金黄色葡萄球菌重组GapC蛋白的GAPDH活性及免疫原性分析

GAPDH Activity and Immunogenicity of Staphylococcus aureus Recombinant GapC Protein

摘要:

为研究金黄色葡萄球菌(Staphylococcus aureus)表面GapC蛋白的GAPDH活性、免疫原性及免疫保护作用, 应用PCR方法扩增出S. aureus的gapC基因, 插入到pQE-30载体相应位点, 构建重组质粒pQE/gapC。将其导入宿主菌E.coli M15(pREP4)后, IPTG诱导表达。重组蛋白纯化后进行GAPDH活性检测, 并与灭活全菌体分别免疫健康家兔。然后, 应用ELISA方法检测血清中IgG抗体水平及IFN-g、IL-4细胞因子浓度, 并用1.0×108CFU/mL S. aureus菌株Wood46对免疫家兔攻毒。SDS-PAGE结果显示, GapC蛋白在E. coli M15(pREP4)中获得表达; 经GAPDH活性检测及Western Blot检测, 重组蛋白具有较高的GAPDH活性和抗原特异性; 经ELISA检测, GapC蛋白及全菌体组兔血清中IgG抗体水平迅速升高, 并在加强免疫后第28天达到最高(1:64 000), 加强免疫后第14 d, 血清中细胞因子IFN-g和IL-4浓度与对照组相比, 显著升高(P<0.05), 而全菌体免疫组升高不明显(P>0.05); 攻毒结果为蛋白免疫组家兔获得一定的免疫保护(4/5)。以上结果表明, 表达的重组GapC蛋白具有GAPDH活性、较好的免疫原性及免疫保护力, 可作为深入研究S. aureus基因工程疫苗的良好靶向。

Abstract:

In order to characterize the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, immunogenicity and immunoprotection of the Staphylococcus aureus (S. aureus) surface protein GapC, gapC gene of S. aureus was amplified from strain BMSA/855/23-1 by PCR, and was inserted into pQE-30 vector subsequently. The recombinant plasmid, designated as pQE/gapC, was transformed into E. coli strain M15 (pREP4). The recombinant GapC fusion proein was successfully expressed in E. coli M15 induced with IPTG and its GAPDH activity was confirmed by GAPDH activity assay. Then, the recombinant GapC protein, inactivated S. aureus whole cell and placebo (PBS) were administrated to healthy rabbits respectively. The IgG antibody titers, concentration of IFN-g and IL-4 cytokines in immunized rabbit sera were measured with Enzyme-Linked Immunosorbnent Assay (ELISA). Finally, immunized rabbits were challenged with S. aureus strain Wood46 to evaluate the immunoprotection. The IgG antibody titers against GapC and whole cell in rabbit sera reached their peaks at day 28 after boost immunization (1:64 000). The concentration of IL-4 and IFN- g in GapC groups rabbit sera increased significantly (P<0.05) at day 14 after boost immunization, while the concentration of those in whole cell group did not increase (P>0.05) compared with the placebo group. 4 rabbits in 5 of the protein immunized group were protected against challenge with 1×108CFU S. aureus. The results above indicate that the expressed recombinant GapC protein have high GAPDH activity and immunogenicity, can also protect against S. aureus challenge to some extent. S. aureus GapC protein could be an attractive target for further genetic engineering vaccine.