抗TNFα单链抗体复制缺陷型腺病毒载体的构建及表达

Construction and identification of recombinant replicationdefective adenovirus vector Αd/CMV/V5-DEST-TNFα-scFv

目的:构建pAd/CMV/V5-DEST-TNFα-scFv重组腺病毒载体,通过病毒包装、纯化及滴定,获得高纯度高感染性的病毒液,并对TNFα-scFv进行表达、鉴定.方法:以携带TNFα-scFv基因的载体PUC57-Amp为模板,扩增TNFα-scFv目的基因,构建穿梭质粒pDONR221-TNFα-scFv,测序证实质粒含有目的基因.与骨架质粒pAd-CMV-V5-DEST腺病毒骨架载体进行同源重组,形成表达克隆pAd/CMV/V5-DEST-TNFα-scFv.表达克隆转染293细胞,包装重组腺病毒,并鉴定和病毒滴定.结果:TNFα-scFv基因成功克隆到腺病毒载体中,转染293细胞后成功包装出重组腺病毒,病毒滴度为2.5×1011TCID50/mL,Western blot检测到TNFα-scFv基因在293细胞中高水平表达.结论:成功构建了带有TNFα-scFv基因的重组腺病毒载体,为进一步研究提供实验基础.

AIM：To construct a recombinant replicationdefective adenovirus vector carrying TNFα-scFv and to obtain high-purity virus solution by viral packaging,purification and titration.METHODS：The TNFα-scFv gene was amplified from the PUC57-Αmp vector and cloned into the shuttle plasmid pDONR221.The resulting pDONR221-TNFα-scFv was identified by DNA sequencing and then co-transfected into bacteria carrying the adenoviral backbone plasmid pΑd-CMV-V5-DEST to generate an adenoviral plasmid carrying TNFα-scFv（pΑd/CMV/V5-DEST-TNFα-ScFv） by homologous recombination in bacteria.After the pΑd/CMV/V5-DESTTNFα-ScFv vector was transfected into 293 cells,the transfected 293 cells were infected with adenoviruses.The expression of TNFα-ScFv was detected by cytopathic effect and Western blot.RESULTS：PCR amplification,restriction analysis and DNA sequencing verified that both the recombinant shuttle plasmid pDONR221-TNFα-scFv and the recombinant adenovirus vector pΑd/CMV/V5-DEST-TNFα-scFv were correctly constructed.After amplification and purification,the titer of recombinant adenovirus was 2.5 ×10 11 TCID 50/mL after proliferation in 293 cells.Western blot analysis demonstrated that TNFα scFv was expressed efficiently in 293 cells after infection.CONCLUSION：The recombinant adenovirus vector Αd/CMV/V5-DEST-TNFα-scFv has been successfully constructed,which lays a foundation for further study of gene function and therapy.