肺炎链球菌烯醇化酶与热休克蛋白DnaJ的结合及结合位点鉴定

Determination of binding sites between heat shock protein DnaJ and enolase in Streptococcus pneumoniae

目的：明确肺炎链球菌糖酵解关键酶烯醇化酶（enolase,Eno）和热休克蛋白DnaJ是否有直接结合及结合位点。方法：原核表达并纯化Eno全长蛋白,鉴定其酶活性,利用重组Eno蛋白皮下免疫昆明小鼠制备特异性抗血清,分析其胞外分泌情况;采用生物膜层干涉（biolayer interferometrvy,BLI）技术和直接结合实验鉴定Eno和DnaJ两者之间是否存在直接相互作用;在前者结果阳性的基础上,截短表达Eno蛋白,通过直接结合实验鉴定Eno蛋白结合DnaJ的位点。结果：成功表达重组Eno蛋白,纯度达90%,该蛋白具有酶活性;重组Eno蛋白皮下免疫昆明小鼠后,获得效价达1∶7.68×106的特异性抗血清;Western blot分析显示,7种不同血清型肺炎链球菌培养上清中均在分子量约47 k D处出现特异性反应带;直接结合试验显示,随着Eno蛋白浓度增加,Eno与DnaJ的结合量也相应增加（r=0.920,P=0.000）,其吸光度值从0.222±0.042（12.5μg/ml）增加到0.569±0.099（200.0μg/ml）;BLI技术测定两者结合的亲和常数为926 nmol/L;截短的Eno（aa1-aa100）与DnaJ的结合量2.25±0.38高于其他截短序列的结合量0.94±0.25、1.08±0.09、0.75±0.12,差异具有统计学意义（P=0.000,P=0.001,P=0.000）。结论：肺炎链球菌Eno蛋白与DnaJ蛋白存在直接结合作用,结合位点主要位于其N端的1～100个氨基酸序列。

Objective ：To determine whether the key glycolytic enzyme enolase（Eno） bind heat shock protein DnaJ directly and to analyze the binding sites between Dna3 and Eno in Streptococcus pneumoniae （S.pneumoniae）. Methods：Recombinant Eno protein was obtained through prokaryotic expression system, and its α-enolase activity was analyzed. Kunming mice were immunized with the recombinant protein to prepare the polyclonal antibody to Eno protein and the titer of the antibody was determined by ELISA. The expression levels of Eno protein in S.pneumoniae with different serotypes were determined by Western blot. The direct interaction between DnaJ and Eno was determined by biolayer interferometrvy（BLI） technology and direct binding assay. Truncated Eno protein was expressed and purified, and the binding sites of DnaJ on Eno were determined by direct binding assay. Results：The recombinant protein Eno with high purity（about 90%） was obtained after purification of Ni affinity chromatography and exhibited ct-enolase activity. The antibody titer of anti-Eno in the immunized mice serum reached to 7.68 ~ 106. Western blot showed the same band with Mr 47 000 presented in the supernatant of all the 7 serotypes of S.pneumoniae. The direct binding assay demonstrated that DnaJ bound Eno in a dose-dependent manner（r=0.920,P=0.000） and a concentration dependent binding of Eno to immobilized DnaJ was detected （KD-926 nmol/L） bv BLI. Binding experiments demonstrated that DnaJ bound more truncated Eno （aal-aa100） （2.25 ± 0.38） thanthe other truncated Eno proteins（0.94± 0.25）, （1.08 ± 0.09）, （0.75 ± 0.12） （P=0.000, P=0.001, P=0.000）. Conclusion ： DnaJ interacts with Eno directly in S.pneumoniae and primarily via the binding sites,which are localized within 100 aa of the N- terminal domain of Eno.