| 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 |
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| 引用本文： | 刘家星,汪开毓,陈德芳,刘韬,贺扬,曾宇鲲,蒋洁.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[J].水产学报,2016,40(3):334-343. |
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| 作者姓名： | 刘家星 汪开毓 陈德芳 刘韬 贺扬 曾宇鲲 蒋洁 |
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| 作者单位： | 1. 四川农业大学鱼病研究中心,四川成都611130;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;四川农业大学动物科技学院,四川成都611130;2. 四川农业大学鱼病研究中心,四川成都611130;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. 四川农业大学鱼病研究中心,四川成都611130;四川农业大学动物科技学院,四川成都611130 |
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| 基金项目： | 国家自然科学基金(31502208);国家海洋公益性行业科研专项(201205023);现代农业产业技术体系建设专项(CARS-48);山东省泰山学者建设工程专项;青岛海洋科学与技术国家实验室鳌山科技创新计划(2015ASKJ01) |
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| 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。
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| 关 键 词： | 罗非鱼 无乳链球菌 EF-Tu 克隆 原核表达 抗原性 |
| 收稿时间： | 2015/12/3 0:00:00 |
| 修稿时间： | 2016/1/16 0:00:00 |
Clone, prokaryotic expression and antigenicity detection of moonlighting protein EF-Tu of Streptococcus agalactiae isolated from tilapia |
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| LIU Jiaxing,WANG Kaiyu,CHEN Defang,LIU Tao,HE Yang,ZENG Yukun and JIANG Jie.Clone, prokaryotic expression and antigenicity detection of moonlighting protein EF-Tu of Streptococcus agalactiae isolated from tilapia[J].Journal of Fisheries of China,2016,40(3):334-343. |
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| Authors: | LIU Jiaxing WANG Kaiyu CHEN Defang LIU Tao HE Yang ZENG Yukun and JIANG Jie |
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| Institution: | Sichuan Agricultural University,Fish Disease Research Center,Sichuan Agricultural University,Fish Disease Research Center,Sichuan Agricultural University,Fish Disease Research Center,Sichuan Agricultural University,Fish Disease Research Center,Sichuan Agricultural University,Fish Disease Research Center,Sichuan Agricultural University,Fish Disease Research Center,Sichuan Agricultural University,Fish Disease Research Center |
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| Abstract: | 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. rEF-Tu protein was obtained by prokaryotic expression systems and purified by Ni-NTA-Sefinose Column. The purified rEF-Tu protein immunized rabbits to obtain the polyclonal rabbit anti-rEF-Tu sera for antigenicity detection. The results showed that EF-Tu gene had an ORF with 1 197 bases, encoding 398 amino acids with a C1933H3096N532O615S11 formula, 43.981 ku molecular mass, and a 4.749 theoretical isoelectric poin. Futhermore, the deduced amino acids comprised phosphorylation sites, while do not contain the transmembrane domain and signal peptide sequence. Three conserved domains, namely EF-Tu, EF-Tu-II and EF-Tu-III exsited in the deduced amino acids via NCBI Conseverd Domains tool. Phylogenetic analysis had revealed a 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. rEF-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-rEF-Tu sera can combine both with the tropina and recombinant protein specifically. Adhesion test suggested that rabbit anti-rEF-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 rEF-Tu possesses nice antigenicity and the rabbit anti-rEF-Tu sera can inhibit the S. agalactiae HN0303 adhesion obviously, which suggested that the EF-Tu protein may be a subunit vaccine candidate against S. agalactiae in tilapia. |
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| Keywords: | Tilapia Streptococcus agalactiae EF-Tu Clone prokaryotic expression antigenicity |
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