梅毒螺旋体重组蛋白Tp0453-Gpd的表达及免疫性研究
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摘要
人工合成梅毒螺旋体重组蛋白Tp0453-Gpd基因,转入大肠杆菌中利用IPTG进行诱导表达,表达的重组蛋白分子量为64kD.重组蛋白镍柱纯化后作为抗原,检测人血清样本.结果显示,梅毒患者样本组与非梅毒患者样本组存在非常显著差别,说明重组蛋白可被梅毒抗体特异性识别,有良好免疫反应性,可作为梅毒诊断试剂新抗原.同时,利用该重组蛋白免疫新西兰兔3次,兔血清中可持续检测到高滴度梅毒抗体,显示该重组蛋白有较强免疫原性,可作为梅毒基因工程蛋白疫苗候选蛋白.
In this study,Treponema pallidum(TP)recombinant protein gene Tp0453-Gpd was synthesized and the recombinant protein was expressed in Escherichia coli with 64 kD molecular weight.Recombinant protein was purified by nickel-affinity chromatography column and was used as antigen to analyze the human serum antibody of syphilis patient group and non syphilis patient group,and statistically significant difference for syphilis antibody was observed between two groups. The ELISA results showed that the recombinant protein could be recognized by syphilis antibody specially and had good reactinogenicity,and may be a good antigen for syphilis detection.The rabbit was injected with the recombinant protein Tp0453-Gpd for 3 times and could product syphilis antibody effectively and persistently.It was suggested that recombinant protein Tp0453-Gpd had good immunogenicity and was an ideal candidate for the syphilis vaccine.
In this study,Treponema pallidum(TP)recombinant protein gene Tp0453-Gpd was synthesized and the recombinant protein was expressed in Escherichia coli with 64 kD molecular weight.Recombinant protein was purified by nickel-affinity chromatography column and was used as antigen to analyze the human serum antibody of syphilis patient group and non syphilis patient group,and statistically significant difference for syphilis antibody was observed between two groups. The ELISA results showed that the recombinant protein could be recognized by syphilis antibody specially and had good reactinogenicity,and may be a good antigen for syphilis detection.The rabbit was injected with the recombinant protein Tp0453-Gpd for 3 times and could product syphilis antibody effectively and persistently.It was suggested that recombinant protein Tp0453-Gpd had good immunogenicity and was an ideal candidate for the syphilis vaccine.
引文
[1] TIPPLE C,TAYLOR G P.Syphilis testing,typing,and treatment follow up:a new era for an old disease[J].Curr Opin Infect Dis,2015,28(1):53-60.
[2] GOLDEN M R,MARRA C M,HOLMES K K,et al.Update on syphilis[J].Journal of the American Medical Association,2003,290(11):1510-1514.
[3] KIDDUGAVU M G,KIWANUKA N, WAWER M J,et al.Effectiveness of syphilistreatment using azithromycin and/or benza-thinepenicillin in Rakai,Uganda[J].Sex Transm Dis,2005,32(1):1-6.
[4] PEELING R W,MABEY D,KAMB M L,et al.Syphilis[J].Nat Rev Dis Primers,2017,12(3):17073.
[5] LITHGOW K V,CAMERON C E.Vaccine development for syphilis[J].Expert Rev Vaccines,2017,16(1):37-44.
[6]郑和平,覃晓琳,黄进梅,等.梅毒螺旋体特异性抗原Tp0453及其截短蛋白的克隆、表达及活性检测[J].皮肤性病诊疗学杂志,2014,21(4):283-286.
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[8] VAN VOORHIS W C,BARRETT L K,LUKEHART S A,et al.Serodiagnosis of syphilis:antibodies to recombinant Tp0453,Tp92,and Gpd proteins are sensitive and specific indicators of infection by Treponema pallidum[J].J Clin Microbiol,2003,41(8):3668-3674.
[9]刘双全,吴移谋,赵飞骏,等.梅毒螺旋体Tp0453重组蛋白的表达纯化及免疫活性研究[J].中国皮肤性病学杂志,2005,19(11):644-646.
[10] CAMERON C E,CASTRO C,LUKEHART S A,et al.Sequence conservation of glycerophosphodiester phosphodiesterase among treponema pallidum strains[J].Infect Immun,1999,67(6):3168-3170.
[11]严加林,吴移谋,赵飞骏,等.梅毒螺旋体外膜蛋白Gpd的基因型分析及其重组蛋白的免疫原性鉴定[J].南华大学学报(医学版),2005,33(1):29-32.
[12]严加林.梅毒螺旋体Gpd重组蛋白的表达,纯化及免疫活性研究[D].衡阳:南华大学病原生物学研究所,2006:28-31.
[13] SMITH B C,SIMPSON Y,MORSHED M G,et al.New proteins for a new perspective on syphilis diagnosis[J].J Clin Microbiol,2013,51(1):105-111.
[14] LITHGOW K V,CAMERON C E.Vaccine development for syphilis[J].Expert Rev Vaccines,2017,16(1):37-44.
[15] GOTTLIEB S L,JOHNSTON C.Future prospects for new vaccines against sexually transmitted infections[J].Curr Opin Infect Dis,2017,30(1):77-86.
[16] HAZLETT K R,COX D L,DECAFFMEYER M,et al.TP0453,a concealed outer membrane protein of Treponema pallidum,enhances membrane permeability[J].J Bacteriol,2005,87(18):6499-6508.
[17]赵飞骏,吴移谋,张晓红,等.梅毒螺旋体外膜蛋白Gpd基因的克隆、表达及其免疫活性研究[J].微生物学报,2005,45(5):767-771.
[2] GOLDEN M R,MARRA C M,HOLMES K K,et al.Update on syphilis[J].Journal of the American Medical Association,2003,290(11):1510-1514.
[3] KIDDUGAVU M G,KIWANUKA N, WAWER M J,et al.Effectiveness of syphilistreatment using azithromycin and/or benza-thinepenicillin in Rakai,Uganda[J].Sex Transm Dis,2005,32(1):1-6.
[4] PEELING R W,MABEY D,KAMB M L,et al.Syphilis[J].Nat Rev Dis Primers,2017,12(3):17073.
[5] LITHGOW K V,CAMERON C E.Vaccine development for syphilis[J].Expert Rev Vaccines,2017,16(1):37-44.
[6]郑和平,覃晓琳,黄进梅,等.梅毒螺旋体特异性抗原Tp0453及其截短蛋白的克隆、表达及活性检测[J].皮肤性病诊疗学杂志,2014,21(4):283-286.
[7] LUTHRA A,ZHU G,DESROSIERS D C,et al.The transition from closed to open conformation of Treponema pallidum outer membrane-associated lipoprotein TP0453 involves membrane sensing and integration by two amphipathic helices[J].J Biol Chem,2011,286(48):41656-41668.
[8] VAN VOORHIS W C,BARRETT L K,LUKEHART S A,et al.Serodiagnosis of syphilis:antibodies to recombinant Tp0453,Tp92,and Gpd proteins are sensitive and specific indicators of infection by Treponema pallidum[J].J Clin Microbiol,2003,41(8):3668-3674.
[9]刘双全,吴移谋,赵飞骏,等.梅毒螺旋体Tp0453重组蛋白的表达纯化及免疫活性研究[J].中国皮肤性病学杂志,2005,19(11):644-646.
[10] CAMERON C E,CASTRO C,LUKEHART S A,et al.Sequence conservation of glycerophosphodiester phosphodiesterase among treponema pallidum strains[J].Infect Immun,1999,67(6):3168-3170.
[11]严加林,吴移谋,赵飞骏,等.梅毒螺旋体外膜蛋白Gpd的基因型分析及其重组蛋白的免疫原性鉴定[J].南华大学学报(医学版),2005,33(1):29-32.
[12]严加林.梅毒螺旋体Gpd重组蛋白的表达,纯化及免疫活性研究[D].衡阳:南华大学病原生物学研究所,2006:28-31.
[13] SMITH B C,SIMPSON Y,MORSHED M G,et al.New proteins for a new perspective on syphilis diagnosis[J].J Clin Microbiol,2013,51(1):105-111.
[14] LITHGOW K V,CAMERON C E.Vaccine development for syphilis[J].Expert Rev Vaccines,2017,16(1):37-44.
[15] GOTTLIEB S L,JOHNSTON C.Future prospects for new vaccines against sexually transmitted infections[J].Curr Opin Infect Dis,2017,30(1):77-86.
[16] HAZLETT K R,COX D L,DECAFFMEYER M,et al.TP0453,a concealed outer membrane protein of Treponema pallidum,enhances membrane permeability[J].J Bacteriol,2005,87(18):6499-6508.
[17]赵飞骏,吴移谋,张晓红,等.梅毒螺旋体外膜蛋白Gpd基因的克隆、表达及其免疫活性研究[J].微生物学报,2005,45(5):767-771.