Recombinant duck viral enteritis virus, preparation method and applications
A duck virus enteritis, recombinant virus technology, applied in the directions of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of application limitation, poor immunogenicity, slow immunity generation, etc., and achieve a simple construction method. , High protective efficacy, good cross-protection effect
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Embodiment 1
[0037] Embodiment 1, the construction of GFP transfer vector plasmid
[0038] 1. Obtain the GFP expression cassette
[0039] The GFP expression cassette is an expression cassette of green fluorescent protein (green fluorescent protein), and its sequence is shown in SEQ ID NO:2. The GFP expression cassette contains pHCMV promoter, green fluorescent protein gene GFP and termination sequence. Using the DNA of the recombinant strain pHA2 corr as a template and Primer GFP-UL55 F and Primer GFP-UL55 R as primers, the GFP expression cassette was amplified by gradient PCR.
[0040] Primer GFP-UL55 F (SEQ ID NO: 4): 5'-TATCCCGGGTTAACCGGGCTGCATCCGAT-3';
[0041] Primer GFP-UL55 R (SEQ ID NO:5): 5'-ATACCCGGGCGAAGTTATGCGGCCATTTA-3'.
[0042] The reaction system of the gradient PCR method is: 10 × PCR Buffer 5 μL, dNTPs (2.5 mM each) 4 μL, Mg 2+ (25 mM) 3 μL, each 2 μL of upstream and downstream primers (10 pmol / μL ), 0.5 μL of ExTaq enzyme, 2 μL of DNA template, dd H 2O 31.5 μL (PCR ...
Embodiment 2
[0056] Embodiment 2, the acquisition and identification of GFP recombinant duck viral enteritis virus
[0057] The 9-10-day-old SPF chicken embryos were taken, and primary and secondary chicken embryo fibroblasts (Primary chicken embryo fibroblasts, CEF) were prepared and cultured according to conventional methods. Inoculate a single layer of chicken embryo fibroblasts with duck plague chicken embryonized attenuated strain C-KCE at a multiplicity of infection (MOI) of 0.05, and culture for about 72 hours. Collect the cells when 95% of the cells have lesions, and follow the routine Methods Viral DNA was extracted from diseased cells, DNA was quantified by a gel scanner, and the DNA concentration was adjusted to 0.1-0.2 μg / μL. The DNA of the transfer vector pDEV-GFP (UL55) was extracted from the recombinant bacteria by alkaline lysis, quantified by Eppendorf Biophotometer (or quantified by agarose electrophoresis measurement), and the concentration was adjusted to 700 ng / μL. Tr...
Embodiment 3
[0058] Example 3. Construction of avian influenza virus hemagglutinin (HA) gene expression cassette and its transfer vector
[0059] 1. Obtaining the hemagglutinin (HA) gene of avian influenza virus
[0060] The hemagglutinin (HA) gene sequences of H5N1 avian influenza published on GenBank since 2010 were used for multiple sequence alignment using MEGA software to obtain consensus sequences. The base at each position of the consensus sequence is the one with the highest frequency among the four bases (A, C, G, T) at the same position in the HA sequence. Remove the basic amino acid cleavage site between the HA1 and HA2 fragments in the consensus sequence, and then refer to the H5N1 avian influenza virus hemagglutinin (HA) gene sequence of the newly popular 2.3.2.1 lineage (GenBank: AB700635.1; JN986881 .1; JN986882.1; JN646713.1; JN646716.1; HQ020376.1; CY098758.1; JF975561.1; ), the optimized design obtained the avian influenza virus hemagglutinin (HA) gene of the present inv...
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