猪繁殖与呼吸综合征病毒分离株E11105 N基因的序列分析与原核表达

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。

Sequence Analysis and Prokaryotic Expression of N Gene of PRRSV E11105 Strain

In order to study the N protein structure,function of PRRSV and its role in the viral pathogene-sis,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 bi-ology related software.We constructed a recombinant vector pCold Ⅰ-N.The recombinant plasmid pColdⅠ-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 E11105 with North American type strain VR-2332,the European type strain Lelystad virus (LV),highly pathogen-ic PRRSV strain JXA1 in 2006 in China and Chinese strain CH-1a 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%.Phy-logenetic tree showed that the N gene of E11105 strain and the American type VR-2332,the highly patho-genic JXA1 strain are closely related.There were no transmembrane domains for N gene encoding protein of E11105.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.