产气荚膜梭菌Alpha毒素突变体的表达及单克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2025.01.040

Abstract

Abstract: 【Objective】 The purpose of this experiment was to obtain the recombinant mutant of Clostridium perfringens alpha-toxin (CPA),evaluate its virulence and antigenicity,and then prepare monoclonal antibodies against CPA,and evaluate the characteristics of the monoclonal antibodies. 【Method】 The CPA gene fragment containing 6 amino acid mutations (aspartic acid mutation at positions 56 and 130 to glycine,tyrosine mutation at positions 275,307 and 331 to phenylalanine,aspartic acid mutation at position 336 to aspartic acid) was synthesized.The synthetic fragment was cloned into pET-30a (+) vector,and transformed into Escherichia coli BL21 (DE3) competent cells for induction expression and purification,and the recombinant protein rCPA_m6 was obtained.The toxicity and immunogenicity were detected.The indirect ELISA assay based on rCPA_m6 for the detection of CPA antibody was established.BALB/c mice were immunized with inactivated natural CPA as immunogen,and the monoclonal antibody was prepared and its function was identified. 【Result】 Recombinant protein rCPA_m6 could be expressed in both soluble and inclusion form in Escherichia coli BL21 (DE3) competent cells. The results of toxicity test showed that all mice survived after 100 μg/mice rCPA_m6 challenge.The results of immunogenicity analysis showed that after 1×minimum lethal dose (MLD) of natural CPA,all mice died in control group,and the survival rate of mice in 10 and 20 μg/mice rCPA_m6 immune groups was 100%.An indirect ELISA method for the detection of CPA antibody was successfully established using rCPA_m6,and 4 hybridoma cell lines secreting anti-CPA monoclonal antibody were screened,named 6E3,6B8,10A11 and 13D10,respectively.The titers of the 4 cell supernatants were all ≥1∶3 200,among which the monoclonal antibody 6B8 cell supernatants could neutralize the natural CPA and react with rCPA_m6. 【Conclusion】 The recombinant protein rCPA_m6 with good safety and immunogenicity was successfully obtained,and the CPA monoclonal antibody 6B8 had certain neutralizing activity as well as high specificity and sensitivity.The results provided a candidate antigen for the development of CPA subunit vaccine,as well as a material basis for the treatment of CPA intoxication and the establishment of antigen/antibody detection methods.

Key words: Clostridium perfringens alpha-toxin (CPA); mutant; prokaryotic expression; monoclonal antibodies

CLC Number:

S852.61

HAN Fengye, LIU Ying, PAN Chenfan, ZHANG Qianyi, CHEN Xiaoyun, ZHU Zhen, YIN Chunsheng, WEN Yongjun, WANG Fengxue, DU Jige. Expression and Preparation of Monoclonal Antibodies of Clostridium perfringens Alpha-Toxin Mutant[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(1): 442-450.

References

[1] FLORES-DÍAZ M,ALAPE-GIRÓN A.Role of Clostridium perfringens phospholipase C in the pathogenesis of gas gangrene[J].Toxicon,2003,42(8):979-986.
[2] XU C,SHE Y,FU F,et al.Study of the structure and biological activity of the amino-terminus of the α-toxin from Clostridium welchii type A[J].Current Microbiology,2019,76(10):1175-1185.
[3] POLETTI G,MASSARI E,FABBRI A,et al.Massive intravascular hemolysis associated with Clostridium perfringens bacteremia[J].Blood Research,2021,56(1):5.
[4] LEIBLEIN M,WAGNER N,ADAM E H,et al.Clostridial gas gangrene—A rare but deadly infection:Case series and comparison to other necrotizing soft tissue infections[J]. Orthopaedic Surgery,2020,12(6):1733-1747.
[5] NAVARRO M A,MCCLANE B A,UZAL F A.Mechanisms of action and cell death associated with Clostridium perfringens toxins[J].Toxins (Basel),2018,10(5):212.
[6] NAGAHAMA M,MUKAI M,MORIMITSU S,et al.Role of the C-domain in the biological activities of Clostridium perfringens alpha-toxin[J].Microbiology and Immunology,2002,46(10):647-655.
[7] PRESEAU T,DEVIENDT J,DUTTMAN R,et al.Clostridium perfringens in gas gangrene:Still a smoked gun![J].Journal of Translational Internal Medicine,2020,8(1):54-56.
[8] LIU S,YANG X F,ZHANG H,et al.Amentoflavone attenuates Clostridium perfringens gas gangrene by targeting alpha-toxin and perfringolysin O[J].Frontiers in Pharmacology,2020,11:179.
[9] BHATTACHARJEE P,KEYEL P A.Cholesterol-dependent cytolysins impair pro-inflammatory macrophage responses[J].Scientific Reports, 2018,8(1):6458.
[10] VAN IMMERSEEL F,ROOD J I,MOORE R J,et al.Rethinking our understanding of the pathogenesis of necrotic enteritis in chickens[J].Trends in Microbiology,2009,17(1):32-36.
[11] MOUSTAFA S,ZAKARIA I,MOUSTAFA A,et al.Bacteriological and serological investigation of Clostridium perfringens in lambs[J].Scientific Reports,2022,12(1):19715.
[12] GAO X W,MA Y Y,WANG Z,et al.Oral immunization of mice with a probiotic Lactobacillus casei constitutively expressing the α-toxoid induces protective immunity against Clostridium perfringens α-toxin[J].Virulence,2019,10(1):166-179.
[13] WANG Y,MIAO Y,HU L P,et al.Immunization of mice against alpha,beta,and epsilon toxins of Clostridium perfringens using recombinant rCpa-b-x expressed by Bacillus subtilis[J].Molecular Immunology,2020,123:88-96.
[14] GOOSSENS E,VERHERSTRAETEN S,VALGAEREN B R,et al.The C-terminal domain of Clostridium perfringens alpha toxin as a vaccine candidate against bovine necrohemorrhagic enteritis[J].Veterinary Research,2016,47(1):52.
[15] 孙刘妹,徐义刚,唐丽杰,等.产气荚膜梭菌α毒素单克隆抗体的制备[J].黑龙江畜牧兽医,2017,3:153-155. SUN L M,XU Y G,TANG L J,et al.Preparation of monoclonal antibody against α toxin of Clostridium perfringens[J].Heilongjiang Animal Husbandry and Veterinary Medicine,2017,3:153-155.(in Chinese)
[16] 许崇波,马从林,赵宝华,等.抗A型产气荚膜梭菌α毒素单克隆抗体杂交瘤细胞系的建立与中和效应[J].中国兽医学报,1999,5:464-466. XU C B,MA C L,ZHAO B H,et al.Establishment of hybridoma cell lines secreting monoclonal antibodies against alpha-toxin of Clostridium perfringens type A and netralization effect of produced mcAbs[J].Chinese Journal of Veterinary Science,1999,5:464-466.(in Chinese)
[17] SOLANKI A K,BHATIA B,KAUSHIK H,et al. Clostridium perfringens beta toxin DNA prime-protein boost elicits enhanced protective immune response in mice[J].Applied Microbiology and Biotechnology,2017,101(14):5699-5708.
[18] 林晓佳.A型产气荚膜梭菌类毒疫素苗的临床应用和保护效果评价[D].泰安:山东农业大学,2016. LIN X J.Clinical application and protective effect evaluation of Clostridium perfringens type A toxoid vaccine[D].Tai’an:Shandong Agricultural University,2016.(in Chinese)
[19] 胡波,魏后军,范志宇,等.兔流行性腹胀病样品中产气荚膜梭菌的分离鉴定[J].江苏农业科学,2020,48(6):160-163. HU B,WEI H J,FAN Z Y,et al.Isolation and identification of Clostridium perfringens in samples of rabbit epidemic bloating disease[J].Jiangsu Agricultural Sciences, 2020,48(6):160-163.(in Chinese)
[20] 杜吉革,薛麒,朱真,等.产气荚膜梭菌α毒素突变体的表达及免疫保护力评价[J].中国兽医学报,2019,39(2):265-270. DU J G,XUE Q,ZHU Z,et al.Expression and protective efficacy of Clostridium perfringens α toxin derivative[J].Chinese Journal of Veterinary Science,2019,39(2):265-270.(in Chinese)
[21] LI J,ADAMS V,BANNAM T L,et al.Toxin plasmids of Clostridium perfringens[J].Microbiology and Molecular Biology Reviews,2013,77(2):208-233.
[22] 中国兽药典委员会.中华人民共和国兽药典2020年版(三部)[M].北京:中国农业出版社,2016. COMMISSION OF CHINESE VETERINARY PHARMACOPOEIA.Veterinary Pharmacopoeia of the Peoples Repubilc of China 2020 Edition (Volume Ⅲ)[M].Beijing:China Agriculture Press,2016.(in Chinese)
[23] SAKURAI J,NAGAHAMA M,ODA M,Clostridium perfringens alpha-toxin:Characterization and mode of action[J].Biochemical Journal,2004,136(5):569-574.
[24] FERREIRA M R,MOREIRA G M,CUNHA C E,et al.Recombinant alpha,beta,and epsilon toxins of Clostridium perfringens:Production strategies and applications as veterinary vaccines[J].Toxins (Basel),2016,8(11):340.
[25] 杜吉革,朱真,薛麒,等.重组产气荚膜梭菌α毒素突变体的表达与免疫保护力评价[J].中国兽药杂志,2018,52(9):1-8. DU J G,ZHU Z,XUE Q,et al.Expression and protective efficacy of Clostridium perfringens α toxin derivative[J].Chinese Journal of Veterinary Drug,2018,52(9):1-8.(in chinese)
[26] ALAPE-GIRÓN A,FLORES-DÍAZ M,GUILLOUARD I,et al.Identification of residues critical for toxicity in Clostridium perfringens phospholipase C,the key toxin in gas gangrene[J].Eurepean Journal of Biochemistry,2000,267(16):5191-5197.
[27] SINGH A,RAWAT P,CHOUDHURY D,et al.Immunogenic and neutralization efficacy of recombinant perfringolysin O of Clostridium perfringens and its C-terminal receptor-binding domain in a murine model[J].Journal of Immunology Research,2022,70(2):240-255.
[28] JEPSON M,BULLIFENT H L,CRANE D,et al.Tyrosine 331 and phenylalanine 334 in Clostridium perfringens alpha-toxin are essential for cytotoxic activity[J].FEBS Letters,2001,495(3):172-177.
[29] ALEJANDRA W P,MIRIAM IRENE J P,FABIO ANTONIO G S,et al.Production of monoclonal antibodies for therapeutic purposes:A review[J].International Immunopharmacology,2023,120:110376.
[30] LOGAN A J,WILLIAMSON E D,TITBALL R W,et al.Epitope mapping of the alpha-toxin of Clostridium perfringens[J].Infection and Immunity,1991,59(12):4338-4342.
[31] LIU Z L,BLASCHEK H P.Monoclonal antibody-based ELISA for detection of Clostridium perfringens alpha-toxin[J].Journal of Food Protection,1996,59(6):621-625.
[32] FORTI K,CAGIOLA M,PELLEGRINI M,et al.Generation of recombinant Baculovirus expressing atoxic C-terminal CPA toxin of Clostridium perfringens and production of specific antibodies[J].BMC Biotechnology,2020,20(1):7.

Expression and Preparation of Monoclonal Antibodies of Clostridium perfringens Alpha-Toxin Mutant

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