伊氏锥虫ISG 65基因表达、生物信息学分析及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2025.11.031

摘要/Abstract

摘要： 【目的】表达伊氏锥虫(Trypanosoma evansi)不变表面糖蛋白65(invariant surface glycoprotein 65,ISG 65)，分析其生物学特性及反应原性，并制备伊氏锥虫ISG 65蛋白多克隆抗体，为该类病原体检测方法的建立奠定基础。【方法】PCR扩增伊氏锥虫新疆株ISG 65基因，对测序列进行相似性比对及系统进化树构建，运用生物信息学工具对ISG 65基因编码蛋白的理化特性、亲/疏水性、跨膜区、信号肽、磷酸化位点及亚细胞定位进行预测分析。通过同源重组方法将ISG 65基因片段转染大肠杆菌BL21(DE3)感受态细胞，经IPTG诱导表达重组ISG 65蛋白(rISG 65)，镍柱亲和层析法纯化rISG 65蛋白；利用SDS-PAGE和Western blotting验证ISG 65蛋白纯化效果。将纯化后的rISG 65蛋白免疫昆明小鼠制备多克隆抗体，采用间接ELISA方法测定多克隆抗体效价及重组蛋白反应原性。【结果】伊氏锥虫新疆株ISG 65基因PCR扩增产物大小为657 bp，与冈比亚布氏锥虫(登录号：XM_011773430.1、XM_011773437.1)相似性最高，为99.06%，与冈比亚布氏锥虫(登录号：XM_011773430.1)亲缘关系最近并聚为一支。生物信息学分析显示，ISG 65蛋白属于亲水性蛋白，含有436个氨基酸，理论等电点为8.02。预测ISG 65蛋白存在35个磷酸化位点；二级结构中α-螺旋、无规则卷曲、β-转角、延伸链占比分别为53.21%、33.03%、2.98%和10.78%；ISG 65蛋白有14个B细胞抗原表位，具有较好的免疫原性。本试验成功构建pET-28a-ISG 65重组质粒；SDS-PAGE结果显示，重组菌pET-28a-ISG 65-BL21在37 ℃ 8 h、1.0 mmol/L IPTG中高效表达，纯化后得到的蛋白大小约24 ku。SDS-PAGE和Western blotting结果显示，均获得与预期大小一致的24 ku条带。间接ELISA检测结果显示，获得的ISG 65鼠源多克隆抗体效价为1∶1 638 400。【结论】经原核表达的重组ISG 65蛋白具有良好的免疫原性，能与伊氏锥虫阳性血清发生特异性结合反应，并具有良好的生物学特性。本研究可为后续伊氏锥虫ISG 65蛋白的生物学特性研究，以及ELISA诊断方法的建立提供重要参考数据和理论支撑。

关键词: 伊氏锥虫; ISG 65蛋白; 原核表达; 多克隆抗体

Abstract: 【Objective】 This study aimed to express the invariant surface glycoprotein 65 (ISG 65) of Trypanosoma evansi (T.evansi),analyze its biological characteristics and immunogenicity,and prepare polyclonal antibody against T.evansi ISG 65 protein,so as to lay a foundation for the subsequent development of detection methods for this disease pathogen. 【Method】 ISG 65 gene of T.evansi Xinjiang strain was amplified by PCR.Similarity alignment of the measured sequences was performed and phylogenetic tree was constructed.Bioinformatics tools were used to predict and analyze the physicochemical properties,hydrophilicity and hydrophobicity,transmembrane region,signal peptide,phosphorylation sites,and subcellular localization of the ISG 65 protein.After homologous recombination,the ISG 65 gene fragment was transfected into E.coli BL21(DE3) competent cells,recombinant ISG 65 protein (rISG 65) was induced by IPTG and purified by nickel column affinity chromatography.The purification of the ISG 65 protein was verified by Western blotting and SDS-PAGE.The polyclonal antibodies were obtained by immunizing Kunming mice with the purified rISG 65 protein.The titer of the polyclonal antibodies and the reactivity of the recombinant protein were determined using indirect ELISA and Western blotting. 【Result】 The PCR amplification product of ISG 65 gene of T.evansi Xinjiang strain was 657 bp in size,and had the highest similarity with Trypanosoma brucei gambiense (accession No.:XM_011773430.1 and XM_011773437.1) at 99.06%.It was closely related to Trypanosoma brucei gambiense (accession No.:XM_011773430.1) and clustered into one branch.Bioinformatics analysis revealed that ISG 65 protein was a hydrophilic protein consisting of 436 amino acids with a theoretical isoelectric point of 8.02.ISG 65 protein was predicted to have 35 phosphorylation sites.The secondary structure comprised alpha helix (53.21%),random coil (33.03%),beta turn (2.98%),and extended strand (10.78%).ISG 65 protein had 14 B cell antigen epitopes,which showed good immunogenicity.pET-28a-ISG 65 recombinant plasmid was successfully constructed in this experiment.SDS-PAGE results showed that the recombinant pET-28a-ISG 65-BL21 was highly expressed in 1.0 mmol/L IPTG at 37 ℃ for 8 h,yielding a protein of approximately 24 ku after purification.The results of indirect ELISA detection showed that the titer of the obtained ISG 65 polyclonal antibody was 1∶1 638 400. 【Conclusion】 The recombinant ISG 65 protein expressed in prokaryotic cells exhibited good immunogenicity,could specifically bind to T.evansi positive serum,and possessed favorable biological characteristics.This study provided important reference data and theoretical support for future research on the biological characteristics of T.evansi ISG 65 protein and the establishment of ELISA diagnostic methods.

Key words: Trypanosoma evansi; ISG 65 protein; prokaryotic expression; polyclonal antibody

中图分类号:

S852.72

金敏, 王美玲, 刘燕, 周娜, 赵雪晴, 党文盈, 古丽博斯坦, 阿布都卡迪尔, 巴依娜, 巴音查汗·盖力克. 伊氏锥虫ISG 65基因表达、生物信息学分析及多克隆抗体制备[J]. 中国畜牧兽医, 2025, 52(11): 5359-5370.

JIN Min, WANG Meiling, LIU Yan, ZHOU Na, ZHAO Xueqing, DANG Wenying, Gulibositan, Abudukadier, Bayina, GAILIKE·Bayinchahan. Expression,Bioinformatics Analysis,and Preparation of Polyclonal Antibodies for ISG 65 Gene of Trypanosoma evansi[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(11): 5359-5370.

参考文献

[1] DESQUESNES M,GONZATTI M,SAZMANG A, et al.A review on the diagnosis of animal trypanosomoses[J].Parasites & Vectors,2022,15(1):64.
[2] KAMYINGKIRD K,CHALERMWONG P,SACEHAN V,et al.Investigation of Trypanosoma evansi infection in bullfighting cattle in Southern Thailand[J].Veterinary World,2020,13(8):1674-1678.
[3] GEREM B,HAMID M,ASSEFA A.Prevalence and associated risk factors of Trypanosoma evansi in camels in Ethiopia based on parasitological examinations[J].Veterinary Medicine International,2020,2020:6172560.
[4] KIM J,LI Z,RADWANSKA M,et al. Recent progress in the detection of Surra,a neglected disease caused by Trypanosoma evansi with a One Health Impact in large parts of the tropic and sub-tropic world[J].Microorganisms, 2023,12(1):44.
[5] 薛淑梅.牛伊氏锥虫病的诊断与治疗[J].现代畜牧科技，2016,3:85.XUE S M.Diagnosis and treatment of bovine Trypanosoma evansi[J].Modern Animal Husbandry Science & Technology,2016,3:85.(in Chinese)
[6] 张中鹏.肉牛伊氏锥虫病的流行病学、临床症状、实验室检查与防治措施[J].现代畜牧科技，2020,4:104-105.ZHANG Z P.Epidemiology,clinical symptoms,laboratory tests,and prevention and control measures of bovine Trypanosoma evansi[J].Modern Animal Husbandry Science & Technology,2020,4:104-105.(in Chinese)
[7] 马志成.牛伊氏锥虫病的诊断与防治[J].养殖技术顾问，2014,2:172.MA Z C.Diagnosis and prevention of bovine Trypanosoma evansi[J].Technical Advisor for Animal Husbandry,2014,2:172.(in Chinese)
[8] 刘志强,郭会玲,金映红,等.新疆边境地区马、驼锥虫病流行病学调查[J].草食家畜，2019,1:60-64.LIU Z Q,GUO H L,JIN Y H,et al.Epidemiological investigation on trypanosomiasis in horse and camels in Xinjiang border region[J].Grass-Feeding Livestock,2019,1:60-64.(in Chinese)
[9] ZIEGELBAUER K,OVERATH P.Organization of two invariant surface glycoproteins in the surface coat of Trypanosoma brucei[J].Infection and Immunity,1993,61(11):4540-4545.
[10] DESQUESNES M,DARGANTES A,LAI D H,et al.Trypanosoma evansi and Surra:A review and perspectives on transmission,epidemiology and control,impact,and zoonotic aspects[J].BioMed Research International,2013,2013:321237.
[11] LA GRECA F,MAGEZ S.Vaccination against trypanosomiasis:Can it be done or is the trypanosome truly the ultimate immune destroyer and escape artist?[J].Human Vaccines,2011,7(11):1225-1233.
[12] ZIEGELBAUER K,RUDENKO G,KIEFT R,et al.Genomic organization of an invariant surface glycoprotein gene family of Trypanosoma brucei[J].Molecular and Biochemical Parasitology,1995,69(1):53-63.
[13] ZIEGELBAUER K,MULTHAUP G,OVERATH P.Molecular characterization of two invariant surface glycoproteins specific for the bloodstream stage of Trypanosoma brucei[J].Journal of Biological Chemistry,1992,267(15):10797-10803.
[14] JACKSON D G,WINDLE H J,VOORHEIS H P.The identification,purification,and characterization of two invariant surface glycoproteins located beneath the surface coat barrier of bloodstream forms of Trypanosoma brucei[J].Journal of Biological Chemistry,1993,268(11):8085-8095.
[15] CHUNG W L,LEUNG K F,CARRINGTON M,et al.Ubiquitylation is required for degradation of transmembrane surface proteins in trypanosomes[J].Traffic (Copenhagen,Denmark),2008,9(10):1681-1697.
[16] LEUNG K F,RILEY F S,CARRINGTON M,et al.Ubiquitylation and developmental regulation of invariant surface protein expression in trypanosomes[J].Eukaryotic Cell,2011,10(7):916-931.
[17] ZIEGELBAUER K,OVERATH P.Identification of invariant surface glycoproteins in the bloodstream stage of Trypanosoma brucei[J].Journal of Biological Chemistry,1992,267(15):10791-10796.
[18] KUMAR R,SETHI K,BATRA K,et al.Exploring the potential of invariable surface glycoprotein (ISG65) as promising antigen for diagnosis of Trypanosoma evansi infection[J].Veterinary Parasitology, 2023,314:109866.
[19] ALSFORD S,ECKERT S,BAKER N,et al.High-throughput decoding of antitrypanosomal drug efficacy and resistance[J].Nature, 2012,482(7384):232-236.
[20] KHAN W,HAFEEZ M A,LATEEF M,et al.Parasitological,molecular,and epidemiological investigation of Trypanosoma evansi infection among dromedary camels in Balochistan province[J].Parasitology Research, 2023,122(8):1833-1839.
[21] 甘露.马虻传伊氏锥虫YLi-Te株PFR基因克隆表达及免疫效果观察[D].乌鲁木齐：新疆农业大学，2023.GAN L.Cloning and expression of PFR gene and observation of immune effect of YLi-Te strain of Trypanosoma evansi transmitted by Tabanidae[D].Urumqi:Xinjiang Agricultural University,2023.(in Chinese)
[22] 金敏,甘露,杨敏,等.新疆部分养马区域马匹血源性和虻源性伊氏锥虫PCR检测[J].动物医学进展,2024,45(10):119-122.JIN M,GAN L,YANG M,et al.PCR detection of equine blood-derived and Tabanus-derived Trypanosoma evansi in some areas of Xinjiang[J].Progress in Veterinary Medicine,2024,45(10):119-122.(in Chinese)
[23] 祖力亚·克力木江,努尔·库尔玛那里,阿不来,等.伊犁河谷地域虻的鉴定及其携带伊氏锥虫的检测[J].动物医学进展,2021,42(9):141-144.ZULIYA·KELIMUJIANG,NUER·KUERMANALI,ABULAI,et al.Identification of Tabanus in Yili River Valley and detection of carried Trypanosoma evansi[J].Progress in Veterinary Medicine,2021,42(9):141-144.(in Chinese)
[24] 周勇志,周金林,沈杰,等.伊氏锥虫、马媾疫锥虫、布氏锥虫、刚果锥虫的18S rDNA部分序列测定与系统发育关系[J].中国预防兽医学报,2006,28(2):178-180.ZHOU Y Z,ZHOU J L,SHEN J,et al.Sequence and phylogenetic analysis of partial 18S rDNA gene for Trypanosoma evansi,Trypanosoma equiperdum,Trypanosoma brucei and Trypanosoma congolense isolates[J].Chinese Journal of Preventive Veterinary Medicine,2006,28(2):178-180.(in Chinese)
[25] RUDRAMURTHY G R,SENGUPTA P P,METILDA B,et al.Development of an enzyme immunoassay using recombinant invariant surface glycoprotein (rISG) 75 for serodiagnosis of bovine trypanosomosis[J].Indian Journal of Experimental Biology, 2015,53(1):7-15.
[26] AUDAGNOTTO M,DAL PERARO M.Protein post-translational modifications: In silico prediction tools and molecular modeling[J].Computational and Structural Biotechnology Journal,2017,15:307-319.
[27] BATISTA F,MOREIRA R S,FILHO V B,et al.Shotgun proteomics of detergent-solubilized proteins from Trypanosoma evansi[J].Journal of Proteomics,2024,304:105231.
[28] 陈永艳,杜承,王晓钧.伊氏锥虫病的诊断及防控研究现状[J].中国兽医学报,2024,44(7):1571-1578.CHEN Y Y,DU C,WANG X J.Current status of research on diagnosis,prevebtion and control of Surra[J].Chinese Journal of Veterinary Science,2024,44(7):1571-1578.(in Chinese)
[29] 曾晴勤,李如松,李红英,等.基于核苷三磷酸二磷酸水解酶的马锥虫病间接ELISA抗体检测方法的建立[J].农业生物技术学报,2025,33(1):220-227.ZENG Q Q,LI R S,LI H Y,et al.Establishment of an indirect ELISA antibody detection method for equine trypanosomosis based on nucleotide triphosphate diphosphohydrolase[J].Journal of Agricultural Biotechnology,2025,33(1):220-227.(in Chinese)