口疮病毒O2O基因的克隆、表达及多抗制备

羊口疮是由口疮病毒(orf virus,ORFV)引起的接触性、嗜上皮性的传染病。020基因是该病毒重要的酶活力调节蛋白。根据Gen Bank中020基因的序列设计引物,从ORFV-F10株感染的病料中提取DNA,PCR扩增获得目的基因。然后将其亚克隆至p ET-32a(+)原核表达载体,构建重组表达质粒p ET-32a-ORF-020。鉴定正确后转化BL21感受态细胞,进行IPTG诱导表达。表达产物进行SDS-PAGE和Western-blot分析。最后将纯化的020蛋白免疫BALB/c雌鼠,制备多抗血清。SDS-PAGE结果表明,ORFV 020基因在体外获得正确表达,大小约37 k Da。Western-blot结果表明,制备的多抗血清能够与020蛋白发生特异性反应具有良好的反应原性。

Cloning and expression of the Orf virus 020 gene and its polyclonal antibody preparation

Orf is an infectious disease caused by orf virus (ORFV). Animals can be infected by ORFV through contact and it is preferred to epithelial infection. The ORFV 020 gene is an important enzyme regulating protein activity of the virus. In this study, specific PCR primers were designed according to the sequence of the Orf 020 gene in GenBank. After the viral DNA was extracted from ORFV infected clinical samples, The Orf 020 gene was amplified by PCR and subcloned to pET-32a prokaryotic expression vector. The recombinant plasmid was identified and named as pET-32a-ORF-020, which was then transformed into E.coli competent cell BL21 and induced by IPTG. The expressed proteins were identified by SDS-PAGE and Western blot. The ORF 020 protein was then purified and immunized to BALB/c mice for antiserum preparation. The results of SDS-PAGE showed that the Orf 020 gene was correctly expressed in vitro. The protein size was about 37 kDa. The western-blot analysis showed that the prepared antiserum reacted specifically with the ORF 020 protein.