表达增强型绿色荧光蛋白的C型禽偏肺病毒反向遗传系统的构建及优化

doi:10.16431/j.cnki.1671-7236.2024.05.030

Abstract

Abstract: 【Objective】 Avian metapnemovirus (aMPV) is a member of Metapnemovirus genus in the family Paramyxoviridae.The swollen head syndrome and decreased egg production caused by aMPV had caused serious harm to the breeding industry.In order to effectively prevent aMPV infection and further study its pathogenesis,enhanced green fluorescent protein (EGFP) was inserted into aMPV subtype C (aMPV/C) to construct recombinant aMPV expressing EGFP.Meanwhile,this study used different vectors and promoters to construct aMPV minigenome to optimize the construction of aMPV reverse genetic system (RGS).【Method】 EGFP was inserted into the non-coding region between P and M proteins in pBluescript-aMPV complete RGS,and rescued to observe the expression of EGFP.The titer and genetic stability of the recombinant virus were also determined.In order to optimize the construction of RGS,pBluescript SK(＋) with T7 promoter and pcDNA3.1 with T7 and CMV promoters were used to construct aMPV minigenome.Firstly,the leader and trailer regions at both ends of the aMPV/C genome and the cDNA encoding EGFP were amplified by PCR and gelled for recovery.Secondly,each glue recovery fragment was seamlessly cloned and connected with the pre-designed homologous arm in the order of leader-EGFP-trailer,and sequenced for verification.Finally,the fully connected minigenome was reverse-inserted into the two vectors,and the aMPV/C minigenome (pBR-aMPV/C and pc-aMPV/C) were constructed.In the RGS rescue experiment,two aMPV/C minigenomes were co-transfected into BHK-21 cells with three plasmids expressing N,P and L proteins,and the expression of EGFP was detected by inverted fluorescence microscopy.【Result】 The rescued aMPV-EGFP recombinant virus was sequenced,and the results showed that EGFP had been successfully inserted into the aMPV genome and was stably expressed in the aMPV-EGFP recombinant virus within three generations.The virus titer of aMPV-EGFP recombinant virus reached 10^5.5 TCID₅₀/mL at 96 h after infection.The sequencing results of two recombinant plasmids,pBR-aMPV/C and pc-aMPV/C,also proved that minigenome containing EGFP had been constructed.The three recombinant plasmids were then co-transfected into the cells with the helper plasmids.After transfection for 24 h,the expression of green fluorescence was observed in the cells,which proved that the aMPV-EGFP recombinant virus and the two minigenomes,pBR-aMPV/C and pc-aMPV/C,successfully expressed EGFP.【Conclusion】 In this study,two aMPV minigenomes were successfully constructed and the aMPV-EGFP recombinant virus was rescued.The recombinant virus had good genetic stability.When CMV and T7 were used as promoters in aMPV/C RGS,the rescue efficiency was the same.Both pBluescript SK(＋) and pcDNA3.1 could be used for aMPV/C RGS construction,and the result of this study provided a variety of methods for aMPV/C RGS construction.

Key words: Avian metapneumonovirus subtype C; minigenome; reverse genetic system; CMV promoter; enhance green fluorescent protein; T7 promoter

CLC Number:

S852.65

GUO Yu, CHENG Jing, ZUO Yuzhu, FAN Jinghui, JIANG Haijun. Construction and Optimization of Reverse Genetic System of Avian Metapneumovirus Subtype C Expressing Enhanced Green Fluorescent Protein[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(5): 2091-2100.

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Construction and Optimization of Reverse Genetic System of Avian Metapneumovirus Subtype C Expressing Enhanced Green Fluorescent Protein

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