嗜吞噬细胞无浆体MSP4蛋白的原核表达及其多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2024.09.031

摘要/Abstract

摘要： 【目的】利用原核表达系统表达嗜吞噬细胞无浆体(Anaplasma phagocytophilum，AP) MSP4蛋白，并制备抗MSP4蛋白的多克隆抗体，为嗜吞噬细胞无浆体检测方法的建立提供试验材料和理论依据。【方法】根据GenBank公布的MSP4基因优化并合成MSP4基因，将其插入pET-30a载体，构建pET-30a-MSP4重组质粒，转化大肠杆菌BL21(DE3)感受态细胞，对影响表达效果的因素(温度和IPTG)进行优化，选择最佳诱导表达条件，经SDS-PAGE分析蛋白表达情况，重组蛋白经Ni-IDA亲和层析纯化，并进行SDS-PAGE和Western blotting鉴定。用纯化后的重组蛋白MSP4免疫新西兰大白兔制备兔源多克隆抗体，利用间接 ELISA和Western blotting检测多克隆抗体效价及特异性。【结果】本研究成功构建pET-30a-MSP4重组质粒，在37 ℃、0.8 mmol/L IPTG诱导条件下蛋白表达量较高，表达的重组蛋白分子质量约为28 ku，以包涵体形式存在。Western blotting鉴定结果显示，其免疫原性较好。获得的多克隆抗体效价分别为1∶64 000(家兔1)和1∶128 000(家兔2)，且能与表达的重组蛋白MSP4发生特异性反应，具有较好的免疫原性。【结论】本研究成功表达纯化了嗜吞噬细胞无浆体MSP4蛋白,并制备了其多克隆抗体，可为嗜吞噬细胞无浆体抗原抗体检测方法的建立及疫苗效果评估提供参考，也为MSP4蛋白功能研究奠定基础。

关键词: 嗜吞噬无浆体; MSP4蛋白; 原核表达; 多克隆抗体

Abstract: 【Objective】 The purpose of this study was to express the protein MSP4 of Anaplasma phagocytophilum (AP), then prepare the polyclonal antibody against MSP4 protein, so as to provide experimental materials and theoretical basis for the establishment of plasma free detection method for phagocytic cells. 【Method】 According the gene sequence published in GenBank, MSP4 gene was optimized and synthesized.Then the MSP4 gene was inserted into pET-30a vector to construct recombinant plasmid pET-30a-MSP4 and then was transformed into Escherichia coli BL21(DE3) competent cells.The factors affecting the expression effect (temperature and IPTG) were optimized to select the best induced expression conditions.The expression of protein was analyzed by SDS-PAGE.The recombinant protein was purified by Ni-IDA affinity chromatography, and was identified by SDS-PAGE and Western blotting.The polyclonal antibody against MSP4 was prepared by immunizing New Zealand White rabbits with the purified recombinant protein MSP4, and then the titer and specificity of the polyclonal antibody were determined by indirect ELISA and Western blotting. 【Result】 The results showed that the pET-30a-MSP4 recombinant plasmid was successfully constructed.The protein expression level was high under the induction conditions of 37 ℃ and 0.8 mmol/L IPTG, and it was mainly expressed in the form of inclusion bodies with molecular weight of about 28 ku.Western blotting showed that it had good immunogenicity.The titer of rabbit-derived polyclonal antibodies were 1∶64 000 (rabbit 1) and 1∶128 000 (rabbit 2), respectively and the prepared polyclonal antibodies had high specificity. 【Conclusion】 In this study, MSP4 recombinant protein was obtained, and polyclonal antibody was successfully prepared, which could provide reference for the establishment of a method for detecting antigen and antibody of AP and the evaluation of vaccine efficacy, and also laid the foundation for the study of MSP4 protein function.

Key words: Anaplasma phagocytophilum; MSP4 protein; prokaryotic expression; polyclonal antibody

中图分类号:

S852.61

崔艳艳, 王冠, 郝俊芳, 李志强, 余复昌, 史柯, 席丽, 齐萌. 嗜吞噬细胞无浆体MSP4蛋白的原核表达及其多克隆抗体制备[J]. 中国畜牧兽医, 2024, 51(9): 4036-4042.

CUI Yanyan, WANG Guan, HAO Junfang, LI Zhiqiang, YU Fuchang, SHI Ke, XI Li, QI Meng. Prokaryotic Expression of Anaplasma phagocytophilum MSP4 Protein and Preparation of Its Polyclonal Antibodies[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(9): 4036-4042.

参考文献

[1] DUMLER J S,CHOI K S,JOSE G C,et al.Human granulocytic anaplasmosis and Anaplasma phagocytophilum[J].Emerging Infectious Diseases,2005,11(12):1828-1834.
[2] SNORRE S,ERIK G G,CORNELIA S.Anaplasma phagocytophilum—A widespread multi-host pathogen with highly adaptive strategies[J]. Frontiers in Cellular and Infection Microbiology,2013,3(3):31.
[3] EROL U,SAHIN O F,ALTAY K.Molecular survey of Anaplasma phagocytophilum and related strains in sheep and goats from Sivas;With a high prevalence of Anaplasma phagocytophilum-like 1[J].Turkiye Parazitoloji Dergisi,2022,46(4):293-300.
[4] AKTAS M,COLAK S.Molecular detection and phylogeny of Anaplasma spp.in cattle reveals the presence of novel strains closely related to A.phagocytophilum in Turkey[J]. Ticks and Tick-borne Diseases, 2021,12(1):101604.
[5] ZOBBA R,MURGIA C,DAHMANI M.Emergence of Anaplasma species related to A.phagocytophilum and A.platys in Senegal[J].International Journal of Molecular Sciences, 2022,24(1):33.
[6] ABDELSALAM M A,FELEFEL W,FADL S.Molecular prevalence and associated infection risk factors of tick-borne protozoan and rickettsial blood pathogens in small ruminants[J]. BMC Veterinary Research,2023,19(1):138.
[7] WANG Y,CHEN C,ZHANG L.Molecular characterization of msp2/p44 of Anaplasma phagocytophilum isolated from infected patients and Haemaphysalis longicornis in Laizhou bay,Shandong province,China[J].Clinical Trial, 2013,8(10):e78189.
[8] 赵庆亮,杨素芳,梁田,等.嗜吞噬细胞无形体msp4蛋白的生物信息学分析及表达鉴定[J].生物技术通报, 2015,31(1):220-224. ZHAO Q L,YANG S F,LIANG T,et al.Expression and bioinformatics analysis of the msp4 protein of Anaplasma phagocytophilum[J].Biotechnology Bulletin,2015,31(1):220-224.(in Chinese)
[9] HUANG H,WANG X,CHI K,et al.Porin activity of Anaplasma phagocytophilum outer membrane fraction and purified P44[J].Journal of Bacteriology,2007,189(5):1998-2006.
[10] MAZUECOS L,ALBERDI P,JARGUIN H A,et al.Frankenbacteriosis targeting interactions between pathogen and symbiont to control infection in the tick vector[J].iScience, 2023,26(5):106697.
[11] VILLAR M,AYLLON N,KOCAN K M,et al.Identification and characterization of Anaplasma phagocytophilum proteins involved in infection of the tick vector,Ixodes scapularis[J].PLoS One, 2015,10(9):e0137237.
[12] YAN Y Q,WANG K L,CUI Y Y,et al.Molecular detection and phylogenetic analyses of Anaplasma spp.in Haemaphysalis longicornis from goats in four provinces of China[J].Scientific Reports, 2021,11(1):14155.
[13] FUENTE D L L,MASSUNG R F,WONG S J,et al.Sequence analysis of the msp4 gene of Anaplasma phagocytophilum strains[J]. Journal of Clinical Microbiology, 2005,43(3):1309-1317.
[14] RAMOS I A S,HERRERA H M,MENDES N S,et al.Phylogeography of msp4 genotypes of Anaplasma marginale in beef cattle from the Brazilian Pantanal[J].Revista Brasileira de Parasitologia Veterinaria，2019,28(3):451-457.
[15] NOAMAN V.Epidemiological study on Anaplasma phagocytophilum in cattle:Molecular prevalence and risk factors assessment in different ecological zones in Iran[J].Preventive Veterinary Medicine, 2020,183:105118.
[16] ZHOU S,HUANG L Y,LIN Y,et al.Molecular surveillance and genetic diversity of Anaplasma spp.in cattle (Bos taurus) and goat (Capra aegagrus hircus) from Hainan island/province,China[J]. BMC Veterinary Research, 2023,19(1):213.
[17] 田万年,张子健.吉林省部分地区蜱和羊携带羊泰勒虫和嗜吞噬细胞无浆体分子流行病学调查[J].中国兽医杂志, 2023,59(6):9-15. TIAN W N,ZHANG Z J.Molecular epidemiological investigation on ovine Theileria and Anaplasma phgocytophilum infections in ticks and sheep in selected regions of Jilin province[J]. Chinese Journal of Veterinary Medicine,2023,59(6):9-15.(in Chinese)
[18] DREHER U M,DE L F J,HOFMANN L R,et al.Serologic cross-reactivity between Anaplasma marginale and Anaplasma phagocytophilum[J].Clinical Vaccine Immunology,2005,12(10):1177-1183.
[19] CONTRERAS M,ALBERDI P,MATEOS H L,et al.Anaplasma phagocytophilum MSP4 and HSP70 proteins are involved in interactions with host cells during pathogen infection[J]. Frontiers in Cellular and Infection Microbiology, 2017,7:307.
[20] DE LA FUENTE J,MORAGA F A,ALBERDI P,et al.A quantum vaccinomics approach for the design and production of MSP4 chimeric antigen for the control of Anaplasma phagocytophilum infections[J].Vaccines, 2022,10(12):1995.
[21] 陈云雨.大肠杆菌FtsZ蛋白原核表达及多克隆抗体的制备与鉴定[J].生物工程学报,2019，6：1117-1125. CHEN Y Y.Bacterial expression,preparation and identification of polyclonal antibody against Escherichia coli FtsZ[J].Chinese Journal of Biotechnology,2019，6:1117-1125.(in Chinese)
[22] 梁之瑄,梅晨,张雪,等.副鸡禽杆菌lpxM蛋白的原核表达及免疫原性研究[J].中国畜牧兽医,2024,51(2):719-727. LINAG Z X,MEI C,ZHANG X,et al.Prokaryotic expression and immunogenicity of lpxM protein of Avibacterium paragallinarum[J].China Animal Husbandry & Veterinary Medicine,2024,51(2):719-727.(in Chinese)
[23] 刘茹,李小龙,张晓倩,等.LbCas12a蛋白的原核表达及活性检测[J].中国畜牧兽医,2022,49(5):1621-1629. LIU R,LI X L,ZHANG X Q,et al.Prokaryotic expression and acticity identification of LbCas12a protein[J].China Animal Husbandry & Veterinary Medicine,2022,49(5):1621-1629.(in Chinese)
[24] ZHANG W Q,LU J,ZHANG S W,et al.Development an effective system to expression recombinant protein in E.coli via comparison and optimization of signal peptides:Expression of Pseudomonas fluorescens BJ-10 thermostable lipase as case study[J].Microbial Cell Factories, 2018,17(1):50.
[25] 袁娣,邵怡,张同瑶,等.刚地弓形虫SRS29C截短型基因的克隆及原核表达质粒的构建[J].天津农学院学报,2021,28(3):50-55. YUAN D,SHAO Y,ZHANG T Y,et al.Cloning and construction of prokaryotic expression plasmid of truncated SRS29C gene of Toxoplasma gondii[J]. Journal of Tianjin Agricultural University,2021，28(3):50-55.(in Chinese)
[26] 杨敏璇,朱焯安,陈嘉俊,等.鲫鱼肌间刺形成基因SOST的克隆表达研究[J].仲恺农业工程学院学报，2019，32(2):58-62. YANG M X,ZHU Z A,CHEN J J,et al.Cloning and prokaryotic expression of the SOST gene of Carassius auratus[J].Journal of Zhongkai University of Agriculture and Engineering，2019,32(2):58-62.(in Chinese)
[27] 索南吉,李沛轩,刘转霞,等.双峰驼Cyp-A的原核表达及多克隆抗体制备[J].中国畜牧兽医，2023,50(7):2896-2905. SUO N J,LI P X,LIU Z X,et al.Prokaryotic expression and polyclonal antibody of Cyp-A in bactrian camel[J].China Animal Husbandry & Veterinary Medicine,2023,50(7):2896-2905.(in Chinese)
[28] 徐栋生，陈蕴，金坚.信号肽对肝细胞生长因子HGF在CHO中表达及分泌的影响[J].中国细胞生物学学报，2016，38(12):1473-1479. XU D S,CHEN Y,JIN J.Effect of signal peptide on the expression and secretion of hepatocyte growth factor in CHO[J]. Chinese Journal of Cell Biology,2016，38(12):1473-1479.(in Chinese)