肺炎链球菌表面蛋白A的原核表达、纯化及其多克隆抗体的制备  

Expression and purification of pneumococcal surface protein A and preparation of its polyclonal antibodies

作　　者：徐悦玥 蔡冉[1] 马颖[2] 尚彦红 袁敬宇 张素芬[2] 李越希[1] 

机构地区：[1]南京医科大学基础医学院生物化学与分子生物学系,江苏南京210002 [2]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《中国生物制品学杂志》2015年第4期372-376,382,共6页Chinese Journal of Biologicals

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

摘　　要：目的 原核表达、纯化肺炎链球菌表面蛋白A（pneumococcal surface protein A,PspA）,并制备多克隆抗体。方法 应用ANTHEWIN、DNAstar等分子生物学软件,对PspA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pGEX-4T-2和pET28a（＋）中,构建重组表达质粒pGEX-4T-2-pspa和pET28a（＋）-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PspA重组蛋白GST-PspA和His-PspA,以His-PspA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pGEX-4T-2-pspa和pET28a（＋）-pspa经双酶切鉴定构建正确;表达的两种重组蛋白GST-PspA和His-PspA相对分子质量分别〈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〈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PspA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。Objective To express pneumoeoecal surface protein A （PspA） in prokaryotie cells, purify the expressed prod- uet and prepare its polycolonal antibodies. Methods The amino acid sequence of PspA was analyzed by ANTHEWIN and DNAStar software, based on which the epitope-rieh region （amino acids 33 - 109） was screened. The pspa gene sequence modified by prokaryotie cells-preferred eodon was synthesized chemically and inserted into vectors pGEX-4T-2 and pET28a （＋）. The constructed recombinant plasmids pGEX-4T-2-psps and pET28a （＋）-pspa were transformed to E. coli BL21 （DE3） and induced with IPTG respectively. The expressed recombinant proteins GST-PspA and His-PspA were puri- fied, and His-PspA was used as an immunogen for immunization of New Zealand white rabbits by subcutaneous injection in several sites on back. The serum antibody titer was determined by indirect ELISA, while the specificity by Western blot. Results Restriction analysis proved that recombinant plasmids pGEX-4T-2-pspa and pET28a （＋）-pspa were constructed correctly. The expressed recombinant proteins GST-PspA and His-PspA, with relative molecular masses of about 33 000 and about 18 000 respectively, existed in forms of inclusion bodies. The purified target protein bands showed no degradation, of which the purities were both about 95%, protein concentrations were 2 and 0. 2 mg/ ml respectively. The prepared rabbit antisera reached a titer of 1 ： 200 000 and a good specificity. Conclusion PspA was successfully ex- pressed in a fusion form in prokaryotie cells and purified, of which the high titer rabbit antisera with good specificity was prepared. It laid a foundation of further development of rapid detection technique for Streptococcus pneumoniae.

关 键 词：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 

分 类 号：R378.1[医药卫生—病原生物学]