| Goose parvovirus is the etiological agent of Derzsy’s disease, which is referred to as Gosling plaque in China. This disease is characterized by anorexia, prostration and death in 30-day-old goslings, and is a serious threat to the goose industry worldwide. Diagnosis and prophylactic vaccination are the potential ways to reduce the GPV infection burden. Currently the attenuated vaccine is the the only choice on the goose parvovirus vaccine market. The weak attenuated vaccine strain was produed from goose embryo or Muscovy duck embryo continuous passage of the domestication and had no pathogenicity of adult Geese and gosling. However, there are high production costs, exogenous pathogenic difficult to control system, weaker protection forces and the reversion to virulence of disadvantages to gosling plague pandemic control. Therefore the development of a new type of Goose parvovirus vaccine, to solve the defects of the existing vaccine has become a pressing matter of the moment.In the Shandong area, one Goose Parvovirus Strain was isolated and identificated, and its VP3 gene sequence was analyzed. A strain of GPV was collected and identified from gosling hepar of affected young layer by PCR, and its biological characteristics was studied by the virus pathogenicity to goose embryos and the protection of egg-yolk antibodies test. The VP3 gene of the virus was cloned and sequenced. The Goose Parvovirus Strain "BZ" strain was isolated and identificated succesfully; the sequence of its VP3 gene was subjected to the NCBI (Accession No. GQ253324); the homology of the VP3 gene of the "BZ" strain and the other 13 strains at home and abroad is 95.8% or more, and there are the close genetic relationship and the little variation between them. The strain has a strong pathogenicity to 3 day old goslings, the LD50 (0.2 mL) of goose embryo allantoic fluid is 10-2.68 to 3 day old Gosling, the LD50 (0.2 mL) of the fifth generation of goose embryo culture is 10-6.32 to the goose embryo.In order to build a subunit vaccine candidate strain of Goose parvovirus, the prediction of Goose parvovirus VP3 protein antigen epitope distributions was done by the software optimization analysis, and the primers of antigenic epitopes of enrichment region were designed. The target VP3 DNA fragment was got by PCR and was oriented inserted into pET32a expression vector. The new expression vector was named to be pET32a-VP3 and transformed into E.coli BL21 expression strains. Then the E.coli BL21 (pET32a-VP3) was induced to access the target VP3-2 protein. The neutralizing titer of the antiserum from SPF chicken immunized with VP3-2 protein was measured to test its immunogenicity. The results showed that the prokaryotic expression vector of pET32a-VP3 was successfully constructed, and the objective protein was in the presence of soluble form. The anti-blood of the protein (PD50) can reach 10-1.91. Conclusion:the soluble VP3-2 protein with good immunogenicity was successfully obtained.Through the study of feeding method and control of dissolved oxygen, promote the high expression of Cap protein. Control oxygen levels by fermentation automatic detection system, determination of glucose content by using the biosensor analyzer, determination of acetic acid by HPLC, Cell density was measured using a spectrophotometer.Tthe effect of different dissolved oxygen (DO) levels and DO stage control strategies on production of VP3-2 protein were investigated, and the results indicated that the DO level controlled at 60%(0-4 h) and 30%(4-10 h) was more effective for reduction of acetate accumulation and improvement of cell density and VP3-2 protein production. Furthermore, the DO feedback feeding was used and the DO level was maintained at 60%(0-4 h) and 30%(4-10 h) by adjusting the feeding rate of glucose, the accumulation of acetate decreased to 0.716 g/L that was decreased by 39.12% compared with that with DO level of 15%, and the optical density of cell and production of VP3-2 protein were 2.256 and 85.64 mg/L, which were 1.28-times and 1.32-times higher than these obtained with DO level of 15%, respectively.The existence form of VP3-2 protein after high density fermentation was inclusion body. Inclusion bodies in the process of dilution, CBS (pH9.6) is the buffer solution, and the urea (6M) as the buffer solution is the inclusion body. VP3-2 proteins with immunogenicity are obtained by dilution, and Gosling plague subunit vaccine antigen industrialization preparation process was completed successfully. Gosling plague propolis inactivated vaccine was prepared with VP3-2 protein as antigen. The relevant test and potency test was done, and the tests were in line with the current vaccine standards. The VP3-2 protein has the potential to develop new gosling plague vaccine. |
PDF Full Text Request