Biological Function And Immune Protection Of Three Differentially Expressed Proteins From Trichinella Spiralis

Trichinosis is a zoonotic disease widely distributed in the world,which seriously endangers the public health.The life cycle of Trichinella spiralis including newborn larva,muscle larva and adult.Each stage of the parasite locate in different tissue of the host.The interaction between the parasite and the host is very complex.The differentially expressed proteins may play important roles in invasion and infection.Proteomics data of our privious study showed that T.spiralis GLIPR1L2,TER1 and ScoAL-β were differentially expressed among newborn larva,muscle larva and adult worms.In this thesis,the effects on the function of peripheral blood mononuclear cells（PBMCs）,cytokines secretion,and the protection potentialities were studied in rats,the results may provide a basis for the prevention of this patasite.1 Cloning and expression of T.spiralis GLIPR1L2,TER1 and ScoAL-βTotal RNA of T.spiralis was used as a template for RT-PCR.Three pairs of specific primers were designed based on gene sequences of glioma pathogenic protein-like protein 2（GLIPR1L2,GenBank accession number:Tsp₀6690）,Trans-2-enoyl-CoA reductase 1（TER1,GenBank accession number:Tsp₀3549）and succinate-CoA ligase β subunit（ScoAL-β,GenBank accession number:Tsp₀3823）.After gel recovery and purification,the PCR products were connected to the expression vector pET-28a（+）or pET-32a（+）,and the recombinants were identified by enzyme digestion and sequencing.The results showed that,the obtained sequence of GLIPR1L2（591 bp）was found to encode a protein of 196 amino acids（21.979 kDa）.The obtained sequence of TER1（1083 bp）was found to encode a protein of 360 amino acids（39.858 kDa）.The obtained sequence of ScoAL-β（1308 bp）was found to encode a protein of 435 amino acids（47.189 kDa）.The recombinant plasmids was transformed into E.oli BL21 and induced by IPTG.The expression rGLIPR1L2,rTER1 and rScoAL-β was analyzed by SDS-PAGE.Results showed that these genes were successfully expressed mainly in the inclusion body as a fusion protein of 25 kDa,57 kDa and 65 kDa.Western blot results showed that these recombinant proteins were successfully recognized by the sera of mice experimentally infected with T.spiralis,and the native protein（GLIPR1L2,TER1 and ScoAL-β）from T.spiralis could be detected by sera from rats immunized with the recombinant rGLIPR1L2,rTERl and rScoAL-β.2 Effects of rGLIPR1L2,rTERl and rScoAL-β on the functions rat PBMCsRat PBMCs were isolated and incubated with the recombinant protein rGLIPR1L2/rTERl/rScoAL-β,the bindings were confirmed by immunofluorescence assay（IFA）.Three recombinant proteins of different concentrations（10,20 and 40 μg/mL）were incubated with rat PBMCs,respectively.After 24 h,the migration chamber was used to study the effects of cell migration,total nitric oxide kit was used to detect the release of NO,and apoptosis was detected by apoptosis kit;after 48 h,the ability of fitc-dextran phagocytosis was detected by flow cytometry;after 72 h,cck-8 kit was used to detect cell proliferation.The results showed that all the three recombinant proteins could bind to PBMCs in vitro.Two recombinant proteins,rGLIPR1L2 and rTER1,can promote the proliferation and migration of rat PBMCs,enhance the secretion of NO and phagocytosis of the cells,and increase the ratio of apoptosis at a certain concentration.While the recombinant protein rScoAL-β inhibited the proliferation,migration and phagocytosis of rat PBMCs,but had no significant effect on NO secretion and apoptosis.3 Effects of recombinant proteins on the cytokines expression in vivoTo investigate the effects of recombinant proteins rGLIR1L2,rTERl and rScoAL-β on cytokines secretion in rats,sixty rats were divided into six groups,with n=10 in each group.Groups 1 and 2 were intraperitoneally injected with 300 μg of pET-28a and pET-32a empty vector protein.group 3 with equal volume of PBS,and groups 4,5 and 6 with 300 μg of recombinant protein rGLIRlL2,rTERl and rScoAL-β.Serum from each rat was collected before injection（Day 0）,1,2,3,4,5 and 6 days after injection.The dynamics of IL-4,IL-9,IL-17,TGF-β,and IFN-y were tested by ELISA kit.The results showed that the secretion of IL-4 in the rGLIRlL2 group was significantly higher than that in control group;the secretion of IFN-y in the rTERl group was significantly higher than that in control group,and the secretion level of IL-17 was significantly lower than that in the control group;the secretion level of IL-17 in the rScoAL-β group was significantly lower than that in control group.4 Immune protective effects of recombinant protein rGLIPR1L2,rTERl and rScoAL-βTo investigate the immune protective effects of recombinant protein rGLIPR1L2,rTER1 and rScoAL-β,one hundred and twenty mice were divided into six groups,with n=20 in each group.Groups 1 and 2 were empty vector protein immune groups,group 3 was PBS blank immune control groups,and groups 4,5 and 6 were rGLIR1L2,rTER1 and rScoAL-β immune groups.The mice were immuned with emulsified 25 μg empty vector protein,the same volume of PBS and 25 μg of recombinant proteins for two times with two weeks interval.One week post the second vaccination,the mice were challenged with 200 muscle larvaes by gavage injection.Specific serum antibodies including IgG,IgG1 and IgG2a were detected at day 0（before immunization）,day 7（7 days after first immunization）,day 21（7 days post second immunization）,day 28（7 days post infection）,day 35,42,49,and 56.The adult worms were collected and counted at the 7th day after challenge,and the muscle larvae were collected and counted at 35th day post infection.The results showed that compared with the control groups,the recombinant proteins rGLIPR1L2,rTER1 and rScoAL-β could cause a significant increase in specific IgG antibodies,and continued to a high level after the infection.The changes of IgG1 and IgG2a in the three recombinant protein groups were similar to that of IgG,except that the IgG1 subtype increased faster and at a higher level than IgG2a after immunization.The number of adult worms in the rTERl group was significantly lower than that in control groups,while the number of adults in the rGLIPR1L2 and rScoAL-β groups was not significantly different from the control groups.The number of muscle larvae in the three recombinant protein immunized groups was significantly lower than that in control groups,which indicated that GLIPR1L2、TER1 and ScoAL-β have the potential values for vaccines.