Duck alpha interferon and its mutant, preparation method and application
A technology of alpha interferon and mutant, which is applied in the preparation of the duck interferon alpha mutant by using the silkworm baculovirus expression system, the preparation and application of the duck interferon alpha mutant, and can solve the problem of late start of research and duck interference. Issues such as the lack of prime research
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Embodiment 1
[0081] Example 1 Expression and detection of DuIFN-α original sequence in silkworm bioreactor
[0082] 1. Experimental method
[0083] 1.1 Obtaining the original sequence of duck interferon alpha
[0084] 1.1.1 In vitro stimulation of duck peripheral blood mononuclear cells (PBMC)
[0085] Ducks were collected aseptically for peripheral anticoagulation, and lymphocyte separation medium was used to separate peripheral blood mononuclear cells and red blood cells. Duck T cells were further isolated by the specific adsorption of nylon wool. In a 24-well cell culture plate, add 1 mL of 8×10 6 mL -1 The above-mentioned PBMCs were incubated at 37℃ for 1-2h to make antigen-presenting somatic cells (APCs) adherent. Separated T lymphocytes (8×10) were added to the above-mentioned 24-well plate with APC cells 6 mL / well) and red blood cells (4×10 6 mL / well) and phytohemagglutinin (PHA) at a final concentration of 5 μg / mL, incubated at 37 °C for 4 h, and collected the cells.
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Embodiment 2
[0112] Example 2 Expression and detection of optimized sequences of DuIFN-α in silkworm bioreactors
[0113] 1.1 Construction of codon-optimized mutant gene of duck interferon alpha
[0114] By analyzing all the amino acid sequences of duck interferon alpha on NCBI, its consensus sequence was obtained. After comparing with the original amino acid sequence of duck interferon alpha cloned in Example 1, the amino acid sequence of the consensus sequence was consistent with the original amino acid sequence, and its amino acid sequence was consistent with the original amino acid sequence. The sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.
[0115] The present invention utilizes OptimumGene TM The technology optimizes the above-mentioned duck alpha interferon consensus sequence gene, and modifies the gene sequence according to the codon preference of the bioreactor silkworm, which affects the GC content and CpG dinucleotid...
Embodiment 3
[0135] Example 3 Expression and detection of DuIFN-α-O in silkworm bioreactor after single amino acid site mutation
[0136] 1. Experimental method
[0137] 1.1 Construction of duck interferon alpha mutant gene
[0138] The present invention uses the gene sequence of DuIFN-α-O as a template, and designs multiple pairs of primers to carry out site-directed mutation of the sequence. The site-directed mutation is carried out by the method of fusion PCR. For the method of fusion PCR, see the aforementioned "2. Experimental method".
[0139] The mutation sites are P2A, L19T, P25L, R35P, D38N, N51D, H64N, L73I, Q80R, D95K, H108D, Q117R, H123Y, R127Q, L136S, R142C, I148T, L159F, D170E or R182C; the resulting duck alpha interference The mutants were named DuIFN-α-O-M1 (P2A, L19T, P25L, R35P, D38N, N51D, H64N, L73I, Q80R, D95K, H108D, Q117R, H123Y, R127Q, L136S, R142C, I148T, L159F, D170E or R182C) mutant.
[0140] Primers required for amino acid single-site, double-site and multi-s...
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