猪乳铁蛋白肽LFcinP基因在巴斯德毕赤酵母中组成型表达及其抑菌活性检测

为获得具有生物活性作用的猪乳铁蛋白肽LFcinP基因在巴斯德毕赤酵母中进行组成型表达,检索NCBI数据库公布的牛、羊、猪、骆驼等动物的乳铁蛋白及其抗菌肽的氨基酸序列,运用ExPASY在线网站,对猪乳铁蛋白保守区域所编码的蛋白进行理化性质分析,参照巴斯德毕赤酵母使用密码子偏好性,优化设计合成138 bp编码猪乳铁蛋白肽LFcinP基因,通过pGAPHαM酵母组成型分泌表达载体将其整合到巴斯德毕赤酵母GS115菌株,获得表达猪乳铁蛋白肽LFcinP基因重组酵母菌株GS115p/GAPHαM-LFcinP。SDS-PAGE和Westernblot分析表明,获得5.4 ku乳铁蛋白肽;分析96 h的蛋白电泳带结果表明,重组蛋白占酵母菌上清蛋白35.9%,表达量为48.5μg.mL-1;动态生长曲线法抑菌试验结果表明,重组猪乳铁蛋白肽具有一定的抑菌作用。     

鸡肾型传染性支气管炎病毒QD株S1基因的克隆与序列分析

One nephropathogenic infectious bronchitis virus （IBV） strain was isolated from Qingdao city, named as QD isolate.S1 gene of the strain was amplified, cloned and sequenced. The S1 gene of QD isolate was composed of 1620 nucleotides, and a spike glycoprotein cleavage recognition site was Arg-Arg-Phe-Arg-Arg. The nucleotide acid similarities among the nine IBV vaccine strains and the QD strain were 78.8% to 82.2%. Phylogenetic analysis based on the S1 genes showed that QD strain and the vaccine strains belonged to different clusters, and showed larger evolutionary distances, but showed the smaller evolutionary distances with the field nephropathogenic IBV strains in China. The result showed that nephropathogenic IBV strains were widely popular in China, and the QD strain could be used as the infectious bronchitis vaccine candidate strain.     

鸡传染性支气管炎病毒SD03株N基因的克隆、序列分析及N蛋白理化性质分析

试验旨在确定国内鸡肾型传染性支气管炎病毒(IBV)流行毒株核蛋白N与市场上现用疫苗株、近年国内外分离株的差异性.本研究利用自山东某鸡场病鸡肾脏组织分离获得、经PCR鉴定为IBV阳性的毒株(命名为SD03株),参考NCBI上部分IBV毒株N基因的保守区域设计引物,进行RT-PCR扩增,构建重组pMD18-T-N/DH5α菌,测序获得N基因序列.与参考株进行了核苷酸及氨基酸同源性分析,结合DNAStar软件与http://www.expasy.org/网站对该基因编码的蛋白进行理化性质分析.结果显示,SD03株为IBV肾型毒株,与LDT3、partridge/GD/S14/2003毒株(肾型)核苷酸及氨基酸同源性均在99.0%以上.与其他国内外肾型经典毒株Ma5、W93、Gray、Holte株氨基酸同源性为90.0%～94.6%,与国内外呼吸型疫苗株H120、H52、M41氨基酸同源性为89.7%～91.0%.本研究深入分析了当前流行株SD03株N蛋白的理化特性,为N蛋白亚单位疫苗在不同系统中表达及蛋白纯化提供了理论依据,为中国现阶段IBV的防控奠定了一定的理论基础.