cell 293T with Lipofect2000. Then the supematant of 293T was used to infect L929 cells. After zeocin selection, a stable cell line B-CAM/Lu/L929 expressing the human B-CAM/In was established. The expression stability and efficiency of the target molecule were identified by RT-PCR, Western blotting, and indirect immunofluorescent staining. Results The full-length sequence encoding B-CAM/Lu was obtained and the retrovirus expression vector pEGZ/ B-CAM/Lu was constructed correctly, mRNA transcription and protein expression of the target molecule in the transgenic cell L929 were confirmed by RT-PCR and Western blouing assay. Moreover, indirect immunofluorescent staining confirmed that the site of B-CAM/Lu protein in cell line B-CAM/Lu/L929 was on the cell membrane. Conlusion The cell line overexpressing B-CAM/Lu is useful as an antigen for inducing anti-B-CAM/ha McAb.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2007年第4期445-448,共4页
Immunological Journal
基金
国家科技部中小企业技术创新基金项目(04C26223200112)
