H3N2亚型犬流感病毒HA1基因的优化表达与间接ELISA检测方法的建立  

Optimized expression of the HA1 gene of the H3N2 subtype canine influenza virus and establishment of an indirect ELISA for its antibody detection

作　　者：秦海斌 包喜军 朱骞 陈舒 贺星亮 QIN Haibin;BAO Xijun;ZHU Qian;CHEN Shu;HE Xingliang(Nanjing Police University/Ministry of Public Security Key Laboratory of Police Dog Technology,Nanjing 210012,China)

机构地区：[1]南京警察学院/警犬技术公安部重点实验室,江苏南京210012

出　　处：《畜牧与兽医》2024年第11期114-120,共7页Animal Husbandry & Veterinary Medicine

基　　金：中央高校基本科研业务费专项资金项目(LGZD202406);公安部科技强警基础工作专项(2022JC20)。

摘　　要：为开展H3N2亚型犬流感的血清流行病学调查,本研究拟原核表达犬流感病毒(CIV)血凝素1(HA1)基因,建立一种灵敏、准确、稳定的ELISA检测方法。对地方毒株的HA1基因进行氨基酸序列优化,采用化学合成法合成目的基因,插入pET30a中并转化至大肠杆菌表达,对蛋白进行纯化、鉴定,建立CIV间接ELISA方法。结果显示:优化后的HA1基因序列密码子对原核表达系统的CAI指数升至0.88,无复杂二级结构,蛋白可获得高效表达;原核表达后蛋白主要存在于包涵体中,经纯化、复性后的蛋白具备作为诊断抗原的应用价值;建立的间接ELISA方法检测国内采集的1690份犬血清,显示H3N2亚型CIV整体阳性率为9.23%,与商品化试剂盒符合率为97.9%。提示:建立的间接ELISA方法可靠,具备良好的应用前景。To conduct a seroepidemiological survey of canine influenza(CI)H3N2 virus infections in dogs,the HA1 gene of CIV was prepared by prokaryotic expression,and an indirect ELISA was established.Firstly,the HA1 gene of the H3N2 local strain was downloaded from Gene Bank.The gene was artificially synthesized after its amino acid sequences were optimized by the commercialized software the MaxCodonTMOptimization Program.The synthesized gene was inserted into the expression vector pET30a,identified,and transformed into the host strain DE3 for protein expression.The expressed protein was purified and identified.And an indirect ELISA was established with the recombinant HA1 protein to conduct serological investigation.The results of the prokaryotic expression assay showed that,with this technique,the target gene was quickly obtained and the amino acid codons were easily optimized.The codon adaptation index(CAI)of the optimized sequences for the prokaryotic expression system rose to 0.88,and the complex protein secondary structure decreased.The expressed HA1 protein mainly existed in the inclusion bodies.After purification and renaturation,the recombinant protein could be used as a diagnosis antigen to establish an indirect ELISA to be used in the subsequent serological survey.The positive rate of the H3N2 subtype CIV was 9.23%in 1690 serum samples.To summarized,this study provided a rapid and efficient method to prepare diagnostic antigens and to establish an indirect ELISA for serological survey of the canine H3N2 virus.

关 键 词：犬流感 H3N2 血凝素1 原核表达 间接ELISA 

分 类 号：S852.21[农业科学—基础兽医学]