Preliminary Study Of Subunit Vaccine Of Ankara Disease And Preparation Of Infectious Bursal Virus-Like Particles

Ankara disease is a highly contagious bird disease,it predominantly infects 3-6 weeks old chicks with a mortality rate of 20-80%.Sick chicken shows sudden death,accompanied by the symptoms of a large number of pericardial effusion,liver enlargement,bleeding and comprehensive hepatitis.So the disease is also known as pericardial hydropneumatic syndrome.Ankara virus belongs to the avian adenovirus class I subgroup serotype 4 and the viral particles consist only of nucleic acids and nucleocapsid.The virus's nucleocapsid is mainly composed of 240 Hexon,12 Penton and 12 Fiber.Penton and Fiber2(Fiber's head section)are important immunogenic genes that bind to viral receptors on the cell surface and play a very important role in the process of virus-infected cells.Infectious bursal disease is a contagious infectious disease caused by infectious bursal disease virus.The disease is prone to flocks of 3-6 weeks age,it not only lead to high mortality rate,but also cause long-term and severe immunosuppression to infected chickens.Its main feature is the swelling of the bursa of Fabricius,rapid contraction after bleeding.The kidney,spleen,lymph nodes,bursa of Fabricius and other important immune organs of the sick chicken are seriously damaged.Infectious bursal disease virus is a double-stranded RNA virus that belongs to the reovirus family.IBDV particles are 30-60 nm 20-dimensional stereospecific structures.Viral particles are capsule-free and consist only of nucleic acids and nucleocapsid.The envelope of the viral nucleocapsid is almost entirely composed of VP2 protein,which is an important structural and functional protein of IBDV.At the same time,VP2 gene is also the main immunogenicity gene of IBDV.Ankara disease and infectious bursa diease are very similar in susceptible chicks age,morbidity,latency,mortality and they cause significant damage to immune system.Inflammation of Ankara disease causes serious damage to the immune system and improves the susceptibility of flocks to infectious bursa disease.The infectious bursal disease causes a chronic and severe immunosuppression in infected flocks,which is susceptible to mixed infection with the Ankara disease.Ankara disease and infectious bursal disease is not only easy to mixed infection but also improved the susceptibility of flocks to various viral diseases such as avian influenza,newcastle disease,infectious anemia infectious bronchitis and various bacterial diseases such as Escherichia coli,Salmonella,Pasteurella and so on.However,there are no commercially available vaccines for the Ankara disease on the market.In order to provide the prevention and treatment of the Ankara disease and the infectious bursa disease,and reduce the poultry industry's huge loss and significant hazards,the main contents of this paper are as follows:1.Construction of three recombinant baculoviruses of Ac-Penton,Ac-Fiber2 and Ac-VP2The Penton,Fiber2 gene was cloned into the pFastBac HTb donor plasmid,and the VP2 gene was cloned into the pFastBacl donor plasmid.Then,the correct recombinant plasmids were transformed into DH10Bac to obtained the correct Bacmid-Penton,Bacmid-Fiber2,Bacmid-VP2 by four resistance and blue-white selection.Three bacmids were transfected into Sf9 cells respectively,and Ac-Penton,Ac-Fiber2,Ac-VP2 were obtained after 3-5 days when the cell lesions were observed.2.Expression,optimization and purification of Penton,Fiber2 and VP2The three recombinant strains were inoculated with healthy Sf9 cells,and lesions were observed after 3 days.The expression of Penton,Fiber2 and VP2 was identified by Western blot and indirect immunofluorescence assay,and the protein expression conditions were optimized and the minimum dose was explored.Then,the three proteins were subjected to extensive expression and purification.3.Penton,Fiber2 related subunit vaccine's preparation and primary studyCombine Penton,Fiber or the equal mixture of the two with the adjuvant ISA 71 VG by mass ratio of 3:7 to prepare the vaccine to immunize SPF chickens respectively.Challenge assay showed that the three vaccines were able to achieve an 100%protection rate for Ankara disease.Lesion observation and tissue biopsy can show that the three vaccines have a good immune protection effect And all the three vaccines can stimulate the immune chickens to produce highly efficient and durable antibodies,in which Penton protein as antigen vaccine can produce the highest antibody titer,and can maintain the longest positive antibody.4.Purification and electron microscopic observation of IBDV virus-like particlesThe VP2 protein was expressed in Sf9 cells in a large amount,and the IBDV virus-like particles were collected in the supernatant of the cell sample and the culture medium supernatant,followed by sucrose density gradient centrifugation.Western blot analysis showed that the infectious bursal disease virus particles were in the band with sucrose density gradient of 30%.Take the corresponding strip denitrification treatment,then 30-60nm and form complete IBDV virus-like particles could were observed by electron microscopy.