Recombinant carrot antifreeze protein gene CaAFP and application thereof
A technology of antifreeze protein and carrot, which is applied in application, genetic engineering, plant genetic improvement, etc. It can solve the problems of difficult analysis and purification of target protein, and achieve the effect of simple extraction process, simple operation, and reduced damage
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Embodiment 1
[0045] Example 1 Construction of genetically engineered strain Pichia pastoris-pPIC9K-CaAFP1 and strain Pichia pastoris-pPIC9K-CaAFP2
[0046] Add restriction enzymes EcoR I and Not I to the two gene fragments with artificially synthesized amino acid sequences of SEQ ID NO.01 and SEQ ID NO.02, respectively, and then connect them with the vector plasmid pMD18 to obtain Recombinant plasmids pMD18-T-antifreeze1 and pMD18-T-antifreeze2.
[0047] The plasmids pMD18-T-antifreeze1 and pMD18-T-antifreeze2 were double digested with restriction enzymes EcoR I and Not I, and then the genes CaAFP1 and CaAFP2 were obtained by gel recovery; then they were ligated to the same restriction using T4 DNA ligase. The plasmid pPIC9K digested with endonuclease EcoR I and Not I was used to construct recombinant expression vectors pPIC9K-CaAFP1 and pPIC9K-CaAFP2, and transform them into E. coli JM109, and select them on the LB solid medium plate containing ampicillin resistance Positive recombinants were...
Embodiment 2
[0049] Example 2 Expression of Shake Flask Level Genes CaAFP1 and CaAFP2
[0050] Using the Pichia pastoris-pPIC9K transformed with the empty pPIC9K vector as the experimental control, single colonies of Pichia pastoris-pPIC9K-CaAFP1, strain Pichia pastoris-pPIC9K-CaAFP2 and Pichia pastoris-pPIC9K were respectively inoculated into 50 mL of BMGY liquid medium. Culture in a 250mL shake flask at a constant temperature of 200rpm and 28℃ shaking to the logarithmic growth phase, namely OD 600 For 3~6. At room temperature, the yeast cells were collected after centrifugation at 3000×g for 5 min. The supernatant was discarded, and the cells were resuspended in a 500 mL shake flask with 100 mL BMMY liquid medium. Then, the culture was continued at 200 rpm and 28°C and the expression was induced with methanol.
[0051] After the induction of expression started, 100% methanol was added to the fermentation broth to maintain induction every 12h until the final concentration of methanol in the ...
Embodiment 3
[0055] Example 3 Expression of CaAFP1 and CaAFP2 genes in a 7L fermentor
[0056] Pichia pastoris-pPIC9K-CaAFP1, Pichia pastoris-pPIC9K-CaAFP2 and Pichia pastoris-pPIC9K single colonies were respectively inoculated into a 250mL shake flask containing 50mL seed medium, and cultured at 30℃, 200rpm on a shaker 20h, as the fermentation tank culture seed liquid.
[0057] The seed liquid was inoculated in a 7L fermentor with an inoculum volume fraction of 10%, and the initial liquid volume was 2.5L. During the fermentation process, the dissolved oxygen concentration is maintained above 10%. The whole fermentation process is divided into three stages: glycerol batch culture, glycerol flow addition (including transient glycerol and methanol mixed addition) and methanol induction culture. First, culture was performed in batches at 28°C and pH 6.0 for 10 hours until the glycerol was exhausted (shown as a sudden increase in DO). In the second stage, the DO-Stat method was used to feed a ba...
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