斯氏艾美耳球虫3-磷酸甘油醛脱氢酶重组蛋白对兔的免疫保护效果评价

doi:10.11843/j.issn.0366-6964.2023.06.035

Abstract

Abstract: The aim of this study was to evaluate the immunoprotective effect of Es-GAPDH, a recombinant protein of Eimeria stiedae, on rabbits and to lay the foundation for the development of a recombinant subunit immunization vaccine for rabbits.The transcript levels of Es-GAPDH at different developmental stages of E.stiedae were analyzed by relative fluorescence quantitative PCR, and Es-GAPDH protein was prokaryotically expressed and purified. Then 42 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 rEs-GAPDH immunization 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 rEs-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. After the secondary immunization, except for the unimmunized and unchallenged group, each rabbit in the other groups was orally infected with 1×10⁴ sporulated oocysts of E.stiedae. The clinical manifestations of each group were observed after the attack. Blood samples were collected and the animals were weighed at regular intervals every week. Twenty-one days after infection, the liver pathological changes were observed by autopsy, and the relative weight gain, feed to gain ratio, liver index, oocyst excretion, biochemical indexes, specific IgG antibodies and cytokines of each experimental group were measured and counted. The results showed that Es-GAPDH was transcribed at all developmental stages of E.stiedae, and there were differences in transcript levels, with the highest transcript level in the sporulating oocyst stage. The immunoprotection test showed that typical symptoms of rabbit hepatic coccidiosis appeared in the unimmunized and challenged group after infection, while rEs-GAPDH immunized group showed no significant symptoms. rEs-GAPDH immunized group showed 87.09% oocyst reduction, and significantly greater relative weight gain than the other three challenged groups (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 unimmunized and challenged group (P<0.05), and histopathological sections also showed that the liver tissue was less destroyed and the number of parasites was lower in the immunized group compared with the unimmunized and challenged group. It can be concluded that the recombinant protein rEs-GAPDH of E.stiedae can reduce weight loss and oocyst excretion, trigger cellular and humoral immune responses in the host, have certain immunoprotective effects, and can be used as a candidate antigen for E.stiedae recombinant subunit vaccine.

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

CLC Number:

ZHENG Ruoyu, REN Yongjun, XIAO Jie, BAI Xin, PU Jiayan, CHEN Hao, YANG Guangyou. Evaluation of the Immunoprotective Effect of Recombinant Protein of Eimeria stiedae 3-Phosphoglyceraldehyde Dehydrogenase on Rabbits[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2581-2595.

References 45

[1]	INDRASANTI D,INDRADJI M,HASTUTI S,et al.Body weight,oocyte elimination and blood profile of rabbit after challenge test using Eimeria stiedai[J].Anim Prod,2019,20(1):53-59.
[2]	DARZI M M,MIR M S,KAMIL S A,et al.Pathological changes and local defense reaction occurring in spontaneous hepatic coccidiosis in rabbits (Oryctolagus cuniculus)[J].World Rabbit Sci,2007,15(1):23-28.
[3]	YAKHCHALI Y,TEHRANI A.Eimeriidosis and pathological findings in New Zealand white rabbits[J].J Biol Sci,2007,7(8):1488-1491.
[4]	赵孝民.γ-射线辐射对兔球虫孢子化卵囊致病性及免疫原性的影响[J].山东农业大学学报,1986,17(2):1-10.ZHAO X M.The effects of gamma rays on the pathogenicity and immunogenicity of sporulated oocysts of Eimeria sliedai[J].Journal of Shandong Agricultural University,1986,17(2):1-10.(in Chinese)
[5]	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.
[6]	SIROVER M A.New insights into an old protein:The functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase[J].Biochim Biophys Acta,1999,1432(2):159-184.
[7]	SIROVER M A.On the functional diversity of glyceraldehyde-3-phosphate dehydrogenase:Biochemical mechanisms and regulatory control[J].Biochim Biophys Acta,2011,1810(8):741-751.
[8]	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.
[9]	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.
[10]	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.
[11]	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.
[12]	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.
[13]	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 coadsorbtion with GM-CSF on alum[J].Vaccine,2000,18(19):2033-2038.
[14]	TALLIMA H,DVO?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.
[15]	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,2004,1741(1-2):85-94.
[16]	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.
[17]	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.
[18]	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.
[19]	李祥瑞,汪志楷.斯氏艾美耳球虫生活史的研究[J].南京农业大学学报,1989,12(2):97-101.LI X R,WANG Z K.Study on life cycle of Eimeria stiedai[J].Journal of Nanjing Agricultural University,1989,12(2):97-101.(in Chinese)
[20]	SHEN N X,ZHANG H J,REN Y J,et al.A chitinase-like protein from Sarcoptes scabiei as a candidate anti-mite vaccine that contributes to immune protection in rabbits[J].Parasit Vectors,2018,11(1):599.
[21]	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)
[22]	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)
[23]	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)
[24]	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)
[25]	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)
[26]	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.
[27]	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.
[28]	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.
[29]	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.
[30]	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.
[31]	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)
[32]	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.
[33]	CHA S J,PARK K,SRINIVASAN P,et al.CD68 acts as a major gateway for malaria sporozoite liver infection[J].J Exp Med,2015,212(9):1391-1403.
[34]	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.
[35]	CHAPMAN H D.Milestones in avian coccidiosis research:a review[J].Poult Sci,2014,93(3):501-511.
[36]	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.
[37]	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.
[38]	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.
[39]	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.
[40]	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-gamma and TNF-alpha[J].J Immunol,1996,157(2):798-805.
[41]	蒋保余,王文青,曲自刚,等.柔嫩艾美耳球虫在鸡巨噬细胞中的培养及子孢子感染巨噬细胞细胞因子的mRNA水平[J].动物医学进展,2020,41(11):33-40.JIANG B Y,WANG W Q,QU Z G,et al.Cultivation of Eimeria tenella in chicken macrophages and mRNA levels of macrophage cytokines infected by sporozoites[J].Progress in Veterinary Medicine,2020,41(11):33-40.(in Chinese)
[42]	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.
[43]	MIN W,LILLEHOJ H S.Isolation and characterization of chicken interleukin-17 cDNA[J].J Interferon Cytokine Res,2002,22(11):1123-1128.
[44]	索勋,孔繁瑶,李安兴,等.斯氏艾美耳球虫子孢子的移行途径[J].畜牧兽医学报,1994,25(3):252-255.SUO X,KONG F Y,LI A X,et al.Migration of the sporozoies of eimeria stiedai (sporozoa:eimeriidae)[J].Acta Veterinaria et Zootechnica Sinica,1994,25(3):252-255.(in Chinese)
[45]	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.

Evaluation of the Immunoprotective Effect of Recombinant Protein of Eimeria stiedae 3-Phosphoglyceraldehyde Dehydrogenase on Rabbits

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