联合应用泛素分子基因的汉滩病毒嵌合基因GnS0.7、GcS0.7重组腺病毒的构建及鉴定  

Construction and identification of recombinant adenoviruses containing fusion genes of ubiquitin and Hantaan virus Gn or Gc and S0.7

作　　者：张亮[1] 于澜[1] 叶伟[1] 李璞媛[1] 张芳琳[1] 程林峰[1] 

机构地区：[1]第四军医大学微生物学教研室,西安710032

出　　处：《传染病信息》2015年第2期83-87,共5页Infectious Disease Information

基　　金：国家自然科学基金面上项目(31070810);国家自然科学基金青年项目(81001344)

摘　　要：目的构建含有泛素(ubiquitin,Ub)基因的汉滩病毒嵌合基因Gn S0.7和Gc S0.7重组腺病毒。方法通过PCR扩增获得Ub基因,分别克隆入本室已构建的重组腺病毒转移载体p Shuttle-p CAG-Gn S0.7和p Shuttle-p CAG-Gc S0.7中,进一步通过双酶切将转移载体中的嵌合基因分别克隆入腺病毒载体,得到重组腺病毒载体r Ad-p CAG-Ub-Gn S0.7和r Adp CAG-Ub-Gc S0.7,使用PacⅠ线性化后转染HEK293细胞,包装后获得重组腺病毒,感染HEK293细胞后用免疫荧光法(indirect immunofluorescence assay,IFA)检测表达产物。结果限制性内切酶酶切鉴定、PCR扩增和测序结果显示,各重组腺病毒转移载体及重组腺病毒载体构建正确。IFA检测到各重组腺病毒中目的蛋白的表达。结论成功制备了含有Ub基因的汉滩病毒嵌合基因Gn S0.7和Gc S0.7重组腺病毒,为进一步研究联合Ub基因的汉滩病毒重组腺病毒的免疫学特性奠定了实验基础。Objective To construct and identify recombinant adenoviruses containing fusion genes of ubiquitin （Ub） and Hantaan virus Gn or Gc and S0.7. Methods The fragment of Ub was amplified by PCR and inserted into the pShuttle-pCAG-GnS0.7 and pShuttle-pCAG-GcS0.7 to create new transfer vectors pShuttle-pCAG-Ub-GnS0.7 and pShuttle-pCAG-Ub-GcS0.7, respectively. The pCAG-Ub-GnSO.7 and pCAG-Ub-GcS0.7 fragments were then cloned into Adeno-XTM Viral vector using PI-Sce I and I-Ceu I diges- tion. The recombinant adenovirus vectors, rAd-pCAG-Ub-GnS0.7 and rAd-pCAG-Ub-GcS0.7 linearized by Pac I were transfected into HEK 293 cells to package the recombinant adenoviruses. Finally, the expression of target proteins in HEK 293 cells were detected by indirect immunofluorescence assay （IFA） after being infected by recombinant adenoviruses. Results The recombinant transfer vec- tors and recombinant adenovirus vectors were confirmed by enzyme digestion, target gene PCR and sequencing. The expressions of tar- get proteins of recombinant adenoviruses were successfully detected by IFA. Conclusions The recombinant adenoviruses containing fusion genes of Ub, Hantaan virus Gn or Gc and S0.7 are successfully constructed, which can be used in further study on the immuno- logical characteristics of the Hantaan virus recombinant adenoviruses.

关 键 词：汉坦病毒 腺病毒科 泛素 

分 类 号：R373.32[医药卫生—病原生物学]