| 鸭疫里默氏杆菌AS87-01740基因生物信息学分析及其原核表达 |
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| 引用本文： | 单敏,李涛,何晶,李文海,王小兰,罗荣廷,于圣青.鸭疫里默氏杆菌AS87-01740基因生物信息学分析及其原核表达[J].南方农业学报,2017,48(11). |
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| 作者姓名： | 单敏 李涛 何晶 李文海 王小兰 罗荣廷 于圣青 |
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| 作者单位： | 1. 广西大学 动物科学技术学院,南宁 530004;中国农业科学院 上海兽医研究所,上海 200241;2. 中国农业科学院 上海兽医研究所,上海,200241;3. 广西大学 动物科学技术学院,南宁,530004 |
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| 基金项目： | 国家自然科学基金项目，上海市科技兴农计划项目，中央级公益性科研院所科研基金项目 |
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| 摘 要： | 【目的】了解鸭疫里默氏杆菌AS87-01740基因分子生物学特性,确定其编码蛋白抗原性,为鸭疫里默氏杆菌亚单位疫苗研发提供新的靶标抗原选择。【方法】采用PCR克隆鸭疫里默氏杆菌AS87-01740基因,以DNASTAR和NCBI数据库分别进行基因生物信息学分析;利用p Cold-TF原核表达载体在大肠杆菌BL21感受态细胞中进行诱导表达,并分别以SDS-PAGE和Western blotting检测融合蛋白的表达形式及其抗原特异性。【结果】从鸭疫里默氏杆菌基因组扩增获得的AS87-01740基因编码框全长618 bp,编码205个氨基酸,理论分子量约21.87 kD,理论等电点(p I)8.2,正电荷氨基酸总数17个,负电荷氨基酸总数16个。AS87-01740蛋白序列在同种属分离株中,与2型血清型分离株(11845、YM、GD和CH-2)有很高的相似性(99%),与1型血清型分离株(CH-1和CH-3)的相似性为92%;在不同种属中,与金黄杆菌(Chryseobacterium sp.)、动物溃疡伯格菌(Bergeyella zoohelcum)和脑膜炎败血伊丽莎白菌(Elizabethkingia meningosepticum)的相似性分别为61%、60%和57%。经异丙基硫代-β-D半乳糖苷(IPTG)诱导表达获得的融合蛋白分子量约77 kD,以可溶性表达形式为主。Western blotting检测结果表明,融合蛋白能与鸭疫里默氏杆菌阳性血清发生特异性免疫反应。【结论】AS87-01740基因编码蛋白在不同鸭疫里默氏杆菌分离株中具有很高的保守性,且能与其阳性血清发生特异性免疫反应,是研发鸭疫里默氏杆菌疫苗的潜在抗原。
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| 关 键 词： | 鸭疫里默氏杆菌 AS87-01740基因 生物信息学 原核表达 免疫原性 |
Bioinformatics analysis for gene AS87-01740 in Riemerella anatipestifer and its prokaryotic expression |
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| SHAN Min,LI Tao,HE Jing,LI Weng-hai,WANG Xiao-lan,LUO Rong-ting,YU Sheng-qing.Bioinformatics analysis for gene AS87-01740 in Riemerella anatipestifer and its prokaryotic expression[J].Journal of Southern Agriculture,2017,48(11). |
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| Authors: | SHAN Min LI Tao HE Jing LI Weng-hai WANG Xiao-lan LUO Rong-ting YU Sheng-qing |
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| Abstract: | Objective]This study was conducted to analyze the molecular biological characteristics of gene AS87-01740 in Riemerella anatipestifer and determine the antigenicity of its encoding protein in order to provide new target an-tigens for research and development of R. anatipestifer subunit vaccine.Method]Gene AS87-01740 in R. anatipestifer was cloned by PCR,and gene bioinformatics analysis was carried out based on DNASTAR and NCBI database. pCold-TF prokaryotic expression vector was used to induce expression in competent cells of Escherichia coli BL21. Meanwhile,the expression form and antigen specificity of fusion protein were detected by SDS-PAGE and Western blotting respectively.Result]Gene AS87-01740 obtained from R. anatipestifer genome amplification contained an open reading frame of 618 bp in total length,encoded 205 amino acids,and its theoretical molecular weight was 21.87 kD,theoretical isoelectric point (pI)8.2,total positively charged amino acids 17,and total negatively charged amino acids 16. Among isolated strains of the same species,AS87-01740 protein sequence had extremely high similarity with serotype 2 isolates(11845,YM,GD and CH-2)(>99%),and 92%similarity with serotype 1 isolates(CH-1 and CH-3). However,in different species,its similarity with Chryseobacterium sp.,Bergeyella zoohelcum and Elizabethkingia meningosepticu was 61%,60%and 57%respectively. The molecular weight of fusion protein obtained by isopropyl β-D-thiogalactoside(IPTG) induction was77 kD ,which was mainly expressed in soluble form. The results of Western blotting revealed that specific immune response could be observed between fusion protein and R. anatipestifer positive serum.Conclusion]Encoding protein of gene AS87-01740 is highly conserved in different R. anatipestifer isolated strains ,and can trigger specific immune response to R. anatipestifer positive serum. It is a potential antigen for R. anatipestifer vaccine research and development. |
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| Keywords: | Riemerella anatipestifer gene AS87-01740 bioinformatics analysis prokaryotic expression immuno-genicity |
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