一种犬细小病毒IgG抗体检测试剂盒的研制
发布时间：2018-10-08 20:06
【摘要】：本研究旨在研究一种犬细小病毒IgG抗体检测试剂盒,通过采集一疑似被犬细小病毒(CPV)感染的病犬粪便,用猫肾细胞(F81)进行病毒分离和培养,进行核酸和血清学鉴定。将F4代CPV病毒感染F81细胞后不同时间收集细胞和培养上清,分别采用免疫过氧化物酶单层细胞染色法(IPMA)和PCR方法检测病毒的增殖动态,病毒分别用无血清培养基和有血清培养基进行体外培养,IPMA测定病毒滴度。设计特异性引物,PCR扩增CPV分离株VP2基因,将PCR产物克隆至p MD18-T载体,重组载体进行酶切鉴定和序列测序,核酸序列采用DNAStar7.0和Mega5.0软件进行相似性和进化分析。结果表明,成功分离到一株CPV病毒,命名为CPV-NY,该毒株在F81细胞中增殖能产生显著细胞病变效应(CPE)。IPMA可从感染后8 h的F81细胞中检出病毒,PCR能从感染后8 h培养上清中检出CPV核酸。该毒株F4代采用有血清培养基72 h后,增值滴度可达106.25/mL,无血清培养基培养的病毒增值滴度可达105.36/mL。该毒株VP2基因开放阅读框(ORF)为1755 bp,编码584aa,进化分析显示,该毒株属于CPV2a型。为制备一株高致病性的CPV毒株(NY株,基因型为2a型)重组VP2蛋白,构建重组原核表达载体pET28a-CPV-VP2,转化E.coli BL21(DE3)表达菌株,通过不同温度,不同IPTG浓度,不同诱导时间等条件的优化进行表达蛋白,采样SDS-PAGE和Western Blot分析蛋白的抗原性。结果表明,重组CPV-NY毒株VP2蛋白的分子量约为72 kDa,该蛋白以包涵体形式存在,在37℃,IPTG诱导浓度为0.2 mmoL/L,诱导4 h表达量最高。该蛋白不仅能与His标签mAb反应,也能与CPV特异性阳性血清反应,说明其具有良好的抗原性,目的蛋白与佐剂混合、乳化制备免疫原,免疫家兔制备VP2蛋白多克隆抗体。采用免疫过氧化物酶单层细胞染色法(IPMA)检测抗体的免疫活性、抗体滴度及病毒滴度。结果表明,所制备的多克隆抗体滴度为1600,病毒滴度为107 TCID50/mL,该抗体与体外培养的CPV及稳定表达VP2蛋白的细胞呈特异性反应,CPV型VP2蛋白的多克隆抗体免疫活性和特异性良好,进而研制出一种犬细小病毒IgG抗体检测试剂盒,并对该试剂盒的组装、批内批间重复性、保存期以及对临床血清的检测进行了实验。实验证明该IPMA检测试剂盒重复性好,保存期长,灵敏度高,为CPV的诊断和疫苗研发奠定了基础。
[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，
本文编号：2258095
[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，
本文编号：2258095
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