| Doctor candidate: PENG DAXINAdvisor: Prof LW XTUFANIn order to develop Marck抯 disease (MD) genetic engineering vaccines which are safe, convenient for use and with high protective efficacy, We designed and completed a series of experiments to reach the goal. Novel nonessential fragments of fowlpox virus (FPV) were identified, and the best one was selected by comparison to construct transfer vector, synthetic promoter, which is 3.5 times stronger than the P7.5 promoter of vaccinia virus was constructed and used for vector construction; recombinant FPVs expressing MDV gB or coexpressing MDV gB and type II interferon were constructed and assayed; recombinant viruses were evaluated for their protective efficacy in vaccination trials with specific-pathogen-free(SPF) and commercial chickens.1. Optimization of newly identified nonessential fragments of Fowlpox virus for vector constructionThe LacZ gene from plasmid pSV-Gal was inserted into the downstream of P7.5 to form plasmid pB7.5. For construction of Fowlpox virus (FPV) transfer vectors, the P7.5-LacZ cassettes from pB7.5 was removed and inserted into nonessential regions of FPV in plasmid pFPV18, pFPV7s and pFPV9s to form pFBI8, pFB7s and pFB9s. These transfer vectors were lransfected with DOS PER liposome into chicken embryo fibroblast(CEF) monolayers pre-infected with Chinese vaccine strain 282E4 of FPV. Recombinant viruses were purified by selection of plaques expressing ~ -galactosidase in secondary CEF cultures overlaid with agar containing X-Gal. The viruses were propagated and titrated by the plaque assay in CEF. The level of the ~ -galactosidase expressed by rFPVs in CEF extraction was assayed and compared at 72 hours post infection. Results suggested that the pFPV7s was the best nonessential fragment suitable for foreign gene insertion, in terms of the stability and intensity of foreign gene expression in recombinant viruses.MDV gB ~LU~ gB ~UX~ Y ~2.Synthesizing and selecting stronger promoters for transfer vector constructionFour olignucleotides were synthesized according to the essential structure of the vaccina virus promoter P7.5 and were used to generate an early (E) and a late(L) promoter. Ten artificial promoters designated Psl-1O were cloned into plasmid pUC18 by using different numbers and combinations of early and late promoters in tandem, selected by colony hybridization and identified by restriction endnuclease cleavage patterns and sequence analysis. The LacZ gene was removed and inserted into the downstream of different promoters, then the Ps-LacZ cassettes were inserted into pFPV7s respectively to form transfer vectors pFBS1-10. rFPVs containing different promoters were constructed through the method of homologous recombination. Extracts of CEF infected with rFPVs were prepared at 10, 24, 36, 48 and 72 hours post infection. The quantity and level of P -galactosidase expression were assayed by colorimetry in the presence or absence of cytosine arabinoside. Results showed that the LLEE combination was the strongest synthetic promoter which drove expression of P -galactosidase 3.5 times higher than that of P7.5.3.Construction of recombinant fowlpox (rFPV) virus expressing MDV gB under the control of the strong promoterFPV insertion vector was constructed with promoter LLEE and P11 -LacZ marker gene cassette in opposite direction which flanked with nonessential fragment from FPV7s. MDV CVI 988 gB was inserted into the downstream of LLEE to form transfer vector pFGBS. Recombinant fowlpox viruses were selected by blue plaque and the expression of MDV gB by rFPV was confirmed by indirection immunofluorescence assay (IFA). Expression level of MDV gB driven by promoter LLEE in rFPV was 3 times higher than that of MDV gB driven by viccina virus P7.5 promoter.4.Construction of recombinant fowlpox virus (rFPV) coexpressing MDV gB and type II interferonRecombinant fowlpox viruses coexpressing MDV gB and chicken type II interferon(IFN-II) gene was constructed by using diff... |
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