Recombinant lactococcus lactis for intracellular expression of peanut allergen and application thereof

A technology of Lactococcus lactis and peanut allergens, applied in the field of bioengineering, can solve the problems of high cost of purification of expression products, limitation of clinical application of E. coli prokaryotic expression system, complex structure, etc.

Inactive Publication Date: 2014-03-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Recombinant lactococcus lactis for intracellular expression of peanut allergen and application thereof
  • Recombinant lactococcus lactis for intracellular expression of peanut allergen and application thereof
  • Recombinant lactococcus lactis for intracellular expression of peanut allergen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Codon Optimization of Arah2 Gene Sequence

[0019] Firstly, the signal peptide sequence of Arah2's amino acid sequence (genbank: AAN77576.1) was predicted using the signal peptide prediction software SignalP 4.1 Server, and the predicted sequence was removed from the gene sequence (genbank: AY158467.1) corresponding to the above amino acid sequence to obtain 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), such as SEQ ID No.1.

[0022] Optimized gene sequence (nArah2), SEQ ID No.2.

[0023] ...

Embodiment 2

[0025] Example 2 Construction of recombinant expression vector pNZ1

[0026] Using the plasmid pUC57-nArah2 (synthesized by Shanghai Sangon) as a template, the nArah2 gene was amplified by PCR using primers Pra1F 5'-CATGCCATGGGTCAACAATGGGAATTACAAG-3' and Pra1R: 5'-CGGGGTACCTTAATAACGATCACGACCAC-3'. The PCR reaction system (total volume: 50 μL) is: 10×KOD plus buffer: 5 μL; dNTP: 5 μL; MgSO4: 2 μL; DNA template: 2 μL; upstream primer / downstream primer (20 μM): each 1 μL; KOD plus: 0.5 μL; wxya 2 O: 33.5 μL. Reaction program: 95°C for 30s (denaturation), 61°C for 30s (annealing), 68°C for 50s (extension), 30 cycles of amplification. The purified nArah2 gene fragment was detected and recovered by 1.0% agarose gel electrophoresis. The recovered and purified amplified fragment and vector pNZ8148 (Mierau and Kleerebezem.10years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis.Appl Microbiol Biotechnol.2005,68(6):705-717) were digested with double enzymes...

Embodiment 3

[0027] Example 3 Preparation of recombinant Lactococcus lactis

[0028]The above constructed plasmid pNZ1 was electroporated to transform L. lactis NZ9000 (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 will be named L.lactis NZNH, 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 building method and application of a recombinant lactococcus lactis for intracellular expression of a peanut allergen Arah2. A natural gene sequence of the peanut allergen Arah2 is subjected to codon optimization, so as to obtain an optimized gene nArah2, and a recombinant vector and lactococcus lactis engineering bacteria aiming at an intracellular expression mode of the nArah2 are built. A recombination strain disclosed by the invention is taken as an oral vaccine to immunize the mice, and the immunoreaction of mice anaphylaxis can be effectively regulated. Therefore, the recombinant lactococcus lactis can be developed as a mucosal vaccine to be applied to prevention and treatment of peanut allergy.

Description

【Technical field】 [0001] The invention relates to a recombinant Lactococcus lactis strain expressing peanut allergen Arah2 in cells, 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 tole...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74A61K48/00A61K39/35A61P37/08C12R1/01
Inventor 张秋香陈卫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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