徐悦玥;蔡冉;马颖;尚彦红;袁敬宇;张素芬;李越希;

• 1:南京医科大学基础医学院生物化学与分子生物学系
• 2:中国药科大学生命科学与技术学院

摘要(Abstract)：

目的原核表达、纯化肺炎链球菌表面蛋白A(pneumococcal surface protein A,Psp A),并制备多克隆抗体。方法应用ANTHEWIN、DNAstar等分子生物学软件,对Psp A氨基酸序列进行分析,筛选出抗原表位富集区(第33~109个氨基酸),选用原核生物偏爱的密码子优化基因序列,化学合成全新的基因序列pspa,插入质粒p GEX-4T-2和p ET28a(+)中,构建重组表达质粒p GEX-4T-2-pspa和p ET28a(+)-pspa,转化大肠埃希菌BL21(DE3),IPTG诱导表达。分别纯化带有GST标签和His标签的Psp A重组蛋白GST-Psp A和His-Psp A,以His-Psp A作为免疫原,经背部多点免疫新西兰大耳白兔,间接ELISA法检测血清抗体效价,Western blot法检测血清抗体特异性。结果两种重组表达质粒p GEX-4T-2-pspa和p ET28a(+)-pspa经双酶切鉴定构建正确;表达的两种重组蛋白GST-Psp A和His-Psp A相对分子质量分别约为33 000和18 000,均为可溶性表达,纯化后目的蛋白条带均无降解,纯度约为95%,蛋白浓度分别为2和0.2 mg/ml;制备的兔抗血清效价较高,可达1∶200 000,且特异性较好。结论原核表达并纯化了肺炎链球菌Psp A融合蛋白,并制备了特异性良好的高效价兔抗血清,为下一步建立肺炎链球菌快速检测技术奠定了基础。

关键词(KeyWords)： 肺炎链球菌表面蛋白A;原核细胞;基因表达;纯化;多克隆抗体

Abstract:

Keywords:

基金项目(Foundation): 国家传染病重大专项（No. 2013ZX10004804鄄003）;;全军十二五重大项目（No. AWS11C001）;;

作者(Author): 徐悦玥;蔡冉;马颖;尚彦红;袁敬宇;张素芬;李越希;

Email:

DOI: 10.13200/j.cnki.cjb.000850

参考文献(References)：

• [1]张敏,朱小燕,王四利,等.肺炎链球菌感染的分布及耐药性分析[J].检验医学,2013,28(12):1147-1149.
• [2]秦颖,余宏杰.2012年WHO关于肺炎链球菌疫苗立场文件的解读[J].中华预防医学杂志,2014,48(2):85-90.
• [3]Bravo LC,Asian Strategic Alliance for Pneumococcal Disease Prevention(ASAP)Working Group.Overview of the disease burden of invasive pneumococcal disease in Asia[J].Vaccine,2009,27(52):7282-7291.
• [4]Hung IF,Tantawichien T,Tsai YH,et al.Regional epidemiology of in vasive pneumococcal disease in Asian adults:epidemiology,disease burden,serotype distribution,and antimicrobial resistance patterns and prevention[J].Int J Infect Dis,2013,17(6):e364-e373.
• [5]Lin TY,Shah NK,Brooks D,et al.Summary of invasive pneumococcal disease burden among children in the Asia-Pacific region[J].Vaccine,2010,28(48):7589-7605.
• [6]Yu H.Progress on detection and resistance of Streptococcus pneumoniae[J].J Pediatr Pharm,2009,15(3):1-3.(in Chinese)俞蕙.肺炎链球菌检测方法及耐药研究[J].儿科药学杂志,2009,15(3):1-3.
• [7]饶春梅,张中和,王勇.社区获得性肺炎病原学调查及检测方法的进展[J].中国呼吸与危重监护杂志,2012,11(1):101-103.
• [8]Melin MM,Hollingshead SK,Briles DE,et al.Development of antibodies to Psp A families 1 and 2 in children after exposure to Streptococcus pneumoniae[J].Clin Vaccine Immunol,2008,15(10):1529-1535.
• [9]Crain MJ,Waltman WD 2nd,Turner JS,et al.Pneumococcal surface protein A(Psp A)is serologically highly variable and is expressed by all clinically important capsular serotypes of Streptococcus pneumoniae[J].Infect Immun,1990,58(10):3293-3299.
• [10]Zhang Q,Guo Z,Mac Donald NE.Vaccine preventable community-acquired pneumonia in hospitalized children in Northwest China[J].Pediatr Infect Dis J,2011,30(1):7-10.
• [11]Wang YJ,Vuori-Holopainen E,Yang Y,et al.Relative frequency of Haemophilus influenzae type b pneumonia in Chinese children as evidenced by serology[J].Pediatr Infect Dis J,2002,21(4):271-277.
• [12]Fu P,He LY,Wang AM,et al.Drug resistance and proportion of five common pathogens isolated from children′s respiratory tract in children′s hospital of Fudan University from 2006 to2009[J].Chin J Evidence Based Pediatr,2010,5(5):371-376.(in Chinese)付盼,王磊燕,王爱敏,等.2006至2009年复旦大学附属儿科医院呼吸道感染患儿5种常见细菌构成比和耐药性分析[J].中国循证儿科杂志,20l0,5(5):371-376.
• [13]Wang YJ,Liu J,Fang F,et al.Microbiological etiology in children with community acquired pneumonia[J].Chin J Contemp Pediatr,2010,12(3):184-187.(in Chinese)王应建,刘洁,方芳,等.儿童社区获得性肺炎病原微生物分布研究[J].中国当代儿科杂志,2010,12(3):184-187.
• [14]Song YJ,Wang LX,Hong YQ,et al.Analysis of pathogen in Huai-an adults with community-acquired pneumonia[J].J Med Postgra,2010,23(12):1279-1281.(in Chinese)宋应健,王立新,洪永青,等.淮安地区成人社区获得性肺炎感染病原体分析[J].医学研究生学报,2010,23(12):1279-1281.
• [15]张隽.老年人社区获得性肺炎的特点[J].中国医刊,2009,44(9):15-17.
• [16]Yao KH,Yang YH.Streptococcus pneumoniae diseases in Chinese children:past,present and future[J].Vaccine,2008,26(35):4425-4433.
• [17]Pimenta FC,Ribeiro-Dias F,Brandileone MC,et al.Genetic diversity of Psp A types among nasopharyngeal isolates collected during an ongoing surveillance study of children in Brazil[J].J Clin Microbiol,2006,44(8):2838-2843.
• [18]Hollingshead SK,Becker R,Briles DE.Diversity of Psp A:mosaic genes and evidence for past recombination in Streptococcus pneumoniae[J].Infect Immun,2000,68(10):5889-5900.
• [19]Mc Daniel LS,Mc Daniel DO,Hollingshead SK,et al.Comparison of the Psp A sequence from Streptococcus pneumoniae EF5668 to the previously identified Psp A sequence from strain Rx1 and ability of Psp A from EF5668 to elicit protection against pneumococci of different capsular types[J].Infect Immun,1998,66(10):4748-4754.