本文選題：豬圓環(huán)病毒2型 + Cap基因��； 參考：《安徽農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：豬圓環(huán)病毒(Porcine circovirus,PCV)是圓環(huán)病毒屬的代表種,可分為PCV1和PCV2。PCV1沒(méi)有致病性;PCV2引起的斷奶仔豬多系統(tǒng)衰竭綜合征(Postweaning multisystemic wasting syndrome,PMWS)是豬的免疫抑制病之一,給世界養(yǎng)豬業(yè)造成了嚴(yán)重的經(jīng)濟(jì)損失。目前,臨床上還沒(méi)有有效的方法控制和消滅PCV2,市場(chǎng)上流通的疫苗也不能起到很好的預(yù)防效果。因此,建立一種血清學(xué)檢測(cè)方法,快速、及時(shí)掌握PCV2的流行情況,做好預(yù)防措施就顯得十分關(guān)鍵。研究利用PCV2 ORF2的原核表達(dá)蛋白作為抗原,建立間接ELISA檢測(cè)方法,并初步應(yīng)用于PCV2血清抗體的臨床檢測(cè)。首先,根據(jù)實(shí)驗(yàn)室保存的PCV2鄂州株(Genebank未登錄)基因序列設(shè)計(jì)特異性引物,分別在上、下游引物加入Sac I和Hind III酶切位點(diǎn),擴(kuò)增出PCV2鄂州株的ORF2基因,將該片段克隆至pET-28a原核表達(dá)載體,經(jīng)PCR、酶切鑒定,表明成功構(gòu)建了陽(yáng)性重組表達(dá)質(zhì)粒pET-ORF2。將其轉(zhuǎn)化入大腸桿菌Rosetta中,經(jīng)IPTG誘導(dǎo)表達(dá),SDS-PAGE電泳分析初步確定重組蛋白為34.5KDa,主要以包涵體形式表達(dá)。最佳誘導(dǎo)條件為:37℃條件下,搖菌至OD值0.4-0.6左右時(shí)加入終濃度1mmol/L IPTG,誘導(dǎo)5h時(shí)目的蛋白表達(dá)量最高,可達(dá)0.46mg/mL。表達(dá)產(chǎn)物經(jīng)His-tag鎳柱純化后進(jìn)行Western-blot分析。結(jié)果表明表達(dá)的重組蛋白能夠與PCV2標(biāo)準(zhǔn)陽(yáng)性血清發(fā)生特異性反應(yīng),具有良好的反應(yīng)原性。其次,用純化的重組蛋白作為包被抗原,建立了PCV2抗體間接ELISA檢測(cè)方法。其中,抗原包被濃度為4.6μg/mL,37℃孵育2h后4℃過(guò)夜;5%SMP/PBST,37℃封閉2h;待檢血清的最佳稀釋度為1∶40,37℃孵育1h;HRP-羊抗豬IgG稀釋度為1∶3 000,37℃孵育1h;TMB顯色15min。經(jīng)統(tǒng)計(jì)學(xué)分析確定陰陽(yáng)性臨界值為0.421。并對(duì)優(yōu)化后的間接ELISA方法的特異性、重復(fù)性進(jìn)行測(cè)定并與商品化的試劑盒進(jìn)行陽(yáng)性符合率比較。結(jié)果顯示,建立的間接ELISA方法與CSFV、PRRSV、FMDV、PRV、TGEV、PEDV的陽(yáng)性血清無(wú)交叉反應(yīng),特異性好;變異系數(shù)最大為8.71%;與商品化試劑盒比較,陽(yáng)性符合率達(dá)到96.7%。用建立的ELISA方法檢測(cè)豬場(chǎng)送檢血樣223份,總陽(yáng)性檢出率為61.9%(138/223),其中保育豬陽(yáng)性檢出率為58.3%(63/108),育肥豬陽(yáng)性檢出率為65.2%(75/115)。結(jié)果表明建立的間接ELISA方法能用于臨床PCV2抗體樣本檢測(cè)并為ELISA診斷試劑盒的開(kāi)發(fā)奠定了基礎(chǔ)。
[Abstract]:Porcine circovirus (PCV) is a representative species of the genus circovirus. It can be divided into two groups: PCV1 and PCV2.PCV1. The postweaning multisystemic wasting syndrome induced by PCV2 is one of the immunosuppressive diseases in pigs, which has caused serious economic losses to the world pig industry. At present, there is no effective method to control and eliminate PCV2 in clinic, and the marketable vaccine can not play a good preventive effect. Therefore, it is very important to establish a serological detection method to grasp the prevalence of PCV2 quickly and timely. To establish an indirect ELISA detection method using prokaryotic expression protein of PCV2 ORF2 as antigen, and to apply it to the clinical detection of PCV2 serum antibody. Firstly, specific primers were designed according to the sequence of PCV2 Ezhou strain (Genebank) kept in laboratory. The ORF2 gene of PCV2 Ezhou strain was amplified by adding Sac I and Hind III sites into the upstream and downstream primers, respectively. The fragment was cloned into the prokaryotic expression vector of pET-28a and identified by PCR and restriction endonuclease digestion. The positive recombinant expression plasmid pET-ORF2 was successfully constructed. It was transformed into Escherichia coli Rosetta and expressed by IPTG induced by SDS-PAGE. The recombinant protein was identified as 34.5KDa. it was mainly expressed as inclusion body. The optimal induction conditions were as follows: at 37 鈩，

本文編號(hào)：1927417