Cloning，sequence analysis and expression of P gene of Newcastle disease virus HN09-69 strain | 新城疫病毒HN09-69株P基因的克隆、序列分析及表达

中国人. 对新城疫病毒(NDV)HN09-69株P基因进行克隆、序列分析和表达鉴定，为研究P和V蛋白的功能，及NDV新流行毒株的免疫预防提供理论依据。以HN09-69株NDV为研究对象，根据GenBank上发布的相关P基因参考序列设计引物，进行RT-PCR扩增、克隆和序列测定分析，将P基因的核苷酸序列与相关参考株的P基因序列进行比对分析并构建进化树。将P基因克隆到原核表达载体PET28a(+)中，得到重组质粒rPET28a(+)-P，将重组表达质粒转化到BL21（DE3）中诱导表达，用SDS-PAGE电泳和Western blotting检测表达情况。扩增出了HN09-69株P基因的ORF，序列长度为1 188 bp。NDV HN09-69株核苷酸与弱毒株La Sota、clone-30同源性分别为82.5%和82.4%，与强毒株F48E8同源性为84.9%，与鸭源，鹅源NDV核苷酸同源性分别为97.9%～98.4%和96.0%～98.0%。SDS-PAGE电泳和Western blotting检测结果表明，重组P蛋白分子质量约为54 ku,符合预期结果，在大肠杆菌中主要以可溶性的形式表达。NDV HN09-69株与SDWF-02和SD-09鸭源强毒分离株同源性高，亲缘关系近。重组P蛋白在大肠杆菌中得到了高效表达。

英语. In order to provide support for the prevention new epidemic Newcastle disease virus (NDV) and forming the foundation for study the function of P and V protein, the P gene of NDV HN09-69 was amplified, analyzed and expressed. Specific primers were designed according to the reported sequences of NDV in GenBank and the P gene of HN09-69 strain was amplified and sequence was analyzed. Sequence of P gene of HN09-69 strain was compared with relevant reference strains and the phylogenetic tree was constructed. The P gene was cloned into the prokaryotic expression vector PET28a (+). The recombinant plasmids rPET28a (+)-P was constructed, identified and transformed into BL21（DE3）competent cells and induced to expression by IPTG. The expressed protein was detected by SDS-PAGE and Western blotting. The complete open read frame (ORF) of P gene was amplified by RT-PCR and contains 1 188 bp in size. The homology of nucleotides was 82.5%, 82.4% and 84.9%, respectively. Comparied with duck, goose source NDV, the homology of nucleotides was 97.9%-98.4%, 96.0%-98.0%, respectively. The results of SDS-PAGE and Western blotting assay show that the recombinant P protein is about 54 ku in size in accordance with the expected result. The NDV HN09-69 strain and Shandong, Heilongjiang virulent strains of source in ducks have high homology and close relationship. The recombinant P protein has high effective expression in E. coli._