牛源抗金黄色葡萄球菌α溶血素单链抗体研制
|
摘要
由金黄色葡萄球菌引起的奶牛乳腺炎给养殖业造成了严重损失,抗生素是治疗奶牛乳腺炎的首选方法,但易出现耐药菌株。而目前针对金黄色葡萄球菌的疫苗也不能对奶牛乳腺炎提供足够的保护,疫苗的主要作用机理是激发机体产生抗体,故被动免疫针对金黄色葡萄球菌毒力因子的抗体也许能对感染起到一定的治疗作用。本研究首先扩增得到影响其致病性的重要毒力因子-α溶血素(Hla)的编码基因,然后将其转化到E.coli中进行表达并纯化,再以此纯化产物为抗原,从牛源噬菌体单链抗体表达文库中筛选到了一株针对α溶血素(Hla)高亲和力的scFv,最终将该scFv进行纯化。本研究一方面为治疗金黄色葡萄球菌所致奶牛乳腺炎提供一种新思路,另一方面也为分子水平上研究α溶血素的致病机制提供了有效的阻断分子。
Staphylococcus aureus-induced bovine mastitis has caused significant losses in the dairy industry.At present,antibiotics are primarily used for the treatment of S.aureus-induced mastitis,but this strategy can increase the prevalence of antibiotic-resistant bacterial strains.However,the current vaccines have not provided adequate protection against S.aureus infection in cattle.Therefore,passive immunization with antibodies against S.aureus virulence factors may be effective.In this study,α hemolysin(Hla) gene,which encodes the most important virulence factor in S.aureus infection,was firstly amplified,and transformed into E.coli for expression and purification.After three rounds of selection,one single-chain variable region fragment(scFv) that bound to Hla with high affinity was obtained from bovine scFv phage display library.Finally,the scFv was purified.For one thing,this study provides an effective blocking molecule for studying the pathogenesis of Hla at the molecular level;for another,it provides a therapeutic candidate for the treatment of S.aureus-induced bovine mastitis.
Staphylococcus aureus-induced bovine mastitis has caused significant losses in the dairy industry.At present,antibiotics are primarily used for the treatment of S.aureus-induced mastitis,but this strategy can increase the prevalence of antibiotic-resistant bacterial strains.However,the current vaccines have not provided adequate protection against S.aureus infection in cattle.Therefore,passive immunization with antibodies against S.aureus virulence factors may be effective.In this study,α hemolysin(Hla) gene,which encodes the most important virulence factor in S.aureus infection,was firstly amplified,and transformed into E.coli for expression and purification.After three rounds of selection,one single-chain variable region fragment(scFv) that bound to Hla with high affinity was obtained from bovine scFv phage display library.Finally,the scFv was purified.For one thing,this study provides an effective blocking molecule for studying the pathogenesis of Hla at the molecular level;for another,it provides a therapeutic candidate for the treatment of S.aureus-induced bovine mastitis.
引文
[1] 孙慧琴,熊天雪,张艳玲,等.奶牛乳房炎疾病研究进展[J].现代农业科技,2018(5):218-226.
[2] Wilke G A,Bubeck Wardenburg J.Role of a disintegrin and metalloprotease 10 in Staphylococcus aureus alpha-hemolysin-mediated cellular injury[J].Proceedings of National Academy Science of the United States of America,2010,107(30):13473-13478.
[3] Elbers A R,Miltenburg J D,De Lange D,et al.Risk factors for clinical mastitis in a random sample of dairy herds from the southern part of The Netherlands[J].Journal of Dairy Science,1998,81(2):420-426.
[4] Ezzat Alnakip M,Quintela-Baluja M,Bohme K,et al.The immunology of mammary gland of dairy ruminants between healthy and inflammatory conditions[J].Journal of Veterinary Medicine,2014(10):659801.
[5] Senoh M,Miyoshi S K,Fouz B,et al.The cytotoxin-hemolysin genes of human and eel pathogenic Vibrio vulnificus strains:comparison of nucleotide sequences and application to the genetic grouping[J].Microbiology & Immunology,2013,49(6):513-519.
[6] Holliger P,Hudson P J.Engineered antibody fragments and the rise of single domains[J].Nature Biotechnology,2005,23(9):1126-1136.
[7] Schaefer J V,Honegger A,Plückthun A.Construction of scFv fragments from hybridoma or spleen cells by PCR assembly[A].Kontermann R,Dübel S.Antibody engineering[M].Berlin,Heidelberg:Springer Berlin Heidelberg,2010:21-44.
[8] Wang M,Zhang Y,Zhu J.Anti-staphylococcus aureus single-chain variable region fragments provide protection against mastitis in mice[J].Applied Microbiology and Biotechnology,2016,100(5):2153-2162.
[9] Mcelroy M C,Harty H R,Hosford G E,et al.Alpha-toxin damages the air-blood barrier of the lung in a rat model of Staphylococcus aureus-induced pneumonia[J].Infection and Immunity,1999,67(10):5541-5544.
[10] Herbelin C,Poutrel B,Gilbert F B,et al.Immune recruitment and bactericidal activity of neutrophils in milk of cows vaccinated with staphylococcal alpha-toxin[J].Journal of Dairy Science,1997,80(9):2025-2034.
[11] Byoung Sun C,San M J,Hyun-Mi K,et al.Protective effects of recombinant staphylococcal enterotoxin type C mutant vaccine against experimental bovine infection by a strain of Staphylococcus aureus isolated from subclinical mastitis in dairy cattle[J].Vaccine,2008,26(17):2081-2091.
[12] 张冰.查尔酮抗金黄色葡萄球菌分选酶A和α-溶血素的作用研究[D].长春:吉林大学,2018.
[13] Brodsky I E,Monack D.NLR-mediated control of inflammasome assembly in the host response against bacterial pathogens[J].Seminars in Immunology,2009,21(4):199-207.
[14] Chahnaz K,Chamberland R R,Allen I C,et al.Staphylococcus aureus α-hemolysin mediates virulence in a murine model of severe pneumonia through activation of the NLRP3 inflammasome[J].Journal of Infectious Diseases,2012,205(5):807-817.
[15] 丁海峰,李小申,黄慧,等.基因敲除技术研究进展[J].黑龙江畜牧兽医,2018(5):50-54.
[16] Rossi A,Kontarakis Z,Gerri C,et al.Genetic compensation induced by deleterious mutations but not gene knockdowns[J].Nature,2015,524(7564):230-233.
[2] Wilke G A,Bubeck Wardenburg J.Role of a disintegrin and metalloprotease 10 in Staphylococcus aureus alpha-hemolysin-mediated cellular injury[J].Proceedings of National Academy Science of the United States of America,2010,107(30):13473-13478.
[3] Elbers A R,Miltenburg J D,De Lange D,et al.Risk factors for clinical mastitis in a random sample of dairy herds from the southern part of The Netherlands[J].Journal of Dairy Science,1998,81(2):420-426.
[4] Ezzat Alnakip M,Quintela-Baluja M,Bohme K,et al.The immunology of mammary gland of dairy ruminants between healthy and inflammatory conditions[J].Journal of Veterinary Medicine,2014(10):659801.
[5] Senoh M,Miyoshi S K,Fouz B,et al.The cytotoxin-hemolysin genes of human and eel pathogenic Vibrio vulnificus strains:comparison of nucleotide sequences and application to the genetic grouping[J].Microbiology & Immunology,2013,49(6):513-519.
[6] Holliger P,Hudson P J.Engineered antibody fragments and the rise of single domains[J].Nature Biotechnology,2005,23(9):1126-1136.
[7] Schaefer J V,Honegger A,Plückthun A.Construction of scFv fragments from hybridoma or spleen cells by PCR assembly[A].Kontermann R,Dübel S.Antibody engineering[M].Berlin,Heidelberg:Springer Berlin Heidelberg,2010:21-44.
[8] Wang M,Zhang Y,Zhu J.Anti-staphylococcus aureus single-chain variable region fragments provide protection against mastitis in mice[J].Applied Microbiology and Biotechnology,2016,100(5):2153-2162.
[9] Mcelroy M C,Harty H R,Hosford G E,et al.Alpha-toxin damages the air-blood barrier of the lung in a rat model of Staphylococcus aureus-induced pneumonia[J].Infection and Immunity,1999,67(10):5541-5544.
[10] Herbelin C,Poutrel B,Gilbert F B,et al.Immune recruitment and bactericidal activity of neutrophils in milk of cows vaccinated with staphylococcal alpha-toxin[J].Journal of Dairy Science,1997,80(9):2025-2034.
[11] Byoung Sun C,San M J,Hyun-Mi K,et al.Protective effects of recombinant staphylococcal enterotoxin type C mutant vaccine against experimental bovine infection by a strain of Staphylococcus aureus isolated from subclinical mastitis in dairy cattle[J].Vaccine,2008,26(17):2081-2091.
[12] 张冰.查尔酮抗金黄色葡萄球菌分选酶A和α-溶血素的作用研究[D].长春:吉林大学,2018.
[13] Brodsky I E,Monack D.NLR-mediated control of inflammasome assembly in the host response against bacterial pathogens[J].Seminars in Immunology,2009,21(4):199-207.
[14] Chahnaz K,Chamberland R R,Allen I C,et al.Staphylococcus aureus α-hemolysin mediates virulence in a murine model of severe pneumonia through activation of the NLRP3 inflammasome[J].Journal of Infectious Diseases,2012,205(5):807-817.
[15] 丁海峰,李小申,黄慧,等.基因敲除技术研究进展[J].黑龙江畜牧兽医,2018(5):50-54.
[16] Rossi A,Kontarakis Z,Gerri C,et al.Genetic compensation induced by deleterious mutations but not gene knockdowns[J].Nature,2015,524(7564):230-233.