重组丙型肝炎病毒腺病毒载体疫苗rAd/E2的构建及鉴定

目的构建含丙型肝炎病毒(Hepatitis C virus,HCV)E2基因的复制缺陷型重组腺病毒载体疫苗,并进行鉴定。方法以含HCV(1a亚型)E2基因的质粒pEGFP-HCV/E1E2为模板,PCR扩增E2基因,扩增产物与pGEM-T载体连接,测序正确后双酶切亚克隆至穿梭质粒pAdTrack-CMV上,构建重组穿梭质粒pAdTrack-CMV/E2,Pme I酶切线性化后,与骨架质粒pAdEasy-1在BJ5183细菌内同源重组,获得重组腺病毒质粒pAd/E2,PacⅠ酶切线性化后,脂质体LipofectamineTM 2000介导转染HEK293细胞进行包装,获得重组腺病毒rAd/E2,在HEK293细胞内扩增,利用报告基因GFP的表达监测病毒包装及感染效率,PCR、RT-PCR和Western blot对重组腺病毒进行鉴定,并测定各代病毒的滴度。结果测序结果证明HCV E2基因序列正确;重组腺病毒质粒pAd/E2经酶切证明重组成功;PCR、RT-PCR及Western blot结果均证实重组腺病毒rAd/E2构建正确;第4代重组腺病毒的滴度为7.5×108 pfu/ml。结论已成功构建了重组HCV腺病毒rAd/E2,为丙型肝炎疫苗的研制提供了新的途径。

Construction and Identification of Recombinant Adenovirus with E2 Gene of Hepatitis C Virus

Objective To construct and identify a replication-defective recombinant adenovirus with E2 gene of hepatitis C virus(HCV).Methods E2 gene was amplified by PCR using plasmid pEGFP-HCV / E1E2 containing E2 gene of HCV subtype 1a as a template and inserted into vector pGEM-T.The constructed recombinant plasmid pGEM-T / E2 was identified by sequencing then subcloned to shuttle plasmid pADTrack-CMV.The constructed recombinant shuttle plasmid pADTrack-CMV / E2 was linearilized with PmeⅠ,then subjected to homologous recombination with skeleton plasmid pAdEasy-1 in BJ5183 cells.The obtained recombinant adenovirus plasmid pAd / E2 was linearilized with Pac Ⅰ and transfected to HEK293 cells with LipofectamineTM2000 for packaging.The obtained recombinant adenovirus rAd / E2 was proliferated in HEK293 cells,then monitored for packaging and infection efficacies by expression of report gene GFP,identified by PCR,RT-PCR and Western blot,and determined for titers of various passages.Results Sequencing result proved correct sequence of HCV E2 gene.Restriction analysis proved that recombinant adenovirus plasmid pAd / E2 was constructed successfully.PCR,RT-PCR and Western blot proved that rAd / E2 was constructed correctly.The titer of rAd / E2 of passage 4 was 7.5 × 108 pfu / ml.Conclusion The rAd / E2 was constructed successfully,which provided a novel route for preparation of HCV vaccine.