Gene Cloning, Prokaryotic Expression And Characterization Of Enolase From Taenia Solium

The larval stage of the Taeniidae Taenia solium mainly infects muscle tissues, which can causecysticercosis in pigs and humans, thereby affecting the production of pigs and the health of human beings.Due to the influence of parasitic environment, T.solium is mainly based on glycolysis to maintain substanceand energy metabolism. Enolase (ENO, enzyme classification number, EC4.2.1.11) is a multifunctionalprotein and ubiquitously present in a wide range of living organisms. It not only do affect substance andenergy metabolism of cells as a glycolytic enzyme, but also has a variety of functions in pathogen invasion,as an immune stimulating protein and heat shock protein. Therefore, this reaserch studied the cloning,expression and characterization of enolase gene. We have achieved the following results.1. Making use of the cDNA of T.solium as a template, we first amplified enolase gene. Real-time PCRindicated that enolase at a high expression level in adult worms. We also used bioinformatics softwares toanalysis the gene sequence. The sequence analysis showed that the size of ORF was1302bp and encoded433amino acids. The theoretical molecular mass of encoded protein was46.56ku. We also analyzed theputative plasminogen binding motif （238GKVKIGMDVAASEFYQDGKYNLDF261） of T.soliumenolase.2. We constructed the recombinant expression plasmid pET-30a（+）-ENO successfully, and the size ofrecombinant protein rTsENO was about53ku, which were highly expressed in a soluble form. Westernblotting analysis showed that rTsENO was recognized by the serum of swine infected with porcinecysticercosis. Ligand-blotting analysis indicated that rTsENO could bind to plasminogen well. It was alsoobserved that rTsENO had the capacity to generate plasmin and to enhance the plasmin generation by thetissue-type plasminogen activator. In addition,6-ACA could inhibit the binding of rTsENO to plasminogen.3. We immunized rabbits with the rTsENO. After4times immunization, a high titer of specific IgGantibodies was produced in rabbits. Immunohistochemical results showed that rTsENO mainly expressed inthe adult worm tegument and reproductive organs.4. Kinetic measurement the activity of rTsENO was carried out at different conditions. The resultsshowed that2-PGA and PEP as substrates gave a specific activity of nearly60.72U（mg protein）-1and22.64U（mg protein）-1, respectively. pH-dependent activity measurements gave a maximum at pH7to7.5irrespective of the direction of catalysis. Only in the presence of metal ions, rTsENO had the enzymeactivity. In addition, it had the highest enzyme activity at the presence of Mg2+. The activity of this rTsENOwas inhibited by NaCl, MgCl2and CaCl2in the detection range of ions concentration. When2-PGA wasused as a substrate, the KCl from50to200mmol/L showed activating effects. Whereas, when PEP wasused as a substrate in the detection range of ion concentration, the KCl showed effects of inhibition.In conclusion, the recombinant expression T.solium enolase（rTsENO）can be recognized by the serumof swine infected with porcine cysticercosis, has a good plasminogen binding property and enzyme activity.Therefore, these lay a theoretical basis for further elucidating its roles in the metabolism, invasion andpathogenesis of T.solium.