Cloning Of Hexokinase Gene From Eimeria Tenella, Prokaryotic Expression And Eukaryotic Expression In Vitro

Energy metabolism is necessary for every organisms, With the implement whole genome sequencing research in Apicomplexan parasites has been recently performed, such as Plasmodium, Toxoplasma and Cryptosporidium, the overviews of biochemical pathways in these Apicomplexan parasites have indicated that lack or only incomplete oxidation pathway, and the Tri-carboxylic acid cycle were limited in them.Glycolysis pathway is the main way for producing energy in these protozoan parasites.Hexokinase (EC2.7.1.1) which phosphorylates hexoses (six-carbon sugars,including glucose) to form hexose phosphated is the first regulating enzyme in thet glycolytic pathway, is essential to the energy metabolism. Inhibiting hexokinase activities by small chemicals can block carbohydrate and energy metabolism of parasites, and thus interfere parasites survival.Previous experimental results showed that T.gondii will not live when the HK gene was koncked out using RNAi, which identified the important functions of HK in its survival. As we known, in Eimeria tenella no any studies on the hexokinase have been made up to date,and the characteristics of this enzyme that allow designing a rational strategy of selective inhibition are unknown.In this study, the gene on coding the hexokinase in Eimeria tenella was predicted using bioinformatics like gene Annotation method,was cloned from E.tenella sporozoites total RNA by RT-PCR. The sequencing results show that the amplified Ethk ORF in its full length is1284bp, and encode a protein contains427amino acids, which display34.4%～46.8%similarity to other protozoans parasites.Prokaryotic expression system and eukaryotic expression system were used for recombinant expression of EtHK in order to study its kinetic properties. The pMAL-c2x was selected for EtHK prokaryotic expression, and the recombinant expression plasmid pMAL-c2x-EtHK was constructed and translated into E.coli Rosseta(DE3).The induced expressing results showed that the recombinant EtHK is Partially in soluble type by this expression system under16℃. The eukaryotic expression plasmid pPIC9K-EtHK was constructed and translated into GS115, and the expression results show that recombinant EtHK can be produced by the methanol induced.