禽白血病病毒K亚群HB2018003株感染性克隆构建

Construction of an infectious clone of the avian leukosis virus subgroup K HB2018003 strain

为了获得背景明确清晰的禽白血病病毒K亚群(ALV-K)毒株,对地方性ALV-K毒株HB2018003采用酶切和同源重组两种方法进行感染性克隆构建及病毒拯救。使用PCR方法获得目的基因,经酶切、连接和同源重组后成功构建重组质粒Topo-003-ABC和Topo-003-K12,测序结果表明无任何突变,进而将重组质粒转染进鸡胚成纤维细胞(DF-1)中,经群特异性抗原P27 ELISA、PCR和间接免疫荧光(IFA)鉴定,结果均表明成功拯救出病毒,将其命名为R-rHB2018003(同源重组法)和E-rHB2018003(酶切法)。本研究为深入探讨ALV-K的致病机制奠定了基础。

In order to obtain an avian leukosis virus subgroup K(ALV-K) with a clear background, in this study, infectious cloning and virus rescue were performed on endemic ALV-KHB2018003, which was constructed using two methods: digestion and homologous recombination. The target gene was obtained by PCR, and the recombinant plasmids Topo-003-ABC and Topo-003-K12 were successfully constructed after restriction enzyme digestion, ligation and homologous recombination. The sequencing results showed no mutations, and then the recombinant plasmid was transfected into the host cell DF-1, which was identified by the group-specific antigen P27 ELISA, PCR and indirect immunofluorescence(IFA);all of which indicated that the viruses named R-rHB2018003(homologous recombination) and E-rHB2018003(enzyme digestion) were successfully rescued. This study laid a foundation for further exploring the pathogenic mechanism of ALV-K.