鸭肠炎病毒核衣壳蛋白基因的克隆与原核表达

Cloning and Prokaryotic Expression of Nucleocapsid Protein Gene of Duck Enteritis Virus

目的：对DEV贵州分离株NP基因进行克隆与序列分析，构建NP基因的原核表达载体，分析NP基因原核表达产物的免疫反应性。方法：根据GeneBank登载的DENNP基因序列设计引物，对DEV贵州分离株进行PCR扩增、克隆和测序，采用生物信息学软件程序分析NP蛋白的氨基酸序列；将该基因插入到原核表达载体pET32a上进行原核表达和Western Blotting分析。结果：DEV贵州分离株NP基因全长759bp，核苷酸序列与参考株一致；NP基因编码蛋白相对分子量为27．1kDa，理论pI为5．89，肽链上第10．15、88．92和182．186区段及其附近区域可能是B细胞表位优势区；构建得到的重组质粒pET32-NP可表达出一条大小约为48kDa的蛋白，且能与兔抗DEN-IgG发生特异性结合。结论：NP基因在DEN基因组中高度保守，其原核表达产物具有良好的免疫反应性。

Objective： To analysis NP gene sequence of DEV Guizhou strain and construct its prokaryotic expression vector, and to identify the reactiongenicity of the prokaryotic expression products. Method： The NP gene of DEV Guizhou strain was amplified by PCR, which the primers were designed on the basis of NP gene sequence of DEV CH strain. And the PCR products were cloned and sequenced, and analyzed by SAPS program and DNAstar Protein software, and then inserted into the prokaryotic vector pET32a and transformed into E.scherichia coli BI21. The target protein were expressed with induction of IFrG and separated on SDS- PAGE, and the reactiongenicity of the expressed fusion protein were identified by Western Blotting. Result： The complete sequence of NP gene of DEV Guizhou strain was 759 bp in length encoding for 252 amino acid, which share 100% homology at nucleotide level with DEV CH strain. The molecular weight of the deduced protein is 27.1 kDa and the pI is 5.89, and its predominant epitopes are probably located in the region of 10 - 15, 88 - 92 and 182 - 186 peptides. The prokaryotic expression vector pET32 - NP was constructed, and SDS - PAGE showed that the fusion protein had a molecular weight 48 kDa, which＇could combine specially with rabbit anti - DEV IgG on Western Blotting. Conclusion： The NP gene is highly conservative in genome of DEN＇, the prokaryotic expression products of this gene had a fine reactiongenicity of immunity, which will be the foundation for the study on function of NP protein.