Cloning, The Recombinant Protein Expressing, Purifying And The Activity Of Malate Dehydrogenase Gene Of Veillonella Dispar

Veillonella dispar is a common kind of Gram-negative anaerobiccoccus in the mouth,is an earlier colonized bacteria in plaque.Malatedehydrogenase play a regulatory role of the acid production metabolismin Veillonella dispar,also play an important role in caries occurrence anddevelopment.This experiment is to Veillonella dispar as the researchobject,to clone and express the malate dehydrogenase(MDH) gene ofVeillonella dispar,and to purify and assay activity in MDH of therecombinant protein.The first part of this study,to extract genomic DNA of Veillonelladispar,MDH gene was amplified by Polymerase Chain Reaction(PCR),thePCR product and the pET-28a-c(+) Vector were digeste by drestrictionenzyme EcoRⅠand SalⅠ,and were inserted by T4DAN ligase,totransform E.coli DH5α.The sequence was performed after PCRidentification and restriction enzyme digestion. The PCR product wasspecific，and the size of the product was1139bp. The sequencing datawere analyzed by BLAST software and the result indicated that itcontained MDH,and it was proved to have a full reading framework,through the analysis of the gene sequences and amino acids.And thesequence has been submitted to the NCBI GenBank, and the accessionnumber was KC421072.The second part of this study，to transform theidentitied recombinant plasmid MDH-pET-28a-c(+) in E.coli BL21(DE3) for expression of recombinant,also to search the optimized conditions.Therecombinant plasmid MDH-pET-28a-c(+) was successfully expressed inE.coli BL21(DE3)，and the recombinant protein was about36kb.We setdifferent concentration gradient of inducer、the same induction time anddifferent induction time gradient、the same induction concentration，successfully figure out the best induced condition is0.5mM IPTGexpresse for6hours. And the ultrasound pyrolysis appraisement proteinexpression in the form of soluble protein. Finally，to get high-levelexpression of recombinant protein in optimal conditions, then todetermine the purification and activity of recombinant MDH, and it activevalue is0.4403U/ml.The MDH gene of Veillonella dispar was successfully cloned inthis study,and it is proved to have a full reading framework, through theanalysis of the gene sequences and amino acids.The optimum conditionfor induction of MDH protein was selected. Further in Veillonella of thedetermination of malate dehydrogenase activity，to provide conditions forfurther research of MDH，to provide new ideas of MDH for caries diseasediagnosis and treatment.