| Trichinella infection can specifically induce the host to produce Th2 cytokines. As a Th2 cytokine, Interleukin-4(IL-4) plays an important role in inflammatory diseases and intestinal worm infection. The lactic acid bacteria belongs to the probiotic, it can regulate the internal environment of gastrointestinal, improve the immunity. It can also be an expression vector to present the target protein. We have proved that it could against the parasitic infections and parasitism by Trichinella spiralis after oral immunization in mice BALB/c with right routes and programs. To verify the effect that against to parasitics of Trichinella spiralis between IL-4 and lactobacillus, and explore new ideas for the coming out of the new anti-Trichinella drugs, we performed the following experiments:We digested the p MD18-T-m IL-4 plasmid in E. coli which synthesized by biotechnology company to get the m IL-4 fragments. We connected the m IL-4 fragment to p SIP409-pgs A, and we extracted the plasmids which come from the bacteria, digested the plasmids and recycled the production, then transformed it into the lactobacillus competent state. We coated the broth on the MRS solid medium plates(20mg/m L Em), and placed it in the incubator, cultured it at 30?, until there were single colonys which have a good form(at least 24h). We extracted the plasmid which came from the positive clones that were screened and did the restriction enzyme digestion to them. We found a band at 435 bp that proved that the m IL-4 were transformated in lactobacillus successfully. We found a band at 58 k Da by SDS-PAGE and Western-blotting, these proved that the recombinant strain had reactionogenicity.The female mice BALB/c were randomly divided into three groups, each group had 20 mice. The first group was Control, which inoculatd with PBS; the second group was lactobacillus group, which inoculated with the lactobacillus plantarum; recombinant group was the third one, which inoculated with the recombinant murine interleukin-4 lactobacillus?The recombinant lactobacillus was built by myself. The first immunization was noted as-21d; at days-21,-20,-19, three times in a row as the primary immune. After that, did the bosster immunization at days-14,-13,-7 and-6.Each mouse was inoculated 500 spiralis via oral-gastric at 0d to establish the Trichinella infection model. At days-1, 7, 14, we use the flow cytometry(FCM), enzyme-linked immune sorbent assay(ELISA) and lymphatic cell proliferation assay to detect the differentiation and the propagation of lymphocyte in mouse spleen(SPL), Peyer Peyer(PP) and mesenteric lymph nodes(mesenteric lymph nodes, MLN). It was found that in the recombinant group, the level of IFN-? was lower than that in the other two groups, the expression level of CD4+ T cell surface marker, IL-4 and IL-10 in the recombinant group was significantly higher than that in the other two groups. The lymphatic cells of mice in recombinant group had shown the strongest multiplication capacity, it shown significantly higher than the LAB NC8 group(P<0.05) and very significantly higher than the control group(P<0.01). At days-1, 7, 14, we detected the Ig G1 in serum of mice, we found that in the 7th day, the level of Ig G1 in serum of recombinant groupshown significantly higher than the LAB NC8 group(P<0.05) andvery significantly higher than control group(P<0.001), the LAB NC8 group shown very significantly more than the control group(P<0.01). In the 14 th day, the level of Ig G1 in serum of recombinant group shown very significantly higher than the LAB NC8 group(P<0.01) and very significantly higher than the control group(P<0.001). In the 21 th day, the level of Ig G1 in serum of recombinant group shown very significantly higher than the LAB NC8 group(P<0.01) andvery significantly higher than the control group(P<0.001), and the LAB NC8 group shown very significantly higher than the control group(P<0.01). We also detected the SIg A in the intestinal contents of mice and we found that,the level of Ig G1 in serum of recombinant groupshown significantly higher than the LAB NC8 group(P<0.05) andvery significantly higher than the control group(P<0.001). The recombinant group shown significantly higher than the LAB NC8 group(P<0.05) and significantly higher than the control group(P<0.05) in the 14 th day. In the 21 th day the recombinant group shown significantly higher than the control group(P<0.05). We did the intestinal adult worms counts at day 7. From the data we know that the recombinant group had least counts of the worms in the three groups, it shown significantly lower than the LAB NC8 group(P <0.05) and very significantly lower than the Control group(P<0.01), the LAB NC8 group shown significantly lower than the Control group the Control group(P<0.05). On the 30 th day we did the detection of muscle larva. Where in the recombinant group shown the least number of larvae in the three groups, it shown very significantly lower than the LAB NC8 group(P<0.01) and very significantly lower than the Control group(P<0.01); the LAB NC8 group shown significantly lower than the Control group(P<0.05). In summary, we believe that the lactobacillus which recombinant m IL-4 can effectively promote the expulsion of intestinal Trichinella adult worms, prevent the parasites migrate in host and improve the intestinal mucosal immunity. |
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