Novel natural antibacterial peptide HKABF and its preparation and application
A technology of antimicrobial peptides and production methods, applied in the field of preparation of drugs for the treatment of infectious diseases, can solve the problems of low efficacy, low seedling survival rate, lack of disease prevention and control measures, etc., and achieve the effect of strong antibacterial effect
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Embodiment 1
[0030] Example 1: Extraction and RT-PCR amplification of total RNA from hippocampal hatching bags
[0031] Extraction of total RNA and synthesis of cDNA: Homogenize hippocampal hatching bag tissue during pregnancy in Trizol, extract total RNA from hippocampus by one-step hot phenol method, and extract and remove protein. To obtain a total RNA concentration of 1.04 μg / μl, its A 260 / A 280 =1.90, two clear bands of 18s and 28s can be seen through 1% formaldehyde denaturing gel electrophoresis detection, and the ratio is greater than 1 (see figure 2 ), indicating that the integrity of total RNA was good. Take 1 μg of total RNA with SMART IV oligonucleotide (5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3′) and CDSIII / 3′PCR primer (5′-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T) 30 N -1 N-3′) was reverse transcribed to synthesize the first strand to obtain 10 μl of cDNA first strand product. 2μl of the first-strand product with 5’PCR primer (5’-AAGCAGTGGTATCAACGCAGAGT-3’), CDS III / 3’PCR p...
Embodiment 2
[0032] Embodiment 2: Analysis of hippocampal antimicrobial peptide gene sequence
[0033]BLAST homology analysis showed that the ORF of the new hippocampal antimicrobial peptide gene was 234bp in length, encoding a precursor protein of 78 amino acid residues, including a signal peptide of 19 amino acids at the N-terminus, a Proregion region of 12 amino acids at the C-terminus and a middle Mature peptide of 47 amino acids. Among them, the Lys at the carboxyl terminus of the mature peptide is amidated. This compositional pattern and C-terminal amidation of hippocampal antimicrobial peptide precursor protein are typical characteristics of the primary structure of antimicrobial peptides. The mature peptide contains 6 basic amino acids and the amidation of the C-terminus makes the molecule positively charged. The predicted isoelectric point of the mature peptide is 8.95 and the molecular weight is 4762 Daltons. This mature peptide has only 55% homology with other antimicrobial p...
Embodiment 3
[0034] Example 3: Construction of recombinant hippocampal antimicrobial peptide yeast expression plasmid
[0035] A pair of primers were synthesized according to the sequences at both ends of the hkabf gene, the upstream primer contained the Xho I cleavage site, and the downstream primer contained the Not I cleavage site.
[0036] Upstream primer (B1):
[0037] 5'-CCTTAGGCCCCTCGAGaaaagaCTGTCGTGCAGATTTGGACGC-3';
[0038] Downstream primer (B2):
[0039] 5'-GCCGAGCTCTGCAGCGGCCGCtcaCTTCTTGCCGACACTAACACC-3'.
[0040] Using the pcDNA3.0 plasmid containing the hkabf gene as a template, and using B1 and B2 as primers for PCR amplification, a specific amplified single band was obtained, and the product size was about 188 bp. The PCR amplified product was digested with Xho I / Not I and cloned into the methanolic yeast expression vector pPICZαA to obtain the recombinant expression vector pPICZαA-hkabf (the construction process is as follows Figure 7 shown). The expression vector wa...
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