多杀性巴氏杆菌转铁结合蛋白TbpA的原核表达及免疫原性
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摘要
为研究多杀性巴氏杆菌转铁结合蛋白TbpA的免疫原性,本试验利用PCR从牛源A型多杀性巴氏杆菌HB01基因组中扩增了TbpA的编码基因tbpA,并将其克隆至原核表达载体pET-30α(+)上,转化至大肠杆菌BL21中进行诱导表达。SDS-PAGE检测结果显示,rTbpA蛋白成功表达。Western blot证实该蛋白能够与抗多杀性巴氏杆菌HB01血清发生阳性反应。将rTbpA免疫小鼠后,分别于免疫后14,28 d采血,利用ELISA试验检测血清中的抗体滴度,结果显示血清中的抗rTbpA蛋白的IgG抗体显著升高(P<0.05)。感染试验结果显示,免疫rTbpA蛋白能够保护70%的小鼠抵抗多杀性巴氏杆菌的致死性攻击,并且病理切片结果表明,免疫小鼠的肺部损伤相对于对照组小鼠显著降低。本试验为筛选新型的多杀性巴氏杆菌免疫原性蛋白提供依据。
To investigate the protective efficacy of the recombinant transferrin-binding protein A(rTbpA) of bovine Pasteurella multocida serogroup A,the TbpA coding gene tbpA harboured in the genome of P.multocida HB01 was amplified and cloned into the prokaryotic expression plasmid pET-30α(+),transformed into E.coli BL21.After induction,the TbpA was successfully expressed in E.coli.Western blot demonstrated that the rTbpA displayed a positive reaction to the anti-P.multocida HB01 serum.The ELISA titers of rTbpA antigen specific IgG in the serum from the mice vaccinated with rTbpA were significantly increased at the 14 th and 28 th day post-immunization(P< 0.05).The rTbpA contributed a lot to the elimination of lung lesions resulted from homologous challenge and provided 70% protection to the bacteria infection mice.The TbpA could be used as an effective component of subunit vaccine against the infection of Pasteurella multocida in cattle and buffalo.
To investigate the protective efficacy of the recombinant transferrin-binding protein A(rTbpA) of bovine Pasteurella multocida serogroup A,the TbpA coding gene tbpA harboured in the genome of P.multocida HB01 was amplified and cloned into the prokaryotic expression plasmid pET-30α(+),transformed into E.coli BL21.After induction,the TbpA was successfully expressed in E.coli.Western blot demonstrated that the rTbpA displayed a positive reaction to the anti-P.multocida HB01 serum.The ELISA titers of rTbpA antigen specific IgG in the serum from the mice vaccinated with rTbpA were significantly increased at the 14 th and 28 th day post-immunization(P< 0.05).The rTbpA contributed a lot to the elimination of lung lesions resulted from homologous challenge and provided 70% protection to the bacteria infection mice.The TbpA could be used as an effective component of subunit vaccine against the infection of Pasteurella multocida in cattle and buffalo.
引文
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[11] HUANG X,LIY,FU Y,et al.Cross-protective efficacy of recombinant transferrin-binding protein A of Haemophilus parasuis in guinea pigs[J].Clin Vaccine Immunol,2013,20(6):912-919.
[12] KIM T J,LEE J I.Cloning and expression of genes encoding transferrin-binding protein A and B from Actinobacillus pleuropneumoniae serotype 5[J].Protein Expr Purif,2006,45(1):235-240.
[13] WEST D,REDDIN K,MATHESON M,et al.Recombinant Neisseria meningitidis transferrin binding protein A protects against experimental meningococcal infection[J].Infect Immun,2001,69(3):1561-1567.
[14] SHIVACHANDRA S B,YOGISHARADHYA R,KUMAR A,et al.Recombinant transferrin binding protein A (rTbpA) fragments of Pasteurella multocida serogroup B:2 provide variable protection following homologous challenge in mouse model[J].Res Vet Sci,2015,98:1-6.
[15] PENG Z,LIANG W,LIU W,et al.Genomic characterization of Pasteurella multocida HB01,a serotype A bovine isolate from China[J].Gene,2016,581(1):85-93.
[16] SHIVACHANDRA S,VISWAS K,KUMAR A.A review of hemorrhagic septicemia in cattle and buffalo[J].Anim Health Res Rev,2011,12(1):67-82.
[17] SCHRYVERS A B,GONZALEZ G C.Receptors for transferrin in pathogenic bacteria are specific for the host-s protein[J].Can J Microbiol,1990,36(2):145-147.
[2] WILSON B A,HO M.Pasteurella multocida:from zoonosis to cellular microbiology[J].Clin Microbiol Rev,2013,26(3):631-655.
[3] 宫强,孔梁宇,牛明福.禽多杀性巴氏杆菌ptfa基因壳聚糖纳米DNA疫苗的制备与检测[J].中国兽医学报,2018,38(3):491-495.
[4] CARTER G R.Studies on Pasteurella multocida.Ⅰ.A hemagglutination test for the identification of serological types[J].Am J Vet Res,1955,16(60):481-484.
[5] HARPER M,BOYCE J D,ADLER B.Pasteurella multocida pathogenesis:125 years after pasteur[J].FEMS Microbiol Lett,2006,265(1):1-10.
[6] JACQUES M.Surface polysaccharides and iron-uptake systems of Actinobacillus pleuropneumoniae[J].Can J Vet Res,2004,68(2):81-85.
[7] GONZALEZ G C,CAAMANO D L,SCHRYVERS A B.Identification and characterization of a porcine‐specific transferrin receptor in Actinobacillus pleuropneumoniae[J].Mol Microbiol,1990,4(7):1173-1179.
[8] GRAY-OWEN S D,LOOSMORE S,SCHRYVERS A B.Identification and characterization of genes encoding the human transferrin-binding proteins from Haemophilus influenzae[J].Infect Immun,1995,63(4):1201-1210.
[9] OGUNNARIWO J A,SCHRYVERS A B.Iron acquisition in Pasteurella haemolytica:expression and identification of a bovine-specific transferrin receptor[J].Infect Immun,1990,58(7):2091-2097.
[10] EWERS C,LüBKE-BECKER A,BETHE A,et al.Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status[J].Vet Microbiol,2006,114(3):304-317.
[11] HUANG X,LIY,FU Y,et al.Cross-protective efficacy of recombinant transferrin-binding protein A of Haemophilus parasuis in guinea pigs[J].Clin Vaccine Immunol,2013,20(6):912-919.
[12] KIM T J,LEE J I.Cloning and expression of genes encoding transferrin-binding protein A and B from Actinobacillus pleuropneumoniae serotype 5[J].Protein Expr Purif,2006,45(1):235-240.
[13] WEST D,REDDIN K,MATHESON M,et al.Recombinant Neisseria meningitidis transferrin binding protein A protects against experimental meningococcal infection[J].Infect Immun,2001,69(3):1561-1567.
[14] SHIVACHANDRA S B,YOGISHARADHYA R,KUMAR A,et al.Recombinant transferrin binding protein A (rTbpA) fragments of Pasteurella multocida serogroup B:2 provide variable protection following homologous challenge in mouse model[J].Res Vet Sci,2015,98:1-6.
[15] PENG Z,LIANG W,LIU W,et al.Genomic characterization of Pasteurella multocida HB01,a serotype A bovine isolate from China[J].Gene,2016,581(1):85-93.
[16] SHIVACHANDRA S,VISWAS K,KUMAR A.A review of hemorrhagic septicemia in cattle and buffalo[J].Anim Health Res Rev,2011,12(1):67-82.
[17] SCHRYVERS A B,GONZALEZ G C.Receptors for transferrin in pathogenic bacteria are specific for the host-s protein[J].Can J Microbiol,1990,36(2):145-147.