肺炎链球菌三磷酸甘油醛脱氢酶GAPDH与热休克蛋白DnaJ的相互作用

GAPDH, a glycolytic enzyme, interacts with heat shock protein DnaJ in Streptococcus pneumoniae

目的验证肺炎链球菌(Streptococcus pneumoniae,S.pn)糖酵解关键酶三磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)和热休克蛋白Dna J的相互作用。方法 PCR扩增S.pn D39 GAPDH蛋白编码基因gap全长,将其克隆至p ET-28a(+),重组质粒经PCR、双酶切及测序鉴定正确后转化大肠杆菌BL21(DE3),IPTG诱导GAPDH-6His的表达,经Ni-NTA亲和层析柱纯化后,用SDS-PAGE鉴定其纯度。重组GAPDH蛋白经腹部皮下免疫昆明小鼠获得多克隆抗体。Western blot鉴定GAPDH的保守性。采用大肠杆菌双杂交系统、直接结合实验、生物膜层干涉技术(Bio-layer Interferometry,BLI)验证GAPDH和Dna J的相互作用。结果获得了纯度达90%的GAPDH重组蛋白,并获得了效价达107的抗GAPDH多克隆抗体,Western blot检测显示在7株不同血清型S.pn的全菌裂解液和培养上清中均存在和抗GAPDH抗体反应的特异性条带。大肠杆菌双杂交系统显示GAPDH和Dna J共转化菌株能够在3-AT平板及双选择培养平板上生长,提示二者在细菌胞内有相互作用。直接结合实验和BLI显示随着加入GAPDH蛋白浓度的增加,二者的结合量也增加,提示GAPDH和Dna J的结合具有浓度依赖性。BLI实验也显示GAPDH和Dna J的亲和常数为1.12×10-7mol/L。结论糖酵解关键酶GAPDH在肺炎链球菌中保守表达,且为分泌性蛋白;该蛋白和热休克蛋白Dna J在细胞内存在相互作用,且为直接相互作用。

Objective To investigate the interaction between heat shock protein DnaJ and glyeeraldehyde-3-phosphate dehydrogenase （GAPDH）, a key enzyme of glycolysis, in Streptococcus pneumoniae （S. pn）. Methods The full-length gene of gap was amplified by PCR with specific primers from S. pn D39 and then cloned into the expression vector pET-28a （ ＋ ）. Recombinant pET-28a （ ＋ ）-gap was transformed into E. coli BL21 （ DE3 ） and induced with IPTG to express 6 His-tagged GAPDH. The affinity chromatography column was used to purify recombinant GAPDH, followed by SDS-PAGE to test its purity. The recombinant GAPDH was used to immunize kunming mice to prepare polyclonal antibodies. The antibody titer was tested by ELISA and the conservation of GAPDH was confirmed by Western blotting. The interaction between GAPDH and DnaJ was validated by BacterioMatch II two-hybrid system, direct binding assay and biolayer interferometry （BLI）. Results Recombinant GAPDH was successfully expressed with a purity of 90%, and its polyclonal antibodies （ a titer of 10^7 ） were obtained. Western blotting showed a specific band recognized by GAPDH antibody in the whole cell and culture medium from 7 serotypes of S. pn. BacterioMatch II two-hybrid system suggested that GAPDH interacted with DnaJ in vivo. Both direct binding assay and BLI demonstrated that GAPDH directly interacted with DnaJ in a concentration-dependent manner. BLI also showed that their affinity constant reach to 1.12 × 10^-7 mol/L. Conclusion GAPDH is a highly conserved secretion protein and directly interacts with DnaJ, which is important to determine the effects of DnaJ to GAPDH on its secretion and violence in S. pn.