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

Abstract

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

CLC Number:

S855.3

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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Construction of Infectious Clones of Two ALV-K Recombinant Viruses and Virus Rescue

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