Cloning, Expression And Immunogenicity Of TSP14 And TSP33 Genes Of Echinococcus Granulosus

Echinococcosis is caused by the metacestode of Echinococcus granulosus and is distributed worldwide.China is one of the countries and regions with high incidence of the disease.The disease is dangerous,with a 10-year mortality rate of 94%.The prevention and control measures are mainly the monthly administration of praziquantel in dogs.Although it is effective,it is difficult to adhere to it for a long time at the grass-roots level,and it is difficult for stray dogs to manage it.The Eg95 vaccine against intermediate host is effective for the prevention and control of pathogens during the migration period.The number of definitive host host and intermediate host is up to 45:1,so the cost of prevention and control is very high.Therefore,to establish an effective immune prevention method for the definitive host is a key way to prevent and control Echinococcosis.Studies show that the four-transmembrane protein family has good immunogenic value of paresite.Therefore,this experiment intends to carry out preliminary cloning,prokaryotic expression and immunogenicity studies of Eg-TSP14 and Eg-TSP33,with a view to provid a theoretical basis for whether Eg-TSP14 and Eg-TSP33 have the potential as vaccine candidate antigens.Objective: To clone Eg-TSP14 and Eg-TSP33 genes,construct recombinant pET-32a-TSP14 and pET-32a-TSP33 plasmids,transform them into E.coli for prokaryotic expression,and immunize mice with the recombinant protein for their immunogenicity.initial research.Methods:(1)Using the cDNA of adult Echinococcus granulosus,protoscolex,and cyst wall as templates,amplification was carried out using quantitative PCR technology to analyze the differential expression of the two genes at different developmental stages of the worm.(2)Cloning the Eg-TSP14 and Eg-TSP33 genes and constructing the expression vectors pET-32a-TSP14 and pET-32a-TSP33.The successfully constructed vector was transformed into E.coli competent cell BL21,and IPTG was added to induce expression.The expressed protein was purified and quantified.(3)The purified protein was subjected to Western Blot to verify its reactivity.(4)Immunize mice with recombinant protein,collect mouse spleen and serum,determine the changes of cytokines by fluorescence quantitative PCR and the changes of antibodies by indirect ELISA.Results:(1)The results of real-time PCR showed that Eg-TSP14 was expressed in the cyst wall and the protozoa,and the expression level in the cyst wall was higher than that in the protozoa.Eg-TSP33 gene was expressed in adult,cyst wall,and protozoan,and the expression of TSP33 gene in cyst wall was higher than that in protozoon and adult stage.(2)The cloned Eg-TSP14 is 270 bp in size and Eg-TSP33 is 420 bp in size.The constructed recombinant vector was identified by double enzyme digestion,and then induced by IPTG and SDS-PAGE.The recombinant protein rEg-TSP14 was 27 kDa in size.The size of the recombinant protein rEg-TSP33 was 32 kDa,which was consistent with the expected result.Analysis of the expression form analysis showed that both were expressed in the form of inclusion bodies in the precipitate.The concentration of the purified recombinant protein rEg-TSP14 was 1.4 mg / mL,the concentration of recombinant protein rEg-TSP33 was 1.7 mg /mL.(3)Western blot results showed that both recombinant protein rEg-TSP14 and rEg-TSP33 can react with canine positive sera and have good reactogenicity.(4)Recombinant protein rEg-TSP14 and rEg-TSP33 caused increased serum antibody levels in mice.(5)Cytokine test results showed that rEg-TSP33 can cause changes in Th1 type cytokines,which is significantly different from the control group(P <0.05),and rEg-TSP14 can cause changes in Th2 type cytokines.Conclusion: Eg-TSP14 and Eg-TSP33 can be expressed at different developmental stages of the worm,and may play an important role in the development of E.g..Their immunogenicity studies have found that both proteins can bind to mouse antibodies and induce The mice developed a negotiable response.The results of measuring cytokines speculate that Eg-TSP33 can enhance the host's anti-infection ability,which is conducive to eliminating pathogens early in the infection,while Eg-TSP14 may play a role in E.g.immune evasion,which is conducive to E.g.establishing infection in the host.