医学分子生物学杂志
醫學分子生物學雜誌
의학분자생물학잡지
FOREIGN MEDICAL SCIENCES
2010年
2期
104-108
,共5页
崔亚利%王虹%尹楠林%龚艺%牛司强%尹一兵%张雪梅
崔亞利%王虹%尹楠林%龔藝%牛司彊%尹一兵%張雪梅
최아리%왕홍%윤남림%공예%우사강%윤일병%장설매
肺炎链球菌%SPD0873假想蛋白%表达纯化%多克隆抗体
肺炎鏈毬菌%SPD0873假想蛋白%錶達純化%多剋隆抗體
폐염련구균%SPD0873가상단백%표체순화%다극륭항체
streptococcus pneumoniae%SPD0873%expression and purification%polyclonal antibody
目的 通过构建原核表达载体,获得纯化的肺炎链球菌S.pn重组假想蛋白SPD0873,并制备多克隆抗体,进一步分析其在常见S.pn菌株中的保守性.方法 分离培养D39型肺炎链球菌,获取其基因组DNA.利用PCR方法扩增去除信号肽的spd0873序列,采用基因体外重组法将spd0873序列克隆到原核表达载体pET-32(a)内,测序鉴定.将重组质粒转化到E.coli Rossetta(DE3)中,经IPTG诱导大量表达融合6个组氨酸标签的SPD0873重组蛋白,经Ni-NTA树脂纯化后,获得的重组蛋白用SDS-PAGE和Western 印迹鉴定;将鉴定后纯化的蛋白免疫BALB/C小鼠制备多克隆抗体,并用间接ELISA检测多克隆抗体的效价,Western 印迹方法分析多克隆抗体的特异性,同时,鉴定该蛋白在5种常见肺炎链球菌分离株的保守性.结果 克隆的spd0873序列与GenBank中的数据相符,并实现了SPD0873蛋白高水平的可溶表达.纯化蛋白免疫BALB/C小鼠获得高滴度、高特异性的的多克隆抗体,Western 印迹验证SPD0873蛋白在5株常见肺炎链球菌菌株中均有表达.结论 成功制备了高滴度、高特异性的SPD0873蛋白多克隆抗体,同时,检测到SPD0873蛋白在5种常见的肺炎链球菌菌株中非常保守,为研究该蛋白的生物学功能及肺炎链球菌多肽联合疫苗的研制奠定了基础.
目的 通過構建原覈錶達載體,穫得純化的肺炎鏈毬菌S.pn重組假想蛋白SPD0873,併製備多剋隆抗體,進一步分析其在常見S.pn菌株中的保守性.方法 分離培養D39型肺炎鏈毬菌,穫取其基因組DNA.利用PCR方法擴增去除信號肽的spd0873序列,採用基因體外重組法將spd0873序列剋隆到原覈錶達載體pET-32(a)內,測序鑒定.將重組質粒轉化到E.coli Rossetta(DE3)中,經IPTG誘導大量錶達融閤6箇組氨痠標籤的SPD0873重組蛋白,經Ni-NTA樹脂純化後,穫得的重組蛋白用SDS-PAGE和Western 印跡鑒定;將鑒定後純化的蛋白免疫BALB/C小鼠製備多剋隆抗體,併用間接ELISA檢測多剋隆抗體的效價,Western 印跡方法分析多剋隆抗體的特異性,同時,鑒定該蛋白在5種常見肺炎鏈毬菌分離株的保守性.結果 剋隆的spd0873序列與GenBank中的數據相符,併實現瞭SPD0873蛋白高水平的可溶錶達.純化蛋白免疫BALB/C小鼠穫得高滴度、高特異性的的多剋隆抗體,Western 印跡驗證SPD0873蛋白在5株常見肺炎鏈毬菌菌株中均有錶達.結論 成功製備瞭高滴度、高特異性的SPD0873蛋白多剋隆抗體,同時,檢測到SPD0873蛋白在5種常見的肺炎鏈毬菌菌株中非常保守,為研究該蛋白的生物學功能及肺炎鏈毬菌多肽聯閤疫苗的研製奠定瞭基礎.
목적 통과구건원핵표체재체,획득순화적폐염련구균S.pn중조가상단백SPD0873,병제비다극륭항체,진일보분석기재상견S.pn균주중적보수성.방법 분리배양D39형폐염련구균,획취기기인조DNA.이용PCR방법확증거제신호태적spd0873서렬,채용기인체외중조법장spd0873서렬극륭도원핵표체재체pET-32(a)내,측서감정.장중조질립전화도E.coli Rossetta(DE3)중,경IPTG유도대량표체융합6개조안산표첨적SPD0873중조단백,경Ni-NTA수지순화후,획득적중조단백용SDS-PAGE화Western 인적감정;장감정후순화적단백면역BALB/C소서제비다극륭항체,병용간접ELISA검측다극륭항체적효개,Western 인적방법분석다극륭항체적특이성,동시,감정해단백재5충상견폐염련구균분리주적보수성.결과 극륭적spd0873서렬여GenBank중적수거상부,병실현료SPD0873단백고수평적가용표체.순화단백면역BALB/C소서획득고적도、고특이성적적다극륭항체,Western 인적험증SPD0873단백재5주상견폐염련구균균주중균유표체.결론 성공제비료고적도、고특이성적SPD0873단백다극륭항체,동시,검측도SPD0873단백재5충상견적폐염련구균균주중비상보수,위연구해단백적생물학공능급폐염련구균다태연합역묘적연제전정료기출.
Objective To obtain purified SPD0873 protein produced by prokaryotic expression system,prepare polyclonal antibody by immunizing BALB/C mice,and further investigate if SPD0873 protein expresses in 5 most common serotypes of S.pneumoniae.Methods Template DNA was isolated from cultured S.pneumoniae D39,from which a truncated spd0873 gene with N-terminal removal of its signal peptide was amplified by PCR. The PCR fragments were then cloned into pET-32(α)expression vector,followed by sequencing analysis. Recombinant protein SPD0873 was over-expressed and purified from the host of Rossetta(DE3),and identified by SDS-PAGE and Western blot. Purified protein was introduced to BALB/C mice to obtain polyclonal antibody. Antibody titers were evaluated by indirect ELISA. The antiserum was used to analyze the expression and conservative features of SPD0873 protein in 5 different S.pneumonia strains.Results The spd0873 gene was successfully cloned into pET-32(α),as confirmed by gene sequencing. The recombinant SPD0873 protein was successfully over-expressed in soluble manner,as shown by SDS-PAGE and Western blot. Specific antiserum obtained from BALB/C mice showed high titers and high specificity in confirming conservative expression of SPD0873 protein in 5 common strains of S.pneumoniae.Conclusion High titers and specific polyclonal antibodies against SPD0873 were obtained by immunizing BALB/C mice. Furthermore,SPD0873 protein was demonstrated to be exceptionally conserved among 5 different serotypes of S.pneumoniae,suggesting that SPD0873 is a highly-conserved protein antigen,which is beneficial for analysis of its biological function,thus making SPD0873 a promising vaccine protein candidate.