新城疫病毒HN基因克隆及其在大肠杆菌中的表达

CLONING OF HN GENE OF NEWCASTLE DISEASE VIRUS AND ITS EXPRESSION IN E. COLI

用RT-PCR方法扩增新城疫病毒HN基因并将其克隆至T载体,测定其核苷酸序列。尔后用PCR扩增球状头部编码区,并将其克隆至pSOC质粒soc基因3'端EcoRⅠ位点,重组质粒pSOC-HN转化E.coliBL-21(DE3)感受态细胞,以终浓度1mmol/L的IPTG诱导表达,在SDS-PAGE凝胶上可检测到分子量约67KD的融合蛋白特异条带,west-blot证实表达产物与NDV抗血清具有良好的反应性。

The HN gene with a length of 1 713 bp of Newcastle Disease Virus (NDV) was amplified by RT-PCR, the PCR product was purified and cloned into pGEM-T vector, and the recombinant plasmid was sequenced. According to the results of HN gene sequencing another pair of primer containing ECOR I restriction site was designed to amplify the HN fragment coding for the globular domain, the main functional region of HN protein. The HN fragment was then inserted at the 3' terminus of soc gene of pSOC plasmid, The recombinant plasmid pSOC-HN was used to transform E. coli BL-21(DE 3) to induce HN protein expression with 1 mmol/L IPTG. A SOC-HN fusion protein band with a molecular weight of 67 KD was detected on the SDS-PAGE gel and nitrocellulose membrane. The expression products can react specifically with antisera against Newcastle Disease Virus.