摘要
试验旨在克隆鸭疫里默氏杆菌协同溶血素蛋白(cooperative hemolysin protein,CAMP)基因,并对其进行原核表达和生物信息学分析。以RA贵州血清2型分离株RA-SS-8基因组为模板扩增并克隆鸭疫里默氏杆菌CAMP基因,构建pET-28a-CAMP重组原核表达质粒,转化大肠杆菌Rosetta(DE3)感受态细胞,重组菌用IPTG进行诱导表达及纯化,利用SDS-PAGE和Western blotting分析表达蛋白的特征,并运用相关生物信息学分析软件对CAMP基因进行分析。结果显示,鸭疫里默氏杆菌CAMP基因大小为1026 bp,该基因序列与GenBank中公布的10株RA参考菌株(如RA-LZ01(CP045564.1))CAMP基因的相似性达99.7%。经BamHⅠ和XhoⅠ双酶切鉴定后,获得大小约5369和1026 bp的基因片段,表明pET-28a-CAMP重组表达质粒构建成功。SDS-PAGE和Western blotting分析显示,表达的重组蛋白大小约为37 ku,以可溶形式进行表达,且能与His-tag单克隆抗体在37 ku处产生特异性反应,与预期结果相符。生物信息学分析表明,CAMP蛋白分子式为C_(1670)H_(2659)N_(437)O_(500)S_(12),含341个氨基酸,理论等电点(pI)为5.62,不稳定系数为40.71,表明其为不稳定的弱酸性蛋白。疏水性预测结果显示,该蛋白属于亲水性蛋白。CAMP蛋白无跨膜结构域,无信号肽,有3个O-糖基化位点,无N-糖基化位点,存在34个磷酸化位点,共有17个抗原表位。二级结构和三级结构预测结果显示,CAMP蛋白以α-螺旋和无规则卷曲为主。本试验结果为进一步研究鸭疫里默氏杆菌CAMP蛋白的功能及鸭疫里默氏杆菌病疫苗的研发提供了参考。
The aim of this study was to clone the cooperative hemolysin protein(CAMP)gene of Riemerella anatipestifer,and analyze its prokaryotic expression and bioinformatics.CAMP gene was amplified and cloned from RA Guizhou serotype 2 strain(RA-SS-8),and the recombinant prokaryotic expression plasmid pET-28a-CAMP was constructed.The plasmid was transformed into Escherichia coli Rosetta(DE3)competent cells.After identification,the recombinant bacteria were induced to express and purified with IPTG.SDS-PAGE and Western blotting were used to analyze the characteristics of the expressed protein.CAMP gene was analyzed by bioinformatics analysis software.The results showed that the size of CAMP gene was 1026 bp,and the sequence of CAMP gene was 99.7%simarility with 10 RA reference strains published in GenBank,such as RA-LZ01(CP045564.1).By BamHⅠand XhoⅠdouble digestion,the size of 5369 and 1026 bp fragments were obtained,indicating that the pET-28a-CAMP recombinant plasmid was successfully constructed.SDS-PAGE and Western blotting analysis results showed that the expressed recombinant protein was about 37 ku in soluble form,and could react with His-tag monoclonal antibody at 37 ku,which was consistent with the expected results.Bioinformatics analysis results showed that the molecular formula of CAMP was C_(1670)H_(2659)N_(437)O_(500)S_(12),containing 341 amino acids,the theoretical isoelectric point(pI)was 5.62,and the coefficient of instability was 40.71,indicating that CAMP was an unstable and weakly acidic protein.The results of hydrophobicity prediction showed that CAMP protein was hydrophilic.CAMP protein had no transmembrane domain,no signal peptide,3 O-glycoylation sites,no N-glycoylation sites,34 phosphorylation sites and a total of 17 epitopes.The secondary and tertiary structures showed that the protein was dominated by alpha helix and random coil.The above results might provide reference for further study on the function of CAMP protein and the development of Riemerella anatipestifer vaccine.
作者
梅世慧
陈国权
阎朝华
王娜
周碧君
程振涛
王开功
文明
MEI Shihui;CHEN Guoquan;YAN Chaohua;WANG Na;ZHOU Bijun;CHENG Zhentao;WANG Kaigong;WEN Ming(College of Animal Science,Guizhou University,Guiyang 550025,China;Guizhou Provincial Animal Disease Research Laboratory,Guiyang 550025,China;Guizhou Province Animal Biological Products Engineering Technology Research Center,Guiyang 550025,China)
出处
《中国畜牧兽医》
CAS
北大核心
2021年第12期4348-4360,共13页
China Animal Husbandry & Veterinary Medicine
基金
三穗鸭工程技术研究中心建设项目(黔科合平台人才[2019]5203号)
贵州省科技平台及人才团队计划项目(黔科合平台人才[2018]5253号)
贵州省研究生教育创新计划项目(GZZ2017002)。
