表达鸡传染性喉气管炎病毒gD蛋白的重组嵌合型新城疫病毒La Sota株的构建  

Construction of recombinant chimeric Newcastle disease virus La Sota strain expressing gD protein of infectious laryngotracheitis virus

作　　者：田静格 徐鸣荷 王向东 张意航 李岩[1] 刘俊杰 李星雨 韩城昊 张伯顺 卜德新 于春梅 丛雁方 杨盼盼 乔麒龙 王增 李建丽[1] 李永涛[1] 王白玉 赵军[1] TIAN Jing-ge;XU Ming-he;WANG Xiang-dong;ZHANG Yi-hang;LI Yan;LIU Jun-jie;LI Xing-yu;HAN Cheng-hao;ZHANG Bo-shun;BU De-xin;YU Chun-mei;CONG Yan-fang;YANG Pan-pan;QIAO Qi-long;WANG Zeng;LI Jian-li;LI Yong-tao;WANG Bai-yu;ZHAO Jun(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;Qingdao Vland Biotech INC,Qingdao 266000,China)

机构地区：[1]河南农业大学动物医学院,河南郑州450046 [2]青岛蔚蓝生物股份有限公司,山东青岛266000

出　　处：《中国预防兽医学报》2024年第7期659-665,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金面上项目(32372997)。

摘　　要：为构建表达鸡传染性喉气管炎病毒(ILTV)糖蛋白D(gD)的重组新城疫病毒(NDV)La Sota株,本研究将ILTV强毒株gD基因胞外域与NDV F基因信号肽、跨膜域和胞质尾区融合,并插入到含有NDV基因VII型F和HN基因的嵌合型La Sota株感染性cDNA克隆pLa Sota-VIIF/HN的P和M基因之间,将获得的重组感染性克隆pLa Sota-VIIF/HN-gD与辅助质粒pCIneo-NP-P-L共转染BHK-21细胞,拯救出表达gD蛋白的重组嵌合型NDV La Sota株rLaSota-VIIF/HN-gD,并采用血凝试验鉴定正确后,将重组病毒在9日龄SPF鸡胚中连续传至10代,提取10代重组病毒基因组RNA,反转录成c DNA作为模板,以ILTV gD基因插入位点两侧的鉴定引物进行PCR鉴定;采用第10代重组病毒感染BHK-21细胞,以ILTV gD蛋白多克隆抗体作为一抗进行间接免疫荧光试验(IFA);分别对鸡红细胞吸附的重组病毒感染的鸡胚尿囊液上清和从红细胞上解离下来的纯化的重组NDV病毒粒子经western blot鉴定,并对重组病毒对鸡胚的致病性和在鸡胚中的复制动态进行初步研究。PCR结果显示ILTV gD基因能够在重组病毒中稳定存在;IFA和western blot鉴定结果显示,重组病毒能够正确表达ILTV的gD蛋白,而且gD蛋白嵌合表达在重组病毒颗粒上。鸡胚致病性试验和鸡胚中的复制动态试验结果显示,重组病毒保持了La Sota疫苗株的低致病性和良好的复制特性,rLaSota-VIIF/HN-gD的鸡胚平均死亡时间(MDT)为168 h,1日龄雏鸡脑内接种该重组病毒的致病指数(ICPI)为0.20,鸡胚半数感染量(EID_(50))峰值可达10^(-8.66)/100μL。本研究所制备的重组病毒r LaSota-VIIF/HN-gD为研制基因VII型NDV和ILTV的二联活疫苗奠定了基础。To construct a recombinant Newcastle disease virus expressing the gD protein of infectious laryngotracheitis virus,the ectodomain of the gD gene was fused with signal peptide,transmembrane domain and cytoplasmic tail,and then strategically inserted between P and M gene of the infectious c DNA clone p La Sota-VIIF/HN,in which the F and HN genes of La Sota strain have been swapped with the corresponding genes from genotype VII NDV strain.The resulted recombinant plasmid p La Sota-VIIF/HN-gD was cotransfected with the helper plasmid p CIneo-NP-P-L into BHK-21 cells to rescue the recombinant chimeric NDV La Sota strain expressing the gD protein of ILTV,named rLaSota-VIIF/HN-gD.After 10 passages in 9-day-old SPF chicken embryonated eggs,the recombinant r La Sota-VIIF/HN-gD were identified by PCR,indirect immunofluorescence assay(IFA)and western blot.The PCR was conducted using c DNA reverse transcribed from RNA of 10^(th)passaged recombinant virus,with primers designed to flank the insertion site of the ILTV gD gene.BHK-21 cells infected with the 10^(th)passage recombinant virus were utilized for IFA,employing a polyclonal antibody against ILTV gD protein.Western blot analysis was performed on the supernatant from recombinant virus-infected chicken embryos allantoic fluid adsorbed by chicken red blood cells and purified recombinant NDV virions released from the absorbed chicken red blood cells.The pathogenicity to 9-day-old chicken embryos and the replication dynamics of the 10^(th)passage of the r La Sota-VIIF/HN-gD were also investigated preliminarily.PCR results confirmed the stability of the gD gene within the recombinant virus.The IFA and western blot analyses indicated that the gD protein was not only correctly expressed by r La Sota-VIIF/HN-gD but also successfully incorporated into the recombinant NDV virion.The chicken embryo pathogenicity test and replication dynamics study demonstrated that the r La Sota-VIIF/HN-gD maintained the characteristics of low pathogenicity and robust replication capability,with a

关 键 词：传染性喉气管炎病毒 重组嵌合型新城疫病毒 gD蛋白 二联活疫苗 

分 类 号：S852.65[农业科学—基础兽医学]