Optimized gene of ietalurus punetaus LEAP-2 mature peptide and preparation method of recombinant protein of optimized gene

Abstract

The invention discloses an optimized gene of ietalurus punetaus LEAP-2 mature peptide and a preparation method of recombinant protein of the optimized gene, and further provides a recombinant expression carrier and yeast host cell containing the optimized gene, application of the optimized gene to producing the ietalurus punetaus LEAP-2 mature peptide, and application of the recombinant protein to preparing antibacterial medicines. On the premise of not changing the amino acid sequence of the ietalurus punetaus LEAP-2 mature peptide, the amino acid sequence of the gene of the ietalurus punetaus LEAP-2 mature peptide is optimized according to pichia pastoris preferred codons, and the optimized sequence is SEQ ID NO:2. By expressing the optimized gene in a pichia pastoris host cell, the recombinant ietalurus punetaus LEAP-2 mature peptide is prepared. By means of the preparation method, the LEAP-2 mature peptide can be efficiently prepared, the applied culture conditions are simple, cost is low, and the obtained LEAP-2 mature peptide is good in antibacterial effect.

Description

technical field [0001] The present invention relates to a codon-optimized gene, in particular to an optimized gene after transformation of the channel catfish LEAP-2 mature peptide gene according to the preferred codon of Pichia pastoris, and a method for preparing a recombinant protein coded by it, which belongs to genetic engineering field. Background technique [0002] Looking at the domestic aquaculture industry, seawater and freshwater aquaculture areas are mostly distributed in the waters near coastal harbors and estuaries, and these waters are also the main receiving places for coastal land-based pollutants and marine sewage. According to statistics, the amount of waste water directly discharged into the sea in my country is as high as 8 billion tons every year. Agricultural sewage rich in nutrients, pathogenic microorganisms and organic pesticides also flows into coastal water bodies along with the surface, resulting in deterioration of water quality. Such aquacultu...

AI Technical Summary

Problems solved by technology

The expression of the latter grouper antimicrobial peptide LEAP-2 in Pichia pastoris has the following disadvantages: first, it adopts a precursor peptide gene that theoretically does not have direct biological activity for recombinant expression, and the obtained The activity of the target protein will be affected in theory; the second is that the gene used is a natural cDNA, which has not been optimized according to the codon preference of Pichia pastoris, and the expressed target protein has not been purified, resulting in an unknown expression level; When constructing the eukaryotic expression vector, EcoRI and XbaI restriction sites were used, resulting in the resulting recombinant protein carrying two additional amino acid residues (YV) at the N-terminus, which are important for its activity It is unknown what kind of impact it will have, so there is a possibility of reducing the activity of the product; Fourth, its product is only identified by SDS-PAGE and Western-blot analysis, and does not provide mass spectrometry detection data (generally confirming whether the target protein is correctly expressed requires After detection and analysis by mass spectrometry, the target protein fragment is compared with the theoretical amino acid sequence of the target protein to determine whether the product is a recombinant target protein), so it cannot be completely confirmed that the product is a recombinant target protein

It is worth noting that the culture medium used in the above-mentioned patent of "Clanted grouper antimicrobial peptide LEAP-2 gene, carrier, recombinant strain and protein and its application" is also a traditional culture medium for laboratories containing yeast extract and peptone. However, the cost of this medium is high, and the post-treatment of the fermentation liquid is troublesome. Generally, it is not used when fermenting with a fermenter in production, so it does not have practical production and application value.

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