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

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

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,Gateway R技术构建重组腺病毒载体(pAV.EX1d-TSHR289/6xHis/IRES/eGFP,pAdTSHR289/6xHis),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 subunit（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-KozakSHR289/6xHis/IRES/eGFP-attB2 vector with overlapping PCR. PCR products purified by agarose gel electrophoresis were collected and adenovirus vector pAV.EXld-TSHR289/6xHis/IRES/eGFP（pAd-TSHR289/6xHis） was recombined with Gateway technology. Then recombined adenovirus vector was translated into Escherichia coli Stb13;positive clones were screened out by PCR before sequencing, pAd-TSHR289/6xHis was transferred into HEK295A cells to produce recombinant adenovirus;adenovirus was purified by CsC1 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 eytopathic effect of HEK293A cells was observed by microscope after infection. RT-PCR demonstrated that mRNA expression of TSHR289 in recombi- nant adenovirus transferred cell were 285 times higher compared with those of untransfected cell. The titer of the virus was 3.16 × 10^10 PFU/ml and the positive rate of T4 and TRAb was 7/10 on the lOth week after BALB/c mice being immunized. Conclusions：Recombinant adenovirus vector of overexpressed human TSHR289 can be successfully constructed. The animal＇ s experiments prove that TRAb, which provide the prerequisite for the experiment in vivo of treating Grave＇ s disease.