Molecular Cloning, Expression And Characterization Of A Glutathione S-transferase TsGST Gene From Trichinella Spiralis

GST are eminent detoxification enzymes against various harmful exogenous compounds, like carcinogens, mutagens, and cellular-derived endogenous toxic compounds, like lipid peroxidation products. The GST plays a pivotal role in helminthes antioxidant system by detoxifying a wide range of exogenous and endogenous electrophilic compounds. GST has been identified in some helminthes, such as Dirofilaria immitis, Brugia pahangi, Heligmosomoides polygyrus, Fasciola hepatica and Taenia solium. However, to the best of our knowledge, there has been no report of the characterization and functional analysis of Ts GST.In the present study, the recombination r Ts GST immune serum was achieved by using an E. coli expression system to successfully express the Ts GST. SDS-PAGE and Western blot analyzed the antigenicity and immunogenicity of the recombinant protein; RT-PCR and Western blot tested transcription and translation of Ts GST gene in different worm periods; indirect immunofluorescence assay analyzed the distribution of Ts GST protein in the worm; the effect of recombinant protein on the invasion of intestinal epithelial cells by Trichinella spiralis was observed in vitro and vivo tests; furthermore, the influence of immune serum on Trichinella spiralis invading into the enterocyte and lethal effect of immune serum’s mediated macrophagocyte on Trichinella spiralis could be observed by recombinant protein immune serum vitro test; applying biology informatics analyzed the evolutionary relationships between Ts GST and other parasites or model organism GST; using biochemistry analyzed enzyme activity and the optimum response of recombinant Ts GST. In summary, this study mainly identifies the property and relevant functions in worm internal of recombination protein, which will build foundation for illuminating mutual effect and the theory of invading and pathogenic mechanism of Trichinella spiralis invading into protein and host intestinal epithelial cell. Materials and Methods1. Parasites, Experimental animals and Cell lineThe T. spiralis(T1) were propagated by serial passage in Kunming mice in our department. BALB/c mice aged 4-6 weeks free of specific pathogens were purchased from the Experimental Animal Center of Henan province. Mouse intestinal epithelial cells were Isolated and cultured by our laboratory.2. Identification of the Ts GST recombinan proteinThe recombinant protein was detected by Western blot with sera from the mice infected with Trichinella. Ts GST immune serum was collected from BALB/c mice injected subcutaneously with the recombinant protein. ES antigens and crude extract antigens from muscle larvae were detected by Western blot and ELISA with Ts GST immune serum.3. Transcription level of Ts GST geneTotal RNA were extracted from the IIL, AW, NBL or ML of T. spiralis. RT-PCR was carried out to observe the expression of Ts GST gene in different development stage of Trichinella. Housekeeping gene GAPDH of Trichinella was used as a constitutively expressed standard gene.4. ImmunolocalizationThe different development stage of T. spiralis was tested by IFA using the anti-Ts GST serum to determine whether the Ts GST was located on the surface of the parasite or inner.5. the protective immunity of the recombinant Ts GST protein60 BALB/c mice were randomly distributed into 3 groups of 20 mice each. The immunized group was inoculated subcutaneously with the recombinant Ts GST protein(20μg/mouse), followed by two boosts with the same dose at 10 days intervals. The control group only injected with adjuvant or PBS. Ten days after the last immunization,all mice were challenged orally with 300 muscle larvae. Mice were sacrificed at 7 d and 42 d after challenge infection,the intestinal adult worms and muscle larvae were respectively collected and counted. The reduction rate of AW or ML burden was calculated.6. The effect of anti-Ts GST serum on T. spiralis ML with vitro testsIEC cells were overlaid with the larvae suspended in semisolid medium mixed with anti-Ts GST serum, serum of mice infected with T. spiralis and normal mouse serum by 1:10. The development of larvae and its invasion of intestinal epithelial cells were observed by inverted microscope after incubated at 37°C in 5% CO2 and the invasion rate was calculated. Though observation, the recombination Ts GST protein immune serume mediated antibody depended on the ADCC cytotoxic effects of anti-r Ts GST serum(1:5 to 1:500 dilutions), Pre-immune serum and mouse infection sera on T. spiralis ML. The assay was performed by adding larvae to a suspension of 2×105 peritoneal exudates cells(PEC), incubated at 37 °C in 5% CO2 for 30 h. the cytotoxicity rate was calculated.7. Multiple sequence alignment and phylogenetic analysisPhylogenetic trees based on the Ts GST homologous sequences were constructed using by MEGA6.0. Phylogenies were estimated under the maximum parsimony(MP) method. The amino acid sequences data were aligned using Clustal W.8. Biochemical properties of r Ts GSTr Ts GST enzyme activity was assayed spectrophotometrically using 1m M GSH and 1 m M CDNB as substrates, based on the enzymatic product extinction coefficient(ε = 9600 M-1 cm-1) at 340 nm and at 25 °C. The optimal temperature and p H for r Ts GST activity was determined, the influence of metal ions and various other agents on the r Ts GST activity were determined. Results1. ELISA showed that the Ts GST recombinant protein and crude extract antigens can be recognized with Ts GST immune serum, And Western blot showed that a component crude extract antigens of approximately 24 k Da was recognized with the Ts GST immune serum.2. RT-PCR and Western blot suggested that Ts GST is transcribed and expressed in all developmental stages of T. spiralis.3. The immunofluorescence result showed that, the anti-Ts GST serum reacted with the endogenous Ts GST localized on the entire body of the T. spiralis AW, NBL, IIL and ML. The bright green fluorescence mainly distribute at the cuticle and stichosome.4. The challenge experiment showed that after a challenge infection with T. spiralis larvae, mice immunized with r Ts GST displayed a 35.71% reduction in adult worms and a 38.55% reduction in muscle larvae. The vaccination of mice with r Ts GST induced the Th1/Th2-mixed type of immune response with Th2 predominant(high levels of Ig G1) and partial protective immunity against T. spiralis infection.5. An in vitro invasion assay showed that when anti-r Ts GST serum and normal mouse serum were added to the medium, and the invasion rate of the infective larvae in an intestinal epithelial cell(IEC) monolayer was 31.0% and 78.96%, respectively(P<0.05). ADCC assay showed that anti-r Ts GST serum induced significant death of larvae(70% cytotoxicity) compared to the larvae incubated with pre-immune serum(12% cytotoxicity,P<0.001) and was dose-dependent.6. The phylogenetic tree based on MP method supported the monophyly of the Trichinella group(including T. spiralis, T. pseudospiralis and T. nativa) with high bootstrap value. The comparison between T. spiralis and T. pseudospiralis, and T. nativa with GST revealed 65% and 60% identity at the amino acid level, respectively.7. The purified r Ts GST showed the maximum enzymatic activity at p H 6.5 and 40 °C. The enzymatic Km values for GSH and CDNB was 457 μM and 123 μM, respectively. Conclusions1. Purified of recombinant protein Ts GST good antigenicity and immunogenicity, and Ts GST protein components of crude extract antigens of trichinella spiralis.2. Ts GST m RNA is transcribed in all developmental stages of T. spiralis. The endogenous glutathione transferase Ts GST mainly distribute at the cuticle and stichosome of the worm.3. The anti-Ts GST antibody has obvious prevention function on T. spiralis infective larvae adhere, invade, migrate within the intestinal epithelium and development in vitro and vivo.