表达GoAstV-2 Cap蛋白的重组FAdV-4的构建与鉴定  

Construction and identification of recombinant fowl adenovirus 4 expressing Cap protein of goose astrovirus virus genotype 2

作　　者：李星雨 李岩[1] 杨盼盼 刘俊杰 相梦佳 朱玉涛 邱路遥 乔麒龙 张伯顺 卜德新 韩城昊 于春梅 丛雁方 王增 李建丽[1] 王白玉 赵军[1] LI Xingyu;LI Yan;YANG Panpan;LIU Junjie;XIANG Mengjia;ZHU Yutao;QIU Luyao;QIAO Qilong;ZHANG Boshun;BU Dexin;HAN Chenghao;YU Chunmei;CONG Yanfang;WANG Zeng;LI Jianli;WANG Baiyu;ZHAO Jun(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;Qingdao Vland Biotech Inc.,Qingdao,Shandong 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：《中国兽医学报》2025年第3期443-448,513,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(32372997)。

摘　　要：为构建表达鹅星状病毒基因2型(GoAstV-2)衣壳蛋白(Cap)的重组禽腺病毒4型(FAdV-4),本研究将GoAstV-2的Cap基因表达盒插入到含有FAdV-4感染性克隆p15A-cm-FAdV4-HNJZ中FAdV4基因组的1966 bp自然缺失区,将获得的重组感染性克隆p15A-cm-FAdV4-HNJZ-Cap/GoAstV-2利用限制性内切酶线性化后转染鸡肝癌细胞系(chicken hepatoma cell line,LMH),拯救出表达Cap蛋白的重组病毒rFAdV4-Cap/GoAstV-2。将重组病毒在LMH细胞中连续传至第15代,提取重组病毒基因组DNA,用插入位点两侧的鉴定引物进行PCR和利用抗Cap蛋白多克隆抗体进行间接免疫荧光试验和Western blot对重组病毒进行鉴定,同时对重组病毒在LMH细胞中的复制动态进行了研究。结果显示,GoAstV-2的Cap基因能够稳定存在于重组病毒rFAdV4-Cap/GoAstV-2基因组中,而且获得稳定表达;重组病毒具有良好的体外复制能力。本研究所制备的重组病毒rFAdV4-Cap/GoAstV-2为研制防控鹅FAdV-4和GoAstV-2混合感染的新型高效二联灭活疫苗奠定了基础。To construct a recombinant fowl adenovirus 4(FAdV-4)expressing the Cap protein of goose astrovirus genotype 2(GoAstV-2),the expression cassette of Cap gene was inserted into the natural 1966 bp deletion region of the FAdV-4 genome in the infectious clone p15A-cmFAdV4-HNJZ.The resulted recombinant plasmid p15A-cm-FAdV4-HNJZ-Cap/GoAstV-2 was linearized with restriction enzyme and transfected into chicken hepatoma cell line(LMH)to rescue the recombinant FAdV-4 expressing the Cap protein of GoAstV-2,rFAdV4-Cap/GoAstV-2.After15 passages in LMH cells,the recombinant rFAdV4-Cap/GoAstV-2 was identified by PCR using primers flanking the insertion site of the Cap gene expression cassette and using viral genome DNA extracted from rFAdV4-Cap/GOAstV-2 infected LMH cells as template.LMH cells were infected with 15~(th)passage rFAdV4-Cap/GoAstV-2 and indirect immunofluorescence was performed with a polyclonal antibody against Cap protein as the primary antibody.Western blot was carried out with lysates of rFAdV4-Cap/GoAstV-2 infected LMH cells.The in vitro replication dynamic of the 15~(th)passage of the rFAdV4-Cap/GoAstV-2 was also investigated in LMH cells.The results demonstrated that the Cap gene of GoAstV-2 was presented in the genome of the recombinant virus rFAdV4-Cap/GoAstV-2,and could be expressed stably.The prepared recombinant virus in this study will lay a foundation for developing inactivated bivalent vaccine candidate against co-infection of FAdV-4 and GoAstV-2 in goose.

关 键 词：鹅星状病毒基因2型 禽病毒4型 CAP蛋白 二联灭活疫苗 

分 类 号：S852.2[农业科学—基础兽医学] S852.65[农业科学—兽医学]