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

摘要:

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。

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.