肺炎链球菌PsaA抗原及其在结合疫苗中的应用
|
摘要
作为一种自然界广泛存在的致病菌,肺炎链球菌可引起肺炎、脑膜炎、中耳炎、鼻窦炎和败血症等疾病。过去认为肺炎链球菌荚膜多糖抗原为肺炎链球菌主要致病因子,近年来,随着对肺炎链球菌致病机制的深入研究,发现肺炎链球菌一些自身蛋白可能为肺炎链球菌致病因子,这些蛋白在所有临床分离的肺炎链球菌菌株中均存在,可产生交叉免疫保护作用,而且蛋白疫苗产生的是T细胞依赖性免疫反应,对成人和儿童均有很好的保护作用。将肺炎球菌自身蛋白作为载体与多糖偶联制备蛋白多糖结合疫苗成为现在肺炎球菌疫苗的主要方向。目的:应用基因工程技术在原核E. coli和真核毕赤酵母GS115两种体系中表达和制备肺炎链球菌表面黏附素A(pneumococcal surface adhesin A,PsaA),并与细菌荚膜多糖偶联制备成多糖蛋白结合疫苗,探讨PsaA作为肺炎球菌蛋白载体在结合疫苗中起到增强其它细菌多糖抗原免疫原性的同时,还能获得抗肺炎球菌的抗体反应,从而达到用一种结合疫苗能诱导出针对两种细菌的抗体免疫反应的目的。方法:从肺炎链球菌基因组中扩增psaA基因,将目的基因分别插入原核表达载体pET-28a(+)和真核表达载体pPIC9K,获得非融合表达的重组质粒pET28a-psaA和pPIC9K-psaA,通过转化分别进入大肠杆菌BL21和毕赤酵母GS115中,分别经IPTG和甲醇诱导,同采用DEAE-阴离子交换层析法纯化基因重组rPsaA蛋白。经过比较,将表达纯化较好的rPsaA蛋白与A群脑膜炎荚膜多糖(Group A Meningococcal Polysaccharide, GAMP)偶联成多糖蛋白结合疫苗后,用小鼠动物模型进行免疫实验,检测该疫苗的免疫原性,用ELISA法测定该疫苗在小鼠体内产生的针对肺炎球菌和脑膜炎球菌两种病原菌特异性抗原的抗体水平。结果:在原核E. coli中成功克隆基因重组表达质粒,而且表达的PsaA蛋白在载体上的组氨酸标签之前终止表达,不带有组氨酸标签,保证疫苗的安全性。SDS-PAGE技术分析表明:rPsaA蛋白高效表达,约为菌体蛋白的60%,蛋白分子量约为37 kD,而且蛋白的可溶性好,不形成包涵体,用DEAE-阴离子交换层析法纯化其纯度可达80%以上。纯化到的PsaA蛋白与GAMP多糖偶联成功,应用于小鼠免疫实验,PsaA蛋白载体能显著增强A群脑膜炎荚膜多糖抗原的免疫原性,并同时产生针对肺炎球菌蛋白抗原和脑膜炎球菌多糖抗原的特异性抗体。另外在真核毕赤酵母中也成功表达了rPsaA蛋白,目的蛋白表达量高达90%,纯度约70%。结论:利用基因工程技术在原核表达体系和真核毕赤酵母体系中均成功获得无组氨酸标签的rPsaA蛋白,并将它与A群脑膜炎荚膜多糖成功偶联,应用于小鼠免疫实验,在小鼠体内PsaA蛋白载体能显著增强A群脑膜炎荚膜多糖抗原的免疫原性,并同时产生针对肺炎球菌蛋白抗原和脑膜炎球菌多糖抗原的特异性抗体,提高疫苗的免疫保护效果。为探讨给儿童接种一种疫苗能同时预防肺炎和脑膜炎两种传染病的可能性提供研究基础。
Streptococcus pneumoniae carriage can lead to a wide range of localized and systemic diseases such as otitis media, sinusitis, conjunctivitis, pneumonia, septicemia and meningitis. In recent years, with the development of study on Streptococcus pneumoniae, Pneumococcal surface adhesinA (PsaA), pneumococcal surface protein A (PspA) and pneumolysin (Ply) have all been shown to contribute to pneumococcal virulence, use of these common proteins in a vaccine is a logical alternative to the use of polysaccharide vaccines to ensure not only complete serotype coverage for S.pneumoniae, but also a more vigorous immune response since these proteins generate a T-dependent response with immunologic memory. The conjugates vaccine (rPsaA as a carrier protein conjugated with meningococcal polysaccharide) becomes the main direction at present. Objective: The gene expression and the purification of the pneumococcal surface adhesin A (PsaA) protein and its application as a protein carrier in conjugates vaccine. Method: The gene encoding for the PsaA protein was amplified from the genomic DNA of Streptococcus pneumoniae using PCR. Then the PCR product was respectively cloned into the prokaryotic expression vector pET28a and the eukaryotic expression vector pPIC9K. After that, the recombinant was respectively transformed into host cell E. coli BL21 (DE3) and host cell Pichiapastoris GS115. The expression of the recombinant protein (rPsaA) was respectively induced by IPTG and methanol and purified by using DEAE anion-exchange chromatography. The rPsaA was successfully conjugated with Group A Meningococcal Polysaccharide (GAMP). The mice were immunized subcutaneously with the conjugate and the immune responses against GAMP and PsaA were detected by ELISA. Results: The recombinant PsaA was expressed as a 37-kD soluble protein without His-Tag. The rPsaA was successfully conjugated with GAMP. In addition to the immune response against PsaA, The antibody response against GAMP was significant improved in the mice immunized with conjugate vaccine in comparison with those immunized with GAMP alone.
Conclusions: The recombinant protein PsaA without His-Tag was obtained and conjugated with GAMP. The strong antibody responses against PsaA and GAMP were obtained in the immunized mice at the same time which may provide the protection against pneumonia and meningitis simultaneously.
Streptococcus pneumoniae carriage can lead to a wide range of localized and systemic diseases such as otitis media, sinusitis, conjunctivitis, pneumonia, septicemia and meningitis. In recent years, with the development of study on Streptococcus pneumoniae, Pneumococcal surface adhesinA (PsaA), pneumococcal surface protein A (PspA) and pneumolysin (Ply) have all been shown to contribute to pneumococcal virulence, use of these common proteins in a vaccine is a logical alternative to the use of polysaccharide vaccines to ensure not only complete serotype coverage for S.pneumoniae, but also a more vigorous immune response since these proteins generate a T-dependent response with immunologic memory. The conjugates vaccine (rPsaA as a carrier protein conjugated with meningococcal polysaccharide) becomes the main direction at present. Objective: The gene expression and the purification of the pneumococcal surface adhesin A (PsaA) protein and its application as a protein carrier in conjugates vaccine. Method: The gene encoding for the PsaA protein was amplified from the genomic DNA of Streptococcus pneumoniae using PCR. Then the PCR product was respectively cloned into the prokaryotic expression vector pET28a and the eukaryotic expression vector pPIC9K. After that, the recombinant was respectively transformed into host cell E. coli BL21 (DE3) and host cell Pichiapastoris GS115. The expression of the recombinant protein (rPsaA) was respectively induced by IPTG and methanol and purified by using DEAE anion-exchange chromatography. The rPsaA was successfully conjugated with Group A Meningococcal Polysaccharide (GAMP). The mice were immunized subcutaneously with the conjugate and the immune responses against GAMP and PsaA were detected by ELISA. Results: The recombinant PsaA was expressed as a 37-kD soluble protein without His-Tag. The rPsaA was successfully conjugated with GAMP. In addition to the immune response against PsaA, The antibody response against GAMP was significant improved in the mice immunized with conjugate vaccine in comparison with those immunized with GAMP alone.
Conclusions: The recombinant protein PsaA without His-Tag was obtained and conjugated with GAMP. The strong antibody responses against PsaA and GAMP were obtained in the immunized mice at the same time which may provide the protection against pneumonia and meningitis simultaneously.
引文
[1] Bogaert D, van Belkum A, Sluijter M, et al. Colonisation by Streptococcus pneumoniae and Staphylococcus aureus in healthy children[J]. Lancet, 2004, 363(9424):1871-1872.
[2] Park I. Discovery of a new capsular serotype (6C) within serogroup 6 of Streptococcus pneumoniae[Z]. 2007,1225-1233.
[3] Heidelberger M, Rebers PA. Immunochemistry of the pneumococcal types II. V. and VI. I. The relation of Type VI to Type II and other correlations between chemical constitution and precipitation in antisera to type VI[J]. J Bacteriol, 1960, 80:145-153.
[4] Bronsdon MA, O'Brien KL, Facklam R R, et al. Immunoblot method to detect Streptococcus pneumoniae and identify multiple serotypes from nasopharyngeal secretions[J]. J Clin Microbiol, 2004, 42(4):1596-1600.
[5] Greenwood B. The epidemiology of pneumococcal infection in children in the developing world[J]. Philos Trans R Soc Lond B Biol Sci, 1999,354(1384):777-785.
[6] Lloyd-Evans N, O'Dempsey T J, Baldeh I, et al. Nasopharyngeal carriage of pneumococci in Gambian children and in their families[J]. Pediatr Infect Dis J, 1996, 15(10):866-871.
[7] Zenni MK, Cheatham SH, Thompson J M, et al. Streptococcus pneumoniae colonization in the young child: association with otitis media and resistance to penicillin[J]. J Pediatr, 1995, 127(4):533-537.
[8] Gray BM, Converse GR, Dillon HJ. Epidemiologic studies of Streptococcus pneumoniae in infants: acquisition, carriage, and infection during the first 24 months of life[J]. J Infect Dis, 1980, 142(6):923-933.
[9] Garenne M, Ronsmans C, Campbell H. The magnitude of mortality from acute respiratory infections in children under 5 years in developing countries[J]. World Health Stat Q, 1992, 45(2):180-191.
[10] Whitney CG, Farley MM, Hadler J, et al. Decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine[J]. NEngl J Med, 2003, 348(18):1737-1746.
[11] Hausdorff WP, Siber G, Paradiso PR. Geographical differences in invasive pneumococcal disease rates and serotype frequency in young children[J]. Lancet, 2001, 357(9260):950-952.
[12] Pai R, Moore MR, Pilishvili T, et al. Postvaccine genetic structure of Streptococcus pneumoniae serotype 19A from children in the United States[J]. J Infect Dis, 2005, 192(11):1988-1995.
[13] Kaplan SL, Mason EJ, Wald ER, et al. Decrease of invasive pneumococcal infections in children among 8 children's hospitals in the United States after the introduction of the 7-valent pneumococcal conjugate vaccine[J]. Pediatrics, 2004, 113(3):443-449.
[14] Yu X, Gray B, Chang S, et al. Immunity to cross-reactive serotypes induced by pneumococcal conjugate vaccines in infants[J]. J Infect Dis, 1999, 180(5): 1569-1576.
[15] Audouy SA, van Selm S, van Roosmalen ML, et al. Development of lactococcal GEM-based pneumococcal vaccines[J]. Vaccine,2007,25(13):2497-2506.
[16] Adrian PV, Bogaert D, Oprins M, et al. Development of antibodies against pneumococcal proteins alpha-enolase, immunoglobulin A1 protease, streptococcal lipoprotein rotamase A, and putative proteinase maturation protein A in relation to pneumococcal carriage and Otitis Media[J]. Vaccine,2004,22(21-22):2737-2742.
[17] Nelson AL, Roche AM, Gould JM, et al. Capsule enhances pneumococcal colonization by limiting mucus-mediated clearance[J]. Infect Immun, 2007, 75(1):83-90.
[18] Hammerschmidt S, Wolff S, Hocke A, et al. Illustration of pneumococcal polysaccharide capsule during adherence and invasion of epithelial cells[J]. Infect Immun, 2005, 73(8):4653-4667.
[19] Weiser JN, Bae D, Epino H, et al. Changes in availability of oxygen accentuate differences in capsular polysaccharide expression by phenotypic variants and clinical isolates of Streptococcus pneumoniae[J]. Infect Immun, 2001, 69(9):5430-5439.
[20] Russell H, Tharpe JA, Wells DE, et al. Monoclonal antibody recognizing a species-specific protein from Streptococcus pneumoniae[J]. J Clin Microbiol, 1990, 28(10):2191-2195.
[21] Sampson JS, O'Connor SP, Stinson AR, et al. Cloning and nucleotide sequence analysis of psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp. adhesins[J]. Infect Immun, 1994, 62(1):319-324.
[22] Dintilhac A, Alloing G, Granadel C, et al. Competence and virulence of Streptococcus pneumoniae: Adc and PsaA mutants exhibit a requirement for Zn and Mn resulting from inactivation of putative ABC metal permeases[J]. Mol Microbiol, 1997, 25(4):727-739.
[23] Claverys JP, Granadel C, Berry AM, et al. Penicillin tolerance in Streptococcus pneumoniae, autolysis and the Psa ATP-binding cassette (ABC) manganese permease[J]. Mol Microbiol,1999,32(4):881-883.
[24] Novak R, Braun JS, Charpentier E, et al. Penicillin tolerance genes of Streptococcus pneumoniae: the ABC-type manganese permease complex Psa[J]. Mol Microbiol, 1998, 29(5):1285-1296.
[25] Berry A M, Paton J C. Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumoniae[J]. Infect Immun, 1996, 64(12):5255-5262.
[26] Johnson SE, Dorman CM, Bolanowski SA. Inhibition of myogenin expression by activated Raf is not responsible for the block to avian myogenesis[J]. J Biol Chem, 2002, 277(32):28742-28748.
[27] Novak R, Braun JS, Charpentier E, et al. Penicillin tolerance genes of Streptococcus pneumoniae: the ABC-type manganese permease complex Psa[J]. Mol Microbiol, 1998, 29(5):1285-1296.
[28] Sampson J S, Furlow Z, Whitney A M, et al. Limited diversity of Streptococcus pneumoniae psaA among pneumococcal vaccine serotypes[J]. Infect Immun, 1997, 65(5):1967-1971.
[29] Morrison K E, Lake D, Crook J, et al. Confirmation of psaA in all 90 serotypes of Streptococcus pneumoniae by PCR and potential of this assay for identification and diagnosis[J]. J Clin Microbiol, 2000, 38(1):434-437.
[30] Johnston JW, Myers LE, Ochs MM, et al. Lipoprotein PsaA in virulence of Streptococcus pneumoniae: surface accessibility and role in protection from superoxide[J]. Infect Immun, 2004, 72(10):5858-5867.
[31] Dintilhac A, Alloing G, Granadel C, et al. Competence and virulence ofStreptococcus pneumoniae: Adc and PsaA mutants exhibit a requirement for Zn and Mn resulting from inactivation of putative ABC metal permeases[J]. Mol Microbiol, 1997, 25(4):727-739.
[32] Rapola S, Jantti V, Haikala R, et al. Natural development of antibodies to pneumococcal surface protein A, pneumococcal surface adhesin A, and pneumolysin in relation to pneumococcal carriage and acute otitis media[J]. J Infect Dis, 2000, 182(4):1146-1152.
[33] Hammerschmidt S. Adherence molecules of pathogenic pneumococci[J]. Curr Opin Microbiol, 2006, 9(1):12-20.
[34] Gor D O, Ding X, Li Q, et al. Enhanced immunogenicity of pneumococcal surface adhesin A by genetic fusion to cytokines and evaluation of protective immunity in mice[J]. Infect Immun, 2002, 70(10):5589-5595.
[35] Ogunniyi AD, Folland RL, Briles DE, et al. Immunization of mice with combinations of pneumococcal virulence proteins elicits enhanced protection against challenge with Streptococcus pneumoniae[J]. Infect Immun, 2000, 68(5):3028-3033.
[36] Pimenta FC, Miyaji EN, Areas AP, et al. Intranasal immunization with the cholera toxin B subunit-pneumococcal surface antigen A fusion protein induces protection against colonization with Streptococcus pneumoniae and has negligible impact on the nasopharyngeal and oral microbiota of mice[J]. Infect Immun, 2006, 74(8):4939-4944.
[37] Seo JY, Seong SY, Ahn BY, et al. Cross-protective immunity of mice induced by oral immunization with pneumococcal surface adhesin a encapsulated in microspheres[J]. Infect Immun, 2002, 70(3):1143-1149.
[38] Miyaji EN, Dias WO, Gamberini M, et al. PsaA (pneumococcal surface adhesin A) and PspA (pneumococcal surface protein A) DNA vaccines induce humoral and cellular immune responses against Streptococcus pneumoniae[J]. Vaccine, 2001, 20(5-6):805-812.
[39]谢浩,杨程,陈丽娥.多肽融合标签的移除策略[J].生物化学与生物物理进展, 2009, (10):1364-1369.
[40]林海英,孟春,林子琳,等.肺炎链球菌外膜蛋白PspA和PsaA的免疫原性比较[J].中华微生物学和免疫学杂志, 2010, 30(8):712-716.
[41] Tharpe JA, Russell H. Purification and seroreactivity of pneumococcal surface adhesin A (PsaA)[J]. Clin Diagn Lab Immunol, 1996, 3(2):227-229.
[42] Scott JA, Obiero J, Hall AJ, et al. Validation of immunoglobulin G enzyme-linked immunosorbent assay for antibodies to pneumococcal surface adhesin A in the diagnosis of pneumococcal pneumonia among adults in Kenya[J]. J Infect Dis, 2002, 186(2):220-226.
[43] Scott JA, Mlacha Z, Nyiro J, et al. Diagnosis of invasive pneumococcal disease among children in Kenya with enzyme-linked immunosorbent assay for immunoglobulin G antibodies to pneumococcal surface adhesin A[J]. Clin Diagn Lab Immunol, 2005, 12(10):1195-1201.
[44] Dowell SF, Butler JC, Giebink GS. Acute otitis media: management and surveillance in an era of pneumococcal resistance. Drug-Resistant Streptococcus Pneumoniae Therapeutic Working Group[J]. Nurse Pract, 1999, 24(10 Suppl):1-9, 15-16.
[45]何斌,朱虹.肺炎链球菌表面黏附素A(PsaA)的研究进展[J].微生物学免疫学进展, 2007, (3):84-86.
[46] De BK, Sampson JS, Ades EW, et al. Baculovirus expression, purification and evaluation of recombinant pneumococcal surface adhesin A of Streptococcus pneumoniae[J]. Pathobiology, 1999, 67(3):115-122.
[47]张雪梅,尹一兵,朱旦,等.肺炎链球菌的转化缺陷导致细菌毒力减弱[J].中华微生物学和免疫学杂志, 2005, 25(2):146-151.
[48]林铃,朱为.肺炎球菌疫苗新策略[J].国际生物制品学杂志, 2006(1):5-7.
[49] Robbins JB, Schneerson R, Szu SC. Perspective: hypothesis: serum IgG antibody is sufficient to confer protection against infectious diseases by inactivating the inoculum[J]. J Infect Dis, 1995, 171(6):1387-1398.
[50] Chu CY, Liu BK, Watson D, et al. Preparation, characterization, and immunogenicity of conjugates composed of the O-specific polysaccharide of Shigella dysenteriae type 1 (Shiga's bacillus) bound to tetanus toxoid[J]. Infect Immun, 1991, 59(12):4450-4458.
[51] Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent[J]. J Biol Chem, 1951, 193(1):265-275.
[52] Robbins JB, Schneerson R, Szu SC. Perspective: hypothesis: serum IgG antibody issufficient to confer protection against infectious diseases by inactivating the inoculum[J]. J Infect Dis, 1995, 171(6):1387-1398.
[53] Taylor DN, Trofa AC, Sadoff J, et al. Synthesis, characterization, and clinical evaluation of conjugate vaccines composed of the O-specific polysaccharides of Shigella dysenteriae type 1, Shigella flexneri type 2a, and Shigella sonnei (Plesiomonas shigelloides) bound to bacterial toxoids[J]. Infect Immun, 1993, 61(9):3678-3687.
[54]张萍,王欣茹,侯亚莉,等. 14型肺炎球菌荚膜多糖-破伤风类毒素结合疫苗的研制[J].微生物学免疫学进展, 2007(1):1-4.
[55]章如安,杨晟,邱荣德,等.巴斯德毕赤酵母表达体系研究及进展[J].微生物学通报, 2000(5):371-373.
[56] De BK, Sampson JS, Ades EW, et al. Purification and characterization of Streptococcus pneumoniae palmitoylated pneumococcal surface adhesin A expressed in Escherichia coli[J]. Vaccine, 2000, 18(17):1811-1821.
[57]黄金钟,林海英,蔡倩影,等.肺炎球菌表面蛋白A(PspA)及其荚膜多糖交联物的免疫原性和交叉保护作用[J].生物化学与生物物理进展, 2008, 35(5):536-539.
[58]刘仲荣,曹春来,杨慧兰,等.全长单纯疱疹病毒Ⅱ型糖蛋白D毕赤酵母表达载体的构建[J].实用医学杂志, 2005, (11):1117-1119.
[59]齐连权,付玲,于长明,等.人ω干扰素在巴斯德毕赤酵母中的表达及纯化[J].军事医学科学院院刊, 2003, (4):268-270.
[60] Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J]. FEMS Microbiol Rev, 2000, 24(1):45-66.
[2] Park I. Discovery of a new capsular serotype (6C) within serogroup 6 of Streptococcus pneumoniae[Z]. 2007,1225-1233.
[3] Heidelberger M, Rebers PA. Immunochemistry of the pneumococcal types II. V. and VI. I. The relation of Type VI to Type II and other correlations between chemical constitution and precipitation in antisera to type VI[J]. J Bacteriol, 1960, 80:145-153.
[4] Bronsdon MA, O'Brien KL, Facklam R R, et al. Immunoblot method to detect Streptococcus pneumoniae and identify multiple serotypes from nasopharyngeal secretions[J]. J Clin Microbiol, 2004, 42(4):1596-1600.
[5] Greenwood B. The epidemiology of pneumococcal infection in children in the developing world[J]. Philos Trans R Soc Lond B Biol Sci, 1999,354(1384):777-785.
[6] Lloyd-Evans N, O'Dempsey T J, Baldeh I, et al. Nasopharyngeal carriage of pneumococci in Gambian children and in their families[J]. Pediatr Infect Dis J, 1996, 15(10):866-871.
[7] Zenni MK, Cheatham SH, Thompson J M, et al. Streptococcus pneumoniae colonization in the young child: association with otitis media and resistance to penicillin[J]. J Pediatr, 1995, 127(4):533-537.
[8] Gray BM, Converse GR, Dillon HJ. Epidemiologic studies of Streptococcus pneumoniae in infants: acquisition, carriage, and infection during the first 24 months of life[J]. J Infect Dis, 1980, 142(6):923-933.
[9] Garenne M, Ronsmans C, Campbell H. The magnitude of mortality from acute respiratory infections in children under 5 years in developing countries[J]. World Health Stat Q, 1992, 45(2):180-191.
[10] Whitney CG, Farley MM, Hadler J, et al. Decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine[J]. NEngl J Med, 2003, 348(18):1737-1746.
[11] Hausdorff WP, Siber G, Paradiso PR. Geographical differences in invasive pneumococcal disease rates and serotype frequency in young children[J]. Lancet, 2001, 357(9260):950-952.
[12] Pai R, Moore MR, Pilishvili T, et al. Postvaccine genetic structure of Streptococcus pneumoniae serotype 19A from children in the United States[J]. J Infect Dis, 2005, 192(11):1988-1995.
[13] Kaplan SL, Mason EJ, Wald ER, et al. Decrease of invasive pneumococcal infections in children among 8 children's hospitals in the United States after the introduction of the 7-valent pneumococcal conjugate vaccine[J]. Pediatrics, 2004, 113(3):443-449.
[14] Yu X, Gray B, Chang S, et al. Immunity to cross-reactive serotypes induced by pneumococcal conjugate vaccines in infants[J]. J Infect Dis, 1999, 180(5): 1569-1576.
[15] Audouy SA, van Selm S, van Roosmalen ML, et al. Development of lactococcal GEM-based pneumococcal vaccines[J]. Vaccine,2007,25(13):2497-2506.
[16] Adrian PV, Bogaert D, Oprins M, et al. Development of antibodies against pneumococcal proteins alpha-enolase, immunoglobulin A1 protease, streptococcal lipoprotein rotamase A, and putative proteinase maturation protein A in relation to pneumococcal carriage and Otitis Media[J]. Vaccine,2004,22(21-22):2737-2742.
[17] Nelson AL, Roche AM, Gould JM, et al. Capsule enhances pneumococcal colonization by limiting mucus-mediated clearance[J]. Infect Immun, 2007, 75(1):83-90.
[18] Hammerschmidt S, Wolff S, Hocke A, et al. Illustration of pneumococcal polysaccharide capsule during adherence and invasion of epithelial cells[J]. Infect Immun, 2005, 73(8):4653-4667.
[19] Weiser JN, Bae D, Epino H, et al. Changes in availability of oxygen accentuate differences in capsular polysaccharide expression by phenotypic variants and clinical isolates of Streptococcus pneumoniae[J]. Infect Immun, 2001, 69(9):5430-5439.
[20] Russell H, Tharpe JA, Wells DE, et al. Monoclonal antibody recognizing a species-specific protein from Streptococcus pneumoniae[J]. J Clin Microbiol, 1990, 28(10):2191-2195.
[21] Sampson JS, O'Connor SP, Stinson AR, et al. Cloning and nucleotide sequence analysis of psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp. adhesins[J]. Infect Immun, 1994, 62(1):319-324.
[22] Dintilhac A, Alloing G, Granadel C, et al. Competence and virulence of Streptococcus pneumoniae: Adc and PsaA mutants exhibit a requirement for Zn and Mn resulting from inactivation of putative ABC metal permeases[J]. Mol Microbiol, 1997, 25(4):727-739.
[23] Claverys JP, Granadel C, Berry AM, et al. Penicillin tolerance in Streptococcus pneumoniae, autolysis and the Psa ATP-binding cassette (ABC) manganese permease[J]. Mol Microbiol,1999,32(4):881-883.
[24] Novak R, Braun JS, Charpentier E, et al. Penicillin tolerance genes of Streptococcus pneumoniae: the ABC-type manganese permease complex Psa[J]. Mol Microbiol, 1998, 29(5):1285-1296.
[25] Berry A M, Paton J C. Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumoniae[J]. Infect Immun, 1996, 64(12):5255-5262.
[26] Johnson SE, Dorman CM, Bolanowski SA. Inhibition of myogenin expression by activated Raf is not responsible for the block to avian myogenesis[J]. J Biol Chem, 2002, 277(32):28742-28748.
[27] Novak R, Braun JS, Charpentier E, et al. Penicillin tolerance genes of Streptococcus pneumoniae: the ABC-type manganese permease complex Psa[J]. Mol Microbiol, 1998, 29(5):1285-1296.
[28] Sampson J S, Furlow Z, Whitney A M, et al. Limited diversity of Streptococcus pneumoniae psaA among pneumococcal vaccine serotypes[J]. Infect Immun, 1997, 65(5):1967-1971.
[29] Morrison K E, Lake D, Crook J, et al. Confirmation of psaA in all 90 serotypes of Streptococcus pneumoniae by PCR and potential of this assay for identification and diagnosis[J]. J Clin Microbiol, 2000, 38(1):434-437.
[30] Johnston JW, Myers LE, Ochs MM, et al. Lipoprotein PsaA in virulence of Streptococcus pneumoniae: surface accessibility and role in protection from superoxide[J]. Infect Immun, 2004, 72(10):5858-5867.
[31] Dintilhac A, Alloing G, Granadel C, et al. Competence and virulence ofStreptococcus pneumoniae: Adc and PsaA mutants exhibit a requirement for Zn and Mn resulting from inactivation of putative ABC metal permeases[J]. Mol Microbiol, 1997, 25(4):727-739.
[32] Rapola S, Jantti V, Haikala R, et al. Natural development of antibodies to pneumococcal surface protein A, pneumococcal surface adhesin A, and pneumolysin in relation to pneumococcal carriage and acute otitis media[J]. J Infect Dis, 2000, 182(4):1146-1152.
[33] Hammerschmidt S. Adherence molecules of pathogenic pneumococci[J]. Curr Opin Microbiol, 2006, 9(1):12-20.
[34] Gor D O, Ding X, Li Q, et al. Enhanced immunogenicity of pneumococcal surface adhesin A by genetic fusion to cytokines and evaluation of protective immunity in mice[J]. Infect Immun, 2002, 70(10):5589-5595.
[35] Ogunniyi AD, Folland RL, Briles DE, et al. Immunization of mice with combinations of pneumococcal virulence proteins elicits enhanced protection against challenge with Streptococcus pneumoniae[J]. Infect Immun, 2000, 68(5):3028-3033.
[36] Pimenta FC, Miyaji EN, Areas AP, et al. Intranasal immunization with the cholera toxin B subunit-pneumococcal surface antigen A fusion protein induces protection against colonization with Streptococcus pneumoniae and has negligible impact on the nasopharyngeal and oral microbiota of mice[J]. Infect Immun, 2006, 74(8):4939-4944.
[37] Seo JY, Seong SY, Ahn BY, et al. Cross-protective immunity of mice induced by oral immunization with pneumococcal surface adhesin a encapsulated in microspheres[J]. Infect Immun, 2002, 70(3):1143-1149.
[38] Miyaji EN, Dias WO, Gamberini M, et al. PsaA (pneumococcal surface adhesin A) and PspA (pneumococcal surface protein A) DNA vaccines induce humoral and cellular immune responses against Streptococcus pneumoniae[J]. Vaccine, 2001, 20(5-6):805-812.
[39]谢浩,杨程,陈丽娥.多肽融合标签的移除策略[J].生物化学与生物物理进展, 2009, (10):1364-1369.
[40]林海英,孟春,林子琳,等.肺炎链球菌外膜蛋白PspA和PsaA的免疫原性比较[J].中华微生物学和免疫学杂志, 2010, 30(8):712-716.
[41] Tharpe JA, Russell H. Purification and seroreactivity of pneumococcal surface adhesin A (PsaA)[J]. Clin Diagn Lab Immunol, 1996, 3(2):227-229.
[42] Scott JA, Obiero J, Hall AJ, et al. Validation of immunoglobulin G enzyme-linked immunosorbent assay for antibodies to pneumococcal surface adhesin A in the diagnosis of pneumococcal pneumonia among adults in Kenya[J]. J Infect Dis, 2002, 186(2):220-226.
[43] Scott JA, Mlacha Z, Nyiro J, et al. Diagnosis of invasive pneumococcal disease among children in Kenya with enzyme-linked immunosorbent assay for immunoglobulin G antibodies to pneumococcal surface adhesin A[J]. Clin Diagn Lab Immunol, 2005, 12(10):1195-1201.
[44] Dowell SF, Butler JC, Giebink GS. Acute otitis media: management and surveillance in an era of pneumococcal resistance. Drug-Resistant Streptococcus Pneumoniae Therapeutic Working Group[J]. Nurse Pract, 1999, 24(10 Suppl):1-9, 15-16.
[45]何斌,朱虹.肺炎链球菌表面黏附素A(PsaA)的研究进展[J].微生物学免疫学进展, 2007, (3):84-86.
[46] De BK, Sampson JS, Ades EW, et al. Baculovirus expression, purification and evaluation of recombinant pneumococcal surface adhesin A of Streptococcus pneumoniae[J]. Pathobiology, 1999, 67(3):115-122.
[47]张雪梅,尹一兵,朱旦,等.肺炎链球菌的转化缺陷导致细菌毒力减弱[J].中华微生物学和免疫学杂志, 2005, 25(2):146-151.
[48]林铃,朱为.肺炎球菌疫苗新策略[J].国际生物制品学杂志, 2006(1):5-7.
[49] Robbins JB, Schneerson R, Szu SC. Perspective: hypothesis: serum IgG antibody is sufficient to confer protection against infectious diseases by inactivating the inoculum[J]. J Infect Dis, 1995, 171(6):1387-1398.
[50] Chu CY, Liu BK, Watson D, et al. Preparation, characterization, and immunogenicity of conjugates composed of the O-specific polysaccharide of Shigella dysenteriae type 1 (Shiga's bacillus) bound to tetanus toxoid[J]. Infect Immun, 1991, 59(12):4450-4458.
[51] Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent[J]. J Biol Chem, 1951, 193(1):265-275.
[52] Robbins JB, Schneerson R, Szu SC. Perspective: hypothesis: serum IgG antibody issufficient to confer protection against infectious diseases by inactivating the inoculum[J]. J Infect Dis, 1995, 171(6):1387-1398.
[53] Taylor DN, Trofa AC, Sadoff J, et al. Synthesis, characterization, and clinical evaluation of conjugate vaccines composed of the O-specific polysaccharides of Shigella dysenteriae type 1, Shigella flexneri type 2a, and Shigella sonnei (Plesiomonas shigelloides) bound to bacterial toxoids[J]. Infect Immun, 1993, 61(9):3678-3687.
[54]张萍,王欣茹,侯亚莉,等. 14型肺炎球菌荚膜多糖-破伤风类毒素结合疫苗的研制[J].微生物学免疫学进展, 2007(1):1-4.
[55]章如安,杨晟,邱荣德,等.巴斯德毕赤酵母表达体系研究及进展[J].微生物学通报, 2000(5):371-373.
[56] De BK, Sampson JS, Ades EW, et al. Purification and characterization of Streptococcus pneumoniae palmitoylated pneumococcal surface adhesin A expressed in Escherichia coli[J]. Vaccine, 2000, 18(17):1811-1821.
[57]黄金钟,林海英,蔡倩影,等.肺炎球菌表面蛋白A(PspA)及其荚膜多糖交联物的免疫原性和交叉保护作用[J].生物化学与生物物理进展, 2008, 35(5):536-539.
[58]刘仲荣,曹春来,杨慧兰,等.全长单纯疱疹病毒Ⅱ型糖蛋白D毕赤酵母表达载体的构建[J].实用医学杂志, 2005, (11):1117-1119.
[59]齐连权,付玲,于长明,等.人ω干扰素在巴斯德毕赤酵母中的表达及纯化[J].军事医学科学院院刊, 2003, (4):268-270.
[60] Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J]. FEMS Microbiol Rev, 2000, 24(1):45-66.