摘要
为研究猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)nsp1、nsp5和N蛋白的免疫原性,试验以复制缺陷型人腺病毒5型(AdMax系统)为载体,利用PEDV-GDgh毒株(登录号为MG983755),通过PCR方法扩增出GDgh毒株nsp1、nsp5、N基因,并将其连接到腺病毒穿梭载体上,构建重组穿梭质粒。将3个重组穿梭质粒和腺病毒骨架载体共转染至HEK-293A细胞中获得第1代重组腺病毒,分别命名为rAd-nsp1、rAd-nsp5、rAd-N。用第1代3株重组腺病毒接种HEK-293A细胞,连续传代,通过RT-PCR和Western-blot方法鉴定第3代重组腺病毒,并采用Reed-Muench法计算病毒半数组织培养感染剂量(TCID_(50))。以3株重组腺病毒的第3代病毒液免疫小鼠,收集血清并检测特异性IgG抗体水平。结果表明:试验成功构建出带有nsp1、nsp5、N基因的重组穿梭质粒;分别用重组腺病毒rAd-nsp1、rAd-nsp5、rAd-N感染正常HEK-293A细胞,可产生典型的细胞病变并观察到荧光信号;目的基因正常转录后得到的目的蛋白可正确表达;3株重组腺病毒rAd-nsp1、rAd-nsp5、rAd-N的TCID_(50)依次为1×10^(-8.35)/mL、1×10^(-8.50)/mL、1×10^(-7.66)/mL;免疫小鼠后均可产生针对目的蛋白的特异性抗体。说明试验成功构建的3株正常表达目的蛋白的重组腺病毒均具有免疫原性。
In order to study the immunogenicity of nsp1,nsp5 and N proteins in Porcine epidemic diarrhea virus(PEDV),in this experiment,the replication-deficient human adenovirus type 5(AdMax system) was used as the vector,and the nsp1,nsp5 and N genes of the GDgh strain were amplified by PCR using the PEDV-GDgh strain(accession number MG983755),and the recombinant shuttle plasmids were constructed.Three recombinant shuttle plasmids and adenovirus backbone plasmids were co-transfected into HEK-293A cells to obtain the first-generation recombinant adenovirus,which was named rAd-nsp1,rAd-nsp5 and rAd-N,respectively.The first generation of three recombinant adenoviruses was inoculated with HEK-293A cells,and the third generation of recombinant adenovirus was identified by RT-PCR and Western-blot,and the infection dose of half of the virus in tissue culture(TCID_(50)) was calculated by Reed-Muench method.Mice were immunized with third-generation cultures of 3 strains of recombinant adenovirus;serum was collected and specific IgG antibody levels were measured.The results showed that the recombinant shuttle plasmids with nsp1,nsp5 and N genes were successfully constructed,and normal HEK-293A cells were infected with recombinant adenovirus rAd-nsp1,rAd-nsp5 and rAd-N,respectively,which produced typical cytopathy and observed fluorescence signal.The proteins could be correctly expressed after normal transcription of the target genes;the TCID_(50) of the three recombinant adenoviruses rAd-nsp1、rAd-nsp5、rAd-N was 1×10~(-8.35)/mL,1×10~(-8.50)/mL and 1×10~(-7.66)/mL,respectively.After immunizing mice,specific antibodies against the target proteins could be produced.The results indicated that the three strains of recombinant adenovirus with normal expression of the target proteins were all immunogenic.
作者
杨霞
王爽云
于林洋
徐舸
郑继豪
王志朋
张歆明
刘燕玲
张乐宜
徐铮
宋长绪
YANG Xia;WANG Shuangyun;YU Linyang;XU Ge;ZHENG Jihao;WANG Zhipeng;ZHANG Xinming;LIU Yanling;ZHANG Leyi;XU Zheng;SONG Changxu(College of Animal Science,South China Agricultural University,Guangzhou 510642,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2024年第3期73-78,共6页
Heilongjiang Animal Science And veterinary Medicine
基金
云浮市科技计划项目“猪流行性腹泻免疫防控关键技术研究与应用”(2022020202)。
作者简介
杨霞(1996-),女,硕士研究生,研究方向为动物健康养殖与安全生产,yx15638139126@163.com;通信作者:宋长绪(1965-),男,研究员,博士,研究方向为动物免疫与生物安全,cxsong2004@163.com.
