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目的构建含有人IKK2dn基因的重组腺病毒载体,为转染未成熟树突状细胞(DC)诱导免疫耐受研究奠定基础。方法从质粒pACCMVPLPASR(+)-IKK2dn中酶切出IKK2dn基因,并插入pShuttle-CMV-GFP(-)TEMP载体中构建成腺病毒穿梭质粒,KpnI/HindⅢ酶切鉴定。将pShuttle-CMV-GFP(-)TEMP-IKK2dn转移到pAdxsi载体上,得到pAdxsi-GFP-IKK2dn病毒质粒,XhoI酶切后鉴定。将鉴定正确的质粒用脂质体法转染人胚肾细胞株HEK293细胞,包装成重组病毒颗粒;并在HEK293细胞中反复扩增并纯化,根据报告基因GFP测定病毒滴度。转染宫颈癌细胞株HeLa细胞后采用RT-PCR检测目的基因的表达。结果经酶切和RT-PCR鉴定,得到预期的1060bp条带,证实成功构建了携带IKK2dn基因的重组腺病毒载体,并制备出高滴度(2×1011PFU/ml)的重组腺病毒。结论成功构建了含IKK2dn-cDNA的重组腺病毒,为进一步研究用IKK2dn基因修饰DC诱导免疫耐受等研究奠定了基础。
Objective To construct a recombinant adenovirus vector containing human IKK2dn gene and lay the foundation for the study of immune tolerance induced by transfected immature dendritic cells (DCs). Methods The IKK2dn gene was digested with restriction endonuclease pACCMVPLPASR (+) - IKK2dn and inserted into pShuttle-CMV-GFP (-) TEMP vector to construct adenovirus shuttle plasmid. The recombinant plasmid was identified by KpnI / HindIII digestion. PShuttle-CMV-GFP (-) TEMP-IKK2dn was transferred to pAdxsi vector to obtain pAdxsi-GFP-IKK2dn virus plasmid, which was digested with XhoI and identified. The recombinant plasmids were transfected into human embryonic kidney cell line HEK293 by lipofectamine and packaged into recombinant virus particles. The recombinant plasmids were amplified and purified repeatedly in HEK293 cells, and the virus titer was determined according to the reporter gene GFP. Transfection of cervical cancer cell line HeLa cells after RT-PCR detection of the target gene expression. Results The 1060bp band was confirmed by restriction enzyme digestion and RT-PCR. The recombinant adenovirus carrying IKK2dn gene was successfully constructed and a recombinant adenovirus with high titer (2 × 1011 PFU / ml) was prepared. Conclusion The recombinant adenovirus containing IKK2dn-cDNA was successfully constructed, which lays the foundation for further research on the induction of immune tolerance by IKK2dn gene modified DC.