[1]郭长明,袁橙,张步彩,等.柔嫩艾美耳球虫烯醇化酶基因的克隆表达和抗原性分析[J].江苏农业学报,2017,(03):642-648.[doi:doi:10.3969/j.issn.1000-4440.2017.03.023]
　GUO Chang-ming,YUAN Cheng,ZHANG Bu-cai,et al.Cloning, expression and antigenic analysis of enolase gene of Eimeria tenella[J].,2017,(03):642-648.[doi:doi:10.3969/j.issn.1000-4440.2017.03.023]
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柔嫩艾美耳球虫烯醇化酶基因的克隆表达和抗原性分析()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2017年03期
页码:
642-648
栏目:
畜牧兽医·水产养殖
出版日期:
2017-06-30

文章信息/Info

Title:
Cloning, expression and antigenic analysis of enolase gene of Eimeria tenella
作者:
郭长明1;2; 袁橙1;2; 张步彩2; 朱善元2; 严若峰1; 徐立新1; 宋小凯1; 李祥瑞1
（1.南京农业大学动物医学院，江苏南京210095；2.江苏农牧科技职业学院/江苏省兽用生物制药高技术研究重点实验室，江苏泰州225300）
Author(s):
GUO Chang-ming1;2; YUAN Cheng1;2; ZHANG Bu-cai2; ZHU Shan-yuan2; YAN Ruo-feng1; XU Li-xin1; SONG Xiao-kai1; LI Xiang-rui1
(1.College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China；2.Jiangsu Agri-animal Husbandry Vocational College/Jiangsu Provincial Veterinary Bio-pharmaceutical High-tech Key Laboratory, Taizhou 225300, China)
关键词:
柔嫩艾美耳球虫; 烯醇化酶; 克隆; 表达; 免疫原性
Keywords:
Eimeria tenella; enolase; cloning; expression; immunogenicity
分类号:
S858.312.72+3
DOI:
doi:10.3969/j.issn.1000-4440.2017.03.023
文献标志码:
A
摘要:
从纯化的柔嫩艾美耳球虫第二代裂殖子中提取总RNA，应用RT-PCR技术克隆获得了烯醇化酶基因（GenBank Accession No. GU169333）。烯醇化酶基因开放阅读框长1 338 bp，编码445个氨基酸。应用DNAstar软件对氨基酸序列的亲水区域和疏水区域的分布、抗原指数及T细胞基序进行分析，结果显示该蛋白质亲水性较强，抗原指数高，含较多T细胞表位。同时构建了烯醇化酶原核表达重组质粒pET-28a(+)-ENO。用IPTG对重组质粒pET-28a（+）-ENO进行诱导表达，SDS-PAGE结果显示表达的融合蛋白大小约为5.17×104，主要存在于裂解上清中。Western blotting结果显示该重组蛋白可被抗柔嫩艾美耳球虫的多克隆抗体识别，表明该蛋白质具有较好的免疫原性。
Abstract:
Enolase gene of Eimeria tenella (GenBank Accession No. GU169333) (EtENO) was amplified from total RNA extracted from E. tenella second-generation merozoites by RT-PCR. The sequence of EtENO contains an open reading frame of 1 338 bp and encodes 445 amino acid. DNAstar software analysis showed that EtENO has high antigenic index, lots of hydrophilicity plots and T cell motif. The recombinant plasmid pET-28a(+)-ENO was constructed and transformed into Escherichia coli BL21(DE3) for expression. SDS-PAGE indicated that the fusion protein (5.17×104) was expressed in the supernatant after induction by IPTG. Western blotting revealed that the protein was specifically recognized by polyclonal antibodies against E. tenella, suggesting that the fusion protein is antigenic.

参考文献/References:

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备注/Memo

备注/Memo:
收稿日期：2016-12-27 基金项目：国家自然科学基金国际(地区)合作与交流项目（NSFC-PSF, 中巴）（31661143017）；国家自然科学基金面上项目（31372428、31672545）；江苏高校优势学科建设工程资助项目（PAPD） 作者简介：郭长明（1984-），男，山东临朐人，博士，讲师，主要从事预防兽医学（寄生虫分子免疫和病原微生物方向）研究。(E-mail)gcmscience@126.com 通讯作者：李祥瑞，(E-mail)lixiangrui@njau.edu.cn
更新日期/Last Update: 2017-06-29