Plant expression vector utilizing beer yeast MF-alpha to mediate recombinant protein secretion
A technology of plant expression vector and brewer's yeast, which is applied in the field of plant expression vector to achieve the effect of improving the enrichment effect
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Embodiment 1
[0041] Example 1: Obtaining of exogenous α signal peptide and Egfp gene
[0042] The α signal peptide gene is derived from Saccharomyces cerevisiae MF-α, and the Egfp reporter gene is derived from the plant expression vector pKGWFS7,0.
Embodiment 2
[0043] Example 2: Construction of plant expression vector α signal peptide-Egfp
[0044] 1. The subcloning vector of PUC57-MF-α-Egfp synthesized by Shuoqing Biotechnology Co., Ltd.
[0045] Strategies for constructing plant expression vectors such as figure 1 As shown, look for the upstream of the Egfp reporter gene α signal peptide derived from Saccharomyces MF-α and the derived substance expression vector pKGWFS7,0 plus the enzyme cleavage site SalⅠ restriction site (GTCGAC), and the downstream of the Egfp reporter gene plus enzyme cleavage Site EcoRⅤ restriction site (GATATC). Send to Kunming Shuoqing Biological Company to synthesize the subcloning vector of PUC57-MF-α-Egfp, after the synthesis is successful. Plasmid DNA was extracted by alkaline lysis method, and the recombinant plasmid whose size was consistent with the theoretical value was selected by 1% agarose gel electrophoresis for further double enzyme digestion detection.
[0046] 2. Construction of the interm...
Embodiment 3
[0050] Example 3: Plant expression vector PK-35S-MF-α-Egfp transformed into Agrobacterium and screening of transgenic tobacco and geranium
[0051] Take a small amount of the correctly detected plant expression vector PK-35S-MF-α-Egfp plasmid and add it to the Agrobacterium competent cells, mix gently; add the mixture to the cold electroporation cup, tap the cup body to mix to the bottom of the cup; place the electric cup in the chute of the electrotransformer, and conduct electric shocks with the parameters of 200 ohms and 2.5kV / 0.2cm. Centrifuge at 200rpm at 28°C for 3-5h in a centrifuge tube; centrifuge at 7500rpm for 1min at room temperature, discard the supernatant to 100μL to suspend the cells; spread the bacteria on LB solid culture containing spectinomycin (Spe) antibiotic Cultured at 28°C on the base for 2 days; positive clones were selected and tested by bacterial liquid PCR (the primers used were the upstream primer of α signal peptide and the downstream primer of E...
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