中文摘要：

目的表达B族链球菌C5a肽酶蛋白功能活性区域,为进一步研究及开发疫苗奠定基础.方法设计引物,从GBSⅡ型标准株的基因组DNA中扩增出含有C5a肽酶蛋白不同功能区域基因的4个目的片段,分别插入T载体,再经酶切后,插入原核表达载体PET32a,进行融合表达.对表达蛋白进行免疫原性检测,并通过镍柱大量纯化.结果成功构建4个目的片段的PET表达质粒.经SDS-PAGE检测表达的融合蛋白相对分子质量分别为80 000、78 000、70 000和36 000.蛋白质谱分析证实,表达的4个蛋白为B族链球菌C5a肽酶蛋白的可能性分数分别为82、152、113和79。免疫印迹证实均能与特异性抗C5a肽酶蛋白的抗体反应,纯化后的蛋白SDS-PAGE纯度达90%.结论已成功地表达了可溶性C5a肽酶蛋白4个功能活性区域,为蛋白免疫表位和毒力机制的研究以及亚单位蛋白疫苗的制备奠定了基础.

英文摘要：

Objective To express the functional domain of C5a peptidase of streptococcus group B and lay a foundation of further development of vaccine. Methods Amplify four gene fragments encoding the different functional domains of C5a peptidase of streptococcus group B from the genomic DNA of standard GBS Ⅱ strain by PCR using the designed primers and insert into T vector respectively. Digest the recombinant plasmid with restriction endonuclease and insert tne obtained target gene fragments into prokaryotic expression vector pET32 for fusion expression. The expressed products were determined for immunogenicities and purified by Ni^2· -NTA column chromatography. Results Four recombinant plasmids for expression of C5a peptidase of streptococcus group B was successfully constructed. SDS-PAGE showed that the relative molecular weights of four kinds of expressed products were 80 000, 78 000, 70 000and 36 000, and their probability based mowse scores of CSa peptidase were 82, 152, 113 and 79, respectively. Western blot showedthe specific reactions of all the four kinds of expressed products with antibody against C5a peptidase. After purification, the expressedproduct reached a purity of 90%. Conclusion Four functional domains of C5 a peptidase of streptococcus group B were successfully expressed. It laid a foundation of study on immune epitopes and virulence mechanism of C5a peptidase and development of streptococcusgroup B subunit vaccine.