携带人NFBD1基因shRNA重组逆转录病毒载体的构建及鉴定

Construction and Identification of Recombinant Retrovirus Vector Expressing shRNA Targeting Human NFBD1 Gene

建立逆转录病毒介导的NFBD1基因RNA干扰表达体系,并观察其在宫颈癌SiHa细胞中对NFBD1表达的影响。将人NFBD1基因RNA干扰双链DNA片段重组到pSUPER Retro质粒中,构建携带人NFBD1基因RNA干扰的逆转录病毒载体pSUPER-shRNA-NFBD1,经PT67细胞包装后,产生的重组逆转录病毒感染宫颈癌细胞株SiHa细胞,并用嘌呤霉素筛选产生稳定的细胞克隆,用实时荧光定量PCR和Westernblotting检测细胞中NFBD1mRNA和蛋白表达的变化。重组逆转录病毒质粒经测序鉴定正确;逆转录病毒感染SiHa细胞后用嘌呤霉素筛选出稳定的细胞克隆;实时荧光定量PCR和Westernblot-ting检测人NFBD1mRNA和蛋白表达水平明显低于阴性对照组和未干扰组。携带人NFBD1基因RNA干扰双链DNA片段的逆转录病毒感染SiHa细胞后能明显抑制NFBD1mRNA和蛋白表达,为进一步研究NFBD1在宫颈癌中的作用奠定了基础。

It was to construct a retrovirus-mediated expression system containing double strands DNA for RNA interference on nuclear factor with BRCT domains protein 1 （NFBD1）, and study its influence on the expression of NFBD1 in SiHa cells. A recombinant retrovirus vector pSUPER-shRNA-NFBD1 was generated by cloning a double strands DNA for RNA interference on human NFBD1 into pSUPER Retro vector. SiHa cells were infected with virus superuant from the PT67 clones, and stable SiHa cell clones were generated after screening with puromycin. NFBD1 mRNA and protein of stable infected SiHa cells were examined by Real time PCR and Western blotting. The pSUPER-shRNA-NFBD1 recombinant retrovirus vector had been constructed correctly after sequencing. Stable infected SiHa cell clones were generated after screening with puromycin, and NFBD1 mRNA and protein of infected cells decreased significantly. The pSUPER-shRNA-NFBD1 retrovirus vector showed effective inhibition on the expression of NFBD1 at mRNA and protein levels, which would establish a favourable foundation for further study on the function of NFBD1 in cervical cancer.