A kind of fly maggot antimicrobial peptide, coding gene, carrier, engineering bacteria and application thereof
A technology of genetically engineered bacteria and encoded genes is applied to the application field of food-grade recombinant lactic acid bacteria and the preparation of antibacterial additives. The effect of simple production process
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Embodiment 1
[0045] Example 1: Codon Optimization of Antimicrobial Peptides and Ankyrin Encoding Genes
[0046] Lactic acid bacteria have a strong codon preference. Therefore, in order to enable the insect-derived antimicrobial peptide gene (the nucleotide sequence is shown in SEQ ID NO.1, the amino acid sequence is SEQ ID NO.2) to be highly expressed in lactic acid bacteria cells, it is necessary to optimize its nucleotide series, Remove rare codons. The optimization principles are: TTC→TTT; GTA→GTT; TCC→TCT; GAG→GAA; TGC→TGT; AGA→CGT; AGG→CGT;
[0047] The optimized nucleotide sequence of the fly maggot antimicrobial peptide is shown in SEQ ID NO.3.
Embodiment 2
[0048] Embodiment 2: Construction of antimicrobial peptide and anchor protein expression cassette
[0049] The expression and localization of antimicrobial peptides are achieved through the combined use of multiple expression regulation elements. These expression control elements include promoters, signal peptide sequences, antimicrobial peptide genes, anchor protein sequences, and linking sequences between each expression control element (see figure 1 ), the antimicrobial peptide expression frame is: LDH promoter (SEQ ID NO.4)-signal peptide (SEQ ID NO.5)-anchor protein (SEQ ID NO.6)-fly maggot antimicrobial peptide (SEQ ID NO.3 ).
[0050] In the present invention, the expression of the antimicrobial peptide is finally controlled by a constitutive promoter, which is derived from the lactate dehydrogenase (LDH) promoter of Lactobacillus casei, and the nucleotide sequence is represented by SEQ ID NO.4 Show.
[0051] The signal peptide sequence is the signal peptide of Lacto...
Embodiment 3
[0054] Embodiment 3: the construction of alr gene (alanine racemase) expression cassette
[0055] Glyceraldehyde triphosphate dehydrogenase promoter P of whole gene synthetic lactic acid bacteria GADPH (The nucleotide sequence is shown in SEQ ID NO.8), with plantaractobacillus genomic DNA as template, use primer alrF (SEQ ID NO.10) and alrR (SEQ ID NO.11), PCR amplification obtains alr (three Amino acid racemase) gene (the nucleotide sequence is shown in SEQ ID NO.9).
[0056] With the above P GADPH The gene fragment (SEQ ID NO.8) and the PCR amplification product of alr (SEQ ID NO.9) were used as templates, and the primer P GADPH F (SEQ ID NO.12) and alrR, the promoter P GADPH Fusion with the alr gene to successfully construct the alr gene expression cassette.
[0057] Fusion PCR conditions: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 1 minute, annealing at 58°C for 30 seconds, extension at 72°C for 2 minutes, 32 cycles.
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