Cloning,Expression And Functional Analysis Of Bursin-Thymic Peptide Fusion Protein

Bursin(BS),a small molecule peptide,exists in the bursa of the avian cavity.Thymic peptide(TP),also known as thymosin,is a kind of immune active substance secreted by thymus,the central immune organ of the body.Both of them can be used as immunopotentiators,auxiliary therapeutic agents and vaccine adjuvants for various animals,which can effectively improve the vaccine effect and enhance the immune ability of animals.Studies have proved that the effect of the combination of the two is better than that of the single adjuvant.Therefore,this paper aims to construct a fusion protein adjuvant by using DNA recombination technology.Firstly,according to the molecular structure of BS and TP,a tripeptide linking peptide was designed to link BS and TP.On this basis,the expression vector p CJ101 plasmid was constructed and transferred into E.coli DH5α,and the positive clones were screened.In the experiment of improving immune function of immunosuppressed mice,the experimental mice were divided into immunosuppressed control group,normal group,TP-BS 0.2 mg/kg,0.4 mg/kg,0.8 mg/kg dose group and Ridaxian control group.The phagocytosis of peritoneal macrophages,the transformation of T lymphocytes and B lymphocytes,the activity of NK cells and the level of IL-2 in mice were detected.In the immune response of mice immunized with JEV subunit vaccine,the titer of protective antibody JEV-BD,antibody subtypes(Ig G1 and Ig G2),lymphocyte proliferation,T cell subsets,IFN-γ and IL-4 cytokines were detected.In the immune protection test of TP-BS against H9N2 inactivated vaccine of avian influenza in mice,the immune effects of TP-BS on HA antibodies and antibody subtypes,secretion levels of Th1 and Th2 cytokines,spleen T cell subsets and NK cells were detected.In the test of TP-BS to improve immune function of immunosuppressed mice,TP-BS can improve the phagocytosis of peritoneal macrophages in mice,promote the transformation of T lymphocytes and B lymphocytes in mice,improve the activity of NK cells in mice,and improve the level of IL-2.The reaction degree of TP-BS 0.4mg/kg and 0.8 mg/kg groups was more obvious.In the immune response of mice immunized with JEV subunit vaccine,TP-BS as adjuvant can produce significantly higher response than that of JEV-BD protein and JEVvaccine alone compared with Ig G,Ig G1 and Ig G2 levels.Quantitative analysis of IL-4 and IFN-γ in serum showed that TP-BS could increase the secretion of IL-4 and IFN-γ.After immunization,the number of T lymphocytes in mouse spleen cells increased significantly,and TP-BS could specifically regulate the changes of T lymphocyte subsets.The serum neutralizing antibody titer of mice immunized with TP-BS as adjuvant was significantly higher than that of JEV-BD protein alone,and almost the same as that of JEV Vaccine group,which indicated that TP-BS could significantly increase the neutralizing antibody titer of JEV.The specific immune response of mice immunized with JEV-BD was significantly improved by TP-BS.TP-BS can balance the secretion levels of Ig G1 and Ig G2 a in the immune protection test of mice with inactivated H9N2 avian influenza vaccine.Immunization with inactivated whole virus(WIV)antigen alone can induce the production of Th1 cytokines in mice,but TP-BS adjuvant can significantly improve the secretion level of two types of cytokines in mice immunized with WIV antigen,but cannot promote the secretion level of IL-10.TP-BS adjuvant can obviously promote the differentiation of CD4+ and CD8+ subsets of T cells in spleen lymphocytes of mice immunized with WIV antigen.It can also regulate the proliferation of NK cells in mouse spleen lymphocytes.To sum up,in this experiment,the engineered Escherichia coli expressing thymosin and capsular peptide was successfully constructed.Furthermore,TP-BS can regulate the immune ability of immunocompromised mice,and as an adjuvant,it can improve the immune effect of Japanese encephalitis subunit vaccine and the immune protection effect of inactivated avian influenza H9N2 vaccine on mice.The above results show that TP-BS has significant effect as vaccine adjuvant.