中文摘要：

目的构建分别携带人核因子-κB（NF-κB）亚基p65、p50基因的逆转录病毒载体,并建立稳定产毒的包装细胞株。方法以RcCMV-p65、RcCMV-p50质粒为模板,PCR扩增p65、p50基因编码区全长序列,分别克隆至逆转录病毒空载体pMSCVneo、pMSCVhyg,酶切、测序鉴定。将重组载体及空载体分别转染包装细胞系PT67,以G418或潮霉素筛选产毒细胞株。收集病毒悬液,测定病毒滴度,并瞬时感染NIH3T3细胞及人神经干细胞,72h后分别采用RT-PCR和免疫细胞化学观察p65、p50的表达。结果重组逆转录病毒载体pMSCVneo-p65、pMSCVhyg-p50的酶切、测序鉴定正确。将筛选所得的高效产毒细胞株分别命名为PT67/pMSCVneo-p65、PT67/pMSCVneo、PT67/pMSCVhyg-p50及PT67/pMSCVhyg,测定病毒滴度分别为4×105、6×105、2×105及1×105cfu/ml。pMSCVneo-p65、pMSCVhyg-p50病毒悬液瞬时感染NIH3T3细胞72h后,细胞p65、p50亚基mRNA的表达明显升高;瞬时感染人神经干细胞（hNSC）72h后,部分细胞胞核呈p65和p50阳性染色。结论成功构建了分别携带人p65、p50基因的逆转录病毒载体,建立了稳定、高效、正确生产逆转录病毒的细胞株。为探讨转NF-κB基因神经干细胞在缺血性卒中的运用前景奠定了实验基础。

英文摘要：

Objective To establish recombinant retrovirus vectors containing human NF-κB subunits p65, p50 genes respectively, and their packaging cell clones which stably produce viruses. Methods The whole coding regions of p65 and p50 genes were amplified from plasmids RcCMV-p65 or RcCMV-p50 by polymerase chain reaction（PCR）, and sub-cloned to the retroviral vectors pMSCVneo and pMSCVhyg respectively. The recombinant vectors were identified by the restriction endonuclease digestion and DNA sequencing. The recombinant and control retroviral vectors were transfected into the packaging cell line PT67 by liposome LipofectamineTM2000. The cell clones stably producing retrovirus were isolated following G418 or hygromycin selection. The viral suspension was harvested, and the viral titer was determined by NIH3T3 cells. The transcription or translation of p65 and p50 subunits was detected in the transiently infected NIH3T3 cells or human neural stem cells（hNSC） by RT-PCR or immunocytochemistry at 72 h after infection. Results The recombinant retroviral vectors pMSCVneo-p65 and pMSCVhyg-p50 were proved to encode p65 and p50 genes by the restriction endonuclease digestion and sequencing. The cell clones efficiently producing virus were screened out by G418 or hygromycin, and designated as PT67/pMSCVneo-p65, PT67/ pMSCVneo, PT67/pMSCVhyg-p50 and PT67/pMSCVhyg. And the titers of them were 4 × 105 , 6 × 105 , 2 × 105 and 1 × 105 cfu/ml respectively, p65 or p50 was transcripted at high level in NIH3T3 cells, and positively stained in the nuclei of hNSC at 72 h after NIH3T3 cells and hNSC had been transiently infected by pMSCVneo-p65 or/and pMSCVhyg-p50 retrovirus. Conclusion The recombinant retrovirus vectors containing human NF-κB subunits p65 and p50 genes respectively, and the packaging cell lines which efficiently and correctly produce viruses have been constructed successfully. These provide a basis for the further research on the possibility of neural stem cells transfected with NF-κB genes for brain ischemia.