非洲猪瘟病毒p37蛋白生物信息学分析及其多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2023.01.030

摘要/Abstract

摘要： 【目的】获取ASFV p37蛋白，并制备抗ASFV p37蛋白的多克隆抗体，为ASFV p37蛋白结构和功能研究提供材料。【方法】应用生物信息学工具对ASFV HLJ/2018（GenBank登录号：MK333180.1） p37蛋白进行分析，设计合成p37基因，并构建pET32a-p37重组质粒。将重组质粒转化大肠杆菌BL21（DE3）感受态细胞，经IPTG诱导表达后，通过SDS-PAGE对重组蛋白的可溶性进行分析。收集菌体进行超声破碎，分离沉淀并使用8 mol/L尿素变性后离心，用0.45 μm滤膜过滤离心后的上清，使用镍亲和层析柱纯化蛋白，通过SDS-PAGE、Western blotting对其纯化效果及特异性进行验证。将纯化后的p37蛋白按照50 μg/只免疫小鼠，制备抗ASFV p37蛋白多克隆抗体。利用间接ELISA方法测定制备的多克隆抗体效价；通过Western blotting、间接免疫荧光试验检测该多克隆抗体的特异性。【结果】生物信息学分析表明，p37蛋白为稳定亲水性蛋白，无跨膜区和信号肽。二级结构主要含有α-螺旋（45.28%）、延伸链（15.09%）、无规则卷曲（31.54%）。SDS-PAGE、Western blotting结果显示，经IPTG诱导后，成功表达重组蛋白p37，大小62 ku左右，主要以包涵体形式存在，且能与His单克隆抗体发生反应。间接ELISA结果显示，制备的鼠源多克隆抗体效价为1：256 000。Western blotting结果显示，在42 ku左右出现了特异性条带；间接免疫荧光显示出了特异性的红色荧光，表明该多克隆抗体能与重组杆状病毒表达的p37蛋白发生反应，具有较好的特异性。【结论】本试验成功表达并纯化了ASFV p37蛋白，制备了抗ASFV p37蛋白多克隆抗体，重组蛋白具有较好的免疫原性，能产生较强的免疫反应，为后续ASFV p37蛋白结构功能研究、抗体及相关疫苗研发奠定基础。

关键词: 非洲猪瘟病毒(ASFV); p37蛋白; 蛋白表达; 多克隆抗体

Abstract: 【Objective】 This study was aimed to obtain ASFV p37 protein and prepare polyclonal antibody against ASFV p37 protein to provide materials for structural and functional studies of ASFV p37 protein.【Method】 In this study, bioinformatics tools were used to analyze the p37 protein of ASFV HLJ/2018 (GenBank accession No.:MK333180.1), and the p37 gene was designed and synthesized, and the pET32a-p37 recombinant expression vector was constructed.The recombinant plasmid was transformed into of Escherichia coli BL21 (DE3) competent cells.After induced by IPTG, the soluble expression of the recombinant protein was analyzed by SDS-PAGE.The bacterial precipitate was denatured by 8 mol/L urea, centrifuged, filtered through a 0.45 μm membrane, then purified by nickel affinity chromatography, and their purification effect and specificity were verified by SDS-PAGE and Western blotting.The polyclonal antibody against ASFV p37 protein was prepared by immunizing mice with 50 μg of purified p37 protein per mouse.The titer of polyclonal antibody was determined by indirect ELISA. The specificity of the polyclonal antibody was detected by Western blotting and indirect immunofluorescence assay.【Result】 Bioinformatics analysis showed that the p37 protein was a stable hydrophilic protein with no transmembrane region or signal peptide.The secondary structure mainly contained alpha helix (45.28%), extended stran (15.09%) and random coil (31.54%).SDS-PAGE and Western blotting results showed that the prokaryotic expressed recombinant protein p37 was successfully expressed after induction by IPTG, with a size around 62 ku, mainly in the form of inclusion bodies, and could react with His monoclonal antibody.Indirect ELISA results showed that the titer of the prepared anti-mouse polyclonal antibody reached 1:256 000.Western blotting results showed a specific band at around 42 ku.Indirect immunofluorescence showed a specific red fluorescence, indicating that the polyclonal antibody reacted with the recombinant baculovirus-expressed p37 protein with good specificity.【Conclusion】 ASFV p37 protein was successfully expressed and purified, and a polyclonal antibody against ASFV p37 protein was prepared, indicating that the prepared recombinant protein had good immunogenicity and could generate strong immune response, which laid the foundation for the subsequent structural and functional study of ASFV p37 protein structure function, antibody and related vaccine development.

Key words: African swine fever virus (ASFV); p37 protein; protein expression; polyclonal antibody

中图分类号:

S852.65^＋1

张梦杨, 何健, 刘阳坤, 姚伦广. 非洲猪瘟病毒p37蛋白生物信息学分析及其多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(1): 300-309.

ZHANG Mengyang, HE Jian, LIU Yangkun, YAO Lunguang. Bioinformatics Analysis and Polyclonal Antibody Preparation of African Swine Fever Virus p37 Protein[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 300-309.

参考文献

[1] 王涛, 孙元, 罗玉子, 等.非洲猪瘟防控及疫苗研发:挑战与对策[J].生物工程学报, 2018, 34(12):1931-1942. WANG T, SUN Y, LUO Y Z, et al.Prevention, control and vaccine development of African swine fever:Challenges and countermeasures[J].Chinese Journal of Biotechnology, 2018, 34(12):1931-1942.(in Chinese)
[2] STOIAN A M M, ZIMMERMAN J, JI J, et al.Half-life of African swine fever virus in shipped feed[J].Emerging Infectious Diseases, 2019, 25:2261-2263.
[3] 刘菊枚, 戚南山, 廖申权, 等.猪场重要虫媒及其传播病原研究进展[J].中国畜牧兽医, 2021, 48(5):1725-1734. LIU J M, QI N S, LIAO S Q, et al.Research progress on important insect vectors and their transmission pathogens in pig farms[J].China Animal Husbandry & Veterinary Medicine, 2021, 48(5):1725-1734.(in Chinese)
[4] 何健, 石建州, 刘阳坤, 等.非洲猪瘟病毒研究进展[J].南阳师范学院学报, 2022, 21(1):55-62. HE J, SHI J Z, LIU Y K, et al.Research progress in African swine fever virus[J]. Journal of Nanyang Normal University, 2022, 21(1):55-62.(in Chinese)
[5] ZHOU X, LI N, LUO Y, et al.Emergence of African swine fever in China, 2018[J].Transboundary and Emerging Diseases, 2018, 65(6):1482-1484.
[6] BLOME S, FRANZKE K, BEER M.African swine fever-A review of current knowledge[J].Virus Research, 2020, 287:198099.
[7] 马兴树, 宋金祥.非洲猪瘟病毒免疫及基因工程疫苗研究进展[J].中国畜牧兽医, 2019, 46(11):3404-3413. MA X S, SONG J X.Research progress on immune response and genetic engineering vaccine against African swine fever virus[J].China Animal Husbandry & Veterinary Medicine, 2019, 46(11):3404-3413.(in Chinese)
[8] REVILLA Y, PÉREZ-NUÑEZ D, RICHT J A.African swine fever virus biology and vaccine approaches[J].Advances in Virus Research, 2018, 100:41-74.
[9] EULÁLIO A, NUNES-CORREIA I, SALAS J, et al.African swine fever virus p37 structural protein is localized in nuclear foci containing the viral DNA at early post-infection times[J].Virus Research, 2007, 130(1-2):18-27.
[10] 程晶, 刘文晓, 王晓玥, 等.非洲猪瘟诊断方法研究进展[J].中国畜牧兽医, 2022, 49(5):1985-1993. CHENG J, LIU W X, WANG X Y, et al.Research progress on development of diagnostic methods of African swine fever[J].China Animal Husbandry & Veterinary Medicine, 2022, 49(5):1985-1993.(in Chinese)
[11] URBANO A C, FERREIRA F.African swine fever control and prevention:An update on vaccine development[J].Emerging Microbes & Infections, 2022, 11(1):2021-2033.
[12] GLADUE D P, BORCA M V.Recombinant ASF live attenuated virus strains as experimental vaccine candidates[J].Viruses, 2022, 14(5):878.
[13] GAUDREAULT N N, RICHT J A.Subunit vaccine approaches for African swine fever virus[J].Vaccines (Basel), 2019, 7(2):56.
[14] KAZAKOVA A S, IMATDINOV I R, DUBROVSKAYA O A, et al.Recombinant protein p30 for serological diagnosis of African swine fever by immunoblotting assay[J].Transboundary and Emerging Diseases, 2017, 64(5):1479-1492.
[15] 吴竞, 王西西, 吴映彤, 等.非洲猪瘟病毒p30基因的原核表达及间接ELISA抗体检测方法的建立[J].中国畜牧兽医, 2018, 45(12):3555-3562. WU J, WANG X X, WU Y T, et al.Prokaryotic expression of the p30 gene of African swine fever virus and establishment of indirect ELISA antibody detection method[J].China Animal Husbandry & Veterinary Medicine, 2018, 45(12):3555-3562.(in Chinese)
[16] 曹琛福, 梁云浩, 陶虹, 等.非洲猪瘟病毒p54基因的原核表达及其抗体的间接ELISA检测方法的建立[J].动物医学进展, 2014, 35(2):6-10. CAO C F, LIANG Y H, TAO H, et al.Prokaryotic expression of the p54 gene of African swine fever virus and establishment of indirect ELISA for detection its antibody against p54[J].Progress in Veterinary Medicine, 2014, 35(2):6-10.(in Chinese)
[17] 王彩霞, 冯春燕, 杜方原, 等.非洲猪瘟病毒主要抗原p54-1的原核表达及多克隆抗体的制备与鉴定[J].中国畜牧兽医, 2018, 45(10):2823-2830. WANG C X, FENG C Y, DU F Y, et al.Prokaryotic expression of African swine fever virus p54-1 and preparation and characterization of polyclonal antibodies[J].China Animal Husbandry & Veterinary Medicine, 2018, 45(10):2823-2830.(in Chinese)
[18] 邬旭龙, 彭彬, 姜睿姣, 等.以原核表达的非洲猪瘟病毒pK205R蛋白为包被抗原的间接ELISA抗体检测方法的建立[J].中国预防兽医学报, 2016, 38(10):800-803. WU X L, PENG B, JIANG R J, et al.Development of an indirect ELISA method for the detection of antibodies against African swine fever virus(ASFV) with prokaryotic expressed ASFV pK205R protein as the coating antigen[J].Chinese Journal of Preventive Veterinary Medicine, 2016, 38(10):800-803.(in Chinese)
[19] ZHONG K, ZHU M, YUAN Q, et al.Development of an indirect ELISA to detect African swine fever virus pp62 protein-specific antibodies[J].Frontiers in Veterinary Science, 2022, 8:798559.
[20] 王翠, 许宗丽, 黄溢泓, 等.非洲猪瘟病原学检测技术研究进展[J].中国动物检疫, 2022, 39(2):74-81. WANG C, XU Z L, HUANG Y H, et al.Advance in the etiological detection techniques against ASF[J].China Animal Health Inspection, 2022, 39(2):74-81.(in Chinese)
[21] HEATH C M, WINDSOR M, WILEMAN T.Membrane association facilitates the correct processing of pp220 during production of the major matrix proteins of African swine fever virus[J].Journal of Virology, 2003, 77(3):1682-1690.
[22] 王俊娟, 李欣芮, 陈成, 等.花生主要过敏原Ara h 1线性B细胞表位的预测及鉴定[J].食品科学, 2021, 42(17):106-112. WANG J J, LI X R, CHEN C, et al.Prediction and identification of linear B cell epitope on the major peanut allergen Ara h 1[J].Food Science, 2021, 42(17):106-112.(in Chinese)