Preparation method and application of animal-derived cationic antimicrobial peptides
A technology of antimicrobial peptides and cations, which is applied in the field of preparation of animal-derived cationic antimicrobial peptides, can solve the problems that the safety and effectiveness of protein poultry and animals have not been proven, and achieve the improvement of feed remuneration and breeding profits, high antibacterial activity, The effect of broad-spectrum antibacterial action
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Embodiment 1
[0032] The antimicrobial peptide gallinacin 14 N-terminal 1-24 amino acid residue fragment (MGIFLLFLVLLAVPQAAPESDTVT (SEQ ID NO.3)) and the antimicrobial peptide Spheniscin-2 C-terminal 5-38 amino acid residue fragment (CRLRRGFCARGRCRRFPSIPIGRCSRFVQCCRRVW (SEQ ID NO.4)) hybrid ( The two fragments are linked together) to obtain the following amino acid sequence: MGIFLLFLVLLAVPQAAPESDTVTCRLRRGFCARGRRFPSIPIGRCSRFVQCCRRVW.
[0033] According to the above amino acid sequence, the preferred codons of genetically engineered bacteria, that is, the common codes of highly expressed genes, are selected for amino acid encoding, and then the unsuitable part of the codon tandem organization is appropriately modified, adjusted and optimized by means of DNA analysis software (that is, the expression of the protein is selected). The amino acid codons preferred by the host bacteria to improve expression efficiency), and finally we obtained the nucleotide sequence and amino acid sequence of the o...
Embodiment 2
[0043]Cloning of the antimicrobial peptide gene: the obtained antimicrobial peptide gene fragment (gal-sphe) was cloned into the selected vector IMPACT-CN, and then the plasmid (IMPACT-CN) was transformed into the genetically engineered bacteria (ER2566), containing 100 μg / ml ampicillin and medium coated with 20mg / ml X-gal, 1mM / ml IPTG (Tryptone (tryptone): 10g / L, Yeast Extract (yeast extract): 5g / L, NaCl (sodium chloride ): 10g / L, Agar agar 15g / L) culture (37 ℃, 12-16 hours), screening out the bacterium colony that contains recombinant plasmid (IMPACT-CN-gal-sphe), and bacterium colony is preserved in 4 ℃.
Embodiment 3
[0045] Expression and purification of antimicrobial peptides:
[0046] 1. Bacterial culture: Take a single colony directly, add it to 1L LB liquid medium (containing 100 μg / ml ampicillin), and culture with shaking at 37°C until the OD600 reaches 0.5-0.7. Note: A single colony newly transformed with a recombinant expression plasmid can be picked, first inoculated in 10ml liquid medium (containing ampicillin 100μg / ml), shaken at 37°C for 3-4 hours, and then inoculated in 1L liquid medium (containing ampicillin 100 μg / ml), cultured at 37°C until OD600 reached 0.5.
[0047] 2. Induction of protein expression: Add IPTG at a final concentration of 0.3 mM, shake and culture at 37° C. for 3 h, and induce protein expression. Set up a negative control. Take a sample of 10-20 μl for SDS-PAGE electrophoresis, and take a sample of 1-2 μl for Western blot identification.
[0048] 3. Cell collection: centrifuge at 5000×g at 4°C for 10 minutes, discard the supernatant. Store at -20 / -80°C....
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