马流产沙门菌鞭毛蛋白FliC对马腺疫链球菌SeM蛋白免疫效应分析
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摘要
为研究马流产沙门菌(Salmonella abortus equi)鞭毛蛋白FliC对马腺疫链球菌(Streptococcus equi subsp.equi)SeM蛋白免疫效果的增强效应,为马腺疫新型高效疫苗的研发奠定基础。本研究构建并诱导表达了FliC,经亲和层析纯化目的蛋白,Western blot检测其免疫原性。将该纯化后FliC与SeM蛋白联合免疫C57BL/6小鼠,免疫后对小鼠进行抗体水平、抗体类型和攻毒保护力的检测。结果表明,成功表达、纯化了FliC重组蛋白,SDSPAGE电泳检测显示该蛋白质为52ku。抗体分型检测结果显示产生的抗体主要以IgG1为主;免疫30d后FliC+SeM免疫组的攻毒保护率为87%,高于SeM免疫组的保护率67%。马流产沙门菌来源的鞭毛蛋白FliC能够增强SeM蛋白的免疫保护效果,该结果为进一步更好地利用SeM蛋白奠定了基础。
The aim of this study was to investigate the effects of the flagellin protein FliC of Salmonella abortus equi on immune response of Streptococcus equi subsp.equi SeM and provide basis for the development of a new effective strangle vaccine.The recombinant protein FliC was constructed,expressed and purified using affinity chromatography,and its immunogenicity was analyzed by Western blot.The purified FliC and SeM were used in-combination to immunize C57 BL/6 mice and the serum antibody level,antibody subtype of immunized mice and challenge protection rate were investigated.SDS-PAGE result indicated that the expressed recombinant FliC protein was 52 kDa.The results of antibody typing showed that the main antibodies were mainly IgG1,and the challenge protection rate of FliC+SeM immunization group(87%)was higher than that of SeM immunization group(67%).The flagellin FliC of Salmonellaabortus equicould enhance the immune efficacy of SeM and this result laid experimental foundation for the further utilization of SeM.
The aim of this study was to investigate the effects of the flagellin protein FliC of Salmonella abortus equi on immune response of Streptococcus equi subsp.equi SeM and provide basis for the development of a new effective strangle vaccine.The recombinant protein FliC was constructed,expressed and purified using affinity chromatography,and its immunogenicity was analyzed by Western blot.The purified FliC and SeM were used in-combination to immunize C57 BL/6 mice and the serum antibody level,antibody subtype of immunized mice and challenge protection rate were investigated.SDS-PAGE result indicated that the expressed recombinant FliC protein was 52 kDa.The results of antibody typing showed that the main antibodies were mainly IgG1,and the challenge protection rate of FliC+SeM immunization group(87%)was higher than that of SeM immunization group(67%).The flagellin FliC of Salmonellaabortus equicould enhance the immune efficacy of SeM and this result laid experimental foundation for the further utilization of SeM.
引文
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[18]邵俊高,李善春,苏艳.马链球菌马亚种新疆分离株XZ1SeM蛋白表达和免疫原性分析[J].新疆农业大学学报,2015,38(4):287-291.SHAO J G,LI S C,SU Y.Expression and analysis on immunogenicity of SeM protein of Streptococcus equi subspeciesequi XZ1 strain from Xinjiang[J].Journal of Xinjiang Agricultural University,2015,38(4):287-291.(in Chinese)
[19]杨康,易鸳鸯,赵亚南,等.马流产沙门菌鞭毛重组蛋白FliC的原核表达及对小鼠的免疫保护试验[J].中国兽医科学,2017,47(12):1549-1554.YANG K,YI Y Y,ZHAO Y N,et al.Prokaryotic expression of FliC protein of Salmonella abortus equi and immune protection test in mice[J].Chinese Veterinary Science,2017,47(12):1549-1554.(in Chinese)
[20]程龙飞,林群群,刘荣昌,等.禽源多杀性巴氏杆菌重组外膜蛋白A间接ELISA方法的建立[J].中国兽医学报,2017,37(10):1886-1890.CHENG L F,LIN Q Q,LIU R C,et al.An indirect ELISA for the detection of serum antibodies against avian pasteurella multocida using recombinant outer membrane protein A as coating antigen[J].Chinese Journal of Veterinary Science,2017,37(10):1886-1890.(in Chinese)
[21]TIMONEY J F.The pathogenic equine streptococci[J].Vet Res,2004,35(4):397-409.
[22]EOM J S,KIM J S,IM JANG J,et al.Enhancement of host immune responses by oral vaccination to Salmonella enterica serovar Typhimurium harboring both FliC and FljB flagella[J].PLoS One,2013,8(9):e74850.
[23]包琦锋,刘洪洋,张婷,等.鞭毛蛋白FliC突变体/HPV18L2N融合蛋白的表达与纯化[J].现代生物医学进展,2014,14(1):7-12.BAO Q F,LIU H Y,ZHANG T,et al.Expression and purification of FliC Mutant/HPV 18L2NFusion Proteins[J].Progress in Modern Biomedicine,2014,14(1):7-12.(in Chinese)
[24]HULEATT J W,JACOBS A R,TANG J,et al.Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity[J].Vaccine,2007,25(4):763-775.
[25]王鹤.停乳链球菌GapC蛋白与鼠伤寒沙门氏菌FliC蛋白的融合表达及免疫原性研究[D].大庆:黑龙江八一农垦大学,2012.WANG H.expression and immunogenicity research of fusion protein of S.dysgalactiae GapC and S.typhimurium FliC[D].Daqing:Heilongjiang Bayi Agricultural University,2012.(in Chinese)
[26]严明,刘宇思,张帅,等.鼠伤寒沙门氏菌鞭毛蛋白可增强DnaJ蛋白的免疫反应[J].中国免疫学杂志,2014,30(8):1023-1027.YAN M,LIU Y S,ZHANG S,et al.Salmonella typhimurium flagellin as adjuvants can enhance immune response of DnaJ protein[J].Chinese Journal of Immunology,2014,30(8):1023-1027.(in Chinese)
[2]郭纪珂,王晓钧.马腺疫[J].畜牧兽医科技信息,2013(10):16-18.GUO J K,WANG X J.Strangle[J].Chinese Journal of Animal Husbandry and Veterinary Medicine,2013(10):16-18.(in Chinese)
[3]NEWTON R,WALLER A,KING A.Investigation of suspected adverse reactions following strangles vaccination in horses[J].Vet Record,2005,156(9):291-292.
[4]KEMP-SYMONDS J,KEMBLE T,WALLER A.Modified liveStreptococcus equi(‘strangles’)vaccination followed by clinically adverse reactions associated with bacterial replication[J].Equine Vet J,2007,39(3):284-286.
[5]TIMONEY J F,ARTIUSHIN S C,BOSCHWITZ J S.Comparison of the sequences and functions ofStreptococcus equi M-like proteins SeM and SzPSe[J].Infect Immun,1997,65(9):3600-3605.
[6]FLOCK M,JACOBSSON K,FRYKBERG L,et al.Recombinant Streptococcus equi proteins protect mice in challenge experiments and induce immune response in horses[J].Infect Immun,2004,72(6):3228-3236.
[7]郭志燕,周明旭,段强德,等.细菌鞭毛的致病性及其免疫学应用的研究进展[J].微生物学报,2014,54(3):251-260.GUO Z Y,ZHOU M X,DUAN Q D,et al.Advance on the pathogenicity and immunological application of bacterial flagella—A review[J].Acta Microbiologica Sinica,2014,54(3):251-260.(in Chinese)
[8]宋丽,郭亚鑫,徐慧,等.细菌鞭毛蛋白作为佐剂的研究进展[J].动物医学进展,2015,36(4):92-95.SONG L,GUO Y X,XU H,et al.Progress on bacterial flagellin as an adjuvant[J].Progress in Veterinary Medicine,2015,36(4):92-95.(in Chinese)
[9]兰英华.沙门菌鞭毛蛋白的研究进展[J].国外医学(微生物学分册),2002,25(5):24-26.LAN Y H.Research progress of Salmonellaflagellin[J].Foreign Medicine(Microbiology Section),2002,25(5):24-26.(in Chinese)
[10]刘文博,焦新安,高崧,等.应用噬菌体肽库技术定位沙门氏菌鞭毛蛋白上属特异性共同抗原表位[J].畜牧兽医学报,2003,34(2):183-187.LIU W B,JIAO X A,GAO S,et al.Mapping of the generically-specific common epitope on Salmonella flagellins using phage-displayed peptide libracy[J].Acta Veterinaria et Zootechnica Sinica,2003,34(2):183-187.(in Chinese)
[11]VAN DER WOUDE M W,BUMLER A J.Phase and antigenic variation in bacteria[J].Clin Microbiol Rev,2004,17(3):581-611.
[12]HE Y,XU T T,FOSSHEIM L E,et al.FliC,a flagellin protein,is essential for the growth and virulence of fish pathogen Edwardsiella tarda[J].PLoS One,2012,7(9):e45070.
[13]KNUDSEN M L,JOHANSSON D X,KOSTIC L,et al.The adjuvant activity of alphavirus replicons is enhanced by incorporating the microbial molecule flagellin into the replicon[J].PLoS One,2013,8(6):e65964.
[14]陈志华.鼠伤寒沙门氏菌鞭毛蛋白的制备及其对口蹄疫疫苗的佐剂作用[D].福州:福建农林大学,2010.CHEN Z H.Preparation and adjuvant effects of Salmonella Typhimurium flagellin[D].Fuzhou:Fujian Agriculture and Forestry University,2010.(in Chinese)
[15]王静.沙门菌鞭毛蛋白作为新城疫疫苗佐剂的研究[D].扬州:扬州大学,2014.WANG J.Efficacy of Salmonellaflagellin as a potential adjuvant for Newcastle disease vaccines[D].Yangzhou:Yangzhou University,2014.(in Chinese)
[16]CHAUNG H C,CHENG L T,HUNG L H,et al.Salmonella flagellin enhances mucosal immunity of avian influenza vaccine in chickens[J].Vet Microbiol,2012,157(1-2):69-77.
[17]赵达.鼠伤寒沙门氏菌鞭毛蛋白对牛金黄色葡萄球菌GapC蛋白的免疫增强作用[D].大庆:黑龙江八一农垦大学,2013.ZHAO D.Immune-enhancing effect of S.typhimurium flagellin on Bovine S.aureus GapC protein[D].Daqing:Heilongjiang Bayi Agricultural University,2013.(in Chinese)
[18]邵俊高,李善春,苏艳.马链球菌马亚种新疆分离株XZ1SeM蛋白表达和免疫原性分析[J].新疆农业大学学报,2015,38(4):287-291.SHAO J G,LI S C,SU Y.Expression and analysis on immunogenicity of SeM protein of Streptococcus equi subspeciesequi XZ1 strain from Xinjiang[J].Journal of Xinjiang Agricultural University,2015,38(4):287-291.(in Chinese)
[19]杨康,易鸳鸯,赵亚南,等.马流产沙门菌鞭毛重组蛋白FliC的原核表达及对小鼠的免疫保护试验[J].中国兽医科学,2017,47(12):1549-1554.YANG K,YI Y Y,ZHAO Y N,et al.Prokaryotic expression of FliC protein of Salmonella abortus equi and immune protection test in mice[J].Chinese Veterinary Science,2017,47(12):1549-1554.(in Chinese)
[20]程龙飞,林群群,刘荣昌,等.禽源多杀性巴氏杆菌重组外膜蛋白A间接ELISA方法的建立[J].中国兽医学报,2017,37(10):1886-1890.CHENG L F,LIN Q Q,LIU R C,et al.An indirect ELISA for the detection of serum antibodies against avian pasteurella multocida using recombinant outer membrane protein A as coating antigen[J].Chinese Journal of Veterinary Science,2017,37(10):1886-1890.(in Chinese)
[21]TIMONEY J F.The pathogenic equine streptococci[J].Vet Res,2004,35(4):397-409.
[22]EOM J S,KIM J S,IM JANG J,et al.Enhancement of host immune responses by oral vaccination to Salmonella enterica serovar Typhimurium harboring both FliC and FljB flagella[J].PLoS One,2013,8(9):e74850.
[23]包琦锋,刘洪洋,张婷,等.鞭毛蛋白FliC突变体/HPV18L2N融合蛋白的表达与纯化[J].现代生物医学进展,2014,14(1):7-12.BAO Q F,LIU H Y,ZHANG T,et al.Expression and purification of FliC Mutant/HPV 18L2NFusion Proteins[J].Progress in Modern Biomedicine,2014,14(1):7-12.(in Chinese)
[24]HULEATT J W,JACOBS A R,TANG J,et al.Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity[J].Vaccine,2007,25(4):763-775.
[25]王鹤.停乳链球菌GapC蛋白与鼠伤寒沙门氏菌FliC蛋白的融合表达及免疫原性研究[D].大庆:黑龙江八一农垦大学,2012.WANG H.expression and immunogenicity research of fusion protein of S.dysgalactiae GapC and S.typhimurium FliC[D].Daqing:Heilongjiang Bayi Agricultural University,2012.(in Chinese)
[26]严明,刘宇思,张帅,等.鼠伤寒沙门氏菌鞭毛蛋白可增强DnaJ蛋白的免疫反应[J].中国免疫学杂志,2014,30(8):1023-1027.YAN M,LIU Y S,ZHANG S,et al.Salmonella typhimurium flagellin as adjuvants can enhance immune response of DnaJ protein[J].Chinese Journal of Immunology,2014,30(8):1023-1027.(in Chinese)