【作者】 赵伟；

【导师】 王开；

【作者基本信息】 吉林农业大学 ， 兽医（专业学位）， 2022， 硕士

【摘要】 盖他病毒（Getah virus,GETV）是属于披膜病毒科（Togaviridae）甲病毒属（Alphavirus）的一种虫媒病毒。其宿主为蚊子和多种脊椎动物,主要导致猪和马发病,最近发现也可导致蓝狐发病。马感染GETV后,会出现淋巴结肿大、水肿、皮疹和发热等症状;猪感染GETV后,仔猪会发生关节炎、瘫痪、腹泻等,导致较高的死亡率,母猪出现流产、产死婴、产弱婴等疾病,所以带病猪又是此病的另一大传染源,蓝狐的感染及发病就与食用猪肉产品有关;蓝狐感染GETV后,出现发烧和厌食的症状,也会有神经系统症状出现,最后死亡。血清学检测方法是目前较为可靠的GETV检测方法,如血凝抑制试验（hemagglutination inhibition test,HI）、补体结合试验（Complementfixationtest,CFT）、中和试验（Neutralization test,NT）及间接免疫荧光（indirect immunofluorescence,IFA）等,这些方法经常被用来检测GETV,但这些方法操作复杂,且费时较长。因此,一个快速准确、灵敏度好、特异性强的检测方法是非常重要的。GETV的E2蛋白是其所有蛋白中非常重要的一个结构蛋白,而且有良好的抗原性。本研究利用E2基因序列构建了原核表达质粒,制备大量E2蛋白用来建立检测GETV的间接ELISA方法。构建过程分以下两部分:1.GETV E2基因的克隆表达和多克隆抗体的制备本研究从Gen Bank上参考GETV GS10-2株（Gen Bank登录号:EU015070.1）的E2基因序列,根据序列设计一对可以完整扩增E2基因序列的引物,加入Eco R I和Xho I限制性酶切位点。通过PCR将E2基因序列扩增,双酶切后用连接酶将E2基因序列与原核表达载体p ET-28a连接,后续经测序鉴定,成功构建了p ET-28a-E2重组质粒。然后将质粒转入BL21（DE3）表达菌中后经一系列的优化条件,最终在37℃下0.1m M IPTG浓度诱导6h后通过SDS-PAGE确定成功表达出分子量为46k D的E2蛋白。对高效表达的E2蛋白进行纯化和浓度测定,然后等比例混合弗氏佐剂给实验兔注射以制备多克隆抗体,产生的抗体与E2蛋白抗原经过Western blot分析,证明可以发生特异性反应。2.GETV间接ELISA方法建立及初步应用以纯化后的E2蛋白作为包被抗原,制备的多克隆抗体作为一抗血清,经一系列优化确定最佳间接ELISA反应条件:4℃过夜包被8μg/m L的抗原、待检血清1:100稀释、2.5%脱脂奶粉封闭液封闭1h、抗原抗体作用60min、酶标二抗1:5000稀释作用60min、显色时间25min。使用所建方法对猪瘟病毒（CSFV）、猪繁殖与呼吸障碍综合征病毒（PRRSV）、猪圆环病毒2型（PCV2）、伪狂犬病毒（PRV）的阳性血清样品进行检测,结果都为阴性,说明所建方法特异性良好。检测50份阴性血清后计算其平均值（x?）和标准差（SD）后确定了该方法的临界值,当OD₄₅₀≥x?+3S=0.415时为阳性,当OD₄₅₀≤x?+2S=0.332时为阴性,在两者之间是判为可疑。GETV阳性血清按1:2560稀释时检测结果仍为阳性,说明该方法敏感性较好。批间批内重复性实验结果也较好,变异系数在10%之内。所建方法可以用于对GETV的检测,对实验室保存的吉林省部分猪场采集的300份血清样品使用该方法进行检测,检出GETV阳性47份,阳性率为15.67%。综上,本研究成功建立了检测GETV的间接ELISA方法,并证明建立的间接ELISA方法可进行样品检测,为GETV的检测工作提供依据。更多还原

【Abstract】 Getah virus（GETV）is an arbovirus belonging to the Alphavirus genus of the Togaviridae family.Its hosts are mosquitoes and a variety of vertebrates,mainly causing disease in pigs and horses,and recently discovered that it can also cause disease in blue foxes.After horses are infected with GETV,symptoms such as lymphadenopathy,edema,rash and fever will appear;after pigs are infected with GETV,piglets will develop arthritis,paralysis,diarrhea,etc.The infection and incidence of blue foxes are related to eating pork products;after blue foxes are infected with GETV,symptoms of fever and anorexia will appear,and there will also be Neurological symptoms appear,and eventually death occurs.Serological detection methods are currently more reliable GETV detection methods,such as hemagglutination inhibition test（HI）,complement fixation test（CFT）,neutralization test（NT）and indirect immunofluorescence（indirect immunofluorescence）.These methods are often used to detect GETV,but these methods are complicated and time-consuming.Therefore,a fast,accurate,sensitive and specific detection method is very important.The E2 protein of GETV is a very important structural protein among all its proteins,and has good antigenicity.In this study,a prokaryotic expression plasmid was constructed using the E2 gene sequence,and a large amount of E2 protein was prepared to establish an indirect ELISA method for the detection of GETV.The build process is divided into the following two parts:1.Cloning and expression of GETV E2 gene and preparation of polyclonal antibodyIn this study,referring to the E2 gene sequence of GETV GS10-2 strain（Gen Bank accession number:EU015070.1）from Gen Bank,a pair of primers that can completely amplify the E2 gene sequence was designed according to the sequence,and Eco R I and Xho I were added for restriction enzyme digestion.site.The E2 gene sequence was amplified by PCR,and the E2 gene sequence was ligated with the prokaryotic expression vector p ET-28a with ligase after double enzyme digestion.After subsequent sequencing identification,the p ET-28a-E2recombinant plasmid was successfully constructed.Then,the plasmid was transferred into BL21（DE3）expressing bacteria after a series of optimized conditions,and finally the E2 protein with a molecular weight of 46k D was successfully expressed by SDS-PAGE after induction with0.1m M IPTG concentration at 37℃for 6h.The highly expressed E2 protein was purified and concentration determined,and then mixed with Freund’s adjuvant in equal proportions to prepare polyclonal antibodies,which were analyzed with the E2 protein antigen by Western blot to prove a specific reaction.2.Establishment and preliminary application of GETV indirect ELISA methodThe purified E2 protein was used as the coating antigen,and the prepared polyclonal antibody was used as the primary antibody serum.After a series of optimizations,the optimal indirect ELISA reaction conditions were determined:8μg/m L antigen was coated overnight at4℃,and the serum to be tested was 1:100.Dilution,2.5%nonfat milk powder blocking solution for 1h,antigen-antibody action for 60min,enzyme-labeled secondary antibody 1:5000 dilution for 60min,color development time 25min.Positive serum samples for swine fever virus（CSFV）,porcine reproductive and respiratory syndrome virus（PRRSV）,porcine circovirus type 2（PCV2）,and pseudorabies virus（PRV）were detected using the established method,and the results were all Negative,indicating that the established method has good specificity.The critical value of this method was determined by calculating the mean（x?）and standard deviation（SD）of 50 negative sera,when OD₄₅₀≥x?+3S=0.415 was positive,and when OD₄₅₀≤x?+2S=0.332 Negative,in between is judged suspicious.When the GETV positive serum was diluted by 1:2560,the detection result was still positive,indicating that the sensitivity of the method was good.The inter-assay and intra-assay repeatability results were also good,with the coefficient of variation within 10%.The established method can be used for the detection of GETV.Using this method,300 serum samples collected from some pig farms in Jilin Province were stored in the laboratory for testing,and 47 samples were positive for GETV,with a positive rate of 15.67%.In conclusion,this study successfully established an indirect ELISA method for the detection of GETV,and proved that the established indirect ELISA method can be used for sample detection,which provides a basis for the detection of GETV.更多还原