【摘要】：本研究旨在研究一種犬細小病毒IgG抗體檢測試劑盒,通過采集一疑似被犬細小病毒(CPV)感染的病犬糞便,用貓腎細胞(F81)進行病毒分離和培養(yǎng),進行核酸和血清學鑒定。將F4代CPV病毒感染F81細胞后不同時間收集細胞和培養(yǎng)上清,分別采用免疫過氧化物酶單層細胞染色法(IPMA)和PCR方法檢測病毒的增殖動態(tài),病毒分別用無血清培養(yǎng)基和有血清培養(yǎng)基進行體外培養(yǎng),IPMA測定病毒滴度。設計特異性引物,PCR擴增CPV分離株VP2基因,將PCR產物克隆至p MD18-T載體,重組載體進行酶切鑒定和序列測序,核酸序列采用DNAStar7.0和Mega5.0軟件進行相似性和進化分析。結果表明,成功分離到一株CPV病毒,命名為CPV-NY,該毒株在F81細胞中增殖能產生顯著細胞病變效應(CPE)。IPMA可從感染后8 h的F81細胞中檢出病毒,PCR能從感染后8 h培養(yǎng)上清中檢出CPV核酸。該毒株F4代采用有血清培養(yǎng)基72 h后,增值滴度可達106.25/mL,無血清培養(yǎng)基培養(yǎng)的病毒增值滴度可達105.36/mL。該毒株VP2基因開放閱讀框(ORF)為1755 bp,編碼584aa,進化分析顯示,該毒株屬于CPV2a型。為制備一株高致病性的CPV毒株(NY株,基因型為2a型)重組VP2蛋白,構建重組原核表達載體pET28a-CPV-VP2,轉化E.coli BL21(DE3)表達菌株,通過不同溫度,不同IPTG濃度,不同誘導時間等條件的優(yōu)化進行表達蛋白,采樣SDS-PAGE和Western Blot分析蛋白的抗原性。結果表明,重組CPV-NY毒株VP2蛋白的分子量約為72 kDa,該蛋白以包涵體形式存在,在37℃,IPTG誘導濃度為0.2 mmoL/L,誘導4 h表達量最高。該蛋白不僅能與His標簽mAb反應,也能與CPV特異性陽性血清反應,說明其具有良好的抗原性,目的蛋白與佐劑混合、乳化制備免疫原,免疫家兔制備VP2蛋白多克隆抗體。采用免疫過氧化物酶單層細胞染色法(IPMA)檢測抗體的免疫活性、抗體滴度及病毒滴度。結果表明,所制備的多克隆抗體滴度為1600,病毒滴度為107 TCID50/mL,該抗體與體外培養(yǎng)的CPV及穩(wěn)定表達VP2蛋白的細胞呈特異性反應,CPV型VP2蛋白的多克隆抗體免疫活性和特異性良好,進而研制出一種犬細小病毒IgG抗體檢測試劑盒,并對該試劑盒的組裝、批內批間重復性、保存期以及對臨床血清的檢測進行了實驗。實驗證明該IPMA檢測試劑盒重復性好,保存期長,靈敏度高,為CPV的診斷和疫苗研發(fā)奠定了基礎。
[Abstract]:The purpose of this study was to study a canine parvovirus IgG antibody detection kit. The feces of a sick dog infected with canine parvovirus (CPV) were collected. The virus was isolated and cultured by cat kidney cells (F81) and identified by nucleic acid and serology. After F4 passage CPV virus was infected with F81 cells, the cell culture supernatants were collected at different times. The proliferation of F81 cells was detected by immunoperoxidase monolayer (IPMA) and PCR methods, respectively. The virus titers were determined by using serum-free medium and serum-free medium in vitro. Specific primers were designed to amplify the VP2 gene of CPV isolate. The PCR product was cloned into p MD18-T vector. The recombinant vector was digested and sequenced. The nucleic acid sequence was analyzed by DNAStar7.0 and Mega5.0 software. The results showed that a strain of CPV virus was successfully isolated, named CPV-NY, which proliferated in F81 cells and produced a significant cytopathic effect. (CPE). IPMA could detect CPV nucleic acid from the supernatant of F81 cells at 8 h after infection, and CPV nucleic acid could be detected from the culture supernatant of F81 cells at 8 h after infection. After 72 h of serum-containing medium, the increment titer of F4 strain could reach 106.25% mL, and that of serum-free medium could reach 105.36% mL. The open reading frame (ORF) of the VP2 gene of this strain encodes 584aa for 1755 bp,. Evolutionary analysis shows that the strain belongs to CPV2a type. In order to prepare a highly pathogenic CPV strain (NY strain with 2a genotype) recombinant VP2 protein, the recombinant prokaryotic expression vector pET28a-CPV-VP2, was constructed and transformed into E.coli BL21 (DE3) expression strain. The expression protein was optimized under different induction time, and the antigenicity of the protein was analyzed by SDS-PAGE and Western Blot. The results showed that the molecular weight of VP2 protein of recombinant CPV-NY strain was about 72 kDa,. The protein existed in the form of inclusion body, and the highest expression was induced at 37 鈩，

本文編號：2258095