Chicken antimicrobial peptides Cathelicidins and preparation method and applications thereof
A technology of antimicrobial peptides and acquisition methods, applied in the biological field, can solve the problems of small molecular weight, difficult separation and purification, and high cost of antimicrobial peptides
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Embodiment 1
[0054] Cloning and sequencing analysis of the complete coding gene of chicken cathelicidin1, 2, 3 antimicrobial peptides, including:
[0055] 1. Primer Design
[0056] According to the cathelicidins antimicrobial peptide gene sequence (DQ092350, DQ092351, DQ092352, DQ092353) published in GenBank and the design principles of PCR primers, and with the help of computer software DNASTAR for auxiliary analysis, three pairs of PCR gene-specific primers were designed for Amplify the cDNA sequences of chicken Cathelicidin1, 2, and 3 coding genes, and the primers are respectively CathL1 P1 / P2, CathL2 P1 / P2, and CathL3 P1 / P2 in the table, wherein P1 is a forward primer and P2 is a reverse primer. Primers were synthesized by Shanghai Boshang Company.
[0057] The primer sequences used in this example and Example 2 are shown in Table 1. In the CathL1S F / R, CathL2S F / R, and CathL3S F / R primer pairs, the underlined bases represent the EcoR I and Xho I restriction endonuclease sites introd...
Embodiment 2
[0077] The genetic engineering preparation method of recombinant chicken Cathelicidins antimicrobial peptide fusion protein comprises the following steps:
[0078]1. Preparation of chicken Cathelicidins 1, 2, 3 mature peptide coding sequence and construction of its fusion expression vector (1) Amplification of chicken Cathelicidins 1, 2, 3 mature peptide coding sequence: According to the cloned chicken Cathelicidins 1, 2, 3 antimicrobial peptide gene sequence and deduced amino acid sequence, design 3 pairs of specific primers CathL1S F / R, CathL2S F / R, CathL3S F / R with restriction sites, downstream primers with stop codon TGA, primer sequence See Table 1. Using the positive plasmids pMD-CathL1, pMD-CathL2, and pMD-CathL3 containing chicken Cathelicidins-1, -2, and -3 gene cDNA sequences as templates, the chicken Cathelicidins 1, 2, and 3 mature peptide coding sequences were amplified by PCR ( ~100bp).
[0079] The reaction system is: 10×PCR Buffer (containing Mg 2+ ) 5.0 μL,...
Embodiment 3
[0135] Optimization of Cathelicidins Fusion Protein Expression Conditions and Affinity Chromatography Purification of Expression Products
[0136] 1. Optimization of fusion protein expression conditions
[0137] In order to increase the proportion of soluble components in the total protein expression, by reducing the induction temperature (down to 25°C or 15-20°C), prolonging the induction time (3h-8h), adjusting the concentration of the inducer (the concentration of IPTG is 0.4mmol / L, 0.8mmol / L, 1.0mmol / L, 1.2mmol / L) and other methods to inhibit the formation of inclusion bodies, thereby simplifying the purification operation.
[0138] By using hyperosmotic medium (i.e. LB / SB medium), long-term induction at low temperature (25°C or 15-20°C), and adjusting the concentration of the inducer IPTG, etc., the formation of inclusion bodies can be inhibited and the soluble protein in the expression product can be increased. The purpose of the proportion. Experiments have shown tha...
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