髓细胞瘤型J亚群禽白血病病毒HB2015029株感染性克隆的构建与病毒拯救

Construction of an infectious clone from the ALV-J strain HB2015029 and rescue of virus

为了拯救具有感染性的髓细胞瘤型J亚群禽白血病病毒(avian leukosis virus,ALV)HB2015029株,本研究以含有ALV-J HB2015029株基因组片段的质粒为模板,采用高保真PCR,扩增出3段重叠并含有合适酶切位点病毒基因片段。将获得的基因组片段分别克隆至pTopo-Blunt Simple载体,经过酶切、连接成功构建了含有病毒全基因组的质粒TOPO-029-ABC。将TOPO-029-ABC质粒转染进DF-1细胞进行病毒拯救,结果表明,转染72 h后的细胞上清禽白血病病毒P27抗原呈阳性;将阳性细胞上清,接种新的DF-1细胞培养3 d,P27抗原ELISA检测、特异性PCR和间接免疫荧光试验均呈阳性,表明成功构建了ALV-J HB2015029感染性克隆,并拯救具有感染性的髓细胞瘤型ALV-J,命名为rHB2015029。

In order to rescue the infectious myeloma type ALV-J HB2015029 strain, this study used the plasmid containing the genome fragment of the ALV-J HB2015029 strain as a template, and high fidelity PCR was used to amplify three overlapping viral gene segments containing appropriate enzyme cutting sites. The obtained genome fragments were cloned into the pTopo-blunt Simple vector, and the plasmid TOPO-029-ABC containing the full genome of the virus was successfully constructed by enzyme digestion and ligation. The TOPO-029-ABC plasmid was transfected into DF-1 cells for virus rescue. The results showed that the supernatant of the cells was positive for avian leukosis P27 antigen at 72 h after transfection. The positive cells were super-purified and inoculated with the new DF-1 cells for three days. The results of the ELISA test, specific PCR and indirect immunofluorescence test of theavian leukosis P27 antigen were all positive, indicating that the infectious clone of ALV-J HB2015029 was successfully constructed and the infectious myeloma type ALV-J was rescued, which was named rHB2015029.