编码人IKK2dn重组腺病毒载体的构建及表达验证

Construction of Adenoviral Vector Encoding IKK2dn Gene and Identification of Their Expression

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 the recombinant adenovirus vector encoding IKK2dn gene for using IKK2dn modified immature DC to induce immune tolerance.Methods IKK2dn cDNA was cloned into adenovirus transfer vector pShuttle-CMV-GFP（-）TEMP,and analyzed by restriction endonuclease KpnIHindIII digestion.Then,the obtained plasmid,pShuttle-CMV-GFP（-）TEMP-IKK2dn was transferred into pAdxsi vector to construct pAdxsi-GFP-IKK2dn plasmid,and was identified by XhoI digestion.The correct adenoviral recombinant was then cleaved with PacI and transfected into 293 cells to produce and purify viral particles.The virus was then transfected into hela cells,and the infection titer was monitored by green fluorescence protein（GFP）expression,IKK2dn gene was identified by RT-PCR.Results As confirmed by restriction digestion analysis and RT-PCR,an expectant fragment of 1060bp was observed in proper recombinants,it was proved that we successfully constructed the recombinant adenovirus vector encoding IKK2dn gene.The viral titer checked by GFP was about 2×1011pfu/ml.Conclusions The recombinant adenovirus vector encoding IKK2dn gene has been constructed successfully,which makes it easier to investigate the induction of immune tolerance by immature DC transfected by IKK2dn.