Cloning And Expression Of The Main Structural Protein Of Infectious Bursal Disease Virus

Infectious bursal disease virus(IBDV),the causative agent of infectious bursal disease(IBD),causes immunosuppression in young chickens by destroying the precursors of B lympHocytes in the bursa of Fabricius. And enhanced any other blight susceptibility.In 1957,the disease first broke out in broilers in Gumboro,Telahua state of America.In the 1980s,the variation strain of IBDV was first appeared in America. Acute IBD caused by very virulent infectious bursal disease virus(vvIBDV) was first reported in Europe at the end of 1980s.The acute form of the disease were then transmitted to Japan in the early 1990s,and then rapidly spreaded all over Asia and other major parts of the world.The IBDV has beeen the cause of significant economic losses in poltry industry for a long time.Recent years,molecular biology research of infectious bursal disease virus achieved great advance,but the study of immune and pathogenic mechanism,molecular structure and function of infectious bursal disease virus need further research.Thus,we had identified the function of the main structural protein of infectious bursal disease virus which is valuable for further research such as the preparation of monoclonal antibody to get a rapid diagnosis kit about this kind of virus which is useful for prevention and cure it.The research divide three parts:Part one: Isolation and Identification of Shandong IBDV strainCollecting pathological tissue from inoculated chickens in Shandong province,according to the epidemiology and clinic symptom of infected chickens, virus isolation,results of tissue pathology,agar gel immunodiffusion test(AGID),molecular biology diagnosis,animal regression test,sequence homology analysis,one IBDV strain had been isolated and identified. and we named it as IBDV SD strain.Part two: Expression of IBDV Structural Protein VP2 Gene in BL21(DE3)plysS and Preparation of immune serumIBDV structural protein VP2 gene is protective protein and related with pathogenicity which is foundation of rapid diagnosis kit.By RT-PCR,the structural protein gene VP2 of infectious bursal disease virus (IBDV) was determined from segment A of IBDV.The gene was cloned into expressing vector PGEX-4T-3.The recombinant plasmid named PGEX-VP2 was constructed. The PGEX-VP2 was used to transform into E. coli BL21(DE3) plysS. The results of SDS- PAGE and Western blot indicated that the VP2 protein was expressed in high level which was about 69 KDa , the high titer anti-VP2 serum was also prepared in BALB /c mice immunized with purified VP2 fusion protein inclusion.ELISA results showed that titer was above 1:5120 and Western blot analysis showed that the expressed protein VP2 reacted with polyclonal antibody,It explained that the expressed protein VP2 possess specific satisfactory immunological reaction.had immunological reactive activity.Part three: Clone Expression and Biologic Activity Identification of VP3 Gene of IBDV Shandong strainIn order to study the relataion to pathogenicity or immunogenic of Shandong strain IBDV VP3 gene,By PCR,a structural protein gene VP3 of infectious bursal disease virus (IBDV) was determined from segment A of IBDV.The gene was cloned into expressing vector PET32a.The recombinant plasmid named PET-VP3 was constructed.The PET-VP3 was used to transform into E.coli BL21(DE3).The results of SDS-PAGE and Western blot indicated that the VP3 protein was expressed in high level and mainly existed as inclusion bodies,which was about 57 KDa,had immunological reactive activity.Purify the protein and immune chicken with the protein,and then give IBDV to the chicken, the result shows that IBDV VP3 gene can withstand virus.This study lay on foundation for the development of the serological diagnosis and the preparation of genetically engineering vaccine.