Serine Protease Inhibitor cvt-spi Gene and Its Application
A technology of serine protease and P. platycodonis, which is applied in the fields of genetic engineering, protein engineering, and molecular biology, and can solve problems such as harm to the safe production of crops, pesticide residue environment, pollution, etc., so as to inhibit melanization and weaken immunity. effect of reaction
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Embodiment example 1
[0033] cDNA Cloning of the Serine Protease Inhibitor CvT-SPI Gene in the Teratocytes of Plutella xylostella
[0034] 1.1 Collection of abnormal cells
[0035] After being fed for 3, 5, and 7 days, the parasitized diamondback moths were removed from the cabbage leaves, sterilized with 75% ethanol, and placed in ampicillin (ampicillin) with a final concentration of 50 ng / μl. ) and kanamycin (Kanamycin) HyClone SFX-Insect (Thermo Scientific) cell culture medium, carefully tear the epidermis with dissecting tweezers under a stereo dissecting microscope to prevent damage to the organs of diamondback moth, and dissected Plutella xylostella residues and parasitoid larvae were removed from the Petri dish. Place the culture dish containing the teratocytes and host hemolymph at room temperature for 30 minutes to allow the host blood cells to adhere to the wall of the culture dish. The teratocytes were aspirated one by one with a pipette, and washed 5 times in Hyclone cell culture medi...
Embodiment 2
[0058] Prokaryotic Induced Expression and Purification of Serine Protease Inhibitor CvT-SPI Gene of Braconis xylostella xylostella
[0059] 2.1 Construction of recombinant expression vector of CvT-SPI
[0060] (1) Design primers according to the full length of the obtained CvT-SPI sequence. The forward and reverse primers also have BamH I and XhoI restriction sites respectively to amplify the open reading frame of the gene and remove the signal peptide. Using cDNA as a template, use 50 μl The LA-taq enzyme system was used for PCR amplification, and the amplified PCR product with restriction sites was subjected to agarose gel electrophoresis, and the target band was excised for DNA gel recovery.
[0061] (2) Using the PET-28a expression vector plasmid and the DNA recovered in the first step as DNA templates, prepare 50 μl of a double enzyme digestion system: restriction enzyme BamH I 2.5 μl, restriction enzyme Xho I 2.5 μl, DNA Template or expression vector plasmid 20μl, enzym...
Embodiment example 3
[0076] Activity Detection of Prokaryotic Expression of Recombinant Protein of Serine Protease Inhibitor CvT-SPI Gene of Braconis xylostella Plutella Plutella Teratocytes
[0077] 3.1 Inhibitory activity of the prokaryotic expression recombinant protein of the serine protease inhibitor CvT-SPI gene in the teratogenic cells of Braconis plutella xylostella to commercial serine proteases
[0078] The proteases detected in this experiment were bovine pancreatic chymotrypsin (Sigma), bovine pancreatic trypsin (Sigma), porcine pancreatic elastase (Sigma) and human plasma thrombin (thrombin from humanplasma, Sigma) and subtilisin A (subtilisin A from Bacillus Licheniformis, Sigma), the corresponding substrates are N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Sigma S-7388), Nα-Benzoyl -L-arginine 4-nitroanilide hydrochloride(Sigma B-3133),N-Succinyl-Ala-Ala-Pro-Leu p-nitroanilide(Sigma S-8511),N-(p-Tosyl)-Gly-Pro-Argp- Nitroanilide acetate salt (SigmaT1637), Z-Gly-Gly-Leup-nitroanilide ...
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