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Construction and application of antifreeze peptide expressing food-grade lactobacillus expression system

An expression system and lactic acid bacteria technology, applied in the fields of biology and food, can solve problems such as safety restrictions, affecting human immune function, etc., and achieve the effects of reducing production costs, improving freeze-drying stress resistance, and simplifying construction methods.

Pending Publication Date: 2017-10-17
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since Escherichia coli is a pathogenic bacterium, and the antibiotic markers used by it will cause biological transplantation in the human intestine and affect the normal immune function of the human body, the application of protein or polypeptide products expressed and secreted by Escherichia coli in the food industry Restricted due to security concerns

Method used

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  • Construction and application of antifreeze peptide expressing food-grade lactobacillus expression system
  • Construction and application of antifreeze peptide expressing food-grade lactobacillus expression system
  • Construction and application of antifreeze peptide expressing food-grade lactobacillus expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Firstly, an antifreeze peptide with cell low temperature protection activity was constructed, and the codons of Lactococcus lactis were optimized, and finally the optimal optimized polynucleotide sequence was obtained as follows:

[0060] ccatgggtTGTAAAGGTGCGGATGGAGCCCACGGAGTTAATGGGTGCCCTGGCACCGCTGGGGCTGCAGGCAGCGTAGGTGGGCCTGGATGCGACGGAGGCCACGGTGGTAATGGAGGCAACGGTAATCCGGGCTGTGCCGGTGGCGTCGGCGGTGCGGGAGGGGCGAGTGGCGGGACTGGAGTTGGGGGTCGAGGCGGCAAAGGTGGCTCGGGAACACCGAAAGGTGCGGACGGCGCGCCGGGCGCTCCTTGTAAGGGGGCCGACGGAGCACATGGTGTCAACGGATGTCCGGGGACTGCCGGCGCAGCAGGTTCTGTCGGAGGACCTGGCTGTGACGGTGGCCACGGCGGCAATGGAGGTAATGGGAATCCGGGTTGTGCAGGGGGAGTTGGCGGAGCGGGGGGCGCAAGCGGAGGAACAGGAGTAGGCGGCAGAGGAGGAAAAGGTGGCTCGGGAACGCCAAAAGGCGCTGATGGCGCCCCTGGTGCCCCGCATCATCATCATCATCACTAAGAGCTC(SEQ ID NO.1)。

[0061] The endonucleases used were NcoI and SacI, and the gene synthesis was constructed on the pUC57 vector (purchased from Nanjing GenScript Biotechnology Co., Ltd.).

[0062] Using double enzyme digestion,...

Embodiment 2

[0089] Expression and purification of embodiment 2 antifreeze peptide

[0090] After the antifreeze peptide SF-P was constructed according to Example 1, the expression conditions of SF-P were optimized by SDS-PAGE and Western-Blot.

[0091] The SF-P recombinant strain was inoculated in GM17 liquid medium containing 0.5% glucose, the inoculum size was 1%, and cultured overnight at 30°C, and then inoculated into fresh 0.5 In GM17 liquid medium containing % glucose, culture statically at 30°C for 2-3h to the initial stage of logarithmic growth (OD value 0.4-0.5), adjust pH to 4, 5, 6 respectively, add inducer Nisin (concentration is 25 , 50, 100ng / mL), continued to culture statically at 20, 30, and 40°C for 6, 12, and 24h respectively, centrifuged at 3000r / min, 4°C for 6min, and collected the bacteria. Wash with 0.02mol / l PBS, break the wall with an ultrasonic cell wall breaker, centrifuge at 12000r / min, 4°C for 10min, and collect the supernatant. SDS-PAGE and Western-Blot iden...

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Abstract

The invention discloses construction and application of an antifreeze peptide expressing food-grade lactobacillus expression system, in particular to a preparation method of the antifreeze peptide expressing food-grade lactobacillus expression system. The preparation method includes: setting an artificially-synthesized antifreeze peptide coding gene on a clone vector; subjecting acquired recombinant plasmids to digestion, and connecting to an expression vector with Nisin resistance gene nisr to construct antifreeze peptide expression plasmids; converting to lactococcus lactis competent cells to obtain the antifreeze peptide expressing food-grade lactobacillus expression system. A food-grade genetically engineered bacterium constructed according to the method is high in factor of safety to human and environment and applicable to various industries of medicines, food and the like.

Description

technical field [0001] The invention belongs to the technical field of biology and food. Specifically, the invention designs a preparation method of food-grade lactic acid bacteria capable of expressing antifreeze peptides and the characterization of its antifreeze activity. More specifically, the invention relates to a novel freeze-drying protective agent antifreeze Construction and application of frozen peptides in lactic acid bacteria food-grade expression system. Background technique [0002] Antifreeze peptides (AFP) are a class of polypeptides or glycopeptides produced by some cryogenic organisms to adapt to extreme cold environments. AFP has special antifreeze activity, mainly as follows: (1) can bind to the surface of ice crystals and inhibit the further growth of ice crystals; (2) can lower the freezing point of the solution in a non-colligative form, so that there is a difference between the melting point and the freezing point of the solution value, showing therm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12R1/46
CPCC07K14/00C12N15/74
Inventor 吴金鸿张莉罗静金泉谢凡兰小红王正武
Owner SHANGHAI JIAO TONG UNIV
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