鼠肺炎沙眼衣原体质粒蛋白pgp5的克隆表达鉴定及免疫原性分析
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摘要
目的获取鼠肺炎沙眼衣原体(Chlamydia muridarum)质粒蛋白pgp5的基因及纯化的蛋白并鉴定其免疫原性。方法设计引物,PCR扩增目的基因,将其定向插入到原核表达载体pET28a中,然后将重组质粒转化入大肠杆菌E.coli DH5α中,并用PCR扩增及序列测定等方法对重组质粒进行鉴定。再将重组质粒转化入感受肽Rosetta(DE3)并诱导表达,用镍柱纯化pgp5-his融合蛋白。用纯化后的目的蛋白免疫新西兰家兔,酶联免疫吸附试验(ELISA)测定抗体效价,Western blot、细胞免疫荧光方法检测抗体与pgp5蛋白的结合。结果所获得的pgp5基因片段经测序长度为795 bp,检索确认其序列与GeneBank一致。十二烷基硫酸钠一聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹实验均显示获得相对分子质量约29 000的纯化蛋白。ELISA检测多克隆抗体效价达1∶100 000。细胞免疫荧光检测结果显示抗体可与体外培养的鼠肺炎沙眼衣原体特异性结合。结论成功表达pgp5-his融合蛋白,并制备了高效价、高特异性的抗pgp5抗体,为进一步研究奠定了基础。
Objective To clone the plasmid-encoded pgp5 of Chlamydia muridarum(CM), and evaluate its immunogenicity.Methods Primers were designed, and PCR was used to amplify the gene of plamsid-encoded pgp5.The gene was inserted into the prokaryotic expression vector pET28 a, and then the recombinant plasmid was transformed into E.coli DH5α, followed by sequencing and PCR identification.After the identification,the recombinant plasmid was transformed into Rosetta(DE3)to induce expression of pgp5.ANi-ion affinity chromatography column was used to purify the pgp5-his fusion protein, which was used to immunize New Zealand rabbits for preparation of polyclonal antibodies.The antibody titer was determined by enzyme-linked immunosorbent assay(ELISA), and the binding of the antibody to pgp5 protein was detected by Western blotting and immunofluorescence.Results The length of pgp5 gene was 795 bp, consistent with pgp5 of Chlamydia muridarum in GeneBank.The molecular weight of the purified target proteins was 29 000 determined by SDS-PAGE and Western blotting.Meanwhile, the titer of anti-pgp5 polyclonal antibodies was 1∶100 000 detected by ELISA.Conclusion The pgp5-his fusion protein has been successfully expressed, and a high-titer, highly specific anti-pgp5 antibody is prepared, which lay a foundation for further research.
Objective To clone the plasmid-encoded pgp5 of Chlamydia muridarum(CM), and evaluate its immunogenicity.Methods Primers were designed, and PCR was used to amplify the gene of plamsid-encoded pgp5.The gene was inserted into the prokaryotic expression vector pET28 a, and then the recombinant plasmid was transformed into E.coli DH5α, followed by sequencing and PCR identification.After the identification,the recombinant plasmid was transformed into Rosetta(DE3)to induce expression of pgp5.ANi-ion affinity chromatography column was used to purify the pgp5-his fusion protein, which was used to immunize New Zealand rabbits for preparation of polyclonal antibodies.The antibody titer was determined by enzyme-linked immunosorbent assay(ELISA), and the binding of the antibody to pgp5 protein was detected by Western blotting and immunofluorescence.Results The length of pgp5 gene was 795 bp, consistent with pgp5 of Chlamydia muridarum in GeneBank.The molecular weight of the purified target proteins was 29 000 determined by SDS-PAGE and Western blotting.Meanwhile, the titer of anti-pgp5 polyclonal antibodies was 1∶100 000 detected by ELISA.Conclusion The pgp5-his fusion protein has been successfully expressed, and a high-titer, highly specific anti-pgp5 antibody is prepared, which lay a foundation for further research.
引文
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[7] 孔杰,孙毅娜,赵乐然,等.沙眼衣原体质粒蛋白pgp3多克隆抗体的制备及鉴定[J].中华传染病杂志,2014,32(2):77-79.
[8] 黄浩,王小红.沙眼衣原体的细胞培养与γ-干扰素[J].华中科技大学学报(医学版),2002,31(4):435-437.
[9] 郑蕾,刘原君,郭睿,等.衣原体噬菌体phiCPG1衣壳蛋白Vp1蛋白IN5部分的克隆、表达、鉴定及其对沙眼衣原体抑制作用的研究[J].中华临床感染病杂志,2017,10(3):199-204,226.
[10] 盛彩虹,孙毅娜,孔杰,等.D型沙眼衣原体多形外膜蛋白I的分段克隆表达及其免疫原性分析[J].中华传染病杂志,2016,34(11):675-679.
[11] Carrasco SE,Hu S,Imai DM,et al.Toll-like receptor 3 (TLR3) promotes the resolutionof Chlamydia muridarum genital tract infection in congenic C57BL/6N mice[J].PLoS One,2018,13(4):e0195165.
[12] Carlson JH,Whitmire WM,Crane DD,et al.The chlamydia trachomatis plasmid is a transcriptional regulator of chromosomal genes and a virulence factor[J].Infect Immun,2008,76(6):2273-2283.
[13] Gong S,Yang Z,Lei L,et al.Characterization of Chlamydia trachomatis plasmid-encoded open reading frames[J].J Bacteriol,2013,195(17):3819-3826.
[14] Liu Y,Huang Y,Yang Z,et al.Plasmid-encoded Pgp3 is a major virulence factor for Chlamydia muridarum to induce hydrosalpinx in mice[J].Infect Immun,2014,82(12):5327-5335.
[15] Patton MJ,Chen CY,Yang C,et al.Plasmid negative regulation of CPAF expression is Pgp4 independent and restricted to invasive chlamydia trachomatis biovars[J].MBio,2018,9(1):e02164-2117.
[16] Zhong G.Chlamydial plasmid-dependent pathogenicity[J].Trends Microbiol,2017,25(2):141-152.
[17] Huang Y,Zhang Q,Yang Z,et al.Plasmid-encoded Pgp5 is a significant contributor to chlamydia muridarum induction of hydrosalpinx[J].PLoS One,2015,10(4):e0124840.
[2] Grasse M,Rosenkrands I,Olsen A,et al.A flow cytometry-based assay to determine the phagocytic activity of both clinical and nonclinical antibody samples against Chlamydia trachomatis[J].Cytometry A,2018,93(5):525-532.
[3] Conrad TA,Gong S,Yang Z,et al.The chromosome-encoded hypothetical protein TC0668 is an upper genital tract pathogenicity factor of chlamydia muridarum[J].Infect Immun,2015,84(2):467-479 .
[4] Snavely EA,Kokes M,Dunn JD,et al.Reassessing the role of the secreted protease CPAF in Chlamydia trachomatis infection through genetic approaches[J].Pathog Dis,2014,71(3):336-351.
[5] Last AR,Roberts Ch,Cassama E,et al.Plasmid copy number and disease severity in naturally occurring ocular Chlamydia trachomatis infection[J].J Clin Microbiol,2014,52(1):324-327.
[6] Liu Y,Chen C,Gong S,et al.Transformation of Chlamydia muridarum reveals a role for Pgp5 in suppression of plasmid-dependent gene expression[J].J Bacteriol,2014,196(5):989-998.
[7] 孔杰,孙毅娜,赵乐然,等.沙眼衣原体质粒蛋白pgp3多克隆抗体的制备及鉴定[J].中华传染病杂志,2014,32(2):77-79.
[8] 黄浩,王小红.沙眼衣原体的细胞培养与γ-干扰素[J].华中科技大学学报(医学版),2002,31(4):435-437.
[9] 郑蕾,刘原君,郭睿,等.衣原体噬菌体phiCPG1衣壳蛋白Vp1蛋白IN5部分的克隆、表达、鉴定及其对沙眼衣原体抑制作用的研究[J].中华临床感染病杂志,2017,10(3):199-204,226.
[10] 盛彩虹,孙毅娜,孔杰,等.D型沙眼衣原体多形外膜蛋白I的分段克隆表达及其免疫原性分析[J].中华传染病杂志,2016,34(11):675-679.
[11] Carrasco SE,Hu S,Imai DM,et al.Toll-like receptor 3 (TLR3) promotes the resolutionof Chlamydia muridarum genital tract infection in congenic C57BL/6N mice[J].PLoS One,2018,13(4):e0195165.
[12] Carlson JH,Whitmire WM,Crane DD,et al.The chlamydia trachomatis plasmid is a transcriptional regulator of chromosomal genes and a virulence factor[J].Infect Immun,2008,76(6):2273-2283.
[13] Gong S,Yang Z,Lei L,et al.Characterization of Chlamydia trachomatis plasmid-encoded open reading frames[J].J Bacteriol,2013,195(17):3819-3826.
[14] Liu Y,Huang Y,Yang Z,et al.Plasmid-encoded Pgp3 is a major virulence factor for Chlamydia muridarum to induce hydrosalpinx in mice[J].Infect Immun,2014,82(12):5327-5335.
[15] Patton MJ,Chen CY,Yang C,et al.Plasmid negative regulation of CPAF expression is Pgp4 independent and restricted to invasive chlamydia trachomatis biovars[J].MBio,2018,9(1):e02164-2117.
[16] Zhong G.Chlamydial plasmid-dependent pathogenicity[J].Trends Microbiol,2017,25(2):141-152.
[17] Huang Y,Zhang Q,Yang Z,et al.Plasmid-encoded Pgp5 is a significant contributor to chlamydia muridarum induction of hydrosalpinx[J].PLoS One,2015,10(4):e0124840.
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