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Preparation method and application of recombinant lactococcus lactis capable of displaying peanut allergen on surface

A technology of Lactococcus lactis and peanut allergens, applied in the field of bioengineering, can solve problems such as complex structure, high cost of expression product purification, and limitation of clinical application of Escherichia coli prokaryotic expression system

Inactive Publication Date: 2014-03-05
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 and application of recombinant lactococcus lactis capable of displaying peanut allergen on surface
  • Preparation method and application of recombinant lactococcus lactis capable of displaying peanut allergen on surface
  • Preparation method and application of recombinant lactococcus lactis capable of displaying peanut allergen on surface

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Codon Optimization of Arah2 Gene Sequence

[0019] First, the signal peptide sequence of Arah2's amino acid sequence (genbank: AAN77576.1) was predicted using the signal peptide prediction software SignalP4.1Server, and the predicted sequence was removed from the gene sequence (genbank: AY158467.1) corresponding to the above amino acid sequence. The signal peptide 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] The natural gene sequence before optimization (genbank: AY158467.1), SEQIDNo.1.

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

[0023] Amino acid sequence befor...

Embodiment 2

[0025] Example 2 Construction of recombinant expression vector pNZ3

[0026]L.lactissubsp.cremorisMG1363 (Gasson.PlasmidcomplementsofStreptococcus lactis NCDO712and other lactic streptococci after protoplast-induced curing.Journal of Bacteriology.1983,154(1):1-9) inherent N-acetylmuramidase ACMA gene (for Genebank The cA domain in the published gene sequence, GenBank: U17696.1) serves as the anchor function sequence CWA. Using the genomic DNA of L. lactis subsp. cremorisMG1363 as a template, PCR amplification was performed using primers Pca1F: 5'-TAGGGTACCTCTGGTGGCTCGACAACCACAATTAC-3' and Pca1R: 5'-CCGCAAGCTTTTATTTTATTCGTAGATACTGACCAATT-3' PCR amplification-cwa sequence. The PCR reaction system (total volume: 50 μL) is: 10×KODplus buffer: 5 μL; dNTP: 5 μL; MgSO4: 2 μL; DNA template: 2 μL; upstream primer / downstream primer (20 μM): each 1 μL; KODplus: 0.5 μL; ddH 2 O: 33.5 μL. Reaction program: 95°C for 30s (denaturation), 59°C for 30s (annealing), 68°C for 60s (extension), 3...

Embodiment 3

[0027] Example 3 Preparation of recombinant Lactococcus lactis

[0028] The above constructed plasmid pNZ3 was electroporated to transform L.lactisNZ9000 (Kuipers etal. 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.lactisNZCW, and the empty plasmid strain L.lactisNZ9000 / pNZ8148 will be carried Named L. lactisNZ48.

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Abstract

The invention discloses a construction method and an application of recombinant lactococcus lactis capable of displaying a peanut allergen Arah2 on the surface. An optimized gene nArah2 is obtained by performing codon optimization on a natural gene sequence of the peanut allergen Arah2, and a recombinant vector for a cell wall-anchored expression way of the nArah2 and engineering lactococcus lactis are constructed. A related recombinant strain is used as an oral vaccine for mouse immunization, and the allergic immune response of mice can be effectively adjusted. Therefore, the recombinant strain can be developed into a mucosal vaccine for the prevention and control of peanut allergy.

Description

【Technical field】 [0001] The invention relates to a recombinant Lactococcus lactis strain expressing peanut allergen Arah2 on the surface, 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, with rapid onset, high mortality and [0003] characteristics such as lifetime. 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. [0004] The acquisition of high-purity allergens is the premise and basis for immune tole...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74A61K39/35A61P37/08C12R1/01
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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