A dna fragment with functions of promoter and coding signal peptide and its application in producing β-mannanase
A technology of mannanase and coding signal, which is applied in the production of β-mannanase, the field of DNA fragments, and achieves the effect of good development prospects
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Embodiment 1
[0034] Example 1 Obtaining and Proteome Identification of Bacillus subtilis 7-3-3 Extracellular Secretion Protein
[0035] Take Bacillus subtilis 7-3-3 (preservation number CCTCC NO.M200038) from the -80°C glycerol tube and streak it on the LB solid plate, culture at 37°C for 12h, pick a single colony in 5mL LB liquid medium, and put it in 37°C , cultured at 200rpm for 12h; transfer 3% inoculum by volume to the seed medium, culture at 37°C and 200rpm for 4h; inoculate 10% inoculum by volume in the fermentation medium, use a 500mL Erlenmeyer flask, and fill the fermentation medium The volume is 100mL, 37°C, 200rpm for shake flask fermentation culture.
[0036]1 mL of the fermentation broth was sampled at 55 hours, and the supernatant was obtained after centrifugation at 8000 g at 4°C. Acetone solution containing 10% trichloroacetic acid was used. After pre-cooling at -20°C, 3 mL of acetone solution was mixed with 500 μL of fermentation broth supernatant, and left overnight at ...
Embodiment 2
[0038] Example 2 Screening and cloning promoter and signal peptide coding sequence
[0039] Using the signal intensity method to extract the quantitative information of Bacillus subtilis 7-3-3 extracellular secreted protein, the ratio of the quantitative value of the peptide segment to the sum of the quantitative value of all peptide segments was used as the protein abundance ratio, the data was processed, and the protein was screened. Protein genes with high abundance in the spectrum. Use the SignalP 5.0 online prediction service (http: / / www.cbs.dtu.dk / services / SignalP / ) to predict signal peptides for proteins with higher abundance, select "Gram-positive" for "Organism group", and enter the amino acid sequence of the protein Make predictions. According to the predicted results, the protein with signal peptide was further screened, and finally a protein gene 2454 with high protein expression and signal peptide was screened out. Its promoter was named P2454, 300bp in length, a...
Embodiment 3
[0045] Example 3 Construction of Secreted Expression Vector pN-P2454-SP2454
[0046] Using the Escherichia coli-Bacillus subtilis shuttle plasmid pNW33N as the backbone, the plasmid was digested with restriction endonucleases KpnI and BamHI, and the digested products were purified with a DNA gel recovery kit. The DNA fragments amplified in Example 2 were subjected to the same digestion and purification operations. T4 DNA ligase was used to connect the purified DNA fragment and plasmid pNW33N, and the ligated product was transformed into Escherichia coli DH5α, and positive transformants were selected and entrusted to Beijing Qingke Xinye Biotechnology Co., Ltd. for sequencing verification to obtain the correct recombinant vector. The secreted expression The recombinant vector is named pN-P2454-SP2454, such as figure 2 As shown, the nucleotide sequence of the promoter P2454 and the gene encoding the signal peptide SP2454 are connected between the KpnI and BamHI restriction sit...
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