不可分型流感嗜血杆菌外膜蛋白P6的生物信息学分析
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摘要
目的克隆不可分型流感嗜血杆菌ATCC49247外膜蛋白P6基因,并对其编码蛋白进行分析。方法以标准菌株ATCC49247NTHI DNA为模板,PCR扩增P6目的基因,构建重组质粒pGEX-6P2/P6并测序,利用生物信息软件和GenBank数据库对编码外膜蛋白P6进行同源性分析,预测其主要理化性质、结构功能、B细胞抗原表位和T细胞抗原表位。结果 ATCC49247 P6基因核苷酸序列长度为462 bp,与不可分型流感嗜血杆菌外膜蛋白P6基因核苷酸同源性为99.17%～100%,编码蛋白氨基酸同源性为96.18%～100%。编码蛋白含有153个氨基酸,相对分子质量为16.137 94×10~3,等电点6.09,为亲水性蛋白;该蛋白含有信号肽和3个结构域,无跨膜结构域;综合蛋白质的二级结构、亲疏水性、柔性、表面可及性和抗原指数预测该蛋白含有6个优势B细胞抗原表位,有7个CTL细胞抗原表位及16个Th细胞抗原表位。结论成功克隆外膜蛋白P6基因,生物信息学方法预测分析ATCC49247外膜蛋白P6高度保守,免疫原性强,可作为流感嗜血杆菌候选疫苗和疫苗载体。
Objective To clone the outer membrane protein P6 gene of nontypeable Haemophilus influenzae ATCC49247 and to analyze the protein it encodes using bioinformatics. Methods PCR was used to amplify the P6 gene from the DNA of H. influenzae ATCC49247. The recombinant plasmid pGEX-6 P2/P6 was constructed and subsequently sequenced. Its homology was analyzed, and the main physicochemical properties, structural function, and B-cell epitopes and T-cell epitopes of outer membrane protein P6 of nontypeable H. influenzae were predicted using bioinformatic software and the GenBank database. Results The P6 gene of H. influenzae ATCC49247 was 462 bp in length. The outer membrane protein P6 gene in H. influenzae ATCC49247 and nontypeable H. influenzae had a nucleotide similarity of 99.17-100% and an amino acid similarity of 96.18-100%. Bioinformatic analysis indicated that the protein encoded by the gene contains 153 amino acids with a relative molecular mass of 16.137 94 ku and an isoelectric point of 6.09. The protein is a hydrophilic protein. The protein had a signal peptide, three domains, and no transmembrane domains. Comprehensive analysis of the protein's secondary structure, hydrophobicity, flexibility, surface accessibility, and antigenic index revealed 6 dominant B-cell epitopes, 7 CTL cell epitopes, and 16 Th-cell epitopes. Conclusion The P6 gene of ATCC49247 was successfully cloned, and bioinformatics predicted that the protein encoded by the P6 gene would be highly conserved and have strong immunogenicity. This protein is a potential vaccine or vaccine vector for H. influenzae.
Objective To clone the outer membrane protein P6 gene of nontypeable Haemophilus influenzae ATCC49247 and to analyze the protein it encodes using bioinformatics. Methods PCR was used to amplify the P6 gene from the DNA of H. influenzae ATCC49247. The recombinant plasmid pGEX-6 P2/P6 was constructed and subsequently sequenced. Its homology was analyzed, and the main physicochemical properties, structural function, and B-cell epitopes and T-cell epitopes of outer membrane protein P6 of nontypeable H. influenzae were predicted using bioinformatic software and the GenBank database. Results The P6 gene of H. influenzae ATCC49247 was 462 bp in length. The outer membrane protein P6 gene in H. influenzae ATCC49247 and nontypeable H. influenzae had a nucleotide similarity of 99.17-100% and an amino acid similarity of 96.18-100%. Bioinformatic analysis indicated that the protein encoded by the gene contains 153 amino acids with a relative molecular mass of 16.137 94 ku and an isoelectric point of 6.09. The protein is a hydrophilic protein. The protein had a signal peptide, three domains, and no transmembrane domains. Comprehensive analysis of the protein's secondary structure, hydrophobicity, flexibility, surface accessibility, and antigenic index revealed 6 dominant B-cell epitopes, 7 CTL cell epitopes, and 16 Th-cell epitopes. Conclusion The P6 gene of ATCC49247 was successfully cloned, and bioinformatics predicted that the protein encoded by the P6 gene would be highly conserved and have strong immunogenicity. This protein is a potential vaccine or vaccine vector for H. influenzae.
引文
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[12] 袁秋露, 付玉荣, 伊正君. 结核分枝杆菌BfrB蛋白的生物信息学分析[J]. 中国病原生物学杂志, 2018, 13(2): 131-4.
[13] 王利磊, 杨琰琰, 王洪法, 等. 弓形虫Toxofilin蛋白基因克隆和生物信息学分析[J]. 中国病原生物学杂志, 2017, 12(8): 747-50.
[14] 张贤, 甘小凤, 程正阳, 等. 弓形虫棒状体蛋白2家族成员研究进展[J]. 中国病原生物学杂志, 2015, 10(6): 574-6.
[15] Michel LV, Kalmeta B, McCreary M, et al. Vaccine candidate P6 of nontypeable Haemophilus influenzae is nota transmembrane protein based on protein structural analysis[J].Vaccine, 2011, 29(8): 1624-7.
[16] Michel LV, Snyder J, Schmidt R, et al. Dual Orientation of the Outer Membrane Lipoprotein P6 of Nontypeable Haemophilus influenzae[J]. J Bacteriol, 2013, 195(14): 3252-9.
[17] Cowles CE, Li Y, Semmelhack MF, et al. Thefree and bound forms of Lpp occupy distinct subcellular locations in Escherichia coli[J]. Mol Microbiol, 2011, 79(5): 1168-81.
[18] Pavlovic MD, Jandrlic DR, Mitic NS. Epitope distribution in ordered and disordered protein regions. Part B-Ordered regions and disordered binding sites are targets of T-cell and B-cell immunity[J]. J Immunol Methods, 2014, 407(3): 90-107.
[19] Ferrante A. For many but not for all: how the conformational flexibility of the peptide/MHCII complex shapes epitope selection[J]. Immunol Res, 2013, 56(1): 85-95.
[2] Duclos P. Haemophilus influenzae type b (Hib) Vaccination WHO position paper: July 2013-Recommendations[J]. Vaccine, 2013, 31(52): 6168-9.
[3] Rubach MP, Bender JM, Mottice S, et al. Increasing incidence of invasive Haemophilus influenzae disease in adults, Utah, USA[J]. Emerg Infect Dis, 2011, 17(9):1645-50.
[4] Sunakawa K, Takeuchi Y, Iwata S. Nontypeable Haemophilus influenzae (NTHi) epidemiology[J]. Kansenshogaku Zasshi, 2011, 85(3): 227-7.
[5] 景春梅, 王偲. 2009-2013年重庆地区儿童感染流感嗜血杆菌的耐药性及血清型分析[J]. 第三军医大学学报, 2014, 36(23): 2400-3.
[6] 胡莹, 夏晓玲, 黄海林, 等. 昆明地区儿童下呼吸道感染流感嗜血杆菌分离株的耐药性和血清分型研究[J]. 实验与检验医学, 2013, 31(2): 118-20.
[7] Berenson CS, Murphy TF, Wrona CT, et al. Outer membrane protein P6 of nontypeable Haemophilus influenzae is a potent and selective inducer of human macrophage proinflammatory cytokines[J]. Infect Immun, 2005, 73(5): 2728-35.
[8] Murphy TF. Vaccines for nontypeable Haemophilus influenzae: the future is now[J]. Clin Vaccine Immunol, 2015, 22(5): 459-66.
[9] Khan MN, Kaur R, Pichichero ME. Bactericidal antibody response against P6, protein D, and OMP26 of nontypeable Haemophilus influenzae after acute otitis media in otitis- prone children[J]. FEMS Immunol Med Microbiol, 2012, 65(3): 439-47.
[10] Chang A, Kaur R, Michel LV, et al. Haemophilus influenzae vaccine candidate outer membrane protein P6 is not conserved in all strains[J]. Hum Vaccine, 2011, 7(1): 102-5.
[11] 孟双, 徐冲, 陈丽媛, 等. 生物信息学在生物学研究领域的应用[J]. 微生物学杂志, 2011, 31(1): 78-81.
[12] 袁秋露, 付玉荣, 伊正君. 结核分枝杆菌BfrB蛋白的生物信息学分析[J]. 中国病原生物学杂志, 2018, 13(2): 131-4.
[13] 王利磊, 杨琰琰, 王洪法, 等. 弓形虫Toxofilin蛋白基因克隆和生物信息学分析[J]. 中国病原生物学杂志, 2017, 12(8): 747-50.
[14] 张贤, 甘小凤, 程正阳, 等. 弓形虫棒状体蛋白2家族成员研究进展[J]. 中国病原生物学杂志, 2015, 10(6): 574-6.
[15] Michel LV, Kalmeta B, McCreary M, et al. Vaccine candidate P6 of nontypeable Haemophilus influenzae is nota transmembrane protein based on protein structural analysis[J].Vaccine, 2011, 29(8): 1624-7.
[16] Michel LV, Snyder J, Schmidt R, et al. Dual Orientation of the Outer Membrane Lipoprotein P6 of Nontypeable Haemophilus influenzae[J]. J Bacteriol, 2013, 195(14): 3252-9.
[17] Cowles CE, Li Y, Semmelhack MF, et al. Thefree and bound forms of Lpp occupy distinct subcellular locations in Escherichia coli[J]. Mol Microbiol, 2011, 79(5): 1168-81.
[18] Pavlovic MD, Jandrlic DR, Mitic NS. Epitope distribution in ordered and disordered protein regions. Part B-Ordered regions and disordered binding sites are targets of T-cell and B-cell immunity[J]. J Immunol Methods, 2014, 407(3): 90-107.
[19] Ferrante A. For many but not for all: how the conformational flexibility of the peptide/MHCII complex shapes epitope selection[J]. Immunol Res, 2013, 56(1): 85-95.