CHO (Chinese hamster ovary) cell strain with high-efficiency expression of CD2V protein of African swine fever (ASF)
An African swine fever, high-efficiency expression technology, applied in genetically modified cells, cells modified by introducing foreign genetic material, animal cells, etc., to achieve the effect of easy purification and high expression
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Embodiment 1
[0014] Embodiment 1, the construction of the recombinant plasmid expressing CD2V gene
[0015] 1.1 Cutting and optimization of CD2V gene
[0016] After analyzing the structural domain of the CD2V gene whose nucleotide sequence is SEQ ID NO: 1 (the amino acid sequence of the encoded protein is SEQ ID NO: 2), the C-terminal 150aa of the protein is cut off, so as to better help The protein folds better into tertiary structure, which exposes its antigenic sites better. For the convenience of protein purification and later detection, 6 His amino acids were added to the C-terminus. Since there are many rare codons in the CD2V nucleotide sequence, the nucleotides were optimized for codons, so that the CD2V protein can be efficiently expressed in CHO cells.
[0017] The nucleotide sequence of the optimized gene is SEQ ID NO: 3, and the amino acid sequence after optimization and modification is SEQ ID NO: 4, which can better express the antigenic site.
[0018] 1.2 Construction of 1...
Embodiment 2
[0071] Example 2: Transfection of CHO-K1 cells
[0072] (1) Preparation: Ultraviolet sterilization in a biosafety cabinet for 30 minutes; F-12 culture solution was placed in a 37°C water bath and preheated to 37°C.
[0073] (2) Add 2.5 μg of the recombinant plasmid DNA and P3000 prepared in Example 1 to 125 μl of OPTI-MEM culture medium, and mix well. Add 3.75 μl lipotectamine3000 to 125 μl OPTI-MEM culture solution and mix well. The liposomes were mixed with the recombinant DNA and allowed to stand at room temperature for 10 min.
[0074] (3) Take out the 6-well plate cells that were plated 24 hours ago from the 37°C incubator, discard the supernatant medium, wash the cells three times with pre-warmed OPTI-MEM culture medium, and discard the OPTI-MEM culture medium.
[0075] (4) Add 2 ml of F-12 culture medium with 10% and 1% double antibody fetal bovine serum to each cell well.
[0076] (5) Gently add the mixture of recombinant DNA and liposomes into each well of cells, m...
Embodiment 3
[0079] Embodiment 3: protein purification and detection
[0080] 4.1 Expression and identification of recombinant CD2V protein in CHO cells
[0081] The cells selected in Example 3 were cultured at 37°C for up to 240 hours, and the supernatant was collected. The collected protein solution was subjected to SDS-PAGE protein analysis. It was found that the CD2V gene before optimization was not expressed in CHO cells, but the optimized CD2V gene was expressed in CHO cells, and its protein content was about 3 mg / L.
[0082] 4.2 Chromatographic purification of recombinant CD2V protein
[0083] The collected CD2V supernatant was subjected to affinity chromatography with Ni Sepharose excel medium of GE. The collected supernatant was centrifuged at 5000rpm for 5min and used as the loading solution.
[0084] 4.2.1 Wash the medium with 5 times column volume of distilled water.
[0085] 4.2.2 Wash the medium with 5 column volumes of equilibration buffer.
[0086] 4.2.3 Sample loadin...
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