The Identification Of The Key Amino Acid Residues Of PT8A And Study On Its Immunoadjuvant Activity

Our recent study has been confirmed that an eight peptide,shorted as PT8A(HIDSQKKA),maintained both mucosal and intramuscular adjuvant activities with integrating into the C-terminal of human rotavirus(hRV)VP8~* antigen.The PT8A was originated from the T and B cell epitope(78-85 aa)of heat-labile enterotoxin B subunit(LTB).In this study,three mutants of PT8A were constructed and fused with vp8~* gene at the C-terminal.The proteins containing the intramolecular adjuvant PT8A and those mutants were used to immune the female Balb/c mice via nasal and intramuscular vaccination.The titers of serum IgG were detected by indirect ELISA.The VP8~* specific serum IgG levels were significantly increased in administration of Vp8-CgR5(Q5) and Vp8-ChR8(A8) mutants than that of original Vp8-PT8A.The results verified that the mutations of Q5 and A8 from PT8A enhanced the mucosal adjuvant activity of PT8A.However,the mutation S4 of PT8A were no effect on the adjuvant activity of PT8A.Objective: To find the stronger intramuscular adjuvant from PT8A mutants and to identify the key amino acid residues of PT8A for its adjuvant activity;Methods:1.The Taq polymerase was used to amplify PT8A by PCR to obtain mutant fragments VP8-CgR4,VP8-CgR5 and VP8-ChR8;2.Construction of pET32-VP8-CgR4,pET32-VP8-CgR5 and pET32-VP8-ChR8.The plasmids were verified by PCR and DNA sequencing;3.Expression and purification of VP8-CgR4,VP8-CgR5 and VP8-ChR8 antigens.The proteins were purified with His-tag magnetic bead,then the purified proteins were condensed into 1 mL with PEG8000 after dialysis for 48 hours with TGE buffer.Finally,the proteins were detected by 10 % SDS-PAGE;4.The concentrated proteins of VP8-CgR4,VP8-CgR5 and VP8-ChR8 were removed endotoxin with De endotoxin test kit;5.Nasal immunization of Balb/c female mice;6.Serum collection;7.Detection of VP8~* specific serum IgG by indirect ELISA.Results:1.pET32a-VP8-CgR4,pET32a-VP8-CgR5 and pET32a-VP8-ChR8 mutant recombinant plasmids were successfully constructed;2.The fusion proteins of VP8-PT8A,VP8-CgR4,VP8-CgR5 and VP8-ChR8 were successfully expressed and purified.The stripe size of the fusion proteins were about 40 KD by 10% SDS-PAGE test;3.The ELISA results showed that the levels of VP8~* specific serum IgG were significantly increased in the administration of Vp8-CgR5(Q5)and Vp8-ChR8(A8)mutants mice than that of original Vp8-PT8A vaccination.The other mutation(S4)of PT8A was no effect on the adjuvant activity of PT8A.Conclusions: 1.The mutants of VP8-CgR4,VP8-CgR5 and VP8-ChR8 were constructed and the fusion proteins with His-tag were expressed successfully;2.Compared with PT8A,the adjuvant activity of VP8-CgR5 and VP8-ChR8 had been improved;3.The results verified that the mutations of Q5 and A8 from PT8A enhanced the mucosal adjuvant activity.The other mutation(S4)of PT8A has no effect on the adjuvant activity of PT8A;...