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目的构建人超极化激活环核甘酸门控通道基因4(hHCN4)的重组腺病毒载体pAV-hHCN4-IRES/eGFP,并对其进行鉴定、包装。方法利用Gateway技术构建pAV-hHCN4-IRES/eGFP,酶切鉴定并进行阳性克隆测序。将构建好的质粒转染人胚肾细胞HEK293,检测病毒液滴度。结果测序符合Genbank中的hHCN4的编码序列。转染HEK293细胞见绿色荧光蛋白表达,病毒滴度为1.26×108 pfu/ml。结论成功构建了重组腺病毒载体pAV-hHCN4-IRES/eGFP。
Objective To construct a recombinant adenovirus vector pAV-hHCN4-IRES / eGFP which is highly homologous to human ghrelin 4 (hHCN4), and to identify and package it. Methods Gateway technology was used to construct pAV-hHCN4-IRES / eGFP, identified by restriction enzyme digestion and sequenced. The constructed plasmid was transfected into human embryonic kidney cells HEK293 to detect the virus titer. Results Sequencing was consistent with the coding sequence of hHCN4 in Genbank. Transfection of HEK293 cells revealed green fluorescent protein expression with a virus titer of 1.26 × 108 pfu / ml. Conclusion The recombinant adenovirus vector pAV-hHCN4-IRES / eGFP was successfully constructed.