| Infectious bronchitis(IB),an acute,respiratory viral disease of chicken caused by infectious bronchitis virus(IBV),is one of the major infectious diseases that affects the poultry industry and has been classified as a Class B infectious disease by the Office International Des Epizooties.The IBV strain mutated rapidly.To date,up to 40 serotypes of IBV have been found and new serotypes are still occurring.Studies have shown that there were different dominant serotypes of IBV in different regions.At present,vaccination is still the main effective means to prevent and control the disease.However,traditional inactivated vaccines and attenuated vaccines cannot provide complete and effective protection due to the poor cross-protection between different serotypes of IBV strains.At the same time,current vaccines have defects in safety and immunogenicity.Therefore,the development of a safe and efficient new vaccine which is consistent with the local IBV dominant serotype is of great significance for the prevention and control of IB and the resolution of actual production problems.Virus-like particles(VLPs)are protein particles formed by self-assembly of viral structural proteins.They are safe vaccines candidate because they don’t have viral genomes and thus can’t replicate autonomously.VLPs can stimulate the body to produce effective humoral and cellular immune responses.Compared with other subunit vaccines,it has more effective adjuvant activity and is expected to replace traditional vaccines.It is an ideal vaccine form.Based on the previous research of our group,Guangxi IBV dominant serotype GX-YL5 strain was used as the research object in this study.We finished the optimization of single expression conditions of four structural proteins S,S1,M and E in the eukaryotic insect baculovirus expression system.At the same time,we also explored the best expression condition of recombinant baculovirus rHBM-S,rHBM-M and rHBM-E co-infected,rHBM-S and rHBM-M co-infected and rHBM-M and rHBM-E co-infected.Three IBV VLPs of SME-VLPs,SM-VLPs and ME-VLPs were prepared under the optimal co-infection expression conditions.Moreover,based on the analysis of the optimal protein expression of IBV recombinant S and S1 proteins,the anti-S and anti-S1 protein polyclonal serum were prepared by immunizing New Zealand white rabbits with purified recombinant protein by Ni-NTA resin.The detailed specific research contents are as follows:1.Optimized expression of IBV recombinant S,S1,M and E proteins in insect cellsRecombinant bacmids rHBM-S1 and rHBM-M constructed by our research group were transfected into Sf9 cells to obtain P1 recombinant baculoviruses rHBM-S1 and rHBM-M,which were then identified by PCR.The P1 recombinant baculoviruses rHBM-S1,rHBM-M and rHBM-S,rHBM-E preserved by our research group were conducted 4 times blind passages to obtain P4 viral stocks and determine the virus titer by TCID50.The expression conditions under different time of infection(TOI)and different multiplicities of infection(MOI)were explored.Analysis of protein expression under different conditions in Western blot using Image J software combining with IFA was carried out.The results showed that the optimal expression time of recombinant S,S1,M,E proteins were 84 h,72 h,96 h and 60 h,respectively,and the best MOI were 1,1,3 and 1,respectively.2.Research on co-infection of recombinant baculovirus expressing IBV S,M and E proteins by different combinations in insect cellsOn the basis of exploring the optimal expression conditions of recombinant S,M and E proteins,the experiments of co-infected with different recombinant baculovirus at different MOI were designed,and the expression products were harvested under different TOI,and then Image J software was used to analyze the TOI and MOI of the highest protein expression in Western blot detection.The results showed that the recombinant baculoviruses rHBM-S、rHBM-M and rHBM-E had the highest protein expression after co-infection for 84 h with MOI=3;recombinant baculoviruses rHBM-S and rHBM-M had the highest protein expression after co-infection for 84 h with MOI=1 and MOI=3,respectively;the recombinant baculoviruses rHBM-M and rHBM-E had the highest protein expression after co-infection for 60 h with MOI=4 and MOI=1,respectively.3.Study on the formation of virus-like particles(VLPs)by different structural proteins of IBVAccording to the optimal conditions for co-infection expression of different baculoviruses explored above,three VLPs were prepared by co-infection of three different combinations of baculovirus in suspension cultured Sf9 cells.They are rHBM-S,rHBM-M and rHBM-E(referred to as SME-VLPs),rHBM-S and rHBM-M(referred to as SM-VLPs),rHBM-M and rHBM-E(referred to as ME-VLPs),respectively.Three different VLPs were identified by Western blot,transmission electron microscopy(TEM)and immunoelectron microscopy(IEM).Western blot results showed that the presence of each corresponding protein band(S prorein 190 k Da,M protein 26 k Da,E protein 16 k Da)was detected in all three VLPs.Circular or polygonal particles with diameters ranging from 80 nm to 120 nm were observed in all three VLPs under TEM.Black gold-labeled S protein particles on the surface of the SME-VLPs and SM-VLPs were observed under IEM.4.Expression of IBV S and S1 proteins in eukaryotic system and preparation of polyclonal antibodiesA large amount of S and S1 proteins were obtained according to the above optimal conditions for the expression of recombinant S and S1 proteins and purified by Ni-NTA resin.New Zealand white rabbits were immunized for four times to prepare antisera.The reactivity of antisera with recombinant protein was tested by IFA and Western blot and the antibody titers were determined by ELISA.IFA showed specific fluorescence signal,and the corresponding specific protein band was detected by Western blot.The antibody titer of antiserum against the S protein was 1:12800 by ELISA.In summary,the optimal conditions of different structural proteins of IBV of expression and co-expression were optimized in this study,and IBV SME-VLPs,SM-VLPs and ME-VLPs were assembled successfully in the insect baculovirus expression system by co-infection.In addition,S and S1 protein polyclonal antisera with good reactivity and specificity were prepared using recombinant proteins expressed in eukaryotic expression system.This study laid a good foundation for the development of a new subunit vaccine and VLPs of IBV,and also provided a reference for preparation of polyclonal antibodies of IBV or other coronavirus structural proteins using eukaryotic expression systems. |
