| Infectious bursal disease(IBD)is a serious infectious disease and may harm to immune organs of young chickens.It causes a highly contagious and severe immunosuppression by destroying B-lymphoid cells presented in the bursa of Fabricius,and the chickens are fully susceptible to IBDV infection.The disease is responsible for losses due to impaired growth and death,leading to an increased susceptibility to other pathogens and considerable economic losses in poultry industries worldwide,and brings a lot of difficulties that vaccine failure for disease prevention and control.Therefore,the prevention and control of the disease have important significance for livestock.A gene encoding the structural protein VP2 of infectious bursal disease virus(IBDV)is the main protective antigen which contains the major antigenic regions to elicit neutralizing antibodies.Therefore,the recombinant VP2 is considered the main target protein in the development of a subunit vaccine against IBDV.At present,VP2 protein has been expressed successfully in many systems,with insect cells the most widely used,although the insect expression system has many advantages,such as high expression level,post-translational modification and simple purification.The high cost and labor limit its application in the mass production.By contrast,the prokaryotic expression system is the simplest and most inexpensive methods used for research or commercial purposed,although the inclusion body and inactive product formation are common phenomenon.At present,many tags have been used to promote protein expression and purification in bacterial system.In this study,to improve the expression level of soluble VP2 protein in E.coli cells,9 gene-fusion systems were used,which constructed with p ET21 b vector and different tags.The effect of these 9 tags on the expression and purification of VP2 protein was discussed.In addition,the expression condition of soluble VP2 protein was optimized though technical study,and the expression level of soluble VP2 protein was significantly improved.The VP2 protein was then purified by affinity chromatography,molecular sieve and sucrose gradient centrifugation.And the virus-like particles(VLPs)formed by VP2 can observed though transmission electron microscope,similarly with the result of insect cells described previously.The immune response in chicken revealed that the prepared subunit vaccine has good immunogenicity and protection effect,which was significantly better than the current commercialization IBDV vaccine.The study established a stable basis for further research and development of new subunit vaccine against IBDV.1.To improve and increase expression and solubility of VP2 protein in E.coli,nine soluble tags: Grifin,GST,MBP,Nus A,SUMO,Thioredoxin,?-crystallin,Ars C and Ppi B,were introduced into VP2 to form fusion protein.Seven tags: Grifin,MBP,SUMO,Thioredoxin,?-crystallin,Ars C and Ppi B,have been enhanced successfully expression and solubility of VP2 protein.Follow by compared the efficiency of seven tags to purify protein from E.coli.The result show that the highest purity of MBP-VP2 was obtained in one step by Ni-NTA chromatography,and eluted quantity of ?-crystallin-VP2 was the maximum from Ni-NTA column.By comparing difference of conformation and antigenicity of fusion tags protein and removal tags protein.We find that this seven purified protein possess good antigenicity whether fusion tags or removal tags.Removal tag proteion MBP-VP2 was not affected the ability that VP2 protein still form VLP.In summary,seven tags are convenient and useful fusion tags that can enhance the expression,solubility and improve the purification process of the model heterologous protein and these tags may have a good prospect in protein production.2.In order to study the influence of the relevant methods for VP2 protein expression,the recombinant expression strains of E.coli BL21(DE3)p Lys S(p ET28a-VP2)were constructed.After optimizing the expression condition,the expression level of soluble VP2 was improved obviously.The AGP result revealed that the specific IBDV antibody titer of soluble extracts in recombinant bacterial was 1:64.The VP2 protein was purified by three methods: affinity chromatography,affinity chromatography combined with molecular sieve and affinity chromatography combined with sucrose gradient centrifugation,and the third method had the best purification result,in which the purity of recombinant protein can reach to 90% with Ni-NTA affinity chromatography.Furthermore,the purified VP2 protein can assemble into VLPs,which was similar to natural IBDV virion under the transmission electron microscope.In addition,the transmission electron microscope result showed that the diameter of self-assembled VLPs was different,the VLPs purified by Ni-NTA affinity chromatography had larger diameter than that purified by affinity chromatography method combined with sucrose gradient centrifugation purification.The result of dynamic light scattering indicated the exact diameter of VLPs was 25 nm.In this study,the expression level of soluble VP2 protein was improved significantly,and the preliminary exploring in VLPs formation conditions of VP2 protein laid a foundation for the further preparation of subunit vaccines.3.To study the immunogenicity of both VP2 protein and VLPs,two virus strains(attenuated strain B87,virulent strain 0504)were used as templates to express VP2 protein and prepare subunit vaccine,respectively.For the preparation of immunization vaccines,different concentrations of purified and unpurified VP2 which diluted in PBS were emulsion with equal volume of oil adjuvant according to the biological products standards,the physical character,safety test,sterility test,stability test and effect test of the vaccines were examined.Then 12 groups of chickens(32 days old,10 chickens per group)were immunized with these vaccines and controls though intramuscular injection,booster immunization was given with the same dose at an interval of two weeks.The animal experiments were carried out according to the Animal Ethics Procedures and there was no side-effect occurred during safety trials period.The serum samples were collected each two weeks and the IBDV specific antibody titers were detected by indirect ELISA.All the chickens were oral challenged with IBDV virulent strain at four weeks after the first immunization and monitored for 4 days,then the chickens were sacrificed to examine the pathological changes of buras.The results showed that the protective rate of high concentration of VLPs from B87 and 0504 strain were about 90 %,the middle AGP titer vaccines from B87 strain were about 90 %,the low AGP titer vaccines from 0504 strain were about 100 %.Furthermore,the antibody titer and protective rate of prepared subunit vaccines was obviously better than commercial vaccines.In conclusion,the subunit vaccine described in this study was safe,reliable and has good immunoprotectivity,which can effectively prevent the infection of infectious bursal disease.It has important significance and practice meaning to promote the development of poultry industry.At the same time,the candidate vaccine also have some problems,such as shelf life,immunization program and so on,which should be further studied and solved in the future. |
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