摘要
目的构建针对CD59基因的RNAi逆转录病毒载体并感染HeLa细胞,观察CD59基因抑制后对补体溶破的抵抗作用的变化。方法应用siRNA表达载体介导的RNAi技术,构建3条含特异性CD59基因的重组载体,并以无关序列作为对照,脂质体法转染包装细胞并感染HeLa细胞,RT-PCR和ELISA检测靶细胞上CD59基因的抑制效果,染料释放试验检测CD59功能的改变。结果重组载体经鉴定正确,转染包装细胞成功,RT-PCR和ELISA结果显示能显著抑制CD59基因的表达,染料释放试验显示干扰组对补体溶破的抵抗作用降低。结论特异性沉默CD59基因的逆转录病毒载体构建和筛选成功,并能抑制CD59基因的表达及减弱CD59对补体的抑制功能,为后续进行肿瘤细胞的研究奠定了基础,可望为肿瘤的免疫治疗开辟新途径。
To construct recombinant retroviral vectors that target CD59 gene in order to analyze the role of CD59 in the immune escape of tumors. Three 60 bp sequences encoding CD59 gene shRNA were cloned into pSUPER vector with DNA recombinant technique, while control group was also prepared. The packaging cell Phoenix A was transfected with this recombinant plasmid using liposome; the virus supemants were harvested and used to infect HeLa cells. CD59 mRNA and protein levels were detected by RT-PCR and ELISA, while its function was analyzed by dye release assay. DNA sequencing demonstrated the pSUPER-siRNA expressing vectors were constructed successfully. RT-PCR and ELISA indicated that CD59 mRNA and protein levels were inhibited, while dye release assay suggested that anti-human complement effects of CD59 protection was decreased, pSUPER-siCD59 vectors were successfully constructed and identified, which could effectively and specially knock down the expression of CD59, even the anti-complement effects of CD59 protection. These resuhs may pave the way for studying the role of CD59 in the immune escape of tumors, thus benefiting tumor therapy.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2010年第2期98-101,共4页
Immunological Journal
基金
国家自然科学基金资助项目(30671936)
