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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 identify the pyruvate oxidase activity, expression orientation and conserved expression of spxB gene in Streptococcus pneumoniae and to study some of the mechanisms underlying the effect of this gene on bacterial virulence. Methods The spxB gene of Streptococcus pneumoniae D39 was amplified by PCR and integrated into the expression vector pET-28a (+). After sequencing, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) , And the protein containing His-tagged rSpxB was induced by IPTG. Purification by Ni-NTA affinity chromatography was used to identify the protein purity by SDS-PAGE. The activity of pyruvate oxidase was identified by enzyme-linked immunosorbent assay. The rSpxB protein was used to immunize Kunming mice to obtain polyclonal antibodies. The titer of polyclonal antibody was detected by ELISA. The specificity of the antibody and the conservative expression of the protein were identified by Western blot. Flow Cytometry was used to identify the cell location of spxB. To construct the spxB-deficient bacterium and explore its virulence mechanism through co-culture with Haemophilus influenzae. Results The soluble expression of SpxB protein with pyruvate oxidase activity was achieved. The anti-SpxB protein antiserum with high titer was obtained after immunization of mice. Western blot showed that SpxB protein was expressed at 1, 2, 4, 6B, 14, 19F And 23F serotypes of Streptococcus pneumoniae were expressed, flow cytometry showed that SpxB protein fluorescence signal than the negative control has a right shift, but much lower than the fluorescence signal of the positive control. The hydrogen peroxide produced by the wild strain D39 was significantly higher than that of the spxB-deficient bacteria, and the growth inhibition effect of the D39 strain on Haemophilus influenzae was significantly stronger than that of the spxB-deficient bacteria. Conclusion The protein encoded by S. pneumoniae spxB gene has pyruvate oxidase activity, which is conserved in Streptococcus pneumoniae and mainly expressed in cells. This protein helps the predominant growth of S. pneumoniae when co-growing with Haemophilus influenzae.