| It has been proved that an excellent anti-embryonation and fecundity immunity in host can be induced in mice by immunized with a soluble immature egg 26-28 kDa(SIEA26-28 kDa) antigens or adult worm 31/32 kDa antigens from Schistosoma japonicum(Sj31/32)in our laboratory.It was found that the twentieth protein spot of Sj31/32 kDa possess a high homologue with Cathepsin B endopeptidase of Schistosoma mansoni (SmCB)by mass spectrum and sequence determination.The CB involved in hemoglobin decomposition of host and the growth of Schistosoma japonicum.However, it still posed challenges in its application, due to the difficulties in the purification and preparation of these natural molecular vaccines in batches.Our group focused on screening and identifying the gene coding a natural molecular vaccine, and has developed several DNA recombinant vaccines such as pcDNA3.0/SjCB relating to Sj31/32 kDa which could induce partial protection.Until now, there is not yet a satisfying vaccine which could induce highly immunity against the challenge of schistosome because of diversity of schistosome antigens and complicated immune system of infected host.Recently, researchers not only pay attention to screen and identify new candidate vaccine, but also try to improve the immune protection of DNA vaccines through reforming several conditions such as vector, adjuvant and immunization approach or strategy. A new type of DNA adjuvant encoding cytokines and being able to continually express and exist in form of recombine plasmid has stimulated extensive interesting.IL-18,initially described as an IFN-γ-inducing factor, stimulates the synthesis of IFN-y and IL-12 in T cells and natural killer (NK) cells, leading to the development of Th1-type immune responses,which will enhance immunoprotection against schistosomiasis, especially in prevent reinfection and anti-hepatic fibrosis.Traditionally, it depends on high dosage of adjuvant and injection of frequent intervals to reach a excellent immunization effect, because only by blood circulation it could reach to local immunity place. However, high dosage and frequent injections will cause side effects and imbalance of cytokines in host. Herein, we presented a novel method of construction of a bivalent vaccine pVAX1/SjCB-IL-18 and observation of the possibility of IL-18 acting as an adjuvant in enhancing the immunoprotection efficacy against Schistosomiasis japonicum.Objectives1.To construct monovalent vaccine pVAX1/SjCB,pVAX1/hIL-18 and bivalent vaccine pVAX1/SjCB-hIL-18,respectively.2.To observe whether IL-18 could enhance immunoprotection efficacies against Schistosoma japonicum in mice. Method1.Construction of the monovalent vaccine pVAX1/SjCB, pVAX1/hIL-18 and bivalent vaccine pVAX1/SjCB-hIL-18.SjCB and hIL-18 were amplified, respectively, with pQE30/SjCB and pET32a/hIL-18 as templates by proper primers.And then, the products were spliced to full-length SjCB-hIL-18 by SOE-PCR(splicing by overlap extension).After confirmed,these genes were cloned into the vector pVAX1 and transformed to the E.coli DH5α.Three positive clones were selected, identified and confirmed by sequencing.2. Expression and immunoprotection test of the monovalent vaccine pVAX1/SjCB,pVAX1/hIL-18 and bivalent vaccine pVAX1/SjCB-hIL-18.10 Kunming mice were randomly divided to 5 groups (2 mice per group) named A, B,C, D and E, A group were vaccinated with the bivalent vaccine pVAX1/SjCB-hIL-18,B with pVAX1/SjCB,C with pVAX1/hIL-18,D with blank plasmid pVAX1 as negative control and E with NS as blank control.Plasmids were extracted in mass and Kunming mice immunized by injecting the plasmids in quadriceps.Enzyme immunohistochemistry biopsy of muscle was carried out after 2 weeks. Afterwards,expression and immunoprotection efficacies of each group were observed.50 Kunming mice were randomly divided to 5 groups(10 mice per group).Plasmids were extracted massively and Kunming mice were immunized by injection of 100μL plasmids or 100μL NS through quadriceps.4 weeks later, every mouse was challenged with 20±1 Schistosoma japonicum cercariae via abdomen cutaneous.The worm burden, intrauterine eggs and liver eggs were determinated six weeks after challenge in order to observe the immunoprotection efficacy against Schistosomiasis japonicum.Results1.Restriction enzyme digestion, PCR and sequencing confirmed that recombinant plasmids pVAX1/SjCB, pVAX1/hIL-18 and pVAX1/SjCB-hIL-18 were constructed successfully.2. Immunohistochemistry showed that groups of recombinant plasmids pVAX1/SjCB,pVAX1/hIL-18 and pVAX1/SjCB·hIL-18 gave a very stable brownish color as the positive signals, but it was negative in blank plasmid and NS group.The results demonstrated that the recombinant plasmids were successfully expressed in the muscle cells of Kunming mice.3.immunoprotection experiment showed that reduction rates of the worm burden reduced 41.32%,32.56% and 15.17% in pVAX1/SjCB, pVAX1/hIL-18 and pVAX1/SjCB-hIL-18 group.The reduction rates of uterine eggs per female and per gram of liver reduced 49.55%,43.04% and 27.21%as well as 72.35%,50.08% and 14.86% respectively. The results mentioned above showed significant differences compared with negative control (P<0.05).No significant differences of the worm burden reduction rate and the egg reduction rate per gram of liver (P>0.05) existed between pVAX1/SjCB·hIL-18 group and pVAX1/SjCB group the. However, as to the reduction rate of uterine eggs per female,there was significant difference (P<0.05)between 2 groups mentioned above.There were significant differences as compared bivalent vaccine with 2 monovalent vaccine groups (P<0.05).At the same times,no significant deference in reduction rate of the worm (P>0.05)existed between pVAX1/SjCB group and pVAX1/hIL-18 group.But, the reduction rate of uterine eggs per female and the egg reduction rate per gram of liver were significant differences (P<0.05).Conclusions1.The eukaryotic recombinant plasmids pVAX1/SjCB, pVAX1/ hIL-18,and pVAX1/SjCB·hIL-18 were successfully constructed.2.The eukaryotic recombinant plasmids pVAX1/SjCB,pVAX1/ hIL-18 and pVAX1/SjCB·hIL-18 was successfully expressed in muscle of Kunming mice.3.Both monovalent vaccine pVAX1/SjCB and bivalent vaccine pVAX1/SjCB·hIL-18 can induce relatively satisfying immunoprotection efficacy, while bivalent vaccine pVAX1/SjCB·hIL-18 showed a better efficacy. The results indicated that as an adjuvant, IL-18 could enhance the immunoprotection efficacy against Schistosomiasis japonicum.
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