Bovine lactoferricin peptide constitutive type yeast expression vector and preparation method thereof

A technology of bovine lactoferrin peptide and yeast expression vector, applied in the field of genetic engineering bacteria, can solve problems such as increasing production cost, affecting inducing expression efficiency, affecting yield, etc., and achieves the effect of reducing production cost and solving the effect of glucose inhibition

Active Publication Date: 2014-10-01
XINJIANG XIPU BIOLOGICAL SCI & TECH +1
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Problems solved by technology

However, the glucose effect problem existing in traditional host bacteria will affect the efficiency of induced expression, thereby affecting the yield, and the inducer used for induced expression greatly increases the production cost

Method used

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  • Bovine lactoferricin peptide constitutive type yeast expression vector and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment

[0032] One, the construction of a kind of bovine lactoferrin peptide constitutive yeast expression vector

[0033] 1. Amplification of the TPI promoter gene: using the pYX212 plasmid containing the TPI promoter sequence as a template, the amplification primers are:

[0034] TP1: 5'-TCTTCAAGAATTGGGGA-3' (SEQ ID NO.5);

[0035] TP2: 5'-CTGTATGTGTTTTTTGTAGTTATAG-3' (SEQ ID NO. 6).

[0036] Reaction conditions: pre-denaturation at 94°C for 5 minutes; 30 cycles at 94°C for 30s, 53°C for 30s, and 72°C for 93s; extension at 72°C for 10 minutes. The reaction products were detected by 1.5% agarose gel electrophoresis and recovered by gel recovery kit.

[0037] The sequence of the amplified TPI promoter is shown in SEQ ID NO.1.

[0038] 2. PCR amplification of the pYES2 plasmid sequence without the GAL1 promoter: using the pYES2 plasmid as a template, design primers: Y1: 5'-CCGGATCGGACTACTAGC-3' (SEQ ID NO.7) and Y2:: 5'-ACTAGTGGATCATCCCCAC-3 ' (SEQ ID NO.8), inverse PCR was perfo...

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Abstract

The invention discloses a bovine lactoferricin peptide constitutive type yeast expression vector and a preparation method thereof. The expression vector is obtained by introducing a bovine lactoferricin peptide gene into pYES2 plasmids and utilizing a TPI (triosephosphate isomerase) promoter to substitute a GAL1 (galactokinase 1) promoter of the pYES2 plasmids. The pYES2-TPI-LfcinB recombinant plasmids constructed by the preparation method disclosed by the invention can effectively and stably secrete bovine lactoferricin peptide without using an inducer, so that production cost and production complexity are lowered.

Description

technical field [0001] The invention belongs to the technical field of genetically engineered bacteria, and in particular relates to a bovine lactoferrin peptide constitutive yeast expression vector and a preparation method thereof. Background technique [0002] Antimicrobial peptides (also known as host defense peptides) are a class of amphiphilic small molecule peptides produced under inducible conditions with antimicrobial activity, and are present in all organisms as components of an evolutionarily conserved innate immune response. Antimicrobial peptides are an important part of the host immune defense system. Antimicrobial peptides have broad-spectrum antibacterial effects, and have inhibitory effects on viruses, parasites, and cancer cells. Antimicrobial peptides can be used in medicine and hygiene, food industry and so on. In terms of medicine and health, the biggest difference between antimicrobial peptides and antibiotics is that the application of antimicrobial p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/66C12N1/19C12P21/02C12R1/865
Inventor 查向东车媛媛程林春赵大伟余忠丽
Owner XINJIANG XIPU BIOLOGICAL SCI & TECH
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