Expression And Purification Of Dihydrolipoamide Dehydrogenase And D-Tyr-tRNA^Tyr Deacylase Of Vibrio Alginolyticus And Cross-Protective Research Of Vibrios

In this research, dldh and dtd were cloned and analyzed in bioinformatics from Vibrioalginolyticus strain HY9901, by PCR technique based on the primers designed from theknown dldh and dtd DNA sequences of other Vibrios published in GenBank. Resultsshowed that the full-length of dldh was1428bp and encoded a putative protein of475amino acids. The predicted molecular weight (MW) of DLDH was50.99kDa and anestimated isoelectric point of5.51. BLAST analysis revealed that the DLDH inV.alginolyticus shared a high homology of99%with V. parahaemolyticus and with97%Vibrio harveyi. And the ORF sequence of dtd consisted of425nucleotides encoding DTDof144amino acids with a calculated molecular mass of16.09kDa and an estimatedisoelectric point of4.98, Blast analysis revealed that the DTD in V.alginolyticus shared ahigh homology of95%with V. parahaemolyticus and with96%V.harveyi.To generate proteins, the above two genes were subcloned into pET-32a(+) toconstruct expression plasmids(pET-DLDH, pET-DTD), and then transformed into E.coliBL21cells. Optimization analysis of protein expression conditions showed thatthe DLDH was highest at the temperature of37℃，concentration of IPTG was0.7mmol/Land4hours,and the protein mainly exists in the form of inclusion body,however thecondition of DTD was37℃，concentration of IPTG was0.1mmol/L and5hours.Westernblot analysis revealed that the proteins were correct expression.Use the techniques of splicing by overlapping extension (SOEing) and three-timesPCR,dldh and dtd were fused together into pET-32a(+) to construct expressionplasmids(pET-DLDH-DTD), and then transformed into E.coli BL21cells. Optimizationanalysis of protein expression conditions showed that the DLDH-DTD was highest at thetemperature of37℃，concentration of IPTG was0.4mmol/L and4hours,and the proteinmainly exists in the form of inclusion body. The recombinant DLDH-DTD fusion proteinswere purified by His TrapTMHP column, and then identified by western-blot.Use the three proteins to immunize grouper, then use the Elisa to test antibody titer ofthe immunized grouper. And the results showed that all the vaccinated group weresignificantly higher than that in the negative control (p<0.01), and reached to the highestlevel in the forth week after vaccinated.In the V.alginolyticus-challenged experiment, the highest RPS was occurred in DLDH-DTD(95%), and higher RPS in DLDH(90%), whilethe lowest RPS in DTD(60%), when challenged with V.harveyi the RPS was90%,85%,55%respectively, however the V. parahaemolyticus was90%,85%,50%respectively.Then we use the insertional mutagenesis to contrast the dldh mutant, and investigatedthe changes of this mutant on physiology.The mutant showed a slower growth level andattenuated swarming ability, significant differences of biofilm formation，we can use thisfor vibrios attenuated vaccine.