CN103642746A - Recombinant lactococcus lactis for intracellular expression of peanut allergen and application thereof - Google Patents

Abstract

The invention describes the construction method and application of the recombinant Lactococcus lactis strain expressing peanut allergen Arah2 in cells. The invention optimizes the codons of the natural gene sequence of the peanut allergen Arah2 to obtain the optimized gene nArah2, and constructs a recombinant vector and an engineering bacterium of Lactococcus lactis aimed at the intracellular expression of the nArah2. The recombinant bacterial strain involved in the present invention can be used as an oral vaccine to immunize mice, and can effectively regulate the allergic immune response of mice; therefore, the present invention can be developed as a mucosal vaccine for the prevention and treatment of peanut allergy.

Description

A kind of recombinant Lactococcus lactis expressing peanut allergen in cells and its application

【Technical field】

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】

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.

The acquisition of high-purity allergens is the premise and basis for immune tolerance induction. There are 11 peanut allergens, among which Arah2 is a glycoprotein with a molecular weight of 17-19KDa, which can be specifically recognized by IgE in the serum of more than 90% of patients. It is currently one of the most widely studied and most important peanut allergens. In recent years, the development of molecular biology technology has made it possible to obtain high-purity allergens by biosynthesis. At present, researchers at home and abroad have used different expression systems to produce peanut allergens. For example, Tao Ailin et al. (2012) introduced the use of E. coli expression systems to prepare A method for recombining peanut allergens and mutants; Katrin Lehmann et al. have successfully used Escherichia coli to recombinantly express Arah2 protein with good immunogenicity (Katrin Lehmann, et al. High-yield expression in Escherichia coli, purification, and characterization of properly folded major peanut allergen Arah 2. Protein Expression and Purification, 2003, 31:250–259). 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.

Lactococcus lactis has long been widely used in the production of traditional foods such as yogurt, cheese, and pickles, and is recognized as a safe microorganism. The use of Lactococcus lactis to express foreign proteins can be taken together with bacteria without purification, and the mucosal vaccine using Lactococcus lactis as an expression carrier can effectively induce the body to produce immune response and immune tolerance. Therefore, we use Lactococcus lactis as a mucosal vaccine delivery carrier to express the peanut allergen Arah2 in L. lactis cells, and the constructed recombinant Lactococcus lactis strain is of great significance in the pharmaceutical industry and other fields.

【Content of invention】

The technical problem to be solved by the present invention is to provide a recombinant Lactococcus lactis strain expressing peanut allergen protein nArah2 in cells and its construction method. The recombinant plasmid pNZ1 is obtained by transforming Lactococcus lactis, wherein the Lactococcus lactis used may be L. lactis NZ9000. The strain obtained after transformation and screening is L. lactis NZNH, which has been preserved in the General Microorganism Center (CGMCC) of the China Committee for the Collection of Microorganisms, and its preservation number is CGMCC: 8216. The present invention also relates to a construction method of the recombinant Lactococcus lactis strain, the method comprising the steps of:

a) Codon optimization was performed on the natural gene sequence of the peanut allergen Arah2 to obtain the optimized gene nArah2; b) The optimized gene nArah2 was used to further construct a recombinant expression plasmid; c) The recombinant expression plasmid was used to transform Lactococcus lactis and obtain recombinant lactic acid milk coccus strain, wherein the recombinant expression plasmid constructed is the intracellular expression vector plasmid PNZ1, the construction of the recombinant expression plasmid PNZ1 includes the plasmid pUC57-nArah2 as a template, and the use of primers Pra1F5'-CATGCCATGGGTCAACAATGGGAATTACAAG-3' and Pra1R: 5'-CGGGGTACCTTAATAACGATCACGACCAC- Perform PCR amplification on the nArah2 gene at 3', and the transformation method in step c) is electroporation.

The present invention also relates to a vaccine comprising the recombinant Lactococcus lactis strain, which can be an oral preparation, such as capsules, lozenges, powders or granules.

The present invention also relates to the use of the recombinant plasmid or the recombinant Lactococcus lactis strain in preparing a Lactococcus lactis vaccine that can be used for treating peanut allergy.

In the present invention, the recombinant peanut allergen protein nArah2 is successfully expressed in L. lactis NZ9000 cells, and the results of animal experiments show that the recombinant vaccine expressed in the cell can regulate the allergic systemic immune response and reverse the Th2 immune response to Th1 A shift in the immune response.

Therefore, the optimized peanut allergen nArah2 gene can be used in the form of recombinant plasmid DNA vaccine for the prevention and treatment of peanut allergy; the recombinant Lactococcus lactis strain can be packaged in the form of capsules, lozenges, powder packets, and granules as a new type of vaccine. Oral vaccines are used in the clinical prevention and immunotherapy of peanut allergy.

The recombinant Lactococcus lactis NZNH involved in the present invention was preserved on September 18, 2013 in the General Microbiology Center of China Microbiological Culture Collection Management Committee, address No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Microbiological Research, Chinese Academy of Sciences Institute, the deposit number is CGMCC No.8216.

【Description of drawings】

Fig. 1 Schematic diagram of the structure of Lactococcus lactis recombinant plasmid PNZ1;

Figure 2: Western blot detection of protein expression in the recombinant strain L.lactis NZNH: Lane 1 is the purified nArah2 protein as a positive control, and lanes 2 and 3 are the culture supernatant and cell pellet components of the strain L.lactis NZ48, respectively , lanes 4 and 5 are the culture supernatant and cell pellet components of strain L.lactis NZNH respectively;

Figure 3: Diagram of immunization procedures for animal experiments;

Figure 4: Levels of specific antibodies in serum after challenge: A-C are the levels of specific IgE, IgG2a, and IgG1 respectively; as shown in the figure: Compared with Sham group and Vector group, oral administration of L.lactis NZNH can reduce specificity in serum IgE and IgG1 levels, increasing specific IgG2a levels;

Figure 5: mMCP-1 level in serum after challenge: * indicates significant difference between the experimental group and the Sham group, P<0.05. As shown in the figure, compared with the Sham group, the level of mMCP-1 in the serum of mice in the CYT group was significantly reduced; this indicated that oral administration of L. lactis NZNH could significantly inhibit mast cell degranulation.

【Detailed ways】

The present invention is further elaborated by the following examples. The experimental methods that do not indicate specific conditions in the following examples are basically carried out according to the common molecular cloning manual. If there are no special instructions for various reagents, their concentrations are mass percent concentration.

The codon optimization of embodiment 1 Arah2 gene sequence

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. The signal peptide sequence to obtain the original nucleotide sequence to be optimized.

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).

Natural gene sequence before optimization (genbank: AY158467.1), such as SEQ ID No.1.

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

Amino acid sequence before optimization (genbank: AAN77576.1), SEQ ID No.3.

The optimized amino acid sequence, SEQ ID No.4.

Example 2 Construction of recombinant expression vector pNZ1

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; _ddH2O : 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 Afterwards, a ligation reaction was performed to obtain the recombinant vector pNZ8148-nArah2, which was named pNZ1. See Figure 1 for the plasmid composition.

Example 3 Preparation of recombinant Lactococcus lactis

The plasmid pNZ1 constructed above was transformed into 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 by electroporation. 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 NZNH, and the empty plasmid strain L.lactis NZ9000 will be carried /pNZ8148 named L. lactis NZ48.

Embodiment 4 Western blot analyzes the expression situation of recombinant protein

(1) Induced expression of recombinant L. lactis NZ9000 strain:

Pick a single colony of the correct recombinant strain and insert it into GM17 medium containing 10 μg/ml chloramphenicol (Cm) (composition of GM17 medium per liter: 5g soybean peptone, 5g bactopeptone, 5g beef extract, 2.5g yeast powder, Glucose 5g, β-glycerol disodium phosphate 19g, vitamin C 0.5g, 1mol/L MgSO ₄ 1mL), 30 ℃ static culture overnight; transfer the overnight culture solution to GM17 (Cm 10μg/ ml) liquid medium, cultured at 30°C until OD ₆₀₀ was about 0.6, added nisin (Sigma, purchased from Shanghai Youyang Biotechnology Co., Ltd.) as an inducer, and made the final concentration 50ng/ml, and continued to culture at 30°C for 6 hours .

(2) Protein sample preparation:

After the induction, the bacterial solution was centrifuged at 5,000 g for 10 min at 4°C, and the resulting bacterial pellet was washed twice with PBS and then resuspended in PBS. Sonicate the bacterial suspension, centrifuge at 12,000×g for 30 minutes at 4°C, collect the supernatant, and store it at -80°C for detection; add 100% (w/v) trichloroacetic acid (TCA) to the culture supernatant To a final concentration of 16% (w/v), place on ice for at least 30 minutes, centrifuge at 12,000×g for 30 minutes at 4°C, collect the precipitate and wash it twice with pre-cooled acetone, and finally dissolve the precipitate in 50mM NaOH solution at -80 ℃ for storage until testing.

(3) Western blot analysis of recombinant protein expression:

After the protein samples prepared above were separated by 12% SDS-PAGE gel, the protein was transferred to PVDF membrane (Millipore, purchased from Zhongke Ruitai Biotechnology Co., Ltd.) at 150mA constant flow for 50min; the transfer membrane was passed through TBST (per liter 10×TBS buffer formula: Tris 2.42g, NaCl 8g, adjust pH to 7.6 with HCl; TBST buffer formula: 10×TBS 100ml, distilled water 900ml, Tween-20 0.1%) After washing, wash with TBST (containing 5% skimmed milk powder) at room temperature for blocking for 1.5h; then the transfer membrane was incubated with the primary antibody (1:750 dilution) overnight at 4°C; HRP-labeled goat anti-rabbit secondary antibody (1:1.2× ¹⁰⁴ dilution, purchased from Shanghai Youyang Biotechnology Co., Ltd.) and the transfer membrane were hybridized at room temperature for 1 h, and then the color developing solution (purchased from Beijing Kangwei Century Biotechnology Co., Ltd.) was added dropwise to the transfer membrane, and then the color was developed in the dark. The test results are shown in Figure 2. As shown in the figure: no signal was detected in the supernatant and precipitate of the negative control strain (lane 2 and 3), and about A 19kDa fragment (consistent with the expected size of nArah2 protein), and no corresponding signal was detected in the supernatant fraction (lane 4) of strain L.lactis NZNH.

Example 5 Animal Model Evaluation of Vaccine Immune Effect

The preparation process of the vaccine is as follows: the step of inducing expression of the recombinant strain is as described above. After the induction, the bacterial solution was centrifuged at 5,000 g for 10 min at 4°C, and the resulting bacterial pellet was washed twice with sterile PBS and then resuspended in PBS to a concentration of 1×10 ¹⁰ CFU/ml. The obtained fresh bacterial suspension For immunization of animals.

The animal experiment process is shown in Figure 3: 40 3-5-week-old female C3H/HeJ mice (purchased from Shanghai Slack Experimental Animal Co., Ltd.) were randomly divided into 4 groups (10 mice/group) after one week of adaptive feeding. Animal experiments are divided into three stages:

(1) Prevention stage: 2×10 ⁹ CFU of freshly prepared recombinant strains were intragastrically administered daily for two consecutive weeks from -14 to 0;

(2) Sensitization stage: 12 mg crude peanut protein extract (CPE) and 10 μg CT were administered orally on days 0, 1, 2, 7, 14, and 21;

(3) Provocation stage: On the 28th day, 30 mg of CPE was administered orally for challenge.

Set the first group as the natural group (Naive group), and feed normally throughout the experiment. The second group is the model group (Sham group), which was administered PBS in the prevention stage, and normal sensitization in the sensitization stage. Groups 3 and 4 (named as Vector group and CYT group respectively) were the experimental groups. In the preventive stage, the recombinant strains L.lactis NZ48 and L.lactis NZNH were administered intragastrically, and in the sensitization stage, CPE and CT were administered intragastrically for sensitization. . Mice in all groups were challenged by intragastric administration of CPE on day 28.

On the 35th day, mice serum samples were collected 30-40 minutes after challenge, and stored at -80°C for subsequent detection of allergen-specific antibodies and mMCP-1 levels in serum; after the experiment, mice were sacrificed and spleens were aseptically isolated to prepare spleens The cell suspension was added with 50 μg/mL peanut crude extract (CPE) and co-cultured in a 37°C, 5% CO ₂ incubator for 72 hours. The supernatant was collected and stored at -80°C for the detection of cytokine levels.

The experimental results showed that: compared with the Sham group and the Vector group, the serum specific IgE and IgG1 levels of the mice in the CYT group after challenge decreased, and the specific IgG2a level increased (see Figure 4), indicating that the intracellular expression of lactic acid Lactococcus recombinant vaccine can inhibit Th2 type antibody response to a certain extent and promote the occurrence of Th1 type antibody response; at the same time, mast cell degranulation in mice of CYT group is also inhibited (see Figure 5). Intragastric administration of the recombinant strain L.lactis NZNH can also inhibit the secretion of Th2 cytokine IL-4 in the culture supernatant of mouse splenocytes, promote the production of Th1 cytokines IFN-γ and IL-12 in splenocytes, and increase Th1/ Ratio of Th2 cytokines (see Table 1). Based on the above results, the intracellular expression vaccine of the present invention can reverse the transformation of Th2 type immune response to Th1 type immune response, and has a certain immune regulation effect on peanut allergy.

Table 1: Cytokine levels in splenocyte culture supernatant

* indicates significant difference between the experimental group and the Sham group, P<0.05

Although the patent of the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims (7)

1. the Recombinant Lactococcus lactis bacterial strain of energy recombinant expressed Peanut Allergen nArah2 in cell, it is characterized in that, described bacterial strain is Lactococcus lactis NZNH, on September 18th, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.8216.

2. the vaccine that comprises Recombinant Lactococcus lactis bacterial strain claimed in claim 1, is characterized in that, described vaccine is oral preparations.

3. the vaccine that comprises Recombinant Lactococcus lactis bacterial strain according to claim 2, is characterized in that, described oral preparations is capsule, lozenge, pulvis, lozenge or granule.

4. Recombinant Lactococcus lactis bacterial strain claimed in claim 1 can be used for treating the purposes in the Lactococcus lactis bacteria vaccine of peanut allergy in preparation.

5. a method of preparing Recombinant Lactococcus lactis bacterial strain claimed in claim 1, is characterized in that described method comprises the steps:

A) the natural gene sequence of Peanut Allergen Arah2 is carried out codon optimized with the gene nArah2 that is optimized, as shown in SEQ No.2;

B) utilize optimized gene nArah2 further to build intracellular expression vector plasmid PNZ1;

C) utilize recombinant expression plasmid conversion Lactococcus lactis Screening and Identification to obtain Recombinant Lactococcus lactis bacterial strain.

6. the method for preparing Recombinant Lactococcus lactis bacterial strain according to claim 5, while it is characterized in that building recombinant expression plasmid PNZ1 in step b), comprise and take plasmid pUC57-nArah2 as template, utilize primer Pra1F5'-CATGCCATGGGTCAACAATGGGAATTACAAG-3' and Pra1R:5'-CGGGGTACCTTAATAACGATCACGACCAC-3' nArah2 gene to be carried out to the step of pcr amplification.

7. the method for preparing Recombinant Lactococcus lactis bacterial strain according to claim 6, is characterized in that the method for transformation in step c) is that electricity transforms.

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