联合应用抗原递呈分子HSP70C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

目的构建含有抗原加工递呈分子基因HSP70C的HTNV嵌合基因GnS0.7、GcS0.7重组腺病毒。方法通过PCR扩增获得HSP70C基因,分别克隆入本室已构建的重组腺病毒转移载体pShuttle-pCAG-GnS0.7及pShuttle-pCAGGcS0.7中,进而构建新的重组腺病毒转移载体pShuttle-pCAG-GnS0.7-HSP70C及pShuttle-pCAG-GcS0.7-HSP70C。进一步通过双酶切将转移载体中的目的嵌合基因再分别克隆入腺病毒载体,得到重组腺病毒载体rAd-pCAG-GnS0.7-HSP70C、rAd-pCAG-GcS0.7-HSP70C,使用Pac I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检测表达产物。结果经过酶切、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。免疫荧光实验检测到各重组腺病毒中目的蛋白的表达。结论成功制备了含抗原递呈分子HSP70C基因的汉滩病毒嵌合基因GnS0.7、GcS0.7重组腺病毒,为进一步研究构建的汉滩病毒重组腺病毒的免疫学特性奠定了实验基础。

Construction and identification of recombinant adenoviruses containing fusion genes of HSP70C and hantaan virus Gn or Gc and S0.7

Objective Construction and identification of recombinant adenoviruses containing fusion genes of HSP70 C and hantaan virus Gn or Gc and S0.7. Methods The fragment of HSP70 C was amplified by PCR and inserted into the pShuttlepCAG-GnS0.7 and pShuttle-pCAG-GcS0.7 to create new transfer vectors pShuttle-pCAG-GnS0.7-HSP70 C and pShuttlepCAG-GcS0.7-HSP70 C. The GnS0.7-HSP70 C and GcS0.7-HSP70 C fragments were then cloned into Adeno-XTM Viral vector using PI-Sce I and I-Ceu I digestion. The recombinant adenovirus vectors linearized by Pac I were transfected into HEK 293 cells to package the recombinant adenoviruses. The expression of target proteins in HEK 293 cells were detected by IFA after infected by recombinant adenoviruses. Results The sequence of recombinant transfer vectors and recombinant adenovirus vectors were confirmed by enzyme digestion, target gene PCR and sequencing. The expressions of target proteins from recombinant adenoviruses were successfully detected by IFA. Conclusion The recombinant adenoviruses containing fusion genes of HSP70 C, Hantaan Virus Gn or Gc and S0.7 were successfully constructed which could be used in further study on the immunological characteristics of the Hantaan virus recombinant adenovirus.