Construction and identification of recombinant adenovirus vector pAd-TSHR289/6xHis with overexpressed TSHR A subunit
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Construction and identification of recombinant adenovirus vector pAd-TSHR289/6xHis with overexpressed TSHR A subunit
Journal of Chongqing Medical UniversityVol. 38, Issue 4, Pages: 524-528(2014)
作者机构:
1. 中国中医科学院广安门医院糖尿病研究室,北京,100053
2. 赛业广州生物科技有限公司销售部,广州,510663
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Published:2014,
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Zhu Xiaoyun, Liu Lijuan, Yang Dongjia, et al. Construction and identification of recombinant adenovirus vector pAd-TSHR289/6xHis with overexpressed TSHR A subunit. [J]. Journal of Chongqing Medical University 38(4):524-528(2014)
DOI:
Zhu Xiaoyun, Liu Lijuan, Yang Dongjia, et al. Construction and identification of recombinant adenovirus vector pAd-TSHR289/6xHis with overexpressed TSHR A subunit. [J]. Journal of Chongqing Medical University 38(4):524-528(2014)DOI:
Construction and identification of recombinant adenovirus vector pAd-TSHR289/6xHis with overexpressed TSHR A subunit
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.
关键词
Graves病重组腺病毒促甲状腺激素受体载体构建动物模型
Keywords
Grave’s diseaserecombinant adenovirusthyroid-stumulating hormone receptorvector constructionanimal model