人CD46 RNAi 逆转录病毒载体系统的构建与鉴定

目的:构建并筛选携带针对CD46基因的pSUPER retro RNAi逆转录病毒载体,从而特异、有效地抑制人CD46mRNA水平。方法:利用DNA重组技术,将2条60nt能转录产生靶向CD46小发夹RNA(shRNA)的寡核苷酸序列,定向克隆入逆转录病毒载体pSUPER retro,并转化大肠杆菌JM109。结果:重组载体经PCR及限制性内切酶酶切鉴定初步成功后送测序,结果表明序列正确。结论:特异性沉默CD46基因的pSUPER retro RNAi逆转录病毒载体构建成功,为后续转染Jurkat细胞,研究CD46在T细胞的信号转导中的作用奠定了基础,对进一步研究T细胞相关疾病及开展细胞免疫缺陷的治疗方面提供了新的思路与方向。

Construction and Identification of Retro RNAi System of the Specific Silencing CD46

Objective: To construct recombinant retrovirus encoding shRNA targeted human CD46 and identify its function.Methods: shRNA was synthesized,annealed and inserted into pSUPER plasmids digested with BglⅡand HindⅢ to construct the recombinant pSUPER RNAi plasmids(pSUPER-CD46).Recombinant pSUPER-CD46 plasmid was identified by enzyme digestion and sequencing analysis.The plasmids were transfected into Jurkat cells in vitro and the inhibitory efficiency of target genes expression was observed with RT-PCR and Western blot.Recombinant pSUPER-CD46 vector was identified correct by enzyme digestion and sequencing analysis.Result: The siRNA eukaryotic expression vectors constructed targeting on CD46 could reduce the expressions of target genes and it might be able to use for the exploration of new anti-fibrosis drugs genetically.Conclusion: The eukaryotic expression vectors were constructed successfully.Future work could investigate the relationship between CD59 and CD46 in T cell signal transduction.