Cloning,Expression And Functional Analysis Of Glutamate Dehydrogenase Gene In Eimeria Tenella

Glutamate dehydrogenase?GDH?is present in all organisms and catalyzes the oxidative deamination and reductive amination of L-glutamic acid and?-ketoglutaric acid,which plays an important role in intracellular signal transduction,the Krebs cycle,ammonia metabolism and energy metabolism.Presently,the full length of the GDH gene have been obtained in a variety of bacteria,animals,plants,apicomplexan protozoa and other organisms.It is found that there is a strong conservative domain between them through the alignment of amino acid sequences.This enzyme can participate in the special energy metabolism pathway of Plasmodium.Based on its important biological functions,this study conducted the cloning,expression and enzyme kinetics analysis of the EtGDH gene from Eimeria tenella.In this study,specific primers were designed based on the gene sequence of Eimeria tenella GDH?EtGDH?gene,which was annotated and predicted in the genome database.The sequence of EtGDH gene was amplified by RT-PCR using the second-generation merozoites of E.tenella total RNA as template,and the amino acid sequence of the gene was analyzed by online software.The results showed that the ORF sequence of the EtGDH gene was 1407 bp and encoded 468 amino acids,the molecular weight of the protein was 50.96 kD,the theoretical isoelectric point?PI?was 6.93,without signal peptide and transmembrane domain.Sequence alignment of EtGDH with GDH amino acids of different species showed that the encoded amino acid sequences of these genes were highly conserved.The predicted tertiary stucture of EtGDH peptide was consisted of 15 alpha helixes and 12 beta sheets.In this experiment,pET-32a was selected as the prokaryotic expression vector to connect the positive cloned plasmid,and then the recombinant plasmid pET-32a-EtGDH was transformed into the E.coli Rosetta?DE3?strain.The expression conditions of IPTG induction and induction temperature were optimized.Finally,the expression of pET-32a-EtGDH was under the induction of 1 mM IPTG overnight at 16?.The presence of recombinant protein was detected by SDS-PAGE.The results showed that the expressed protein pET-32a-EtGDH was soluble protein.The soluble protein was purified by Ni column affinity chromatography,and the pure fusion protein was identified as target protein by Western-blot.The enzyme activity of the purified pET-32a-EtGDH recombinant protein was measured,and the effects of pH and temperature on the activity of the enzyme were investigated,and the enzyme kinetics and inhibition kinetics were analyzed.The results showed that the best reaction condition was confirmed as 42? and pH 9.0.The Km of EtGDH to NADP⁺was 42.7±3.17?M;and the Km of EtGDH to L-Glu was 3.52±0.32mM,and the maximum reaction rate of EtGDH was 4.089 nmol/min/mg,which is different from other species.Hexachlorophenol,Bithionol,GW5074,Chloroquine,Apigenin,Vitamin K3 and Tannic acid had a certain inhibitory effect on EtGDH,and their IC₅₀ value was 26.39±0.58?M?158.17±0.71?M?57.78±2.67?M?89.31±3.37?M?65.94±2.45?M?406.19±1.95?M and 12.11±1.69?M,respectively.In this study,the EtGDH gene was cloned successfully for the first time,and its recombinant expression vector was constructed and its expression was successfully induced.The activity of EtGDH in vitro and biochemical characteristics was studied and a new target screening model of EtGDH was established,and some compounds with inhibitory activity were screened,which laid a theoretical foundation for the development of new anti-coccidian drugs and the screening of potential target.