| Summary: | 碩士 === 國立中興大學 === 獸醫學系暨研究所 === 103 === Chicken infectious anemia virus (CIAV) is the causative agent of chicken infectious anemia (CIA). CIA distributes globally, affecting all countries and presenting economic threat especially to the broiler industry and the producers of specific-pathogen (SPF) eggs. Though live vaccines are now in use, the recommended age is over 6 weeks of age and can’t offer complete protection to young chickens. Thus, the purpose of this study was to construct the plasmid which inserted target genes and developed a recombinant fowlpox virus (rFPV) co-expressing VP1 and VP2 genes of CIAV by homologous recombination. The strain of CIAV was sampled from clinical cases and helped determine a high pathogenicity strain after cloning and sequencing. A recombinant plasmid was constructed with TK, GFP, VP1 and VP2 genes of CIAV. TK gene was separated to 2 segments by specific restriction sites and was inserted into the on multiple cloning sites of the pGEX6P-1 vector. The separated TK gene segments flanked GFP gene, VP1 and VP2 genes of CIAV were inserted in the pGEX6P-1 vector to generate a pGEX6P-1/TK/GFP/CIAV VP1/VP2 recombinant plasmid. Next, to generate rFPV, DF1 cells were infected by FPV as parental FPV and then transfected with recombinant plasmid at the same time. After homologous recombination with parental FPV, green fluorescence could be seen by fluorescence microscope when recombination occurred. After observing the first generation of recombinant fowlpox virus, indirect immunofluorescence assay (IFA) tests were performed to confirm the expression of CIAV proteins.
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