Gene Cloning, Prokaryotic Expression And Functional Research Of Enolase From Three Entomopathogenic Nematodes

Entomopathogenic nematodes (EPNs) is a kind of important factor in pest bio-control. Nowadays, it has been used widely in agricultural production . Enolase is a key enzyme in glycolytic pathway. However, recent studies showed that enolase was a multifunctional protein, and participated in the invasion of some pathogens. Gene cloning, expression and functional research of enolase from three kinds of EPNs, included Steinernema feltiae, Steinernema carpocapsae and Heterorhabditis indica, were carried out.1 Using RT-PCR and RACR technique, the full-length enolase cDNA from three EPNs were cloned. Enolase gene from S. feltiae was 1453bp in length, included an open reading frame (ORF) of 1311bp, encoding 436 amino acid residues; The isoelectric point and molecular weight of the protein were predicted as 5.67 and 47309.82, respectively; GenBank accession no. HM067751. Enolase gene from S. carpocapsae was 1405 bp in length, included an open reading frame (ORF) of 1311bp, encoding 436 amino acid residues; The isoelectric point and molecular weight of the protein were predicted as 5.59 and 47154.61, respectively; GenBank accession no. HM067750. Enolase gene from S. feltiae was 1615 bp in length, included an open reading frame (ORF) of 1305 bp, encoding 436 amino acid residues; The isoelectric point and molecular weight of the protein were predicted as 5.43 and 47268.63, respectively; GenBank accession no. HM067750.Multiple sequence alignment indicated that enolase from three EPNs shared more than 75% identity with those of other six nematode species at the amino acid level, showing high homology among them. Phylogenetic tree indicated that H. indica has the closest relationship with C. briggsae, S.feltiae and S. carpocapsae have the closest relationship each other.2 Three enolase genes were cloned into pGEX-6P-1 to construct GST-Tag fusion expression vectors, and were expressed successfully in Escherichia coli, respectively. And then recombinant protein was purified, and a lot of purified protein was harvested. 3 The measurement of biological functions were carried out. Western blot indicated enolase was expressed on the surface of S.feltiae. Enzymatic activity assay of enolase showed that three enolase had bio-activity to catalyze the reversible reaction between 2-PGA and PEP. Immunosuppressive test indicated that three enolase can suppress phagolysis and encapsulation of Galleria mellonella hemocyte. The result of far western blot showed that three enolases can combine with plasminogen from human.