Construction and identification of recombinant adenovirus vector pAd-TSHR289/6xHis with overexpressed TSHR A subunit

Abstract:

Objective：To construct recombinant adenovirus vector，which takes thyroid-stumulating hormone receptor（TSHR） A sub－unit（TSHR289） as immunogen，to construct Grave’s animal model and to explore traditional Chinese medicine prescription for the prevention and treatment of Grave’s disease. Methods：TSHR289 gene was amplified from the plasmid of pSV2neoECE-TSHR289-6H-dhfr to attB1-Kozak-TSHR289/6xHis/IRES/eGFP-attB2 vector with overlapping PCR. PCR products purified by agarose gel electrophoresis were collected and adenovirus vector pAV.EX1d-TSHR289/6xHis/IRES/eGFP（pAd-TSHR289/6xHis） was recombined with GatewayR technology. Then recombined adenovirus vector was translated into Escherichia coli Stbl3；positive clones were screened out by PCR before sequencing. pAd-TSHR289/6xHis was transferred into HEK293A cells to produce recombinant adenovirus；aden－ovirus was purified by CsCl gradient centrifugation and titer of the virus was measured by TCID50. Then mRNA expression of of TSHR289 in HEK293 was measured by RT-PCR. Grave’s model was created by immunizing BALB/c mice with Ad-TSHR289/6xHis. Results：Construction was proved correct by screening positive bacterial. Enhanced green fluorescence protein and cytopathic effect of HEK293A cells was observed by microscope after infection. RT-PCR demonstrated that mRNA expression of TSHR289 in recombinant adenovirus transferred cell were 285 times higher compared with those of untransfected cell. The titer of the virus was 3.16×1010 PFU/ml and the positive rate of T4 and TRAb was 7/10 on the 10th week after BALB/c mice being immunized. Conclusions：Recom－binant adenovirus vector of overexpressed human TSHR289 can be successfully constructed. The animal’s experiments prove that Ad-TSHR289/6xHis can induce autoimmunity and produce TRAb，which provide the prerequisite for the experiment in vivo of treating Grave’s disease.