肺炎链球菌SpxB蛋白的细胞表达定位、保守性分析及其生物学功能初探  

Protein expression localization,conservation analysis and biological functions of SpxB in Streptococcus pneumoniae

作　　者：王哲[1] 王建敏[1] 马峰[1] 张帅[1] 黄远帅[1] 张雪梅[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《第三军医大学学报》2014年第14期1486-1491,共6页Journal of Third Military Medical University

基　　金：重庆市自然科学基金(CSTC 2001BB5137)~~

摘　　要：目的鉴定肺炎链球菌中spxB基因编码蛋白的丙酮酸氧化酶活性、细胞表达定位和保守性表达,并初步研究该基因对细菌毒力影响的部分机制。方法利用PCR方法扩增肺炎链球菌D39菌株的spxB基因全长序列,并将其整合至表达载体pET-28a(+),经测序鉴定后,将重组质粒转化至大肠埃希菌BL21(DE3),以IPTG诱导表达含有His标签的rSpxB蛋白,经Ni-NTA亲和层析柱纯化后,使用SDS-PAGE鉴定蛋白纯度,采用一种商品丙酮酸氧化酶活性质控方法鉴定其酶活性。以rSpxB蛋白免疫昆明小鼠获得其多克隆抗体。采用ELISA检测多克隆抗体效价,采用Western blot鉴定抗体的特异性和蛋白的保守性表达。采用流式细胞术鉴定spxB的细胞表达定位。构建spxB缺陷菌,通过与流感嗜血杆菌进行共同培养初步探索其毒力机制。结果实现了具有丙酮酸氧化酶活性SpxB蛋白的可溶性表达,该蛋白免疫小鼠后获得高效价特异的抗SpxB蛋白抗血清,Western blot鉴定显示SpxB蛋白在1、2、4、6B、14、19F和23F等血清型肺炎链球菌均有表达,流式细胞术检测显示SpxB蛋白的荧光信号较阴性对照有右移,但较阳性对照的荧光信号弱很多。野生菌株D39产生的过氧化氢显著高于spxB缺陷菌,D39菌株对流感嗜血杆菌的生长抑制作用显著强于spxB缺陷菌。结论肺炎链球菌spxB基因编码的蛋白具有丙酮酸氧化酶活性,在肺炎链球菌中有保守表达,且主要表达于细胞内。该蛋白有助于肺炎链球菌与流感嗜血杆菌共同生长时的优势生长。Objective To investigate the enzyme activity and protein expression localization of pyruvate oxidase encoded by spxB gene from Streptococcus pneumoniae（S.pn）,its conservation in different serotypes of S.pn,and its virulence mechanisms.Methods The full-length spxB gene of S.pn D39 was amplified by PCR with specific primers,and then inserted into pET-28a（＋）.Recombinant plasmid pET-28a（＋）-spxB was transferred to E.coli BL21（DE3） and then the positive clones were induced with IPTG to express His-tagged rSpxB recombinant protein.The recombinant protein was purified by affinity chromatography column,and the protein purity was checked by SDS-PAGE.The enzyme activity assay was performed in a commercial pyruvate oxidase quality control method.The KM mice were immunized with the purified rSpxB protein to obtain polyclonal antibody.The antibody titers were determined by ELISA,and the antibody specificity and protein conservation were determined by Western blotting.The antibody was used for protein expression localization of SpxB in S.pn by flow cytometry.The virulence mechanism was studied preliminarily through the construction of spxB-deficient mutant strain and co-culture with Haemophilus influenzae.Results Soluble expression of SpxB with pyruvate oxidase activity was realized.Antiserum of high titer against SpxB protein was obtained from KM mice.Western blotting showed high antiserum specificity to SpxB and the conservation of spxB in the 1,2,4,6B,14,19F,and 23F serotypes of S.pn.Flow cytometry results showed that the fluorescence signal of SpxB shifted to the right compared to the negative control,but much weaker than that of the positive control.The wild D39 strain which produced hydrogen peroxide inhibited the growth of Haemophilus influenzae,while the SpxB-deficient strains showed much weaker activity.Conclusion Pyruvate oxidase encoded by SpxB is highly conserved in 7 different serotypes of S.pn and has mostly intracellular expression.SpxB helps S.pn hold advantages to Haemophilus influenza in co

关 键 词：肺炎链球菌 丙酮酸氧化酶 细胞表达定位 保守性 

分 类 号：R372[医药卫生—病原生物学] R378.12[医药卫生—基础医学]