摘要
为研究PRRSV N蛋白的结构、功能以及N蛋白在病毒致病中的作用,以临床分离PRRSV毒株E11105为研究对象,采用Primer Premier 5.0设计一对特异性引物,经RT-PCR扩增出N基因片段,利用相关分子生物学软件对N基因序列进行分析;将N基因克隆连接到pColdⅠ原核表达载体上,经PCR、双酶切鉴定及序列测定后,得到重组质粒pColdⅠ-N,将pColdⅠ-N转入大肠埃希菌BL21(DE3)感受态细胞中,IPTG诱导表达后用SDS-PAGE蛋白电泳及Western blot验证分析。结果显示,分离株E11105N基因与北美洲型代表株VR-2332、欧洲型代表株Lelystad virus(LV)、中国2006年暴发的高致病性PRRSV代表株JXA1、中国代表株CH-1a的核苷酸序列同源性分别为93.3%、34.7%、99.2%、95.4%,氨基酸序列同源性分别为94.4%、15.3%、94.4%、99.2%;系统进化树显示,E11105株N基因与美洲型代表株VR-2332、中国高致病性JXA1毒株的亲缘关系较近;分离株E11105N基因所编码蛋白不存在跨膜区;二级结构主要以α-螺旋和无规则卷曲为主,分别占20.33%和63.41%;预测该蛋白可能存在5个较为明显的B细胞优势抗原表位。SDS-PAGE蛋白电泳结果表明,重组N蛋白主要存在于菌体沉淀中,分子质量约为16.7ku;Western blot结果显示,带His标签的重组表达蛋白能被His单克隆抗体识别,显色后条带约为16.7ku,与SDS-PAGE蛋白电泳的条带大小一致。
In order to study the N protein structure,function of PRRSV and its role in the viral pathogenesis,we took the isolated PRRSV strain E11105 as the research object and designed a pair of primers by Primer Premier 5.0. Then we amplified N gene by RT-PCR and analyzed N gene sequences by molecular biology related software. We constructed a recombinant vector pCold I -N. The recombinant plasmid pCold I -N was confirmed by PCR, double enzyme digestion and sequencing, and was transfected into Escherichia coli BL21（DE3） competent cells. N protein induced by IPTG was detected and analyzed by SDS-PAGE and Western blot. The results showed that the N gene nucleotide sequence similarities of the isolated El1105 with North American type strain VR-2332, the European type strain Lelystad virus （LV）, highly pathogenic PRRSV strain JXA1 in 2006 in China and Chinese strain CH-1α are respectively 93.3% ,34.7% ,99.2% and 95.4% ,and the amino acid sequence similarities are respectively 94.4% ,15.3% ,94.4% ,99.2%. Phylogenetic tree showed that the N gene of El1105 strain and the American type VR-2332,the highly pathogenic JXA1 strain are closely related. There were no transmembrane domains for N gene encoding protein of El1105. The secondary structure is mainly dominated by alpha helix and irregular coil, which account for 20.33% and 63.41% ,and the N protein was predicted that might have five B cell dominant epitopes. SDS-PAGE showed that recombinant N protein has a 16.7 ku molecular weight and mainly distributes in the cell precipitate. Western blot results showed that the recombinan N protein has an immune response with His antibody,and the band is about 16.7 ku,which is coincident with the result that analyzed by SDS-PAGE.
出处
《动物医学进展》
北大核心
2016年第10期41-47,共7页
Progress In Veterinary Medicine
基金
国家自然科学基金项目(31460668)
贵州省科学技术基金项目(黔科合J字[2015]2046号)
贵州大学大学生"SRT"计划项目(贵大SRT(2014)121号)
贵州大学"本科教学工程"项目(JKSP2013004)
