Bionomics Studies Of Recombinant Fowlpox Viruses Coexpressing GPMV F Gene And GPV VP3 Gene

In the study, the preliminary studies on the physical and chemical properties of rFPV-VP3-F, genetic stability of rFPV-VP3-F, expressing of F gene and VP3 gene in CEF and the expressing of F gene and VP3 gene in the chicken were carried out.The virus was purified, and then the existence and expression of the foreign genes were identified by PCR and Dot-ELISA. In the study of physical and chemical, the recombinant fowlfox virus(rGPV-vp3-F) which has been treated by physical and chemical factors were inoculated into CEF, then the different infection titer were observed between the treatment and untreatment viruses and part of the chemical property were verified. The optimum viral multiplication were definited by observed the effects of different cell number and different MOI. When the cell density is 105/cm2 and MOI is 0.01, the titer of the virus will achived to 3.08×10~5/100ul.In the genetic stability studies of recombinant fowlpox virus, the recombinant fowlpox virus on the CEF were passaged for 20 generations.Then, the 5th,10th,15th and 20th generations of recombinant fowlpox were chosen to identify the F gene and VP3 gene's genetic stability.The result shown that bule plaque percentage of these four generation is 100%. No white plaque were observed which may occur when the foreign genes were loss. Use the 5th,10th,15th and 20th generations of recombinant fowlpox virus DNA as a template for PCR amplification, 460bp F partly amplified gene fragment and 657bp VP3 partly gene fragment were acquired. Using the other two pairs of primer, a total length of the F gene and total length VP3 gene were amplified. After sequencing, the gene order of 5th,10th,15th and 20th generations were compared with the gene order of original generation. F gene of GPMV had two nucleotide changed, but amino acid didn't changed. VP3 gene of GPV had three nucleotide changed and amino acid didn't changed too. Using indirect immunofluorescence test to identify the expression of F gene and VP3 gene of the 5th,10th,15th and 20th generation recombinant fowlpox virus. Results from experiment shown that the two foreign genes of the recombinant fowlpox can be stably expressed in CEF.The recombinant fowlpox have good genetic stability . The foreign gene didn't be lost in the process of passage and F gene and VP3 gene can express corresponding protein correctly.35 days old chicken were inoculated with the rFPV-VP3-F by 106PFU /ml. Blood were collected 7d, 14d, 21d, 28d and 35d after immunization and the serum were seperated. The antibody levels anti-GPV-F were detected through neutralization test and the antibody levels anti-GPV-VP3 were detected through indirect ELISA. The results shown that the recombinant fowlpox virus rFPV-VP3-F can express the corresponding proteins of inserted foreign genes and induce corresponding antibodies in chicken.