人TSHR A亚单位重组腺相关病毒2/9载体的构建及鉴定

Construction and identification of an adeno-associated viral vector serotype 2/9 containing human thyrotropin receptor A subunit

目的构建人促甲状腺素受体(TSHR)A亚单位重组腺相关病毒2/9杂合载体(rAAV2-9-hTSHR289-IRES-ZsGreen),为研究格雷夫斯病建立理想动物模型和探索基因预防提供更佳手段。方法以 Eco RⅠ和 Bam HⅠ将腺相关病毒骨架质粒 pAAV-IRES-ZsGreen和含hTSHR289的pDC315质粒双酶切,回收酶切病毒骨架质粒及目的片段进行连接,产物转化JM109感受态细菌,获取重组腺相关病毒骨架质粒pAAV-hTSHR289-IRES-ZsGreen。经酶切电泳、测序鉴定后,将pAAV-hTSHR289-IRES-ZsGreen、辅助质粒pHelper、包装质粒pAAV-2/9(AAV2-Rep,AAV9-Cap)采用磷酸钙法转染293AAV细胞系,获取大量rAAV2/9-hTSHR289-IRES-ZsGreen,经纯化后荧光显微镜下观察其包装效率,rQ-PCR法测定病毒滴度。将rAAV2/9-hTSHR289-IRES-ZsGreen感染HEK293细胞,ELISA法检测不同时间点hTSHR289蛋白表达水平。结果重组腺相关病毒骨架质粒pAAV-hTSHR289-IRES-ZsGreen经双酶切及测序鉴定,质粒构建正确;荧光显微镜下观察显示转染293AAV细胞72 h,病毒包装效率达92%~94%,rQ-PCR法测定的rAAV2/9-hTSHR289-IRES-ZsGreen病毒滴度为1×10^13 vg/mL;ELISA法显示rAAV2/9介导的 hTSHR289 蛋白表达96 h明显高于72 h及48 h。结论成功构建rAAV2/9-hTSHR289-IRES-ZsGreen重组腺相关病毒载体并能有效转染和表达。

Objective To construct an adeno-associated viral vector serotype2/9 containing human thyrotropin receptor (rAAV2-9-hTSHR289-IRES-ZsGreen) so as to provide a better means for establishing an ideal animal model and the gene prevention against Graves disease. Methods AAV skeleton plasmid pAAV-IRES-ZsGreen and PDC315 plasmid containing hTSHR289 were digested by Eco RⅠ+ Bam HⅠ. The digestion products were connected. And pAAV-hTSHR289-IRES-ZsGreen vector was generated after transformation of JM109 by ligation products. The recombinant plasmid was identified by gel electrophoresis and sequencing. Three plasmids (pAAV-hTSHR289-IRES-ZsGreen,pHelper,and pAAV-2/9) were transfected into 293AAV cell line by calcium phosphate method and a large amount of rAAV2/9-hTSHR289-IRES-ZsGreen was obtained. After purification,the packaging efficiency of recombinant adeno-associated virus was observed by using fluorescence microscope,and its titer was determined by rQ-PCR. HEK293 cells were transfected with rAAV2/9-hTSHR289-IRES-ZsGreen;then the expression level of hTSHR289 was analyzed by ELISA at different time points. Results The success of hTSHR289 gene inserted into the AAV skeleton vector was confirmed by double digestion and sequencing. After 72 h of transfection of 293AAV cells,the packaging efficiency of the virus was 92%-94% under fluorescence microscope.The titer of the recombinant adeno-associated virus was 1×10^13 vg/mL. ELISA assay showed that the expression level of hTSHR289 protein mediated by rAAV2/9 was significantly higher at 96 h than that at 72 h and 48 h. Conclusion The rAAV2/9-hTSHR289-IRES-ZsGreen was successfully constructed. Furthermore,it could be transfected and expressed in cells effectively.