鸭干扰素刺激基因的克隆、表达及其多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2020.11.035

中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (11): 3713-3721.doi: 10.16431/j.cnki.1671-7236.2020.11.035

鸭干扰素刺激基因的克隆、表达及其多克隆抗体的制备

黄惠兰, 李文俊, 谢梓民, 杨惠湖, 崔惠仪, 黄淑坚, 张雪莲

佛山科学技术学院生命科学与工程学院, 佛山 528231

收稿日期:2020-06-09 出版日期:2020-11-20 发布日期:2020-11-20
通讯作者: 黄淑坚, 张雪莲 E-mail:sjhuang.foshan@163.com;zxlsjw0312@163.com
作者简介:黄惠兰(1996-),女,广东梅州人,硕士,研究方向:水禽疫病防控,E-mail:1785334697@qq.com
基金资助:
广东省教育厅青年创新人才项目（2018KQNCX277）；佛山科学技术学院动物新发疫病防控重点实验室"开放基金"项目（KLPREAD201801-09、KLPREAD201801-08）；广东省现代农业产业技术体系创新团队（2019KJ137）；广东省教育厅预防兽医学重点实验室项目（2014KTSPT037）；广东省教育厅普通高校重点领域专项（乡村振兴）（2020ZDZX1004）

Molecular Cloning and Expression of Duck IFIT5 Gene and Preparation of Its Polyclonal Antibodies

HUANG Huilan, LI Wenjun, XIE Zimin, YANG Huihu, CUI Huiyi, HUANG Shujian, ZHANG Xuelian

College of Life Science and Engineering, Foshan University, Foshan 528231, China

Received:2020-06-09 Online:2020-11-20 Published:2020-11-20

摘要/Abstract

摘要： 本研究旨在克隆、表达鸭干扰素刺激基因（interferon-induced proteins with tetratricopeptide repeats 5，IFIT5）并制备其多克隆抗体。根据GenBank公布的绿头鸭IFIT5（登录号：KF956064）序列设计特异性引物，PCR扩增获得鸭IFIT5基因，并构建原核表达重组质粒pET-30a-IFIT5和pET-32a-IFIT5及真核表达重组质粒pcDNA3.1-IFIT5。原核表达获得鸭IFIT5重组蛋白后，进行SDS-PAGE分析，并利用镍柱亲和层析方法对重组蛋白进行纯化。以纯化的鸭IFIT5重组蛋白为免疫原制备兔抗鸭IFIT5多克隆抗体。利用间接ELISA测定多克隆抗体效价、用Western blotting鉴定其特异性；将真核表达重组质粒pcDNA3.1-IFIT5转化BHK21细胞，通过间接免疫荧光法鉴定多克隆抗体的反应性。结果表明：目的蛋白主要以包涵体的形式存在；制备的兔抗鸭IFIT5多克隆抗体效价达1：49 600，可特异性识别鸭IFIT5重组蛋白，间接免疫荧光试验则表明纯化的多克隆抗体可特异性识别BHK21瞬时真核表达的鸭IFIT5蛋白。综上，成功克隆了鸭干扰素刺激基因IFIT5，制备的兔抗鸭IFIT5多克隆抗体可用于鸭IFIT5蛋白的检测，可为鸭IFIT5蛋白的后续研究提供材料支撑。

关键词: 鸭; 干扰素刺激基因(IFIT5); 基因克隆; 蛋白表达及检测

Abstract: The aim of this study was to clone the duck interferon-induced protein with tetratricopeptide repeats 5(IFIT5) gene,express duck IFIT5 protein and prepare the polyclonal antibodies against the duck IFIT5 protein.Primers specific for duck IFIT5 gene were designed according to Mallard duck reference sequence(accession No.:KF956064)available in GenBank.The complete ORF of duck IFIT5 gene was amplified by PCR.Recombinant prokaryotic expression plasmids pET-30a-IFIT5,pET-32a-IFIT5 and eukaryotic expression plasmid pcDNA3.1-IFIT5 were constructed.Recombinant duck IFIT5 proteins were expressed and analyzed by SDS-PAGE.Recombinant duck IFIT5 proteins were purified by Ni-NTA column affinity chromatography.Rabbit anti-duck IFIT5 polyclonal antibodies were prepared from rabbits which had immunized by recombinant protein pET-30a-IFIT5.The titer of polyclonal antibodies was detected by indirect ELISA,and the specificity was identified by Western blotting.Eukaryotic expression recombinant plasmid pcDNA3.1-IFIT5 was transfected into BHK21 cells and the reactivity of polyclonal antibodies was verified by indirect immunoinfluscence assay.The results showed that recombinant duck IFIT5 proteins were expressed in the form of inclusion bodies.The titer of polyclonal antibodies was approximately up to 1:49 600.Rabbit anti-duck IFIT5 polyclonal antibodies could identify recombinant protein pET-32a-IFIT5.Indirect immunofluorescence assay (IFA) confirmed that the purified polyclonal antibodies could identify duck IFIT5 protein by eukaryotic expression system specifically.These results demonstrated that the duck interferon stimulating gene IFIT5 was cloned successfully,rabbit anti-duck IFIT5 polyclonal antibodies could be used in the detection of duck IFIT5 protein,and also provided material support for the further study of duck IFIT5 protein.

Key words: duck; interferon-induced proteins with tetratricopeptide repeats 5(IFIT5); gene cloning; protein expression and detection

中图分类号:

S834

黄惠兰, 李文俊, 谢梓民, 杨惠湖, 崔惠仪, 黄淑坚, 张雪莲. 鸭干扰素刺激基因的克隆、表达及其多克隆抗体的制备[J]. 中国畜牧兽医, 2020, 47(11): 3713-3721.

HUANG Huilan, LI Wenjun, XIE Zimin, YANG Huihu, CUI Huiyi, HUANG Shujian, ZHANG Xuelian. Molecular Cloning and Expression of Duck IFIT5 Gene and Preparation of Its Polyclonal Antibodies[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(11): 3713-3721.

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鸭干扰素刺激基因的克隆、表达及其多克隆抗体的制备

Molecular Cloning and Expression of Duck IFIT5 Gene and Preparation of Its Polyclonal Antibodies

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