蜘蛛拖丝蛋白基因逆转录病毒载体构建与检测

Construction and detection of spider dragline silk protein gene retroviral vector

利用pIRES2-EGF和pMX载体以及前期获得的蜘蛛拖丝蛋白基因(2S)构建逆转录病毒载体pMX-2S-IRES-EGFP。采用磷酸钙法将pMX-2S-IRES-EGFP质粒转染到PlatE细胞并获得重组逆转录病毒。通过小鼠成纤维细胞系NIH3T3检测病毒侵染能力,并采用PCR法检测蜘蛛拖丝蛋白基因在NIH-3T3细胞的整合状况。结果显示,成功构建了逆转录病毒载体pMX-2S-IRES-EGFP;通过感染NIH-3T3细胞测定重组逆转录病毒滴度约为2×105 cfu/mL;PCR鉴定结果显示,目的基因2S-IRES-EGFP已整合入NIH-3T3细胞基因组中。

In order to establish a method of obtaining spider dragline silk protein by animal hair,pMX-2S-IRES-EGFP retroviral vector was constructed with pIRES2-EGFP vectors,pMX retroviral vector and spider dragline silk protein gene which was obtained previously.Recombinant retroviral particles were generated in platE cell by calcium phosphate transfection method.The infection ability of recombinant retroviral particles was detected by infecting into mouse fibroblasts NIH3T3,and spider dragline silk protein gene insertion into the genome could be detected by PCR in NIH-3T3 cells.The result indicated that pMX-2S-IRES-EGFP retroviral vector was constructed successfully and the viral titers were about 2×105cfu/mL through infecting into mouse fibroblasts NIH3T3.Moreover,2S-IRES-EGFP had inserted into the genome of NIH-3T3 cell.The research could be used in the study of producing transgenic spider dragline silk protein gene animals by retroviral transgenic method.