牛IRF7蛋白的原核表达及多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2021.10.034

摘要/Abstract

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。

关键词: 牛; IRF7; 蛋白纯化; 多克隆抗体

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.

Key words: bovine; IRF7; protein purification; polyclonal antibody

中图分类号:

S852.4⁺3

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.

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 and Veterinary Medicine, 2021, 48(10): 3812-3822.

参考文献

[1] 张超, 何金科, 何延华, 等.胎牛血清中牛病毒性腹泻病毒2型毒株分离及脱脂奶粉中抗体的检测[J]. 中国畜牧兽医, 2020, 47(7):2239-2247. ZHANG C, HE J K, HE Y H, et al.Isolation of Bovine viral diarrhea virus type 2 in fetal bovine serum and detection of antibodies in skimmed milk powder[J]. China Animal Husbandry & Veterinary Medicine, 2020, 47(7):2239-2247.(in Chinese)
[2] 马旭升, 何延华, 盛金良, 等.牛病毒性腹泻病毒BVDV-LC株的分离与鉴定[J]. 中国预防兽医学报, 2018, 40(6):546-549. MA X S, HE Y H, SHENG J L, et al.Isolation and identification of BVDV LC strain of Bovine viral diarrhea virus[J]. Chinese Journal of Preventive Veterinary Medicine, 2018, 40(6):546-549.(in Chinese)
[3] 向文杰, 林俊, 宋文凤, 等.3株牛病毒性腹泻病毒的分离鉴定及其基因分型[J]. 中国预防兽医学报, 2019, 41(4):422-425. XIANG W J, LIN J, SONG W F, et al.Isolation, identification and genotyping of three Bovine viral diarrhea viruses[J]. Chinese Journal of Preventive Veterinary Medicine, 2019, 41(4):422-425.(in Chinese)
[4] 陈新诺, 张斌.牛病毒性腹泻病毒的分子生物学研究进展[J]. 中国畜牧兽医, 2017, 44(11):3137-3142. CHEN X N, ZHANG B.Advances in molecular biology of Bovine viral diarrhea virus[J]. China Animal Husbandry & Veterinary Medicine, 2017, 44(11):3137-3142.(in Chinese)
[5] CHEN P G, GUAN Y J, ZHA G M, et al.Swine IRF3/IRF7 attenuates inflammatory responses through TLR4 signaling pathway[J]. Oncotarget, 2017, 8(37):61958-61968.
[6] HATESUER B, HOANG H T, RIESE P, et al.Deletion of IRF3 and IRF7 genes in mice results in altered interferon pathway activation and granulocyte-dominated inflammatory responses to influenza A infection[J]. Journal of Innate Immunity, 2017, 9(2):145-161.
[7] EDVIGE P, GIULIA M, MARCO S, et al.IRF-7:An antiviral factor and beyond[J]. Future Virology, 2013, 8(10):1007-1020.
[8] TING L, GUANG X W, JING L, et al.TARBP2 inhibits IRF7 activation by suppressing TRAF6-mediated K63-linked ubiquitination of IRF7[J]. Molecular Immunology, 2019, 109:116-125.
[9] ANTONCZYK A, KRIST B, SAJEK M, et al.Direct inhibition of IRF-dependent transcriptional regulatory mechanisms associated with disease[J]. Frontiers in Immunology, 2019, 10(2):1176.
[10] WANG Z X, NING Z Y, SUN M H, et al.Interferon regulatory factor 7-(IRF7-) mediated immune response affects Newcastle disease virus replication in chicken embryo fibroblasts[J]. Acta Veterinaria Hungarica, 2014, 62(4):500-511.
[11] YONG F L, LIANG S, YUAN S, et al.Effects of the nuclear localization of the N(pro) protein of classical Swine fever virus on its virulence in pigs[J]. Veterinary Microbiology, 2014, 174(3-4):391-398.
[12] 刘莹.牛副流感病毒3型C和V蛋白通过负调控IRF7拮抗Ⅰ型干扰素表达的研究[D].哈尔滨:东北农业大学, 2017. LIU Y.Cattle parainfluenza 3 C and V protein by negative regulation IRF7 antagonism Ⅰ type of interferon expression research[D].Harbin:Northeast Agricultural University, 2017.(in Chinese)
[13] DEL T E, RAPPUOLI R, DELANY I.Reverse vaccinology:Exploiting genomes for vaccine design[J]. Human Vaccines, 2017, 15(9):65-86.
[14] WU Y, NARUM D L, FLEURY S, et al.Particle-based platforms for malaria vaccines[J]. Vaccine, 2015, 33(52):7518-7524.
[15] KAZMIN D, NAKAYA H I, LEE E K, et al.Systems analysis of protective imune responses to RTS, S malaria vaccination in humans[J]. Proceedings of the National Academy of Sciences of the United States of America, 2017, 114(9):2425-2430.
[16] SHAHNAZARYAN D, KHALIL R, WYNNE C, et al.Herpes simplex virus 1 targets IRF7via ICPo to limit type Ⅰ IFN induction[J]. Scientific Reports, 2020, 10(1):22216.
[17] 时坤.鹿源牛病毒性腹泻病毒分离鉴定及CP型BVDV激活NF-κB分子机制研究[D].长春:吉林农业大学, 2018. SHI K.Isolation and identification of Bovine viral diarrhea virus from deer and study on molecular mechanism of activation of NF-κB by CP-type BVDV[D].Changchun:Jilin Agricultural University, 2018.(in Chinese)
[18] 周玉龙.牛病毒性腹泻病毒诱导的细胞自噬对病毒感染影响机制研究[D].长春:吉林大学, 2017. ZHOU Y L.Effects of autophagy on Bovine viral diarrhea virus infection[D].Changchun:Jilin University, 2017.(in Chinese)
[19] 何延华, 马旭升, 钟发刚, 等.基于RNA-Seq技术分析非致病细胞病变BVDV感染牛单核细胞对NF-κB信号通路的影响[J]. 动物医学进展, 2020, 41(2):39-46. HE Y H, MA X S, ZHONG F G, et al.Effects of RNA-Seq on the NF-κB signaling pathway in bovine monocytes infected with non-pathogenic cytologic disease BVDV[J]. Progress in Veterinary Medicine, 2020, 41(2):39-46.(in Chinese)
[20] FADAJAN Z, FAZELI M.Expression of the rabies virus nucleoprotein and matrix protein in a prokaryotic system at high-levels:An efficacious production method[J]. Gene Reports, 2020, 21(7):100799.
[21] WENBAP P, SEETANG N Y, LUANGTONGKUM T, et al.Construction, expression and purification of a novel CadF-based multiepitope antigen and its immunogenic polyclonal antibody specific to Campylo-bacter jejuni and Campylobacter coli[J]. Protein Expression and Purification, 2021, 180(4):105818.
[22] DARWISH I A, ALMEHIZIA A A, RADWAN A A, et al.Preparation and characterization of two immunogens and production of polyclonal antibody with high affinity and specificity for darunavir[J]. Molecules (Basel, Switzerland), 2020, 25(18):4075.
[23] 李占鸿, 宋子昂, 杨振兴, 等.帕利亚姆病毒VP7蛋白的原核表达及多克隆抗体的制备[J]. 中国兽医科学, 2021, 51(2):176-183. LI Z H, SONG Z A, YANG Z X, et al.Prokaryotic expression of VP7 protein of Pariam virus and preparation of polyclonal antibody[J]. Chinese Veterinary Science, 2021, 51(2):176-183.(in Chinese)