Immunoscreening Of CDNA Library From Trichinella Spiralis And Protective Immunity Induced By The Recombinant Excretory/Secretory Protein Ts-ES-1

Trichinellosis which is due to Trichinella spiralis (T. spiralis) is a food-bornezoonosis. Human infection occurs by eating raw or undercooked meat containinginfective T. spiralis larvae. Moreover, trichinellosis shows complicated clinicalsymptoms and is difficult to be diagnosed correctly. Unfortunately, there has not aneffective vaccine available until now. Therefore, the development of vaccinesagainst Trichinella infection for livestock and humans is an urgent approach tocontrol this disease.One of effective methods is to discover the antigen genes from cDNA libraryby immunoscreening. The adult cDNA library of T. spiralis had beenimmunoscreened with T. spiralis-infected swine sera in our study. After sequencedand analysised of positive clones, one positive clone encoding protein secreted by T.spiralis muscle larvae and adult worms was cloned and characterized. Theimmunogenicity and protective effect of the recombinant protein were evaluated.The T. spiralis adult cDNA library was immunoscreened with sera from swineexperimentally infected with T. spiralis and42positive clones were identified. Afterthe sequence analysis,8-3-4clone encoding a20-kDa protein was selected. It was revealed that the cDNA sequence of the clone was700bp which had an openreading frame of516bp encoding for172amino acid residues with a predictedmolecular mass of19.7kDa. Through the protein structure prediction, the first21amino acids of the protein of172amino acids is a signal peptide. Recombinantprotein should be expressed without the signal peptide (approximately17kDa). Theresults of a GenBank database search revealed that the predicted amino acidsequence had100%identity with the hypothetical protein cDNA of T. spiralis andhad79%identity with a21kDa ES of T. pseudospiralis. The results mentionedaboveconfirmed that the protein was an excretory/secretory protein from T. spiralis.Therefore, this20kDa protein was designated as Ts-ES-1(excretory-secretoryprotein-1of T. spiralis).In order to obtain the recombinant protein of the Ts-Es-1, the ORF fragment ofthe Ts-Es-1gene not including the singnal peptide sequence (453bp) was cloned intothe prokaryotic expression vector pET-28a(+). It was shown that a24kDa fusionprotein (including His-tag amino acid residues) was expressed in E. coli. A highlypurified recombinant protein pET-28a(+)/Ts-Es-1(rTs-Es-1) was obtained byHis-bind resin affinity and refolding.The rTs-Es-1emulsified with ISA50V2was used to immunize female BALB/cmice three times at intervals of2weeks, and the mouse serum samples werecollected from each immunized mouse one week after final immunization. Theantibody titers of the serum samples against rTs-ES-1were measured using ELISA.A high titer of specific IgG antibodies was elicited in all of the immunized mice oneweek after each immunization, and the highest IgG titer was induced after the thirdimmunization. In addition, the results of immunoblotting demonstrated that rTs-ES-1was recognized by sera from human patients with trichinellosis, as well as T.spiralis-infected animal sera from mice, swine and rabbits. These results indicated that the Ts-ES-1is a highly immunogenic antigen and the antigenicity is preserved inthe recombinant molecule. Immunolocalization suggested that the native Ts-ES-1was strongly and exclusively distributed in the stichocytes of T. spiralis musclelarvae stichosomes. RT-PCR results showed that Ts-ES-1mRNA was transcribed atboth the adult and muscle larval stages. The Western blot results demonstrated that atight band at approximately20kDa was detected by anti-Ts-ES-1antisera not onlyin somatic extracts but also in ES products of both T. spiralis adult worms andmuscle larvae, and indicated that the native Ts-ES-1is a secreted protein in bothmuscle larvae and adult stages.Protective immunity against T. spiralis infection induced by rTs-ES-1wasexplored in immunized BALB/c mice. BALB/c mice were divided into three groupsrandomly. The mice in the first group were each vaccinated with rTs-ES-1emulsified with ISA50V2, then boosted twice using the same method at intervals of2weeks. The control groups were inoculated with ISA50V2emulsified with PBSalone, or with PBS only. The immunization regimen in controls is the same as thefirst group. Two weeks after the final boost, mice was each challenged orally with500T. spiralis infective muscle larvae. The reduction in adult worm and musclelarvae burdens was evaluated at5days and45days after T. spiralis muscle larvaechallenge, respectively. The results suggested that mice immunized with rTs-ES-1formulated with the adjuvant ISA50V2induced a27%adult worm reduction and42.1%muscle larvae reduction, which was significantly different from controlgroups (p<0.01)..In order to explore the possible mechanism of protective immunity induced byrTs-ES-1, sera from mice were collected one week after each vaccination andmeasured for rTs-ES-1-specific IgG, IgG1, and IgG2a antibodies by using anindirect enzyme-linked immunosorbence assay (ELISA). One week after each immunization, the production of cytokines IFN-γ, IL-2, IL-4and IL-5in splenocytesfrom mice of each group were detected using an ELISPOT. The results showed that,compared to control groups, the rTs-ES-1elicited a significantly higher level of totalIgG after immunization (p<0.01), The IgG subclass antibody levels were measuredto further assess the efficacy of rTs-ES-1in induction of the Th1or Th2response invivo. The predominant IgG subclass was IgG1, but there was also a significant levelof IgG2a response. The cytokines (IFN-γ, IL-2, IL-4, and IL-5), secreted byimmunized mouse splenocytes upon stimulation of rTs-ES-1in vitro, were measuredusing ELISPOT. The levels of the typical Th1cytokines (IFN-γ, IL-2) and Th2cytokines (IL-4, IL-5) were significantly elevated in mice vaccinated with rTs-ES-1compared to the control groups.In summary, the gene (Ts-ES-1), encoding a20kDa ES of T. spiralis and itsrecombinant protein (rTs-ES-1) were obtained. And rTs-ES-1could induce partialprotective immunity in mice of27%adult worm reduction and42.1%muscle larvaereduction. According to the isotype of IgG1, IgG2a and quantitative analysis of thecytokines (IFN-γ, IL-2, IL-4and IL-6) secreted by immunized mouse splenocytes, itsuggested that rTs-ES-1triggered a Th1/Th2mixed type of immune response, withTh2predominant. Therefore, this indicated that rTs-ES-1could be considered as apotential vaccine candidate for trichinellosis. These studies have layed a foundationfor the development of vaccine against trichinellosis.