大型艾美耳球虫重组蛋白GAPDH对兔的免疫保护效果评价

doi:10.11843/j.issn.0366-6964.2023.10.030

摘要/Abstract

摘要： 本研究旨在评价大型艾美耳球虫重组蛋白（rEm-GAPDH）对兔的免疫保护效果，为大型艾美耳球虫重组亚单位疫苗的研制奠定基础。根据转录组数据筛选出Em-GAPDH基因，进行原核表达与蛋白纯化；将50只45日龄无球虫感染幼兔随机分为5组（空白对照组、不免疫攻虫组、Trx-His-S tag攻虫对照组、Quil-A saponin攻虫对照组和rEm-GAPDH免疫组），分别经颈部皮下注射1 mL PBS、1 mL PBS、1 mL PBS含100 μg pET-32a空载蛋白和1 mg Quil-A、1 mL PBS含1 mg Quil-A、1mL 100 μg rEm-GAPDH含1 mg Quil-A，首免14 d后同等剂量加强免疫，二免14 d后每只兔经口感染1×10⁵个大型艾美耳球虫孢子化卵囊，观察各组兔临床症状，每周固定时间采血和称重，感染14 d后宰杀剖检并测定和统计各组兔的卵囊排出量、相对增重、特异性IgG抗体和细胞因子等。结果成功表达了重组蛋白rEm-GAPDH，大小在59 ku左右，且大部分表达在包涵体。免疫保护试验表明：感染后不免疫攻虫组出现症状，而免疫组症状不明显。rEm-GAPDH免疫组的卵囊减少率达57.16%，相对增重率显著大于不免疫攻虫组（P<0.05），ACI值为144.96。特异性IgG抗体水平、细胞因子（IFN-γ、IL-2、IL-4、IL-10、TGF-β）水平均与对照组存在显著差异（P<0.05）。大型艾美耳球虫重组蛋白rEm-GAPDH可以减少增重损失和卵囊排出，能引发宿主体内的细胞免疫和体液免疫应答，具有一定免疫保护作用，可以作为大型艾美耳球虫重组亚单位疫苗的候选抗原。

关键词: 兔, 大型艾美耳球虫, 三磷酸甘油醛脱氢酶, 重组蛋白, 免疫保护

Abstract: The aim of this study was to evaluate the immunoprotective effect of rEm-GAPDH, a recombinant protein of Eimeria magna, on rabbits and to lay the foundation for the development of a recombinant subunit immunization vaccine for rabbits. Screening of Em-GAPDH gene based on transcriptome data for prokaryotic expression and protein purification.Then 50 45-day-old coccidia-free rabbits were randomly divided into 5 groups (Unimmunized and unchallenged group, unimmunized and challenged group, Trx-His-S tag-challenged group, Quil-A saponin-challenged group and rEm-GAPDH immunized group). 1 mL PBS, 1 mL PBS, 1 mL PBS containing 100 μg pET-32a and 1 mg Quil-A, 1 mL PBS containing 1 mg Quil-A, 1 mL 100 μg rEm-GAPDH and 1 mg Quil-A were respectively injected subcutaneously via the neck to the rabbits, and were booster immunized with the same dose 14 d after the first immunization. The clinical manifestations of each group were observed after orally infected 1×10⁵ sporulated oocysts of Eimeria magna to every rabbit, while blood sample and body weight data were collected at regular intervals every week. After 14 d of the challenge, rabbits were dissected, and oocyst output, relative weight gain, cytokines, and specific IgG antibodies were measured and counted for each group. The results showed that the recombinant protein rEm-GAPDH was successfully expressed, with a size of about 59 ku and mostly expressed in the inclusion bodies. The immunoprotection test showed that typical symptoms of rabbit intestinal coccidiosis appeared in the unimmunized and challenged group after infection, while rEm-GAPDH immunized group showed no significant symptoms. rEm-GAPDH immunized group showed 57.16% oocyst reduction, the relative weight gain rate was significantly greater than the unimmunized and challenged group (P<0.05), specific IgG antibody levels, cytokine (IFN-γ, IL-2, IL-4, IL-10, TGF-β) levels were significantly different from those of the control group (P<0.05). The ACI of rEm-GAPDH immunized group was 144.96. The recombinant protein rEm-GAPDH of E.magna can reduce weight loss and oocyst excretion, trigger cellular and humoral immune responses in the host, and have certain immunoprotective effects, thus can be used as a candidate antigen for E.magna recombinant subunit vaccine.

Key words: rabbit, Eimeria magna, 3-phosphoglyceraldehyde dehydrogenase, recombinant protein, immunoprotection

中图分类号:

郑若愚, 肖洁, 白鑫, 陈浩, 蒲家艳, 任永军, 杨光友. 大型艾美耳球虫重组蛋白GAPDH对兔的免疫保护效果评价[J]. 畜牧兽医学报, 2023, 54(10): 4338-4349.

ZHENG Ruoyu, XIAO Jie, BAI Xin, CHEN Hao, PU Jiayan, REN Yongjun, YANG Guangyou. Evaluation of the Immunoprotective Effect of Recombinant Protein of Eimeria magna 3-Phosphoglyceraldehyde Dehydrogenase on Rabbits[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4338-4349.

参考文献 51

[1]	RYLEY J F, MILLARD B J, LONG P L.Further studies on the life cycle of Eimeria brunetti Levine 1942[J].Z Parasitenk, 1972, 40(1):35-48.
[2]	PAKANDL M, LICOIS D, COUDERT P.Electron microscopic study on sporocysts and sporozoites of parental strains and precocious lines of rabbit coccidia Eimeria intestinalis, E. media and E. magna[J].Parasitol Res, 2001, 87(1):63-66.
[3]	史晓春, 林昆华.扫描电镜对兔大型艾美耳球虫致病性的研究[J].中国兽医寄生虫病, 1995, 3(4):23-27, 19.SHI X C, LIN K H.Study on pathogenicity of Eimeria magna in rabbits using a scanning electron microscope[J].Chinese Journal of Veterinary Parasitology, 1995, 3(4):23-27, 19.(in Chinese)
[4]	方素芳, 关琛, 崔平, 等.不同地理株大型艾美耳球虫单卵囊分离及卵囊产量的比较[J].河北北方学院学报:自然科学版, 2019, 35(11):32-35, 39.FANG S F, GUAN C, CUI P, et al.Isolation of single oocyst of Eimeria magna and comparison of oocyst yield of different geographical strains[J].Journal of Hebei North University:Natural Science Edition, 2019, 35(11):32-35, 39.(in Chinese)
[5]	石团员, 鲍国连, 索勋, 等.妥曲珠利及其复方制剂对人工感染大型艾美耳球虫病兔的治疗[J].中国兽医学报, 2014, 34(12):1935-1939.SHI T Y, BAO G L, SUO X, et al.The curative efficacy of toltrazuril and its compound preparations against artificial infection with Eimeria magna in rabbits[J].Chinese Journal of Veterinary Science, 2014, 34(12):1935-1939.(in Chinese)
[6]	柳南星, 方素芳, 顾小龙, 等.大型艾美耳球虫生物学特性研究[J].动物医学进展, 2018, 39(3):40-44.LIU N X, FANG S F, GU X L, et al.Study on biological characteristics of Eimeria magna[J].Progress in Veterinary Medicine, 2018, 39(3):40-44.(in Chinese)
[7]	任永军, 罗跃军, 叶勇刚, 等.中药提取物对兔大型艾美耳球虫的预防效果观察[J].中国养兔, 2021(3):4-6.REN Y J, LUO Y J, YE Y G, et al.Observation on the preventive effect of traditional Chinese medicine extracts on Eimeria magna in rabbits[J].Chinese Journal of Rabbit Farming, 2021(3):4-6.(in Chinese)
[8]	崔平, 方素芳, 顾小龙, 等.大型艾美耳球虫早熟株的选育及18S rDNA系统发育分析[J].中国兽医学报, 2018, 38(7):1327-1331.CUI P, FANG S F, GU X L, et al.Selection and phylogenetic analysis of 18S rDNA from precocious line of Eimeria magna[J].Chinese Journal of Veterinary Science, 2018, 38(7):1327-1331.(in Chinese)
[9]	BACHENE M S, TEMIM S, AINBAZIZ H, et al.A vaccination trial with a precocious line of Eimeria magna in Algerian local rabbits Oryctolagus cuniculus[J].Vet Parasitol, 2018, 261:73-76.
[10]	TAO G R, SHI T Y, TANG X M, et al.Transgenic Eimeria magna Pérard, 1925 displays similar parasitological properties to the wild-type strain and induces an exogenous protein-specific immune response in rabbits (Oryctolagus cuniculus L.)[J].Front Immunol, 2017, 8:2.
[11]	GUPTA S K, SHUKLA P.Advanced technologies for improved expression of recombinant proteins in bacteria:perspectives and applications[J].Crit Rev Biotechnol, 2016, 36(6):1089-1098.
[12]	SIROVER M A.New insights into an old protein:the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase[J].Biochim Biophys Acta (BBA)-Protein Struct Mol Enzymol, 1999, 1432(2):159-184.
[13]	SIROVER M A.On the functional diversity of glyceraldehyde-3-phosphate dehydrogenase:biochemical mechanisms and regulatory control[J].Biochim Biophys Acta (BBA)-Gen Subj, 2011, 1810(8):741-751.
[14]	WAINE G J, BECKER M, YANG W, et al.Cloning, molecular characterization, and functional activity of Schistosoma japonicum glyceraldehyde-3-phosphate dehydrogenase, a putative vaccine candidate against Schistosomiasis japonica[J].Infect Immun, 1993, 61(11):4716-4723.
[15]	TANG C L, YANG J F, PAN Q, et al.Anti-CTLA-4 monoclonal antibody improves efficacy of the glyceraldehyde-3-phosphate dehydrogenase protein vaccine against Schistosoma japonicum in mice[J].Parasitol Res, 2019, 118(7):2287-2293.
[16]	TANG C L, XIE Y P, YU W H, et al.Effects of regulatory T cells on glyceraldehyde-3-phosphate dehydrogenase vaccine efficacy against Schistosoma japonicum[J].Acta Trop, 2020, 202:105239.
[17]	HUANG W L, GU M J, CHENG W J, et al.Mechanism by which the combination of SjCL3 and SjGAPDH protects against Schistosoma japonicum infection[J].Parasitol Res, 2021, 120(1):173-185.
[18]	ARGIRO L, KOHLSTÄDT S, HENRI S, et al.Identification of a candidate vaccine peptide on the 37 kDa Schistosoma mansoni GAPDH[J].Vaccine, 2000, 18(19):2039-2048.
[19]	ARGIRO L, HENRI S, DESSEIN H, et al.Induction of a protection against S. mansoni with a MAP containing epitopes of Sm37-GAPDH and Sm10-DLC.effect of coadsorption with GM-CSF on alum[J].Vaccine, 2000, 18(19):2033-2038.
[20]	TALLIMA H, DVOR[DD(-*2] ^ÁK J, KAREEM S, et al.Protective immune responses against Schistosoma mansoni infection by immunization with functionally active gut-derived cysteine peptidases alone and in combination with glyceraldehyde 3-phosphate dehydrogenase[J].PLoS Negl Trop Dis, 2017, 11(3):e0005443.
[21]	ERTTMANN K D, KLEENSANG A, SCHNEIDER E, et al.Cloning, characterization and DNA immunization of an Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH)[J].Biochim Biophys Acta (BBA)-Mol Basis Dis, 2005, 1741(1-2):85-94.
[22]	STEISSLINGER V, KORTEN S, BRATTIG N W, et al.DNA vaccine encoding the moonlighting protein Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) leads to partial protection in a mouse model of human filariasis[J].Vaccine, 2015, 33(43):5861-5867.
[23]	HAN K K, XU L X, YAN R F, et al.Vaccination of goats with glyceraldehyde-3-phosphate dehydrogenase DNA vaccine induced partial protection against Haemonchus contortus[J].Vet Immunol Immunopathol, 2012, 149(3-4):177-185.
[24]	LIU L R, HUANG X M, LIU J H, et al.Identification of common immunodominant antigens of Eimeria tenella, Eimeria acervulina and Eimeria maxima by immunoproteomic analysis[J].Oncotarget, 2017, 8(21):34935-34945.
[25]	JOHNSON J, REID W M.Anticoccidial drugs:lesion scoring techniques in battery and floor-pen experiments with chickens[J].Exp Parasitol, 1970, 28(1):30-36.
[26]	赵爱云, 段嘉树.一株和缓艾美耳球虫的致病性和免疫原性[J].北京农学院学报, 2006, 21(1):50-52.ZHAO A Y, DUAN J S.Pathogenicity and immunogenicity a new isolated strain of E. mitis[J].Journal of Beijing Agricultural College, 2006, 21(1):50-52.(in Chinese)
[27]	MCMANUS E C, CAMPBELL W C, CUCKLER A C.Development of resistance to quinoline coccidiostats under field and laboratory conditions[J].J Parasitol, 1968, 54(6):1190-1193.
[28]	田依凡, 赵权, 信彩岩, 等.斯氏艾美耳球虫Rhomboid基因抗原优势区的筛选与鉴定[J].黑龙江畜牧兽医, 2017(3):156-159.TIAN Y F, ZHAO Q, XIN C Y, et al.Screening and identification of antigenic dominant region of the rhomboid gene of Eimeria stiedai[J].Heilongjiang Animal Science and Veterinary Medicine, 2017(3):156-159.(in Chinese)
[29]	孟庆玲, 乔军, 才学鹏, 等.斯氏艾美耳球虫MIC-5与兔IL-15基因在真核细胞中的共表达[J].中国农业科学, 2011, 44(19):4096-4101.MENG Q L, QIAO J, CAI X P, et al.Co-expression of MIC-5 gene of Eimeria stiedai and rabbit IL-15 in eucaryotic cell line[J].Scientia Agricultura Sinica, 2011, 44(19):4096-4101.(in Chinese)
[30]	孟庆玲, 乔军, 才学鹏, 等.携带兔斯氏艾美耳球虫MIC-5基因减毒鼠伤寒沙门菌活疫苗的安全性、稳定性与免疫原性[J].中国兽医学报, 2011, 31(5):659-662.MENG Q L, QIAO J, CAI X P, et al.Safety, stability and immunogenicity of attenuated Salmonella typhimurium carrying MIC-5 gene of Eimeria stieda[J].Chinese Journal of Veterinary Science, 2011, 31(5):659-662.(in Chinese)
[31]	景瑾, 张帅, 仇保丰, 等.斯氏艾美耳球虫热休克蛋白70基因的克隆及表达分析[J].黑龙江畜牧兽医, 2017(4):11-16.JING J, ZHANG S, QIU B F, et al.Cloning and expression analysis of heat shock protein 70 (HSP70) gene from Eimeria stiedai[J].Heilongjiang Animal Science and Veterinary Medicine, 2017(4):11-16.(in Chinese)
[32]	罗跃军, 任永军, 白鑫, 等.斯氏艾美耳球虫重组表面抗原SAG13和SAG14对兔的免疫保护效果初步观察[J].畜牧兽医学报, 2022, 53(3):883-893.LUO Y J, REN Y J, BAI X, et al.Preliminary observation on the immunoprotective effects of recombinant surface antigens SAG13 and SAG14 of Eimeria stiedae in rabbits[J].Acta Veterinaria et Zootechnica Sinica, 2022, 53(3):883-893.(in Chinese)
[33]	PU J Y, XIAO J, BAI X, et al.Prokaryotic expression of Eimeria magna SAG10 and SAG11 genes and the preliminary evaluation of the effect of the recombinant protein on immune protection in rabbits[J].Int J Mol Sci, 2022, 23(18):10942.
[34]	CHEN H, PU J Y, XIAO J, et al.Evaluation of the immune protective effects of rEmMIC2 and rEmMIC3 from Eimeria magna in rabbits[J].Parasitol Res, 2023, 122(2):661-669.
[35]	XIAO J, CHEN H, ZHENG R Y, et al.Recombinant GMA56 and ROP17 of Eimeria magna conferred protection against infection by homologous species[J].Front Immunol, 2023, 13:1037949.
[36]	TERAO Y, YAMAGUCHI M, HAMADA S, et al.Multifunctional glyceraldehyde-3-phosphate dehydrogenase of Streptococcus pyogenes is essential for evasion from neutrophils[J].J Biol Chem, 2006, 281(20):14215-14223.
[37]	JIN H, AGARWAL S, AGARWAL S, et al.Surface export of GAPDH/SDH, a glycolytic enzyme, is essential for Streptococcus pyogenes virulence[J].mBio, 2011, 2(3):e00068-11.
[38]	PEREZ-CASAL J, POTTER A A.Glyceradehyde-3-phosphate dehydrogenase as a suitable vaccine candidate for protection against bacterial and parasitic diseases[J].Vaccine, 2016, 34(8):1012-1017.
[39]	LAMA A, KUCKNOOR A, MUNDODI V, et al.Glyceraldehyde-3-phosphate dehydrogenase is a surface-associated, fibronectin-binding protein of Trichomonas vaginalis[J].Infect Immun, 2009, 77(7):2703-2711.
[40]	ROSINHA G M S, MYIOSHI A, AZEVEDO V, et al.Molecular and immunological characterisation of recombinant Brucella abortus glyceraldehyde-3-phosphate-dehydrogenase, a T-and B-cell reactive protein that induces partial protection when co-administered with an interleukin-12-expressing plasmid in a DNA vaccine formulation[J].J Med Microbiol, 2002, 51(8):661-671.
[41]	王璐, 朱顺海, 赵其平, 等.宿主GAPDH对柔嫩艾美耳球虫子孢子入侵细胞的影响[J].畜牧兽医学报, 2019, 50(10):2088-2096.WANG L, ZHU S H, ZHAO Q P, et al.Effects of host glyceraldehyde-3-phosphate dehydrogenase on Eimeria tenella sporozoites invasion of cells[J].Acta Veterinaria et Zootechnica Sinica, 2019, 50(10):2088-2096.(in Chinese)
[42]	BRUNO S, MARGIOTTA M, PINTO A, et al.Selectivity of 3-bromo-isoxazoline inhibitors between human and Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenases[J].Bioorg Med Chem, 2016, 24(12):2654-2659.
[43]	CHA S J, KIM M S, PANDEY A, et al.Identification of GAPDH on the surface of Plasmodium sporozoites as a new candidate for targeting malaria liver invasion[J].J Exp Med, 2016, 213(10):2099-2112.
[44]	CHAPMAN H D.Milestones in avian coccidiosis research:a review[J].Poult Sci, 2014, 93(3):501-511.
[45]	YUN C H, LILLEHOJ H S, CHOI K D.Eimeria tenella infection induces local gamma interferon production and intestinal lymphocyte subpopulation changes[J].Infect Immun, 2000, 68(3):1282-1288.
[46]	LI G X, LILLEHOJ E P, LILLEHOJ H S.Interleukin-2 production in SC and TK chickens infected with Eimeria tenella[J].Avian Dis, 2002, 46(1):2-9.
[47]	MIYAMOTO T, MIN W, LILLEHOJ H S.Kinetics of interleukin-2 production in chickens infected with Eimeria tenella[J].Comp Immunol, Microbiol Infect Dis, 2002, 25(3):149-158.
[48]	ISOBE T, LILLEHOJ H S.Dexamethasone suppresses T cell-mediated immunity and enhances disease susceptibility to Eimeria mivati infection[J].Vet Immunol Immunopathol, 1993, 39(4):431-446.
[49]	GAZZINELLI R T, WYSOCKA M, HIENY S, et al.In the absence of endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune response dependent on CD4+ T cells and accompanied by overproduction of IL-12, IFN-γ and TNF-α[J].J Immunol, 1996, 157(2):798-805.
[50]	ROBINSON P, OKHUYSEN P C, CHAPPELL C L, et al.Transforming growth factor β1 is expressed in the jejunum after experimental Cryptosporidium parvum infection in humans[J].Infect Immun, 2000, 68(9):5405-5407.
[51]	TIAN L, LI W Y, HUANG X M, et al.Protective efficacy of coccidial common antigen glyceraldehyde 3-Phosphate dehydrogenase (GAPDH) against challenge with three Eimeria species[J].Front Microbiol, 2017, 8:1245.