腺病毒介导的hHCN4表达载体的构建及其转染人胚肾细胞

Construction of adenovirus vector hHCN4 and transfecting it into human embryonic kidney cell

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 recombinant adenovirus vector pAV-hHCN4-IRES/eGFP expressing human hyperpolarization activated cyclic nueleotide gated cation channel 4 （hHCN4） and carry on the identification and package. Methods Gateway technology was used to build pAV- hHCN4-IRES/eGFP, which was followed by enzyme cut identification and sequence analysis of positive clones. The plasmids were transfected into HEK293 cells and virus titer was determined. Results The sequences were in accordance with the coding sequences of hHCN4 in Genbank. The expression of green fluorescent protein was seen in the transfected HEK293 cells. Virus titer was 1.26 × 108 pfu/ml. Conclmion Recombinant adenovirus vector pAV-hHCN4-IRES/eGFP has been successfully constructed.