Construction Of Attenuated Salmonella Typhimurium Expressing Trichinella Spiralis ES Antigen 43ku Gene And Its Evaluation Of Immune Protective Effects

Trichinosis is a kind of zoonoses which is caused by Trichinella spiralis.This parasitic disease could severely jeopardize the development of animal husbandry and human health.Nowadays,only clinical drugs are applied to treat the disease,which usually brings about serious damage to the host.With the development of modern biological technology,the vaccine is produced utilizing genetic engineering technology widely.Among them,live bacteria vector vaccine has the advantages of simple inoculation ways,good immunogenicity and low cost,which offers a new strategy to prevent Trichinosis.Recombinant Attenuated Salmonella Vaccine(RASV),the most advanced exogenous antigen delivery system,it is of much valuable because of delayed the attenuation.This experiment utilized 43 kuES antigen gene of Trichinella spiralis and Salmonella vector pYA3681 to construct recombinant plasmid pYA3681-Ts43,then electroporated the plasmid into Escherichia coli peptide χ6212.After identification by double enzyme digestions and plasmid sequencing,another electroporation to final bacteria χ11802 was needed.After induction of IPTG,Western Blotting assay results showed expression of the target proteins were stable.The specific research contents and results are as follows:(1)Construction of recombinant attenuated Salmonella vaccine to express ES antigen gene 43 ku of Trichinella spiralis40days after challenge,we sacrificed the BALB/c mice,removed the muscle,digested the muscle with pepsin,collected the polypides and kept them in liquid nitrogen,extracted total RNA of Trichinella spiralis in a mortar.Then we reversed the RNA into c DNA.According to the GenBanK gene sequence of 43 kuES,KpnI and SacII enzyme restriction sites,specific primers were designed by Primer5.0.The 43 kuES target genes were amplified and verified to have a length fragment of 1035 bp,as we expected.Collected and purified the target genes for further use.Ligated Ts43 genes to Salmonella vector pYA3681,then electroporated the recombinant plasmid into χ6212.After double enzyme digestions,5825 bp and 1035 bp gene fragments were obtained,as expected.Then the plasmid was electroporated into final Salmonella χ11802.The double enzyme digestions were performed again to make the identification.Finally IPTG induced the expression of the target protein,Western Blotting results indicated a-38KDa-size protein appeared to prove a good immunogenicity for further research about immune protection.(2)Evaluation of immune protective effect of recombinant attenuated Salmonella vaccine χ11802(pYA3681-Ts43)Divided experimental animals into four groups: control group,PBS group,attenuated Salmonella vector group(pYA3681),recombinant attenuated Salmonella(pYA3681-Ts43)group.Each group contained ten four or five-week-old BALB/C mice.Inoculate the mice three times,one time a week for three consecutive weeks.7days after the third immunization,sacrificed five mice,collected the blood and intestinal lavage,detected the levels of IgG、IgA、IL-4、IL-17 a and IFN-γ by Elisa.Flow cytometry was used to detect the levels of CD4+T,IL-4,IL-17 a and IFN-γ in spleen and mesenteric lymph nodes.The rest of the mice were challenged by 300 Trichinella spiralis.And the body weight were recorded for the following 15 days to calculate body weight reduction rate.42 days after challenge,numbers of the worms were needed to calculate reduction rate and tongue muscle were removed to cut into slices for pathological observation of lesions.The experimental results showed that the recombinant attenuated Salmonella vaccines could effectively stimulate the proliferation of lymphocytes in spleen and mesenteric lymph nodes.Elisa assay showed an significant increase in the levels of specificity IgG and IgA,the activation of T lymphocytes and B lymphocytes,the level of IL-4,IL-17 a and IFN-γ.Flow cytometry results indicated that levels of CD4+T,IL-4,IL-17 a and IFN-γ increased significantly too.After TS challenge,under the evidence of slices and worm reduction rate,RASV could inhibit the growth of Trichinella spiralis effectively.By oral administration of recombinant attenuated Salmonella vaccine,the experimental mice could effectively preclude the invasion of Trichinella spiralis.The results obtained could lay a foundation for the development and exploitation of Trichinellosis vaccine and also offer a new strategy to prevent Trichinosis.