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。
【Objective】This study was conducted to analyze the molecular biological characteristics of gene AS87-01740in Riemerella anatipestifer and determine the antigenicity of its encoding protein in order to provide new target antigens for research and development of R.anatipestifer subunit vaccine.【Method】Gene AS87-01740in 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 AS87-01740obtained from R.anatipestifer genome amplification contained an open reading frame of618bp in total length,encoded205amino acids,and its theoretical molecular weight was21.87kD,theoretical isoelectric point(pI)8.2,total positively charged amino acids17,and total negatively charged amino acids16.Among isolated strains of the same species,AS87-01740protein sequence had extremely high similarity with serotype2isolates(11845,YM,GD and CH-2)(>99%),and92%similarity with serotype1isolates(CH-1and CH-3).However,in different species,its similarity with Chryseobacterium sp.,Bergeyella zoohelcum and Elizabethkingia meningosepticu was61%,60%and57%respectively.The molecular weight of fusion protein obtained by isopropylβ-D-thiogalactoside(IPTG)induction was77kD,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 AS87-01740is 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
作者
单敏
李涛
何晶
李文海
王小兰
罗廷荣
于圣青
SHAN Min;LI Tao;HE Jing;LIWeng-hai;WANG Xiao-lan;LUO Rong-ting;YU Sheng-qing(College of Animal Science and Technology,Guangxi University,Nanning 530004,China;Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2017年第11期2058-2063,共6页
Journal of Southern Agriculture
基金
国家自然科学基金项目(31272591)
上海市科技兴农计划项目(2016HNG4-1)
中央级公益性科研院所科研基金项目(2017JB12)
