新城疫病毒HN09-69株P基因的克隆、序列分析及表达

Cloning,sequence analysis and expression of Pgene of Newcastle disease virus HN09-69 strain

【目的】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。【方法】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。【结果】扩增出了HN09-69株P基因的ORF,序列长度为1 188bp。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54ku,符合预期结果,在大肠杆菌中主要以可溶性的形式表达。【结论】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。

【Objective】 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.【Method】 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.【Result】 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.【Conclusion】 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.