Gene Cloning, Expression And Protective Immunity Of Aldolase, Enolase And Hexokinase From Trichinella Spiralis

Trichinosis is a globally spreaded zoonosis caused by Trichinella spiralis. It was one of the class B diseases listed by OIE ?The World Health Organization of Animals?. Energy metabolism is the basic biology process for all creatures. For most parasites, glycolysis is the main path to generate energy. Studies showed that some enzymes involved in the glycolytic pathway for parasites may play important role in immune regulation and immune evasion. These enzymes were considered as potential vaccine candidate molecules. Adolase, enolase and hexokinase are three key glycolytic enzymes, not only play important role in the energy metabolism of the parasite but also do some favor in parasite invasion and immune evasion. To study the protective immunity of the glycolytic enzymes from Trichinella spiralis, following research work were carried out.1. Gene cloning and characterization of aldolase?Tsald?, enolase ?Tseno? and hexokinase 2 ?Tshk2? of Trichinella spiralisThree pairs of primers were designed based on the sequences of Tsald ?GenBank accession No. XM₀03374234?, Tseno ?AF363629.1? and Tshk2 ?XM₀03371674.1?, and used to amplify Tsald, Tseno and Tshk2 genes with RT-PCR from totle RNA of Trichinella spiralis muscle larva. Then the gene fragments were cloned into pMD18-T vector and identified by enzyme digestion. Results showed that two segments sized as 2700bp and 1092bp,2700bp and 507bp,2700bp and 1314bp, were obtained respectively.99%,99% and 95% homologies were found when the amino acid sequences of Tsald, Tseno and Tshk2 were compared with these in GenBank, respectively.2. Prokaryotic expreesion of Tsald, Tseno and Tshk2Three recombinant plasmids of pET32a-Tsald, pET32a-Tseno and pET32a-Tshk2 were constructed and expressed in Escherichia coli BL21. SDS-PAGE indicated that the recombinant proteins were expressed with the molecular weight of 57 kDa,68kDa and 35kDa, respectively. The recombinant protein of Tsald existed in supernatant and inclusion bodies, whereas the Tseno and Tshk2 mainly existed in inclusion bodies. Results of Western blot showed that the recombinant protein of Tsald and Tseno could be recognized by the serum from rat infected with T. spiralis, while Tshk2 could not. In the biochemical activity assay, it was found that the purified recombinant protein of Tsald exhibited enzymatic activity, and the optimum reaction temperature and pH value for the recombinant enzyme was 35 ? and 7.0, respectively.3. Constructions of the eukaryotic recombinant plasmids of pVAX1-Tsald, pVAX1-Tseno and pVAX1-Tshk2Three eukaryotic recombinant plasmids pVAX1-Tsald, pVAX1-Tseno and pVAX1-Tshk2 were constructed and indentified by enzyme digestion, repectively. To learn the immune responses induced by these recombinant plasmids, pVAX1-Tsald, pVAX1-Tseno and pVAXl-Tshk2 were injected into ICR mice intracularly. Western blot showed that the recombinant protein of Tsald, Tseno and Tshk2 could be recognized by the serum from mice injected with pVAX1-Tsald?pVAX1-Tseno and pVAX1-Tshk2, respectively.4. Protective immunites of Tsald, Tseno and Tshk2400 ICR mice were divided into ten groups, with 40 in each group.3 groups were treated with recombinant proteins?Tsald, Tseno and Tshk2?,3 groups were treated with recombinant plasmids ?pVAX1-Tsald, pVAX1-Tseno and pVAX1-Tshk2?, one groups recived the mixture of recombinant proteins ?Tsald+Tseno+Tshk2?, one groups recived the mixture of recombinant plasmids ?pVAX1-Tsald+pVAX1-Tseno+pVAX1-Tshk2?, and two control groups ?pVAXl and PBS?. The animals were immunized intramuscularly 2 times at two-week interval. Two weeks after the last immunization, each mouse was challenged orally with 200 larvae of T. spiralis. Thirty-five days post infection,10 mice from each group were sacrificed and larvae load in muscle tissue was evaluated to measure LPG ?larvae per gram? and reduction ratio of muscle larvae. Sera collected at 0,14,28,45, 64 days after the immunization were assayed for the presence of specific antibodies and cytokines. Subsets of T-lymphocytes ?CD4+ and CD8+? from spleen cells were analyzed by flow cytometric method. Results showed that mice vaccinated with recombinant protein and plasmids induced specific antibodies of IgG, IgA, IgM, but no IgE. Subclasses of IgG antibodies showed that groups immunized with recombinant protein induced a Th1/Th2 immune response, whereas groups immunized with recombinant plasmids showed a dominant Th2 immune response. Concentrations of serum cytokines ?IFN-?, IL-4, TGF?1 and IL17? were detected and showed significant levels of Thl cytokine ?IFN-??, low levels of IL-4 and TGF?1. IL17 level of each experimental group was not significantly increased.Flow cytometric analysis showed significant increase in both CD4+, CD8+T lymphocytes percentages in the groups immunized with recombinant protein ?Tsald, Tseno, Tshk2 and Tsald+Tseno+Tshk2?. Groups immunized with recombinant plasmids ?pVAX1-Tsald, pVAX1-Tseno, pVAX1-Tshk2 and pVAX1-Tsald+pVAX1-Tseno+pVAX1-Tshk2? showed significant increase of CD4+ratio, while a significant decrease of CD8+ subset was noticed after second vaccination. Challenge infection demonstrated that immunized groups had a reduced number of LPG The LPG level of groups immunized with Tshk2 recombinant protein and pVAX1-Tshk2 were not significant compared with control groups ?P>0.05?, while the other immunized groups were significant ?P<0.05?. There were no significant differences among the immunized groups ?except for groups immunized with Tshk2 recombinant protein and pVAX1-Tshk2? on LPG. Groups vaccinated with the mixture of recombinant protein and mixture of recombinant plasmids gave better reduction ?24.8% and 23%? in LPG.