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Preparation of DuCV Nucleic Acid Probe and Subcellular Distribution of Cap Capsid Protein

Author: ZouJinFeng
Tutor: JiangShiJin
School: Shandong Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Duck circovirus Cap gene Baculovirus expression system Indirect immunofluorescence Nuclear localization signal
CLC: S858.32
Type: Master's thesis
Year: 2011
Downloads: 35
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Abstract

Duck circovirus the (Duck circovirus, DuCV) is a the circovirus Division (Circoviridae) circovirus new members of the genus (Circovirus). DuCV infection can lead to the duck feathers messy, growth retardation, weight loss and other symptoms. This study to establish a nucleic acid probe method to detect the circovirus the duck, and epidemiology of this method of duck circovirus investigation, at the same time with the baculovirus - insect cell expression system DuCV Cap gene expression and its The nuclear localization signal. The infectious clone of DuCV template preparation detection DuCV a nucleic acid probe, the study showed that the specificity of the probe, and specific hybridization occurs only with DuCV nucleic acid, with control duck Ⅰ type of viral hepatitis (DHV-1) virus, duck plague virus (DPV) nucleic acid hybridization reaction was negative;, high sensitivity, minimum detectable amount 5pg; easy to use, suitable for batch testing. Application of the probe, total 1025 copies organ of tissue DNA detection of the 196 ducks diseased collected from Shandong, Jiangsu, Sichuan and other places, the result the individual positive rate was 33.2%, in the various organs in the bursa of Fabricius, liver spleen DuCV higher detection rate, the detection rate was 52.3%, 49.2%, 38.5%. Complete the Cap gene was amplified by PCR, and cloned into the correct reading frame the pFastBac TMHTB baculovirus vector will be screened positive recombinant vector pF-CP, transformed to contain a baculovirus shuttle vector and auxiliary vector DH10Bac E. coli competent cells, blue-white screening and PCR amplification, obtained recombinant expression the carrier rBacmid-CP. Liposome-mediated transfection of recombinant baculovirus Sf9 insect cells after 72 hours. Westernblot and indirect immunofluorescence assay (IFA) test results show that the study successfully constructed a recombinant baculovirus expression of the of duck circovirus Cap protein expressed protein prokaryotic expression Cap protein immunized mice collected serum with good reactogenicity . There are 36 DuCV FJ0601 strain Cap protein N-terminal basic amino acid (AA), software PROSITE analysis found that in this area there are two two separatist nuclear localization signal (Bipartite nuclear localization signal), respectively, in the Cap protein N-terminal 2 to 17 AA and 21 to 36 AA. For further study of the of duck circovirus Cap protein biological function, we constructed a series of fusion expression plasmid Cap, its nuclear localization signal. Strong green fluorescent protein (EGFP) to insertion of pFastBac ^{TM HTB carrier build the pF-EGFP recombinant plasmid, and then build pF-EGFP-CP the pF-EGFP-CP-Δ17N (N end missing 2 -17 AA) and pF-EGFP-CP-Δ36N (N end missing 2-36 months AA) three recombinant plasmids were transfected into Sf9 insect cells. The results show the pF-EGFP-CP, pF-EGFP-CP-Δ17N protein located in the nucleus, while the pF-EGFP-CP-Δ36N protein was localized in the cytoplasm, the results confirmed a the PROSITE software predicts DuCV Cap in approved bit signal functional nuclear localization signal.}

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Poultry > Duck
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