| 柔嫩艾美耳球虫抗药性相关基因M20的克隆与表达 |
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| 引用本文： | 程军,董辉,郭涛,韩红玉,姜连连,赵其平,朱顺海,马卫娇,曾艳波,孔春林,李婷,黄兵. 柔嫩艾美耳球虫抗药性相关基因M20的克隆与表达[J]. 中国兽医寄生虫病, 2011, 19(1): 62-67 |
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| 作者姓名： | 程军 董辉 郭涛 韩红玉 姜连连 赵其平 朱顺海 马卫娇 曾艳波 孔春林 李婷 黄兵 |
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| 作者单位： | 1. 上海师范大学生命与环境科学学院,上海,200234;中国农业科学院,上海兽医研究所农业部动物寄生虫学重点开放实验室,上海,200241
2. 中国农业科学院,上海兽医研究所农业部动物寄生虫学重点开放实验室,上海,200241 |
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| 基金项目： | 国家自然科学基金项目，家畜疫病病原生物学国家重点实验室开放基金课题 |
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| 摘 要： | 利用前期构建的柔嫩艾美耳球虫（Eimeria tenella）地克珠利抗药株和马杜拉霉素抗药株与各自母株之间的抑制性消减文库而获得的ESTs序列,选择其中一个差异表达的EST序列（克隆号为M20）,采用RACE技术,以柔嫩艾美耳球虫敏感株孢子化卵囊cDNA为模板,扩增获得了该基因的全长cDNA序列,命名为M20,该基因全长847 bp,包括一个525 bp的开放阅读框,编码174个氨基酸,预测理论分子量约为19 ku。实时荧光定量PCR结果显示,该基因在敏感株中表达量高于地克珠利抗药株和马杜拉霉素抗药株;在不同发育阶段中,未孢子化卵囊表达量高于孢子化卵囊、子孢子以及裂殖子阶段虫体。将该基因克隆于原核表达质粒pET28b中,构建重组质粒pET28b-M20,转化大肠杆菌BL21（DE3）后,经IPTG诱导表达获得了重组蛋白,分子量约约为23 ku,该蛋白以包涵体形式存在,在IPTG诱导8 h后表达稳定,Western blot检测表明,其具有较好的免疫原性。
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| 关 键 词： | 柔嫩艾美耳球虫 抗药性 地克珠利 马杜拉霉素 实时荧光定量PCR |
CLONING AND EXPRESSION OF M20RELATED TO EIMERIA TENELLA DRUG-RESISTANCE |
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| CHENG Jun,DONG Hui,GUO Tao,HAN Hong-yu,JIANG Lian-lian,ZHAO Qi-ping,ZHU Shun-hai,MA Wei-jiao,ZENG Yan-bo,KONG Chun-lin,LI Ting,HUANG Bing. CLONING AND EXPRESSION OF M20RELATED TO EIMERIA TENELLA DRUG-RESISTANCE[J]. Chinese Journal of Veterinary Parasitology, 2011, 19(1): 62-67 |
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| Authors: | CHENG Jun DONG Hui GUO Tao HAN Hong-yu JIANG Lian-lian ZHAO Qi-ping ZHU Shun-hai MA Wei-jiao ZENG Yan-bo KONG Chun-lin LI Ting HUANG Bing |
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| Affiliation: | CHENG Jun1,2,DONG Hui2,GUO Tao1,HAN Hong-yu2,JIANG Lian-lian2,ZHAO Qi-ping2,ZHU Shun-hai2,MA Wei-jiao1,ZENG Yan-bo2,KONG Chun-lin1,LI Ting2,HUANG Bing2 (1.College of Life and Environment Sciences,Shanghai Normal University,Shanghai 200234,2.Shanghai Veterinary Research Institute,CAAS,Key Laboratory of Animal Parasitology of Ministry of Agriculture,Shanghai 200241) |
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| Abstract: | A number of ESTs sequences were obtained by suppression subtractive hybridization(SSH) of Eimeria tenella between drug-resistant strains and their parental strains.From one of ESTs(Clone No.M20),total RNA of the sensitive strain was isolated,and then the full-length cDNA sequence of gene was cloned by RACE.The length of gene is 847 bp,containing a 525 bp open reading frame(ORF),encoding a deduced protein of 174 amino acids(19 ku).Real-time PCR revealed that the gene transcri-ption level in the sensitive str... |
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| Keywords: | Eimeria tenella drug-resistance Diclazuril Maduramisin real-time PCR |
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