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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
纸质出版日期:2014,
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朱晓云, 刘丽娟, 杨东甲, 等. 过表达人TSHR-A亚单位的重组腺病毒Ad-TSHR289/6xHis载体构建与鉴定[J]. 重庆医科大学学报, 2014,38(4):524-528.
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, 2014,38(4):524-528.
朱晓云, 刘丽娟, 杨东甲, 等. 过表达人TSHR-A亚单位的重组腺病毒Ad-TSHR289/6xHis载体构建与鉴定[J]. 重庆医科大学学报, 2014,38(4):524-528. 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, 2014,38(4):524-528. DOI:
目的:构建以促甲状腺激素受体(thyroid stimulating hormone receptor,TSHR)A亚单位(TSHR289)基因片段为免疫原的重组腺病毒载体,以期建立Graves病(Graves’s disease,GD)动物模型,为探讨中药复方对GD的防治作用提供条件。方法:利用重叠PCR技术从 pSV2neoECE-TSHR289-6H-dhfr 质粒中将目的基因TSHR289扩增至载体attB1-Kozak-TSHR289/6xHis/IRES/eGFP-attB2,琼脂糖凝胶电泳回收,GatewayR技术构建重组腺病毒载体(pAV.EX1d-TSHR289/6xHis/IRES/eGFP,pAd-TSHR289/6xHis),转化到大肠杆菌Stbl3中,菌落PCR筛选阳性克隆、测序鉴定。将pAd-TSHR289/6xHis转染HEK293A细胞包装腺病毒,CsCl梯度离心法纯化腺病毒,TCID50法检测病毒滴度。RT-PCR法检测HEK293A中TSHR289的mRNA表达。应用该腺病毒免疫BALB/c小鼠制备GD模型。结果:菌落 PCR 筛选阳性克隆,测序证明构建正确;细胞感染腺病毒后出现明显细胞病变及增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP)表达;RT-PCR 检测证实重组腺病毒转染的细胞中TSHR289的mRNA表达量是未转染的285倍;实验测得病毒滴度为3.16×1010 PFU/ml;BALB/c小鼠免疫第10周T4、TSHR抗体(TRAb)阳性率7/10。结论:成功构建了过表达人TSHR289基因的重组腺病毒载体,动物实验证明该腺病毒诱发自身免疫,产生TRAb。为下一步体内药物治疗GD试验提供了条件。
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.
Graves病重组腺病毒促甲状腺激素受体载体构建动物模型
Grave’s diseaserecombinant adenovirusthyroid-stumulating hormone receptorvector constructionanimal model
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