FpgMBV1-P4基因的原核表达及多克隆抗体制备  

Prokaryotic Expression of FpgMBV1-P4 Gene and Preparation of Its Polyclonal Antibodies

作　　者：刘东伟 宋嘉庆 潘鑫 闫书味 李轲 范沛[3] 高飞[1] 张晓婷[1] 代君丽[1] 李洪连[1,2] LIU Dong-Wei;SONG Jia-Qing;PAN Xin;YAN Shu-Wei;LI Ke;FAN Pei;GAO Fei;ZHANG Xiao-Ting;DAI Jun-Li;LI Hong-Lian(College of Plant Protection,Henan Agricultural University,Zhengzhou 450002,China;State Key Laboratory of Wheat and Maize Crop Science,Zhengzhou 450046,China;Colledge of Biology Engineering,Henan University of Technology,Zhengzhou 450001,China)

机构地区：[1]河南农业大学植物保护学院,郑州450002 [2]小麦玉米作物学国家重点实验室,郑州450046 [3]河南工业大学生物工程学院,郑州450001

出　　处：《农业生物技术学报》2022年第11期2246-2254,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金国际(地区)合作与交流项目(31961143018);河南省科技攻关计划(农业领域)项目(212102110138);2018年度河南省高等学校青年骨干教师培养计划(2018GGJS032);河南农业大学科技创新基金(KJCX2020A14)。

摘　　要：假禾谷镰孢巨型双链RNA病毒1 (Fusarium pseudograminearum megabirnavirus 1, FpgMBV1)是从假禾谷镰孢(Fusarium pseudograminearum)弱毒菌株FC136-2A中发现的一种真菌病毒。FpgMBV1共编码4个蛋白质,其中FpgMBV1-P4由268个氨基酸残基组成,功能未知。本研究设计特异性引物,采用反转录PCR (reverse transcription PCR, RT-PCR)方法从菌株FC136-2A的菌丝中扩增FpgMBV1-P4基因,构建原核表达载体pGEX4T-FpgMBV1-P4并转化大肠杆菌(Escherichia coli) BL21(DE3)菌株,在25℃以0.5mmol/L异丙基硫代半乳糖苷(isopropyl β-D-thiogalactoside, IPTG)诱导表达重组蛋白。结果表明,FpgMBV1-P4基因长为807 bp,重组表达的融合蛋白分子量约为60 kD,与预期大小相符。将该重组蛋白作为抗原免疫新西兰大白兔(Lepus sinensis),获得抗血清,采用间接ELISA测定其效价达1∶160 000,能够特异性检测到假禾谷镰孢菌丝中约36 kD的FpgMBV1-P4蛋白。研究结果为深入分析FpgMBV1-P4的结构和功能及进一步探讨FpgMBV1与假禾谷镰孢菌的相互作用提供了基础。Fusarium pseudograminearum megabirnavirus 1(FpgMBV1) is a mycovirus found in the hypovirulence Fusarium pseudograminearum strain FC136-2A.Four proteins were encoded in FpgMBV1.Among them,FpgMBV1-P4 composed of 268 amino acids with unknown function.Specific primers were designed and used in reverse transcription PCR(RT-PCR) to amplify the FpgMBV1-P4 gene from the hyphae of the strain FC136-2A in this study.Then a prokaryotic expression vector pGEX4T-FpgMBV1-P4 was constructed and transformed into Escherichia coli BL21(DE3) strain,and the recombinant protein was induced at 25 ℃ with 0.5 mmol/L isopropyl β-D-thiogalactoside(IPTG).Results showed that FpgMBV1-P4 in length of 807 bp was successfully amplified and the fusion protein with a molecular weight of about 60 kD was highly expressed.The purified fusion protein was used to immunize Zealand white rabbit(Lepus sinensis) to obtain antiserum.Indirect ELISA results showed the titer of this antiserum reached 1∶160 000.And the antiserum was used to specifically detect the FpgMBV1-P4 protein at about 36 kD from samples of the hyphae of FC136-2A.These results laid a foundation for the structural and functional analysis of FpgMBV1-P4,which would be meaningful in deciphering the hypovirulent mechanism of FpgMBV1.

关 键 词：真菌病毒 假禾谷镰孢巨型双链RNA病毒1(FpgMBV1) P4蛋白 原核表达 多克隆抗体 

分 类 号：S432.1[农业科学—植物病理学]