S.suis 2溶血素SLY的分子克隆及溶血活性和免疫学特性研究

Molecular Cloning,Hemolytic activity,and Immunological characterization of Suilysin from S.suis 2

目的克隆并表达猪链球菌2型(S.suis 2)溶血素重组蛋白SLY,对其溶血活性及免疫学特性进行研究,为探讨SLY在S.suis 2感染中的致病机理及筛选有效的疫苗预防和控制猪链球菌病奠定基础。方法对S.suis2 05ZYH33全基因序列进行生物信息学分析,构建pET32a-sly原核表达质粒并诱导表达出S.suis 2重组溶血素SLY蛋白;采用高浓度尿素裂解包涵体和His-Tag亲和层析对重组SLY蛋白进行纯化并复性;免疫保护实验检验SLY的免疫保护作用。结果 SDS-PAGE显示70kD的SLY蛋白条带;重组SLY蛋白具有溶血活性并受多种因素的影响;SLY对感染S.suis 2的Balb/c小鼠有一定的免疫保护作用。结论优化原核表达体系可以有效提高SLY蛋白的表达量,经提纯并复性的重组SLY具有较高的溶血价,为进一步纯化及分析SLY蛋白的特性提供了必要的前提。

Objective To understand Streptococcus suis serotype 2（S.suis 2）suilysin（SLY）display which function in the severe infection of S.suis 2and identify vaccine candidates,molecular cloned and analyzed hemolytic activity and immunological characterization of SLY.Methods Bioinformatics was adopted to analyze the whole genome sequence of the Chinese strain 05ZYH33 of S.suis 2.A recombinant plasmid pET32a-slycarrying sly gene was developed and transformed into prokaryotic expression host BL21（DE3）strain.Using chelating chromatography to purify SLY.Immunity test was used to test the immune protective effect of SLY.Results Protein expression analysis showed that a 70 kD protein can be observed in SDS-PAGE.After being purified by chelating chromatography,SLY showed clear hemolytic activity and affected by many factors.In an animal model system,we demonstrated that the mice immunized with SLY become to be protected against lethal doses of bacteria.Conclusion Successfully constructed prokaryotic expression system,SLY showed clear hemolytic activity which would be useful for future research on function of SLY.Animal model system means the successful application of proteomics may as a technique for identifying vaccine candidates.