融合PCR技术实现志贺菌候选抗原rstxB-phoE的胞膜表面表达
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摘要
目的实现志贺菌候选抗原rstxB-phoE的胞膜表面表达。方法本研究利用融合PCR技术(SOEPCR)构建可以表达rstxB-phoE的重组菌,并通过蛋白杂交、免疫胶体金电镜等手段证明重组蛋白在细胞中的定位。连续传代鉴定重组株的遗传稳定性。结果一段长21个氨基酸的stxB肽段通过phoE蛋白载体准确递呈到了细菌胞膜表面,重组蛋白的表达对细菌的生长没有产生明显影响,且连续传代结果表明重组株的良好遗传稳定性。结论本实验基于SOE-PCR技术初步构建了可以表面递呈rstxB-phoE重组蛋白并稳定遗传的重组菌株,为开发新型志贺菌疫苗打下基础。其免疫作用需通过动物实验进一步鉴定。
Objective To construct a recombinant strain BL21for the expression of a vaccine candidate rstxBphoE protein on the outer surface of cell membrane. Methods SOE-PCR was used in this study for the construction of a recombinant strain BL21to express rstxB-phoE antigen of Shigella.The localization of the recombinant protein was confirmed by SDS-PAGE,western blotting and the technique of colloidal gold immuneelectron-microscopy.The genetic stability of the recombinant strain was also assessed by continuous generation passage. Results A short recombinant stxB peptide with 21amino acids was identified on the outer surface of cell membrane carried by phoE protein.The expression of the recombinant protein had no significant effect on bacterial growth and the recombinant strain BL21was found to be genetically stable. Conclusions A recombinant strain BL21which can express rstxB-phoE protein on the outer surface of cell membrane was constructed successfully by SOE-PCR and is proved to be genetically stable,which has laid foundation for the development of novel vaccine against shigella.
Objective To construct a recombinant strain BL21for the expression of a vaccine candidate rstxBphoE protein on the outer surface of cell membrane. Methods SOE-PCR was used in this study for the construction of a recombinant strain BL21to express rstxB-phoE antigen of Shigella.The localization of the recombinant protein was confirmed by SDS-PAGE,western blotting and the technique of colloidal gold immuneelectron-microscopy.The genetic stability of the recombinant strain was also assessed by continuous generation passage. Results A short recombinant stxB peptide with 21amino acids was identified on the outer surface of cell membrane carried by phoE protein.The expression of the recombinant protein had no significant effect on bacterial growth and the recombinant strain BL21was found to be genetically stable. Conclusions A recombinant strain BL21which can express rstxB-phoE protein on the outer surface of cell membrane was constructed successfully by SOE-PCR and is proved to be genetically stable,which has laid foundation for the development of novel vaccine against shigella.
引文
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[11]Agterberg M,Adriaanse H,Lankhof H,et al.Outer membrane PhoE protein of Escherichia coli as a carrier for foreign antigenic determinants:immunogenicity of epitopes of foot-andmouth disease virus[J].Vaccine,1990,8(1):85-91.
[12]Agterberg M,Adriaanse H,van Bruggen A,et al.Outermembrane PhoE protein of Escherichia coli K-12as an exposure vector:possibilities and limitations[J].Gene,1990,88(1):37-45.
[13]Janssen R,Tommassen J.PhoE protein as a carrier for foreign epitopes[J].Int Rev Immunol,1994,11(2):113-121.
[2]Pu XY,Pan JC,Wang HQ,et al.Characterization of fluoroquinolone-resistant Shigella flexneri in Hangzhou area of China[J].J Antimicrob Chemother,2009,63(5):917-920.
[3]Wu T,Grassel C,Levine MM,et al.Live attenuated Shigella dysenteriaetype 1vaccine strains overexpressing shiga toxin B subunit[J].Infect Immun,2011,79(12):4912-4922.
[4]Noriega FR,Losonsky G,Lauderbaugh C,et al.Engineered deltaguaB-A deltavirG Shigella flexneri 2astrain CVD 1205:construction,safety,immunogenicity,and potential efficacy as a mucosal vaccine[J].Infect Immun,1996,64(8):3055-3061.
[5]Nassif X,Mazert MC,Mounier J,et al.Evaluation with an iuc:Tn10mutant of the role of aerobactin production in the virulence of Shigella flexneri[J].Infect Immun,1987,55(9):1963-1969.
[6]Coster TS,Hoge CW,VanDeVerg LL,et al.Vaccination against shigellosis with attenuated Shigella flexneri 2a strain SC602[J].Infect Immun,1999,67(7):3437-3443.
[7]Kotloff KL,Pasetti MF,Barry EM,et al.Deletion in the Shigella enterotoxin genes further attenuates Shigella flexneri 2abearing guanine auxotrophy in a phase 1trial of CVD 1204and CVD 1208[J].J Infect Dis,2004,190(10):1745-1754.
[8]Rahman KM,Arifeen SE,Zaman K,et al.Safety,dose,immunogenicity,and transmissibility of an oral live attenuated Shigella flexneri 2avaccine candidate(SC602)among healthy adults and school children in Matlab,Bangladesh[J].Vaccine,2011,29(6):1347-1354.
[9]Kotloff KL,Simon JK,Pasetti MF,et al.Safety and immunogenicity of CVD 1208S,a live,oral DeltaguaBA Deltasen Deltaset Shigella flexneri 2avaccine grown on animal-free media[J].Hum Vaccin,2007,3(6):268-275.
[10]李钧,邵铁娟,濮小英.生物信息学法筛选志贺氏菌疫苗候选抗原[J].微生物学通报,2010,37(7):1028-1034.
[11]Agterberg M,Adriaanse H,Lankhof H,et al.Outer membrane PhoE protein of Escherichia coli as a carrier for foreign antigenic determinants:immunogenicity of epitopes of foot-andmouth disease virus[J].Vaccine,1990,8(1):85-91.
[12]Agterberg M,Adriaanse H,van Bruggen A,et al.Outermembrane PhoE protein of Escherichia coli K-12as an exposure vector:possibilities and limitations[J].Gene,1990,88(1):37-45.
[13]Janssen R,Tommassen J.PhoE protein as a carrier for foreign epitopes[J].Int Rev Immunol,1994,11(2):113-121.