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

摘要/Abstract

摘要： 【目的】禽偏肺病毒(Avian metapneumovrus,aMPV)是副黏病毒科偏肺病毒属成员,其所造成的鸡肿头症和产蛋率下降对养殖业造成严重危害。为更好地预防aMPV并研究其发病机制,本研究将增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)插入C型aMPV(aMPV/C)基因组中,构建表达EGFP的重组aMPV,并使用不同的载体及启动子构建aMPV迷你基因组,来优化aMPV反向遗传操作系统(reverse genetic system,RGS)的构建。【方法】将EGFP插入pBluescript-aMPV RGS的P和M蛋白之间非编码区并进行拯救,观察绿色荧光蛋白的表达情况,同时测定重组病毒滴度及遗传稳定性。为优化RGS的构建,采用含有T7启动子的pBluescript SK(＋)和含有T7、CMV启动子的pcDNA3.1两种载体构建aMPV迷你基因组。首先将aMPV/C基因组两端的先导区(leader)和尾随区(trailer)与EGFP的cDNA进行PCR扩增,并切胶回收;其次将各胶回收片段按照leader-EGFP-trailer的顺序进行无缝克隆连接,并测序验证;最后将连接完整的迷你基因组反向插入两个载体中,构建aMPV/C的迷你基因组(pBR-aMPV/C和pc-aMPV/C)。在拯救迷你基因组的试验中,将两个aMPV/C迷你基因组与3个表达N、P和L蛋白的质粒共转染到BHK-21细胞中,观察绿色荧光蛋白的表达情况。【结果】拯救的aMPV-EGFP重组病毒测序结果显示,EGFP已成功插入aMPV基因组中,并在前3代aMPV-EGFP重组病毒中稳定表达。aMPV-EGFP重组病毒滴度在感染96 h后达到10^5.5 TCID₅₀/mL。而pBR-aMPV/C和pc-aMPV/C两个重组质粒的测序结果也证明了含有EGFP的aMPV迷你基因组构建完成。将3个重组质粒分别与辅助质粒共转染至BHK-21细胞中,转染24 h后观察到细胞中绿色荧光蛋白表达,证明aMPV-EGFP重组病毒及pBR-aMPV/C和pc-aMPV/C两个aMPV 迷你基因均都成功表达了EGFP。【结论】本研究成功构建了两个aMPV迷你基因组并拯救了aMPV-EGFP重组病毒,重组病毒具有良好的遗传稳定性。而CMV和T7作为aMPV/C RGS中的启动子时,二者的拯救效率相同。pBluescript SK(＋)和pcDNA3.1均可用于aMPV/C RGS的构建,本研究为aMPV/C RGS的构建提供了多种方法。

关键词: C型禽偏肺病毒; 迷你基因组; 反向遗传系统; CMV启动子; 增强绿色荧光蛋白; T7启动子

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

中图分类号:

S852.65

郭禹, 程晶, 左玉柱, 范京惠, 姜海军. 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[J]. 中国畜牧兽医, 2024, 51(5): 2091-2100.

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 & Veterinary Medicine, 2024, 51(5): 2091-2100.

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