Prokaryotic Expression And Immunological Analysis Of TSP8 And TSP11 Genes Of Echinococcus Granulosus

Objective: Echinococus granulosus,as the main pathogen of hydatidosis,has caused countless harm to our country and other countries in the world,which has caused great threat to people's lives.Since the understanding of hydatidosis,every country has taken active measures to examine its prevention and treatment,so the immune prevention of hydatidosis has become the focus of current research.Take into account this,the development of a long-term protective vaccine against hydatidosis of the host will promote the prevention and treatment of hydatidosis in China.Up to now,Tetraspanin has been identified as a candidate protein for a variety of parasite host vaccines,which play an important role in a wide range of cellular activities,and has been found to play a decisive role in immune interaction and immune evasion.In this study,the TSP8 and TSP11 of Echinococcus granulosus were amplified and prokaryotic expressed for the first time,and the levels of cytokines and antibodies in mice immunized with the recombinant protein was detected,which laid a foundation for the study of Tetraspanin as a candidate vaccine antigen of Echinococcus granulosus.Methods:(1)Cloning and sequence analysis of the full-length genes of TSP8 and TSP11 of Echinococcus granulosusAccording to the TSP8 and TSP11 full-length genes of Echinococcus granulosus in NCBI Genebank,the specific primers were designed respectively,and the product was cloned into pMD19-T vector and then sequenced.The structure and function of TSP8 and TSP11 full-length genes were analysed by bioinformatics software.The relative transcription of TSP8 and TSP11 genes in protoscolex and adult was analyzed by SYBR Green ? qRT-PCR.(2)Cloning and prokaryotic expression of major antigen genes of TSP8 and TSP11 of Echinococcus granulosusIn order to successfully encode and express TSP8 and TSP11 genes of Echinococcus granulosus,the specific primers were designed for the main antigen region sequence,and the main antigen region fragments were cloned and sequenced.On the basis of correct sequencing,the target fragment and expression plasmid pET-32 a were digested,linked and transformed.The recombinant expression vector pET-32a-TSP was established,and purified on the affinity chromatography column,identified and detected by SDS-PAGE,Western blot immunoassay to detect its antigenicity,and the precise location of TSP8 and TSP11 protein in the body was analyzed by immunolocalization.(3)Analysis of the immunological characteristics of TSP8 and TSP11 of Echinococcus granulosusThe experiment was divided into four groups: the recombinant protein TSP8 and TSP11 group,PBS and adjuvant control group,each group of 15 mice,were immunized with the same volume of Freund's adjuvant and 500 ?g protein content of each mouse,immunized every 15 days,a total of 4 times.The first time was mixed immunization with complete Freund's adjuvant,and the rest was mixed immunization with incomplete Freund's adjuvant.After each immunization,serum was harvested from the tail of three mice in each group.q-PCR method was used to determine the changes of six cytokines in Th1 type(IFN-??TNF-?)and Th2 type(IL-4?IL-5?IL-6?IL-10)immune responses with immune time,and the antibody level was determined by ELISA method.Results: 1.The total length of TSP8 gene was 669 nucleotides,and the encoded protein contains 222 amino acids.The molecular weight of the protein was expected to be 24 kDa.Two potential N-glycosylation site and two protein kinase phosphorylation sites were predicted.There were five transmembrane regions in the amino acid sequence encoding TSP8 gene,and had four dominant B cell antigen epitopes.The results of qRT-PCR showed that TSP8 gene was expressed in protoscolex and adult stages,and the difference was statistically significant(p <0.05).2.The total length of TSP11 gene is 765 nucleotides.The encoded protein contains 254 amino acids and the relative molecular weight is 29.02 kDa.It is predicted that there are three potential N-glycosylation sites,five protein kinase phosphorylation sites and one casein kinase phosphorylation site.There are three transmembrane regions in the amino acid sequence encoding TSP11 gene,and had seven dominant B cell antigen epitopes.At last,qRT-PCR results showed that the TSP11 gene was expressed in both the protoscolex and adult stages,and there was no statistical difference(p>0.05).3.Two weeks after the first immunization,recombinant proteins Eg-TSP8 and Eg-TSP11 could be detected specific IgG antibodies in the serum of mice.After the consequent enhanced immunization,the level of the specific IgG antibody in the serum increased gradually,indicating that the recombinant protein can induce the specific antibody in mice and has better immunogenicity.In the first two weeks of immunization,the expression of IL-5 and IL-10 in spleen tissue of recombinant protein Eg-TSP8 was higher than that in adjuvant group and PBS group,while the expression of cytokines IFN-?,IL-4,IL-5 and IL-10 in spleen tissue of recombinant protein Eg-TSP11 was higher than that in adjuvant group and PBS group(P < 0.01).It can be shown that the recombinant protein Eg-TSP11 induced Th1 /Th2 type immune response in mice,while the recombinant protein Eg-TSP8 mainly preferred Th2 type immune response.Conclusion: The recombinant proteins Eg-TSP8 and Eg-TSP11 have immunogenicity.However,compared with Eg-TSP8,Eg-TSP11 can stimulate Th1 type immune response in mice,which offers the opportunity to become the vaccine antigen of hydatidosis.