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Preparation method of lactococcus lactis engineering bacteria for secretory expression of peanut allergens and application thereof

A technology for Lactococcus lactis and peanut allergens, which is applied in the field of bioengineering, can solve the problems of high purification cost of expression products, complex structure, and limited clinical application of Escherichia coli prokaryotic expression system.

Inactive Publication Date: 2014-03-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, Escherichia coli can produce endotoxins with complex structures and various types, and the subsequent purification of expression products is expensive, which limits the clinical application of Escherichia coli prokaryotic expression system

Method used

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  • Preparation method of lactococcus lactis engineering bacteria for secretory expression of peanut allergens and application thereof
  • Preparation method of lactococcus lactis engineering bacteria for secretory expression of peanut allergens and application thereof
  • Preparation method of lactococcus lactis engineering bacteria for secretory expression of peanut allergens and application thereof

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Experimental program
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Embodiment 1A

[0017] The codon optimization of embodiment 1Arah2 gene sequence

[0018] First, use the signal peptide prediction software SignalP4.1Server to analyze the amino acid sequence of Arah2

[0019] (genbank: AAN77576.1) signal peptide sequence was predicted, and the predicted signal peptide sequence was removed from the gene sequence (genbank: AY158467.1) corresponding to the above amino acid sequence to obtain the original nucleotide sequence to be optimized .

[0020] The original nucleotide sequence was optimized according to the preferred codon table of Lactococcus lactis, and the optimized gene was named nArah2, which was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. and subcloned into the vector pUC57 to obtain pUC57 -nArah2 (synthesized by Shanghai Sangon, where pUC57 is a commercial plasmid).

[0021] Natural gene sequence before optimization (genbank: AY158467.1), such as SEQIDNo.1.

[0022] Optimized gene sequence (nArah2), such as SEQIDNo.2.

[0023] Ami...

Embodiment 2

[0025] Embodiment 2 Construction of recombinant expression plasmid

[0026] First, the plasmid pUC57-nArah2 (synthesized by Shanghai Sangon) was used as a template, and the nArah2 gene was amplified by PCR using primers Pra2F: 5'-CTCGAGCTCCGTCAACAATGGGAATTACAAG-3' and Pra1R: 5'-CGGGGTACC TTAATAACGATCACGACCAC-3'. Using the genomic DNA of Lactococcus lactis subsp.cremoris MG1363 (Gasson.Plasmid complements of Streptococcus lactis NCDO712and other lactic streptococci after protoplast-induced curing.Journal of Bacteriology.1983,154(1):1-9) as a template, use primer Psp1F: 5'-CCG GCCATGGTGAAAAAAAAGATTATCTCAG-3 and Psp1R: 5'-CTCGAGCTCAGCG TAAACACCTGACAAC-3'PCR amplified signal peptide sequence of Usp45 protein-SP Usp45 (-SP Usp45 It is a Genebank published sequence, GenBank: M60178.1). Detect and recover purified nArah2 and -SP by 1.0% agarose gel electrophoresisUsp45 Gene fragments, will recover and purify the amplified fragment-SP Usp45 Perform ligation reaction with nArah2 and...

Embodiment 3

[0027] The preparation of embodiment 3 recombinant Lactococcus lactis

[0028] The plasmid pNZ2 constructed above was electroporated to transform L. lactisNZ9000 (Kuipers et al. Quorum sensing-controlled gene expression in lactic acid bacteria. Journal of Biotechnology. 1998, 64(1): 15-21) competent cells. Pick a single colony for colony PCR and double enzyme digestion verification, and send it to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing verification. The sequenced correct transformant is named L.lactis NZSE, and the empty plasmid strain L.lactis NZ9000 will be carried / pNZ8148 designated L. lactis NZ48.

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Abstract

The invention discloses a preparation method of lactococcus lactis engineering bacteria for secretory expression of peanut allergens and application thereof. The codon optimization is implemented for natural gene sequences of peanut allergens Arah2 to obtain optimized genes, nArah2; recombinant plasmids and recombinant lactococcus lactis strains for secretory expression of nArah2 are constructed. The recombinant strains, disclosed by the invention, are used for immunizing mice as oral vaccines to effectively adjust the immune reaction of mice allergy, so that the recombinant strains can be developed as mucosa vaccines for preventing and treating peanut allergy.

Description

【Technical field】 [0001] The invention relates to a recombinant Lactococcus lactis strain secreting and expressing peanut allergen Arah2, and its construction method and application belong to the technical field of bioengineering. 【Background technique】 [0002] Peanut allergy is one of the most serious and common food allergic reactions, which has the characteristics of rapid onset, high mortality rate and lifelong nature. In recent years, the incidence of peanut allergy has been increasing year by year worldwide. In the United States, 1.1% of the population is allergic to peanuts, and its incidence nearly doubled between 1997 and 2002. Inducing specific oral tolerance, that is, giving patients gradually increasing doses of allergens over a period of time to induce patients to develop immune tolerance to this food antigen, is currently a relatively effective treatment for peanut allergy. [0003] The acquisition of high-purity allergens is the premise and basis for immune...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/29A61K39/35A61P37/08C12R1/46
Inventor 陈卫张秋香任晟诚王刚田丰伟刘小鸣赵国忠赵建新张灏郭敏
Owner JIANGNAN UNIV
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