罗非鱼源无乳链球菌兼职蛋白EF-Tu的克隆、表达及其抗原性检测  

Clone, prokaryotic expression and antigenicity detection of moonlighting protein EF-Tu of Streptococcus agalactiae isolated from tilapia

作　　者：刘家星[1,2,3] 汪开毓[1,2] 陈德芳[1,3] 刘韬[1,2] 贺扬[1,2] 曾宇鲲 蒋洁[1,2] 

机构地区：[1]四川农业大学鱼病研究中心,四川成都611130 [2]四川农业大学动物疫病与人类健康/四川省重点实验室,四川成都611130 [3]四川农业大学动物科技学院,四川成都611130

出　　处：《水产学报》2016年第3期334-343,共10页Journal of Fisheries of China

基　　金：长江学者和创新团队发展计划(IRT0848)~~

摘　　要：为检测罗非鱼源无乳链球菌兼职蛋白EF-Tu(延伸因子Tu,Elongation Factor Tu)的抗原性,本实验克隆了罗非鱼源无乳链球菌HN0303的EF-Tu基因序列,并进行了蛋白相关性质的预测和系统发育树的构建。通过原核表达得到EF-Tu重组蛋白,同时利用纯化的蛋白免疫家兔获得多克隆兔抗EF-Tu重组蛋白血清以用于EF-Tu蛋白抗原性检测。结果显示,罗非鱼源无乳链球菌HN0303 EF-Tu基因有1个由1197个碱基组成的ORF,编码398个氨基酸。生物信息学分析显示其分子式为C_(1933)H_(3096)N_(532)O_(615)S_(11),分子质量为43.981 ku,理论等电点为4.749;具有多个磷酸化位点,不具有信号肽和跨膜区域;具有保守的EFTu结构域、EF-Tu-II结构域和EF-Tu-Ⅲ结构域,且与其他来源无乳链球菌的EF-Tu蛋白具有很高的同源性;具有较高的抗原指数,表明其可形成多个抗原表位。SDS-PAGE检测发现,诱导表达的重组蛋白以包涵体的形式出现在沉淀中,大小约为66.4 ku。Western Blot分析表明,兔抗EF-Tu重组蛋白血清能分别特异性结合菌体蛋白和EF-Tu重组蛋白。同时使用兔抗EF-Tu重组蛋白血清封闭罗非鱼源无乳链球菌HN0303表面的EF-Tu蛋白后,无乳链球菌HN0303粘附EPC(Epithelioma papulosum cyprini,鲤鱼上皮细胞)的能力下降了79.99%±2.43%。本研究表明,原核表达的罗非鱼源无乳链球菌EF-Tu重组蛋白具备较好的抗原性,用其制备的兔抗血清能够较好地抑制罗非鱼源无乳链球菌的粘附,推测其可能为罗非鱼源无乳链球菌亚单位疫苗的候选蛋白。In order to detect the antigenicity of moonlighting protein EF-Tu（Elongation Factor Tu）, EF-Tu gene from Streptococcus agalactiae HN0303 isolated from tilapia was cloned. The related properties of EF-Tu protein were predicted and its phylogenetic tree was also constructed. The r EF-Tu protein was obtained by prokaryotic expression systems and purified by Ni-NTA-Sefinose Column. The purified r EF-Tu protein immunized rabbits were used to obtain the polyclonal rabbit anti-r EF-Tu sera for antigenicity detection. The results showed that EFTu gene had an ORF with 1197 bases, encoding 398 amino acids with a C_（1933）H_（3096）N_（532）O_（615）S_（11） formula, 43.981 ku molecular mass, and a 4.749 theoretical isoelectric point. Futhermore, the deduced amino acids comprised phosphorylation sites, but did not contain the transmembrane domain and signal peptide sequence. Three conserved domains, namely EF-Tu, EF-Tu-II and EF-Tu-III existed in the deduced amino acids via NCBI Conseverd Domains tool. Phylogenetic analysis revealed an exaggerated degree of homology with other S.agalactiae EF-Tu protein. Additionally, high antigen index of the deduced amino acids was predicted using DNAstar-Protean, which means it can form numerous epitopes. r EF-Tu proteins formed into inclusion bodies were found in the pellet and an about 66.4 ku band was observed by SDS-PAGE. Moreover, Western Blot analysis showed that rabbit anti-r EF-Tu sera can combine the tropina with recombinant protein specifically. Adhesion test suggested that rabbit anti-r EF-Tu sera prevented S. agalactiae HN0303 adhering the EPC（Epithelioma papulosum cyprini） with a decrease of 79.99%±2.43%. In this study, our results showed that r EF-Tu possesses nice antigenicity and the rabbit anti-r EF-Tu sera can inhibit the S. agalactiae HN0303 adhesion obviously, which suggested that the EF-Tu protein may become a subunit vaccine candidate against S. agalactiae in tilapia.

关 键 词：罗非鱼 无乳链球菌 EF-TU 克隆 原核表达 抗原性 

分 类 号：Q785[生物学—分子生物学] S917[农业科学—水产科学]