两株ALV-K重组病毒感染性克隆的构建及病毒拯救

doi:10.16431/j.cnki.1671-7236.2022.10.008

摘要/Abstract

摘要： 【目的】为探明引起禽白血病病毒K亚型(ALV-K)两不同毒株之间致瘤性差异的原因,进一步探索ALV-K的致病机理,实现两株ALV-K重组病毒感染性克隆的构建及病毒拯救。【方法】以前期构建的ALV-K毒株HB2018003和HB2015032的感染性克隆为模板,PCR扩增分别获取两毒株单独的5'-端长末端重复序列(5'-LTR)基因片段和剩余目的基因片段。切胶回收目的片段后将两毒株的5'-LTR互换,通过同源重组的方法获得连接产物。将连接产物转化大肠杆菌DH5α感受态细胞后涂布Amp抗性平板过夜培养,挑取PCR鉴定为阳性的菌落提取重组质粒TOPO-003-032和TOPO-032-003并送检测序。将PCR和测序鉴定正确的重组质粒转染至易感细胞DF-1中盲传3代,使用群特异性抗原P27 ELISA、多重 PCR和间接免疫荧光等方法对拯救的病毒进行检测。【结果】测序和重组感染性克隆PCR鉴定的结果表明成功构建了5'-LTR互换的重组质粒,多重PCR结果显示在目的片段大小处出现ALV-K预期条带,未出现ALV-A和ALV-J条带,间接免疫荧光试验结果显示接种拯救病毒的DF-1细胞出现明显的亮绿荧光,阴性对照则没有出现荧光。综合以上结果,说明两株重组病毒拯救成功,并将其分别命名为rHB2022050(TOPO-032-003)和rHB2022051(TOPO-003-032)。【结论】本研究采用同源重组的方法构建了5'-LTR互换的感染性克隆TOPO-003-032和TOPO-032-003,并成功拯救出病毒rHB2022050(TOPO-032-003)和rHB2022051(TOPO-003-032)。

关键词: 禽白血病病毒K亚型(ALV-K); 重组病毒; 感染性克隆; 病毒拯救

Abstract: 【Objective】 The aim of this study was in order to find out the cause of the tumorigenicity difference between two different strains of Avian leukemia virus subgroup K(ALV-K),further consider the pathogenic mechanism of ALV-K,and realize the construction of infectious clones and virus rescue of two recombinant viruses of ALV-K.【Method】 The infectious clones of the previously constructed ALV-K HB2018003 and HB2015032 strains were used as templates,and the separate 5'-long terminal repeat(5'-LTR) gene fragments and the remaining target gene fragments of the two strains were obtained by PCR amplification.After the target fragment was recovered by cutting glue,the 5'-LTR of the two strains were exchanged,and the connecting product was obtained by homologous recombination.After transformed into E.coli DH5α competent cells,the ligation products were coated with Amp resistant plates for overnight culture.The positive colonies identified by PCR were selected to extract recombinant plasmids TOPO-003-032 and TOPO-032-003 and sequenced.The recombinant plasmids identified by PCR and sequencing were transfected into susceptible cells DF-1 for 3 generations blindly,and the rescued virus was detected by group-specific antigen P27 ELISA,multiplex PCR and indirect immunofluorescence.【Result】 The results of sequencing identification and recombinant infectious clone PCR identification showed that the 5'-LTR exchanged recombinant plasmid was successfully constructed.Multi PCR results showed that the expected band ALV-K appeared at the size of the target fragment,and ALV-A and ALV-J bands did not appear.The results of indirect immunofluorescence test showed that DF-1 cells inoculated with rescue virus showed obvious bright green fluorescence,while the negative control did not show fluorescence.Above all,the two recombinant viruses were successfully rescued and named rHB2022050(TOPO-032-003) and rHB2022051(TOPO-003-032),respectively.【Conclusion】 In this study,the 5'-LTR exchanged infectious clones TOPO-003-032 and TOPO-032-003 were constructed by homologous recombination,and the viruses rHB2022050(TOPO-032-003)and rHB2022051(TOPO-003-032) were successfully rescued.

Key words: Avian leukemia virus subtype K (ALV-K); recombination virus; infectious clone; virus rescue

中图分类号:

S855.3

王淼, 陈雪阳, 王兴明, 梁雄燕, 杨玉莹. 两株ALV-K重组病毒感染性克隆的构建及病毒拯救[J]. 中国畜牧兽医, 2022, 49(10): 3764-3770.

WANG Miao, CHEN Xueyang, WANG Xingming, LIANG Xiongyan, YANG Yuying. Construction of Infectious Clones of Two ALV-K Recombinant Viruses and Virus Rescue[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 3764-3770.

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