东方泰勒虫P33蛋白单克隆抗体的制备与鉴定

doi:10.16431/j.cnki.1671-7236.2025.08.028

摘要/Abstract

摘要： 【目的】 P33蛋白是东方泰勒虫(Theileria orientalis)虫体重要的表面蛋白，具有较好的反应原性和免疫原性。本试验旨在通过杂交瘤技术研制针对P33蛋白的特异性单克隆抗体，系统评估其免疫学特性并筛选出能稳定分泌该抗体的单克隆细胞株，为后续建立基于P33抗原的东方泰勒虫血清学诊断方法奠定物质基础。【方法】通过在线软件分析预测P33蛋白信号肽位置和B细胞抗原决定簇，选择其无信号肽及有抗原优势决定簇的基因克隆至原核表达载体pET-30a(+)和pGEX-4T-1中，构建重组表达载体pET-30a-P33和pGEX-4T-1-P33，将重组质粒分别导入大肠杆菌BL21(DE3)和BL21感受态细胞进行诱导表达。使用SDS-PAGE和Western blotting分析表达产物，将过柱纯化后的pET-30a-P33重组蛋白免疫BALB/c小鼠，用pGEX-4T-1-P33重组蛋白筛选特异性单克隆抗体。利用ELISA方法进行抗体亚类鉴定和抗体效价检测；应用Western blotting和间接免疫荧光试验(IFA)检测单克隆抗体的反应原性和特异性。【结果】 PCR及双酶切鉴定结果显示，成功构建pET-30a-P33和pGEX-4T-1-P33 2种表达载体。SDS-PAGE 和Western blotting结果显示，成功制备了大小约 43和57 ku的pET-30a-P33和pGEX-4T-1-P33重组蛋白，并有良好的反应原性。用pGEX-4T-1-P33重组蛋白筛选，获得1株能稳定分泌 P33 蛋白单克隆抗体的杂交瘤细胞株(2H10A6)。单克隆抗体亚类鉴定结果表明,2H10A6株分泌的抗体为IgG1亚类，抗体效价为1∶6 553 600。Western blotting和IFA结果显示，2H10A6杂交瘤细胞株分泌产生的抗体可与东方泰勒虫P33蛋白特异性结合。【结论】本试验经原核表达系统成功获得了东方泰勒虫的P33重组蛋白，并成功制备了其单克隆抗体，该抗体可特异性识别P33蛋白。

关键词: 东方泰勒虫; P33蛋白; 原核表达; 单克隆抗体

Abstract: 【Objective】 P33 protein is an important surface protein of Theileria orientalis,possessing good antigenicity and immunogenicity.The present study aimed to develop specific monoclonal antibodies against P33 protein using hybridoma technology,systematically evaluate their immunological characteristics,and screen monoclonal cell lines with stable antibody-secreting capacity,thereby laying a material foundation for establishing a P33 antigen-based serological diagnostic method for Theileria orientalis. 【Method】 The position of the signal peptide and B cell epitopes of P33 protein was predicted and analyzed by online software.P33 gene without signal peptide but with dominant antigenic epitopes was cloned into prokaryotic expression vectors pET-30a and pGEX-4T-1 to construct recombinant expression vectors pET-30a-P33 and pGEX-4T-1-P33,respectively.The recombinant plasmids were transformation into Escherichia coli BL21 (DE3) and BL21 competent cells for induction of expression.The expressed products were analyzed by SDS-PAGE and Western blotting.BALB/c mice were immunized with the purified pET-30a-P33 recombinant protein,and the specific monoclonal antibody was screened with the pGEX-4T-1-P33 recombinant protein.Antibody subclasses and titer were identified by ELISA.The antigenicity and the specificity of the monoclonal antibody were detected by Western blotting and indirect immunofluorescence assay (IFA). 【Result】 PCR and restriction enzyme digestion results showed that the two expression vectors pET-30a-P33 and pGEX-4T-1-P33 were successfully constructed.The results of SDS-PAGE and Western blotting showed that the recombinant proteins pET-30a-P33 and pGEX-4T-1-P33 with sizes of approximately 43 and 57 ku were successfully prepared and had good antigenicity.A hybridoma cell line (2H10A6) that stably secretes monoclonal antibodies against P33 protein was established through screening with the pGEX-4T-1-P33 recombinant protein.Monoclonal antibody subclass identification results showed that the antibody secreted by 2H10A6 was IgG1 subtype,and the antibody titer was 1∶6 553 600.Western blotting and IFA results showed that the 2H10A6 hybridoma cell line could specifically bind to the P33 protein of Theileria orientalis. 【Conclusion】 The recombinant P33 protein of Theileria orientalis was successfully obtained through a prokaryotic expression system in this study,and monoclonal antibody specifically recognizing the P33 protein was successfully generated.

Key words: Theileria orientalis; P33 protein; prokaryotic expression; monoclonal antibody

中图分类号:

王贤炅, 金钰, 夏莹, 张丙寅, 董思言, 金春梅, 于龙政. 东方泰勒虫P33蛋白单克隆抗体的制备与鉴定[J]. 中国畜牧兽医, 2025, 52(8): 3800-3809.

WANG Xianjiong, JIN Yu, XIA Ying, ZHANG Bingyin, DONG Siyan, JIN Chunmei, YU Longzheng. Preparation and Identification of Monoclonal Antibodies Against Theileria orientalis P33 Protein[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3800-3809.

参考文献

[1] OTA N,MIZUNO D,KUBOKI N,et al.Epidemiological survey of Theileria orientalis infection in grazing cattle in the eastern part of Hokkaido,Japan[J]. Journal of Veterinary Medical Science,2009,71(7):937-944.
[2] GEBREKIDAN H,PERERA P K,GHAFAR A,et al.An appraisal of oriental theileriosis and the Theileria orientalis complex,with an emphasis on diagnosis and genetic characterisation[J].Parasitology Research,2020,119(1):11-22.
[3] WATTS J,PLAYFORD M,HICKEY K.Theileria orientalis:A review[J]. New Zealand Veterinary Journal,2016,64(1):3-9.
[4] EL-ALFY E S,ABBAS I,ELSEADAWY R,et al.Global prevalence and species diversity of tick-borne pathogens in buffaloes worldwide:A systematic review and Meta-analysis[J].Parasites and Vectors,2023,16(1):115.
[5] LUO J,LU W.Cattle theileriosis in China[J].Tropical Animal Health and Production,1997,29(4 Suppl):4S-7S.
[6] 罗金,刘文阁,任巧云,等.microRNA-654调控巨噬细胞游走抑制因子抗东方泰勒虫感染长角血蜱的作用[J].中国兽医科学,2022,52(8):1023-1030. LUO J,LIU W G,REN Q Y,et al.Regulation of microRNA-654 in MIF of Haemaphysalis longicornis for anti-infection of Theileria orientalis[J].Chinese Veterinary Science,2022,52(8):1023-1030.(in Chinese)
[7] 金春梅.牛瑟氏泰勒虫P33主要表面蛋白基因的克隆PCR诊断方法的建立[D].延吉：延边大学,2006. JIN C M.Molecular cloning of major surface protein (P33) of Theileria sergenti and establishment of PCR diagnosis method of Theileria sergenti[D].Yanji：Yanbian University,2006.(in Chinese)
[8] NELSON P N,REYNOLDS G M,WALDRON E E,et al.Monoclonal antibodies[J].Journal of Molecular Pathology,2000,53(3):111-117.
[9] 张楚晗,赵帅阳,朱浩瀚,等.环形泰勒虫Tams1单个B细胞抗体的制备及鉴定[J].中国兽医科学,2024,54(9):1209-1215. ZHANG C H,ZHAO S Y,ZHU H H,et al.Preparation and identification of single B cell antibody against Tams1 of Theileria annulata[J].Chinese Veterinary Science,2024,54(9):1209-1215.(in Chinese)
[10] NEMA S,NITIKA N.Monoclonal antibody:Future of malaria control and prevention[J].Transactions of the Royal Society of Tropical Medicine and Hygiene,2023,117(9):673-674.
[11] ZHANG F,SU R,HAN C,et al.Production and characterization of monoclonal antibodies against Toxoplasma gondii ROP18 with strain-specific reactivity[J].Parasitology,2020,147(9):940-948.
[12] 吉婧,朱明哲,印春生,等.弓形虫GRA1蛋白单克隆抗体的制备与初步鉴定[A].中国畜牧兽医学会兽医寄生虫学分会论文集[C].2021. JI J,ZHU M Z,YIN C S,et al.Preparation and preliminary identification of monoclonal antibodies against Toxoplasma gondii GRA1 protein[A].Proceedings of Veterinary Parasitology Branch of China Association of Animal Husbandry and Veterinary Medicine[C].2021.(in Chinese)
[13] 代琴.弓形虫SAG1单克隆抗体的制备及其生物传感器的初步研究[D].重庆：重庆理工大学,2023. DAI Q.Preparation of monoclonal antibody against Toxoplasma gondii SAG1 and preliminary study of its biosensors[D].Chongqing:Chongqing University of Technology,2023.(in Chinese)
[14] GONDIM L F,WOLF A,VRHOVEC M G, et al.Characterization of an IgG monoclonal antibody targeted to both tissue cyst and sporocyst walls of Toxoplasma gondii[J].Experimental Parasitology,2016,163:46-56.
[15] 杨兴,许应天,张守发,等.牛瑟氏泰勒虫双拷贝p33表面蛋白基因在原核细胞中的串联表达[J].中国预防兽医学报,2010,32(8):647-649. YANG X,XU Y T,ZHANG S F,et al.Tandem expression of two copies surface protein p33 gene from Theileria sergenti in E.coli[J].Chinese Journal of Preventive Veterinary Medicine,2010,32 (8):647-649.(in Chinese)
[16] WANG L X,ZHAO J H,HE L,et al.An indirect ELISA for detection of Theileria sergenti antibodies in water buffalo using a recombinant major piroplasm surface protein[J].Veterinary Parasitology,2010,170(3-4):323-326.
[17] 张守发,许应天,梁晚枫,等.间接荧光抗体试验对牛瑟氏泰勒虫病的诊断[J].中国兽医科技,2003,33(8)：59-61. ZHANG S F,XU Y T,LIANG W F,et al.Diagnosis of bovine Theileria sergenti by indirect fluorescent antibody test[J].Chinese Journal of Veterinary Science and Technology,2003,33(8):59-61.(in Chinese)
[18] SONG K H,SANG B C.Prevalence of Theileria sergenti infection in Korean native cattle by polymerase chain reaction[J].Korean Journal of Parasitology,2003,41(3):141-145.
[19] 许应天,张守发,郑寓硕,等.以 P33重组蛋白为诊断抗原建立牛瑟氏泰勒虫病乳胶凝集试验[J].畜牧兽医学报,2004,35(3):347-349. XU Y T,ZHANG S F,ZHENG Y S,et al.Latex agglutination test of Theileris sergenti by P33 recombinant protein[J].Acta Veterinaria et Zootechnica Sinica,2004,35(3):347-349.(in Chinese)
[20] 金春梅,许应天,张守发,等.牛瑟氏泰勒虫病 PCR诊断方法的建立[J].畜牧与兽医,2007,39(5):46-48. JIN C M,XU Y T,ZHANG S F,et al.Establishment of PCR diagnostic method for Theileria sergenti in cattle[J].Animal Husbandry and Veterinary Medicine,2007,39 (5):46-48.(in Chinese)
[21] 夏晓辉,丁晓双,张晓轩,等.猪附红细胞体 PCR-ELISA检测方法的建立[J].中国兽医科学,2013,43(2):164-169. XIA X H,DING X S,ZHANG X X,et al.Development of PCR-ELISA method for detection of Eperythrozoon suis[J].Chinese Veterinary Science,2013,43(2):164-169.(in Chinese)
[22] 王中光,张守发,曹世诺,等.牛瑟氏泰勒虫环介导等温扩增检测方法的建立[J].中国预防兽医学报,2010,32(2)：108-111. WANG Z G,ZHANG S F,CAO S N,et al.Development of LAMP for detection of Theileria sergenti[J].Chinese Journal of Preventive Veterinary Medicine,2010,32(2):108-111.(in Chinese)
[23] KUMAR S,PALIWAL S,SUDAN V,et al.An endpoint visualization loop-mediated isothermal amplification (LAMP) for detecting bubaline theileriosis[J].Beni-Suef University Journal of Basic and Applied Sciences,2022,11(1):67.
[24] FERNÁNDEZ F J,VEGA M C.Choose a suitable expression host:A survey of available protein production platforms[J]. Advanced Technologies for Protein Complex Production and Characterization,2016,896:15-24.
[25] 白宇,李培德,胡景炎,等.弓形虫卵囊壁蛋白owp1的重组表达和特异性单克隆抗体制备[J].中国动物传染病学报,2016,24(3):60-65. BAI Y,LI P D,HU J Y,et al.Recombinant expression and preparation of monoclonal antibodies to oocyst wall protein 1 of Toxoplasma gondii[J]. Chinese Journal of Animal Infectious Diseases,2016,24(3):60-65.(in Chinese)
[26] 朱丽霖.IFIT5的原核表达及其对ALV-J复制的影响[D].荆州:长江大学,2022. ZHU L L.Prokaryotic expression of IFIT5 and its effect on the replication of ALV-J replication [D].Jingzhou:Yangtze University,2022.(in Chinese)
[27] 闫文娟,崔京涛,解宏杰,等.抗刚地弓形体单克隆抗体制备鉴定及初步应用[J].标记免疫分析与临床,2018,25(10):1542-1546. YAN W J,CUI J T,XIE H J,et al.The preparation of monoclonal antibodies to Toxoplasma gondii and its preliminary application[J].Labeled Immunoassays and Clinical Medicine,2018,25 (10):1542-1546.(in Chinese)
[28] 杨升.抗NcP40单抗制备及牛新孢子虫感染ELISA和胶体金检测方法的建立[D].长春：吉林大学,2018. YANG S.Preparation on monoclonal antibodies against NcP40 and development of ELISA and colloidal gold to detect infection of Neospora caninum in cattles[D].Changchun:Jilin University,2018.(in Chinese)
[29] 王盼举.马驽巴贝斯虫地方虫株抗Bc48蛋白单克隆抗体的制备及CI-ELISA检测方法的建立[D].乌鲁木齐：新疆农业大学,2019. WANG P J.Preparation of monoclonal antibodies against Bc48 protein and established CI-ELISA of Babesia caballi local strains[D].Urumqi:Xinjiang Agricultural University,2019.(in Chinese)
[30] 宋晶晶.马泰勒虫EMA-1单克隆抗体的制备及胶体金免疫层析快速检测试纸条的研发[D].乌鲁木齐:新疆农业大学,2022. SONG J J.Preparation of monoclonal antibodies against EMA-1 and development of rapid serological detection method for Theileria equi infection[D].Urumqi:Xinjiang Agricultural University,2022.(in Chinese)
[31] 张文波.牛羊巴贝斯虫和泰勒虫病的防治[J].中国畜牧业,2024,15:121-122. ZHANG W B.Prevention and control of babesiosis and taylorosis in cattle and sheep[J].Chinese Animal Husbandry,2024,15:121-122.(in Chinese)