The Identification And Immunity Effect Evaluation Of The New Epitope Of RSV

Part 1 Rational design the peptide vaccine against the Respiratory syncytial virus.Objective: To identifiy and optimize the peptide vaccine against the Respiratory syncytial virus by the rational design and molecular simulation.Method: the crystal structure data of proteins derived from the Respiratory syncytial virus and the corresponding neutralizing antibody with the epitope peptide were collected from the Protein Data Bank?PDB?.The interaction between the neutralizing antibody and epitope was analyzed by the molecular simulation.The contributions of the different parts of antigen FFL-001 to stabilize architecture of FFL-001 and bind with the neutralizing antibody were evaluated by the molecular dynamic simulation.Finally,the interaction between the rational designed peptide and the neutralizing antibody were evaluated and verified by the theoretical computation and experiments respectively.Result:1.The structural data of the F protein of the Respiratory syncytial virus were successfully obtained frome the Protein Data Bank,and the cocrystal structure of antibody Motavizumab-epitope complex were also obtained from the PDB.2.It was verified that there existed a section of peptide with high stability in its architecture in the F protein,and almost no obvious change in the architecture of this peptide were observed derived from the deep analysis on states of prefusion and post fusion of the F protein.And it was estimated that this peptide acted as the target for the developed neutralizing antibody of the RSV.3.The results obtained from the structural and energic analysis showed that the binding regions of the Motavizumab-FFL₀01 just located in the Loop region?amino acid sequence 65-89?which link the H2 and H3 helix bundles.And it was verified that there were nine H-bonding and one salt-bridge interactions between Motavizumab and FFL₀01 in this interface.4.The results obtained from the Molecular dynamic simulation showed that the full-length FFL₀01 protein has no considerable structural change as compared to its crystal counterpart.The two-helix bundle H2-H3 is absent for helix H1 relative to FFL₀01,which partially exhibits intrinsic disorder in solution;the peptide segment of Motavizumab-binding site in FFL₀01;only a small helix region can be maintained at peptide N-terminus,while the C-terminus and the middle region of the peptide are fully disorder during the simulations5.The energical evalution showed that the full-length FFL₀01 can bind tightly with ?Gtotal of –23.6 kcal/mol.The value can be decomposed into a very favorable intermolecular interaction between the FFL₀01 and Motavizumab??Gint = –110.8 kcal/mol?as well as a large unfavorable desolvation effect??Gslv = 71.5 kcal/mol?and a small unfavorable entropy effect?–T?S=15.7kcal/mol?two-helix bundle H2-H3 and Motavizumab-binding site exhibited a different energetic profile as compared to the intact FFL₀01 protein when binding to Motavizumab.The two peptide segments incur large entropy penalty?–T?S = 47.2 and 27.0 kcal/mol?and also unfavorable desolvation effect??Gslv = 52.6 and 58.8 kcal/mol?upon the binding.the two-helix bundle H2-H3 with ?Gtotal of 3.4 kcal/mol,the motavizumab-binding site with ?Gtotal of –7.9 kcal/mol?6.Energical evaluation on the truncated and cyclized peptide derived from the motavizumab-binding site showed that different cyclization site significantly influence the cyclized peptide binding to the Motavizumab.And ?Gtotal value between the cyclized PV2 and Motavizumab reached-21.2kcal/mol.The experimental results showed that synthetic cyclized peptide PV2 can bind to the Motavizumab strongly?Kd =16.2n M?Conclusions:1.The Motavizumab binding site of the FFL₀01?amino acid sequence 65-89?were similar to the peptides with the high stability in architecture derived from the F protein.2.Significant difference in the binding affinity between the Motavizumab with the cyclized peptide with different length.The cyclized PV2 with the best performance in binding to the Motavizumab was similar to the full length FFL₀01,it indicated that cyclized PV2 may stable its original configuration that can specically bind with the Motavizumab.3.Experimental results verified that the cyclized PV2 can interact with the Motavizumab with high binding affinity,indicated that the rational designed cyclized PV2 can be the candidate of the peptide vaccine against RSV.Part II The gene clone and Prokaryotic expression of F protein of RSVObjective: To obtain the F protein with full length to immunity the mouse and subsequent to evaluate of the immunity effect.Methods: The expression of the recombinant F protein: The gene of F protein was cloned from the c DNA of the RSV by the RT-PCR,and subcloned into the p ET-28 a vector.The obtained recombinant vector p ET-28a-F were translated into the E.coli BL21.The recombinant protein was expressed in the E.coli under the induction by IPTG,and finally purified by the Ni ion affinity Chromatography.Results:1.c DNA were obtain by the RT-PCR,and subsequently act as the template for PCR to clone the gene of F protein.The obtaind gene was almost 1700 bp by the agarose gel electrophoresis analysis,which is identical to the theoretical prediction.Finally it was verified by the sequence.2.The recombinant F protein were successfully expressed in the E.coli BL21 induced by IPTG.The expressed protein was about 70 k D verified by the SDS-PAGE analysis.3.The recombinant F protein with high purity was obtained by the Ni2+ ion affinity chromatography approach.Conclusions:1.The gene encoding the F protein of RSV were cloned by the gene clone technology.2.The recombinant F protein purified by Ni2+ ion affinity chromatography approach satisfied with requirement of further research.Part III,Evaluate the immunity effect of cyclized PV2 and non-cyclized PV2 peptide.Objective: To evaluate the immunity effect of new type epitope?cyclized PV2?of RSV by nasal drip and intramuscular injection,and compare the immune effect with the corresponding non-cyclized PV2.Methods:1.The RSV virus was cultured and purified,and the TCID50 and the titer of RSV was determined.2.The female Balb/c mice with 6 8 weeks old were selected and divided into nine groups: PBS group?5 mice per group?,Freund's adjuvant group,Gelatin adjuvant group,PV2/gelatin/nasal dropping group,PV2/ Freund's adjuvant /ntramuscular injection group,Cyclized PV2/gelatin/nasal dropping group,Cyclized PV2/Freund's adjuvant/intramuscular injection group,F protein/gelatin/nasal dropping group and F protein/Freund's adjuvant/ intramuscular injection group.3.The immunization were performed every two weeks,and the serum form the mice tail was collected after the immunization,and after ten days of the third immunization,the serum from the orbit of the mice was collected after the mice were sacrificed.4.The indirect ELISA was used to detect the titers of serum specific Ig G antibody in immunized mice.5.The irrigation solution from the lung and nasal form the immuned mice were collected.The titers of Specific Ig G in irrigation solution of lung and nasal induced by new RSV antigen epitope cyclized PV2 and non-cyclized PV2 were also detected by indirect ELISA method.6.The neutralization ability of neutralizing antibody in serum of immunized mice was detected in Vero cells,and the titer of neutralizing antibody in serum was detected by Microneutralization assay.The recognition effect of mouse antiserum to the native F protein of virus was evaluated by indirect immunofluorescence.Results:1.RSV virus were cultured in Vero cells,the virus supernatant was harvested after 7 days culture,and the TCID50 was determined by Karber method.The results showed that the titer of the virus was 10-8.5.2.The titer of specific Ig G antibody in the serum of immunized mice was measured by indirect ELISA method.The results showed that: i)The immune effect of intramuscular injection was better than that of nasal dropping.The titer of mice serum specific Ig G antibody was 1: 40 in the group of PV2/gelatin adjuvant/nasal dropping after three times of immunization,and The titer of mice serum specific Ig G antibody was 1: 158 in the group of PV2/Freund's adjuvant/ntramuscular injection after three times of immunization.The titer of serum specific IgG antibody was 1: 630 in the cyclized PV2/gelatin adjuvant/nasal drip group and 1: 1584 after the third injection of cyclized PV2/ adjuvant/intramuscular injection.The titer of serum specific Ig G antibody in F protein/gelatin adjuvant/nasal drip group was 1: 19952,and that in F protein/Freund's adjuvant/intramuscular injection group was 1: 50118.ii)The ability of cyclized PV2 to induce specific antibody Ig G in mice was significantly better than that of non-cyclized PV2.No specific Ig G was detected by non-cyclized PV2 group after the first immunization and low level of specific Ig G antibody was detected after the second immunization.After the third immunization,a certain level of specific Ig G antibody was detected.At the same time,the specific Ig G antibody could be detected after the first immunization in cyclized PV2 group,and the titer of the antibody in cyclized PV2 group were much higher than that of the non-cyclized PV2 groups,and for the first immunization,the titer of the serum immuned by cyclized PV2 was even better than that immuned by the full-length F protein.3.The titer of specific Ig G antibody in the irrigation solution from lung and nasal of immunized mice were measured by indirect ELISA method.The results showed that: i)specific Ig G antibody with different level were determined in the lung of the mice immuned by three antigens.A certain level of specific Ig G antibody was detected in the nasal of mice immuned by cyclized PV2 and F protein.No specific antibody was detected in the irrigation solution from lung and nasal in mice immuned with the manner of nasal drip.ii)The titer of specific Ig G antibody was 1: 12 in PV2/Freund's adjuvant/ntramuscular injection group.The titer of specific Ig G antibody in irrigation solution from lung of cyclized PV2/Freund's adjuvant/intramuscular injection group was 1: 132.The titer of specific Ig G antibody in irrigation solution from nasal was 1: 5.no specific Ig G in irrigation solution from lung and nasal was detected in cyclized PV2/gelatin adjuvant/nasal dropping groups.In immunized mice with recombinant F protein,the titer of antibody in irrigation solution from lung was 1: 1:891,the titer of antibody in irrigation solution from nasal was 1:8.4.The results derived from the micro-neutralization showed that: i)the titer of neutralization antibody in the PV2/ gelatin adjuvant/nasal drip group was 1: 15.The titer of PV2/ Freund's adjuvant/intramuscular injection was 1: 39.The titer of cyclized PV2/gelatin adjuvant/nasal dropping group was 1: 63.The titer of cyclized PV2/Freund's adjuvant/intramuscular injection group was 1: 158.The titer of F protein/gelatinadjuvant/nasal dropping group was 1: 39.The titer of F protein/Freund's adjuvant/intramuscular injection group was 1: 1584.ii)The serum neutralizing antibody titer of all nasal immunization groups were significantly lower than that of the intramuscular immunization group.?P<0.05?5.The protective effect of neutralizing antibody was determined by indirect immunofluorescence.The results showed that the non-cyclized PV2/gelatin adjuvant/nasal dropping group almost had no signal of fluorescence while the PV2/Freund's adjuvant/intramuscular injection group had a certain fluorescence intensity,but it was weaker than that of the groups immuned with the F protein.The fluorescence intensity of the cyclized PV2 immunized group was much stronger than that of the negative control group,and no obvious difference in fluorescence intensity between the cyclized PV2/ Freund's adjuvant/intramuscular injection group and the Positive control?Motabizumab?.Conclusions:1.Specific Ig G antibody with different levels were determined in the serum of the mice immuned by three antigens.The ability of cyclized PV2 induce specific antibody Ig G in the serum of the immuned mice was significantly higher than that of non-cyclized PV2,but weaker than that of full-length F protein.2.Specific Ig G antibody with different level were determined in the lung of the mice immuned by three antigens.Only cyclized PV2 and F protein can induce the specific Ig G antibody in the nasal of the immuned mice.The ability of cyclized PV2 induce specific antibody Ig G in the lung of the immuned mice was significantly higher than that of non-cyclized PV2,but weaker than that of full-length F protein.3.With the manner of nasal drip,no specific Ig G antibody was determined in the irrigation solution from the lung and nasal of the mice immuned by three antigens respectively.4.The titers of neutralization antibody in the cyclized PV2 group were higher than that non-cyclized PV2 group in both immunity manners,but weaker than that of full-length F protein.And the titers of neutralization antibody in the serum of the nasal drip group was weaker than that of intramuscular injection group?p<0.05?5.In both immunity manner,the obtained antibody in the serum of mice immuned with cyclized PV2 in the recognition ability to the native F protein of RSV were better than that of the non-cyclized PV2...