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A kind of bovine lactoferrin peptide constitutive yeast expression vector and its preparation method

A bovine lactoferrin peptide and yeast expression vector technology, applied in the field of genetic engineering bacteria, can solve the problems of affecting the induction expression efficiency, increasing the production cost, affecting the yield and the like, and achieving the effect of solving the glucose inhibitory effect and reducing the production cost.

Active Publication Date: 2016-04-20
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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  • A kind of bovine lactoferrin peptide constitutive yeast expression vector and its preparation method

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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: 94°C pre-denaturation for 5 minutes; 94°C for 30s, 53°C for 30s, 72°C for 93s, 30 cycles; 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 performed to delete the GAL1 ...

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Abstract

The present invention disclosed a type of yeast expression carrier and its preparation methods formed by a cotton iron protein peptide.The expression carrier is to get the GAL1 promoter of Pyes2 plasmid with the GAL1 promoter of PYES2 plasmid by connecting the milk iron protein peptide gene to the GAL1 promoter.Pyes2-TPI – LFCINB reorganization plasmids built by the present invention can efficiently and stably secrete and express milk iron protein peptide; and no need to use inducer, which reduces the complexity of production costs and production.

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 Patents(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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