| 牛IRF7蛋白的原核表达及多克隆抗体的制备 |
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| 引用本文： | 张江伟,张超,何金科,席静,孙天浩,何延华,陈创夫.牛IRF7蛋白的原核表达及多克隆抗体的制备[J].中国畜牧兽医,2021,48(10):3812-3822. |
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| 作者姓名： | 张江伟 张超 何金科 席静 孙天浩 何延华 陈创夫 |
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| 作者单位： | 1. 石河子大学动物科技学院, 石河子 832000;2. 人兽共患传染性疾病防治协同创新中心, 石河子 832000;3. 新疆第二医学院, 克拉玛依 834000;4. 新疆农垦科学院, 省部共建绵羊遗传改良与健康养殖国家重点实验室, 石河子 832000 |
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| 基金项目： | 西部地区高发人兽共患传染性疾病防治协同创新项目（2013-179） |
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| 摘 要： | 本研究旨在通过原核表达系统表达牛干扰素调节因子7（interferon regulatory factor 7，IRF7）蛋白，并对其进行纯化，进而制备高纯度的牛IRF7兔多克隆抗体。使用生物信息学软件预测并分析牛IRF7潜在的生物学功能，参考GenBank已公布的牛IRF7基因序列（登录号：NM_001105040.1）设计引物，利用PCR技术从牛病毒性腹泻病毒（Bovine viral diarrhea virus，BVDV）感染的MDBK细胞中扩增牛IRF7基因，连接至pMD19-T克隆载体，提取质粒连接至原核表达载体pET-28a （+），转化大肠杆菌DH5α感受态细胞，构建重组质粒pET-28a-IRF7。将重组质粒pET-28a-IRF7转化大肠杆菌BL21（DE3）感受态细胞，经IPTG诱导后，通过SDS-PAGE进行表达产物的分析与鉴定。通过Western blotting鉴定多克隆抗体的特异性，间接ELISA测定兔抗牛IRF7多克隆抗体效价，经BVDV感染细胞后，实时荧光定量PCR检测抗病毒因子IRF7的表达。生物信息学分析发现，IRF7蛋白不存在跨膜结构域，无信号肽，存在较多磷酸化位点，二级结构主要以无规则卷曲为主，与三级结构的三维模型一致，说明该蛋白可能存在很好的抗原潜力。PCR成功扩增出大小为1 497 bp的IRF7基因片段，成功表达牛IRF7蛋白，分子质量约为60 ku，间接ELISA测得兔抗牛IRF7多克隆抗体效价为1∶128 000，并可与牛IRF7蛋白发生免疫反应，感染BVDV毒株的MDBK细胞中IRF7表达量下降；BVDV感染过表达IRF7后的细胞发现，IRF7能显著促进干扰素-β（IFN-β）的表达（P<0.05）。综上，本研究成功表达纯化了牛IRF7蛋白，并制备兔抗牛IRF7多克隆抗体，为阐明牛IRF7在先天免疫抗病毒应答的分子机制提供了材料。
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| 关 键 词： | 牛 IRF7 蛋白纯化 多克隆抗体 |
| 收稿时间： | 2021-04-02 |
Prokaryotic Expression of Bovine IRF7 Protein and Preparation of Polyclonal Antibody |
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| ZHANG Jiangwei,ZHANG Chao,HE Jinke,XI Jing,SUN Tianhao,HE Yanhua,CHEN Chuangfu.Prokaryotic Expression of Bovine IRF7 Protein and Preparation of Polyclonal Antibody[J].China Animal Husbandry & Veterinary Medicine,2021,48(10):3812-3822. |
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| Authors: | ZHANG Jiangwei ZHANG Chao HE Jinke XI Jing SUN Tianhao HE Yanhua CHEN Chuangfu |
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| Institution: | 1. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China;2. Collaborative Innovation Center for Prevention and Control of Zoonotic Infectious Diseases, Shihezi 832000, China;3. Xinjiang Second Medical College, Karamay 834000, China;4. State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding, Xinjiang Academy of Agricultural Reclamation Sciences, Shihezi 832000, China |
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| Abstract: | The purpose of this study was to express bovine interferon regulatory factor 7 (IRF7) protein by prokaryotic expression system, and purify the protein, so as to prepare high purity bovine IRF7 rabbit polyclonal antibody. Bioinformatics software was used to predict and analyze the potential biological functions of bovine IRF7, with reference to the bovine IRF7 gene sequence (GenBank accession No. :NM_001105040.1), primers for bovine IRF7 were designed and PCR was used to amplify bovine IRF7 gene from Bovine viral diarrhea virus (BVDV) infected MDBK cells. The bovine IRF7 gene was attached to the pMD19-T clone vector, and then attached to the prokaryotic expression vector pET-28a(+) to transform into Escherichia coli DH5α competent cells, recombination plasmid pET-28a-IRF7 was constructed. The recombinant plasmid pET-28a-IRF7 was transformed into Escherichia coli BL21 (DE3) competent cells. After induction by IPTG, the expression products were analyzed and identified by SDS-PAGE. The specificity of the polyclonal antibody was identified by Western blotting, and the titer of rabbit anti-bovine IRF7 polyclonal antibody was determined by indirect ELISA. After BVDV infected cells, the expression of the antiviral factor IRF7 was detected by Real-time quantitative PCR. Bioinformatics analysis showed that IRF7 protein had no transmembrane domain, no signal peptide, and many phosphorylation sites. The secondary structure was mainly random coil, which was consistent with the three-dimensional model of the tertiary structure, indicating that this protein might have a good antigenic potential. A 1 497 bp fragment of IRF7 gene was successfully amplified by PCR, and the bovine IRF7 protein was successfully expressed with a molecular weight of about 60 ku. The titer of rabbit anti-bovine IRF7 polyclonal antibody was 1:128 000 by indirect ELISA. The expression of IRF7 was decreased in MDBK cells infected with BVDV strain, and the expression of IFN-β was significantly promoted by BVDV infection of cells overexpressing IRF7 (P<0.05). In conclusion, bovine IRF7 protein was successfully expressed and purified in this study, and rabbit polyclonal antibody against bovine IRF7 protein was prepared, which provided materials for elucidating the molecular mechanism of bovine IRF7 in innate immune antiviral response. |
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| Keywords: | bovine IRF7 protein purification polyclonal antibody |
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