Studies On Biologic Activity And The Change Of Expression Levels Under Different Stress Of GroEL And GroES In Riemerella Anatipestifer

GroEL and GroES, as the important members in the family of heat shock protein, plays an important role in bacterial resistance to external stimuli. Therefore, we constructed the GroEL and GroES recombinant protein from RA CH-1 strain, then we studied the activity of the recombinant proteins and the change of GroEL and GroES genes’ expression levels under different stress conditions. it is indicates that the GroEL and GroES gene have important theoretical and practical value. The results are as follows:1. Expression of RA GroEL and GroES and Co-expression of GroEL gene and GroES geneThe gene of GroEL and GroES from RA was cloned, expression in E.coli and purified. The molecular mass were 65 kDa and 15 kDa by SDS-AGE. respeetively. RA GroEL gene and GroES gene were amplified respectively, and then connected with Linker by overlapping PCR to get the fusion gene GroEL-GroES. The fusion gene cloned into pMD19-T vector in order to obtain the recombinant Plasmid pET-28a(+). The molecular mass of recombinant protein was about 70 kD by SDS-PAGE.2. Homology modelling, docking and activity of GroEL and GroES proteinWe built the three dimensional structure of GroEL and GroES protein by the method of homologous model and validated the rationality of the structure model by Procheck software. The homology model of GroEL and GroES protein were very good composite with the template. Finally, GroEL structure model docked with ATP by Autodock4.0 software. Detailed analysis shows that purine of ATP fits into the hydrophobic binding pocket, surrounded by residues ALA-2, LEU-524 and PRO-525. Additionally, another two hydrogen bonds were observed between phosphate group and residues ASP-4 and ILE-5. In summary, the above molecular docking gives us rational explanation of ATP binding to GroEL, which provided valuable information for further study. Within the scope of the 15-70 ℃, The highest activity was observed at about 55℃. The ATPase activity of GroEL depends on temperature. After that, we found GroES promotes the ATPase activity of GroEL at 30-55℃,however, when temperature was above 55℃, GroES had no obvious effect on ATPase activity of GroEL.3. The expression levels of GroEL and GroES under different stress conditionsThe change of GroEL and GroES genes’expression levels were studied with different stress conditions. Under the condition of different temperatures. GroEL and GroES genes’ expression levels increased with rising heat shock temperature. In 55℃ heat shock, GroEL and GroES genes’expression levels reached its highest, and GroEL and GroES genes’expression levels increased 3.59-fold and 3.62-fold respectively with significant difference (P< 0.05).Under the condition of H2O2 of low concentration 1.25% and 2.5%), GroEL genes’ expression level increased obviously, the expression level was respectively 2.45-fold and 2.22-fold compared with the control group (P< 0.05). The relative expression level of GroES only increased significantly under the condition of 1.25% concentration (P< 0.05), and the expression level was 2.62-fold. But under the condition of 2.5% concentration, the expression level of GroES decreased with no significant difference (P> 0.05). With the action of high concentration of H2O2 (5%), GroEL and GroES genes’expression levels were inhibited (P> 0.05).Different pH cause the change of GroEL and GroES genes’mRNA expression levels after 10 min reacting on RA. In acid conditions (pH= 3), GroEL and GroES genes’mRNA expression levels were respectively 2.105-fold and 4.683-fold (P< 0.05) compared with the control group (pH= 7). In alkaline conditions (pH= 10), the mRNA expression levels of GroEL and GroES raised 4.8% and 51.3% respectively, but there is no significant difference compared with the control group (P> 0.05). The results showed that the acid environment significantly influenced the expression levels of GroEL and GroES genes, and alkaline environment exertd no obviously effect on GroEL and GroES genes.Compared with ordinary TSB culture medium, GroEL and GroES genes are induced in TSB medium containing 3% NaCl, and their expression levels increased with significant difference (P< 0.05); GroEL and GroES genes’expression levels decreased in TSB medium containing 6% NaCl, and the expression levels of GroES decreased significantly (P<0.05).