Identification Mimetic Peptides Of Bursin And Construction Of The SWPV Transfer Vector Expressing The Mimetic Peptide

Bursin is an important bioactive substances in bursa of Fabricius tissues,which was evidenced as a kind of three peptides of small molecules,and the structure of Bursin is Lys-His-Gly-NH2.Studies showed that bursin have the function of inducing birds and mammals lymphocyte proliferation and differentiation,enhancing the body's immune function,improving the level of specificity of antibodies biological activities,etc.Therefore,it can be used as a kind of immune enhancer in practical application.Due to the limited amount of extracted from chicken bursa of Fabricius tissues and Synthesis of high cost.The molecular structure of c-terminal is the amino,it is unable to be produced by prokaryotic and eukaryotic expression;As a result,it is difficult to large-scalely applicating in livestock and poultry industry.In this study,with monoclonal antibodies of bursin as the target molecules,we used phages display technology select the mimetic peptide of bursin,hoping that we can pan immunity enhancement activity of mimetic peptide of bursin and explore mimetic peptide of bursin as adjuvant was applied to the feasibility of veterinary vaccines.1.Selection and identification Mimetic Peptides of bursin.As the purification of Anti-Bursin monoclonal antibody 2D2 and 11C4 seted as target molecule,we performed selections respectively for seven random peptide library and twelve random peptide library affinity elutriation.20 phages containing bursin mimetic epitope were selected from randomly picked 227 plaque through sequencing,indirect ELISA and indirect competitive ELSIA.14 kinds of mimetic epitope were selected for peptide synthesis,through the combination of corresponding phage and monoclonal antibody block by the synthetic peptides.Two specific mimetic peptide of bursin were panned,the amino sequence of which were ANCIFQHPARHC(named M42 peptide)and LTSHGGYIPGGR(named K02 peptide).2.Analyzing core sequence of mimetic peptide of M42.On the basis of mimetic peptide of M42(ANCIFQHPARHC),12 kinds of peptides were synthesized for core sequence analyzing after reducing N-terminal or C-terminal amino acid step-by-step.Indirect competitive ELISA method was used to analyze the blocking effect of M42 phage with monoclonal antibody,the core sequence of M42 peptide(IFQHPA)deduced eventually.3.Influence of secretion of hybridoma antibodies from mimetic peptide of bursin.In order to study the biological activity of M42 mimetic peptide and K02 mimetic peptide,we added different concentrations of bursin,M42 mimetic peptide and K02 mimetic peptide into the hybridoma(Anti-PEDV protein S1-1)8C3 plants culture.Determine its effects on hybridoma antibody secretion by measuring the level of antibodyafter 48 h.The experimental results showed that hybridoma antibody production quantity was increased with the rise of concentration of Bursin,M42 peptide and K02 peptide.As the concentration of M42 mimetic peptide reached 200?g/mL,the highest increased antibody production was 41.75%.This demonstrated that M42 mimetic peptide and K02 mimetic peptide can promote the secretion of antibody like bursin.4.Mimetic peptide of bursin enhancing the body's immune function in mice.In order to study the effecting of enhancing the body's immune function in animals,the same molarity of bursin or M42 peptide or K02 peptide together with the recombinant swine pox virus rSWPV-E11-28 C which expressed PCV2 capsid protein were used to infect 8 weeks ICR mice.Detection of mice serum specific antibody against PCV2 through collecting serum of these mice weekly.The results showed that the PCV2 antibody of group of immune M42 peptide was higher than the controlled group,and the peak antibody levels was presented 1-2 weeks earlier,as well as immune enhancement ability of which was similar to aluminum adhesive adjuvants.According to the result,we inferred that the M42 mimetic peptide selected has immune enhancement effect like bursin which was hopefully used as immune enhancer applied in veterinary vaccines.5.The combined characteristic between spleen lymphocytes of swine and chicken and bursin and M42 mimetic peptide.The FITC labeled Bursin and M42 peptide was interacted with chicken and swine spleen lymphocytes,the result of which was observed under fluorescence microscope.Results showed that bursin and M42 mimetic peptide can be specifically combined with partial spleen lymphocytes.It proved that the M42 mimetic peptide for immune enhancement effect in certain way with the evidence that receptor of bursin may existed in some chicken and swine spleen lymphocytes.6.Constructing of the transfer vector of recombinant swine pox virus expressing M42 mimetic peptide.To explore the possibility and feasibility of mimetic peptide of bursin work as immune enhancer applied in the veterinary vaccines,we synthetic the M42 peptide sequence and cloned it to the transfer vector.With the VV later promoter P28 was selected as the promoter of M42 peptide.We successfully built the transfer vector(named pSW-E11-28C-28M42)which express M42 peptide and PCV2 capsid protein,the study aid the foundation for the next step to build recombinant swine pox virus which express M42 peptide and PCV2 capsid protein laid the foundation.