一株牛肠病毒F型的全基因组分析及抗体检测间接ELISA方法的建立  

Full-genome Analysis of a Bovine Enterovirus Type F and the Establishment of an Indirect ELISA Method for Antibody Detection

作　　者：刘健 于泽海 张玫瑜 李丹 王君 刘芳琴 张群 徐守振 LIU Jian;YU Zehai;ZHANG Meiyu;LI Dan;WANG Jun;LIU Fangqin;ZHANG Qun;XU Shouzhen(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China)

机构地区：[1]青岛农业大学动物医学院,青岛266109

出　　处：《畜牧兽医学报》2025年第2期814-825,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家重点研发计划项目(2018YFD0501403)。

摘　　要：本研究从牛肠病毒(BEV)阳性粪便中通过接种BHK细胞进行蚀斑纯化分离得到一株F型肠道病毒,经过RT-PCR鉴定正确后送生物公司进行全基因测序以及进行TCID_(50)的测定,以MEGA11.0等生物信息学软件对BEV全基因进行分析并构建遗传进化树。以原核表达方式得到含BEV分离株VP3基因的重组蛋白,作为包被抗原,建立检测抗体的间接ELISA方法,并对建立的ELISA方法的特异性、灵敏性、重复性进行测定与用于临床样品的检测。结果显示,BEV分离株的TCID 50为10-6.26·mL^(-1),病毒分离株全长为7439 nt,ORF大小为6504 bp,编码2168个氨基酸,与F型肠道病毒BEV4遗传关系最近且核苷酸相似性最高,故分离到的病毒为F型肠道病毒,暂命名为BEV-QD,与其他F型毒株相比,BEV-QD株存在氨基酸的突变以及缺失并且大多集中在P2、P3区。本次建立的抗体检测间接ELISA方法重复性良好;阳性血清稀释1600倍后结果仍呈现阳性,灵敏性良好;与BVDV等阳性血清不发生特异性结合,特异性良好;检测40份临床样品中,13份为阳性,阳性率为32.5%。本研究分离到一株F型牛肠病毒突变株,建立了一种抗体间接ELISA方法。为牛肠病毒的检测和防控提供了基础手段。In this study,a strain of F-type enterovirus was isolated from bovine enterovirus(BEV)positive feces through plaque purification by inoculating BHK cells.After being correctly identified by RT-PCR,it was sent to a biological company for whole-genome sequencing and TCID_(50) determination.The whole genome of BEV was analyzed and a genetic evolution tree was constructed using bioinformatics software such as MEGA11.0.The recombinant protein containing the VP3 gene of the BEV isolate was obtained through prokaryotic expression and used as a coating antigen to establish an indirect ELISA method for detecting antibodies.The specificity,sensitivity,and repeatability of the established ELISA method were measured and used for the detection of clinical samples.The results showed that the TCID 50 of the BEV isolate was 10-6.26·mL^(-1),and the full length of the virus isolate was 7439 nt,with an ORF size of 6504 bp,encoding 2168 amino acids.It had the closest genetic relationship and the highest nucleotide homology with F-type enterovirus BEV4.Therefore,the isolated virus was an F-type enterovirus,temporarily named BEV-QD.Compared with other F-type strains,the BEV-QD strain had amino acid mutations and deletions,mostly concentrated in the P2 and P3 regions.The indirect ELISA method for antibody detection established in this study had good repeatability;the results remained positive after the positive serum was diluted 1600 times,indicating good sensitivity;it did not specifically bind to positive sera such as BVDV,indicating good specificity;among the 40 clinical samples tested,13 were positive,with a positive rate of 32.5%.This study isolated an F-type bovine enterovirus mutant strain and established an indirect ELISA method for antibody detection,providing a basic means for the detection and prevention of bovine enterovirus.

关 键 词：牛肠道病毒 分离鉴定 TCID_(50) 全基因组分析 ELISA 

分 类 号：S855.3[农业科学—临床兽医学]