CN103642746A - Recombinant lactococcus lactis for intracellular expression of peanut allergen and application thereof - Google Patents
Recombinant lactococcus lactis for intracellular expression of peanut allergen and application thereof Download PDFInfo
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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]
The Recombinant Lactococcus lactis bacterial strain that the present invention relates to cell inner expression Peanut Allergen Arah2, its construction process and application, belong to technical field of bioengineering.
[background technology]
Peanut allergy is one of serious and the most common food anaphylaxis reaction, has rapid onset, lethality rate is high and the feature such as lifelong property.In the last few years, the sickness rate of peanut allergy had the trend rising year by year in the world.In the U.S., there is 1.1% population to peanut allergy, its sickness rate has increased nearly one times between 1997 to 2002.Inducing specific oral tolerance produces the methods for the treatment of of immunological tolerance by giving the anaphylactogen that patient increases dosage gradually within for some time to this kind of food antigens with induction patient, be more effective peanut allergy methods for the treatment of at present.
Obtaining of high purity anaphylactogen is prerequisite and the basis of carrying out immunotolerance induction.Peanut Allergen has 11 kinds, and wherein Arah2 is that a kind of molecular weight is the glycoprotein of 17-19KDa, can be exceeded IgE specific recognition in 90% patients serum, is that research is the most also one of most important Peanut Allergen at present.In the last few years, the development of Protocols in Molecular Biology made to utilize biosynthetic means to obtain highly purified anaphylactogen becomes possibility.At present existing domestic and international investigator utilizes different expression systems to produce Peanut Allergen, as (2012) such as Tao Ailin have introduced the method for utilizing escherichia expression system to prepare a kind of recombinate Peanut Allergen and mutant in mono-kind of patent < < recombinates Peanut Allergen and mutant and its preparation method and application > >; Katrin Lehmann etc. successfully utilizes Recombinant protein expression to have good immunogenic Arah2 albumen (Katrin Lehmann, et al.High-yield expression in Escherichia coli, purification, and characterization of properly folded major peanut allergen Arah2.Protein Expression and Purification, 2003,31:250 – 259).But intestinal bacteria can produce complex structure, miscellaneous intracellular toxin, and the subsequent purification of expression product is with high costs, have limited the clinical application of escherichia coli prokaryotic expression system.
Lactococcus lactis is widely used in the production of the traditional foods such as Yoghourt, cheese, pickles for a long time, is generally regarded as safe microorganism.Utilizing Lactococcus lactis to express foreign protein can take together with thalline without purifying, utilizes Lactococcus lactis can effectively induce body to produce immune response and immunological tolerance as the mucosal vaccine of expression vector simultaneously.Therefore, we utilize Lactococcus lactis (Lactococcus lactis) to be delivery carrier as mucosal vaccine, Peanut Allergen Arah2 is expressed in L.lactis cell, and constructed Recombinant Lactococcus lactis bacterial strain is significant in fields such as medicine industries.
[summary of the invention]
Technical problem to be solved by this invention is to provide Recombinant Lactococcus lactis bacterial strain and the construction process thereof of a kind of cell inner expression Peanut Allergen albumen nArah2, this bacterial strain is to obtain with the recombinant plasmid pNZ1 conversion Lactococcus lactis of the encoding gene of the coding Peanut Allergen albumen nArah2 that contains codon optimization, and the Lactococcus lactis that wherein used can be L.lactis NZ9000.The bacterial strain that after transforming, screening obtains is L.lactis NZNH, this bacterial strain Yi China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, and its deposit number is CGMCC:8216.The invention still further relates to the construction process of described Recombinant Lactococcus lactis bacterial strain, 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; B) utilize optimized gene nArah2 further to build recombinant expression plasmid; C) utilize recombinant expression plasmid conversion Lactococcus lactis Screening and Identification to obtain Recombinant Lactococcus lactis bacterial strain, wherein constructed recombinant expression plasmid is intracellular expression vector plasmid PNZ1, while building recombinant expression plasmid PNZ1, comprise and take plasmid pUC57-nArah2 as template, utilize primer Pra1F5'-CATGCCATGGGTCAACAATGGGAATTACAAG-3' and Pra1R:5'-CGGGGTACCTTAATAACGATCACGACCAC-3' to carry out pcr amplification to nArah2 gene, the method for transformation in step c) is that electricity transforms.
The invention still further relates to the vaccine that comprises described Recombinant Lactococcus lactis bacterial strain, described vaccine can be oral preparations, such as capsule, lozenge, pulvis or granule etc.
The invention still further relates to described recombinant plasmid or Recombinant Lactococcus lactis bacterial strain and can be used for treating the purposes in the Lactococcus lactis bacteria vaccine of peanut allergy in preparation.
The Peanut Allergen albumen nArah2 that recombinates in the present invention successfully expresses in L.lactis NZ9000 cell, and results of animal shows, the recombiant vaccine of intracellular expression mode can regulate hypersensitive systemic immunity reaction, reverses Th2 type immunne response and changes to Th1 type immunne response.
Therefore the form that, the Peanut Allergen nArah2 gene of optimization can recombinant plasmid DNA vaccine is for prevention and the treatment of peanut allergy; Recombinant Lactococcus lactis bacterial strain can capsule, the form of lozenge, powder bag, particulate state packing, is applied to clinical prevention and the immunotherapy of peanut allergy as a kind of novel oral vaccine.
The Recombinant Lactococcus lactis Lactococcus lactis NZNH the present invention relates to, 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.
[accompanying drawing explanation]
The structural representation of Fig. 1 Lactococcus lactis recombinant plasmid PNZ1;
Fig. 2: in recombinant bacterial strain L.lactis NZNH, the Western blot of protein expression situation detects figure: the nArah2 albumen that swimming lane 1 is purifying is as positive control, swimming lane 2,3 is respectively cleer and peaceful cell precipitation component on the culture of bacterial strain L.lactis NZ48, and swimming lane 4,5 is respectively cleer and peaceful cell precipitation component on the culture of bacterial strain L.lactis NZNH;
Fig. 3: experimentation on animals immune programme for children figure;
Fig. 4: excite specific antibody level in rear serum: A-C to be respectively specific IgE, IgG2a, IgG1 level; As shown in the figure: compare with Vector group with Sham group, oral L.lactis NZNH can reduce specific IgE and IgG1 level in serum, increases specific IgG 2a level;
Fig. 5: excite mMCP-1 level: * in rear serum to represent that between experimental group and Sham group, otherness is remarkable, P<0.05.As shown in the figure, compare with Sham group, in CYT group mice serum, mMCP-1 level significantly reduces; This illustrates that oral L.lactis NZNH can significantly suppress mastocyte threshing.
[embodiment]
Carry out by the following examples further to set forth the present invention, the experimental technique of unreceipted actual conditions in lower routine embodiment, substantially all according to common molecular cloning handbook, carry out experimental implementation, if no special instructions, its concentration is mass percent concentration to all ingredients.
First utilize signal peptide prediction software SignalP 4.1 Server to predict the signal peptide sequence of the aminoacid sequence of Arah2 (genbank:AAN77576.1), in the gene order corresponding with above-mentioned aminoacid sequence (genbank:AY158467.1), remove the signal peptide sequence that prediction obtains, obtain parent nucleotide sequence to be optimized.
According to Lactococcus lactis preference codon table, parent nucleotide sequence is optimized, unnamed gene after optimization is nArah2, by Sangon Biotech (Shanghai) Co., Ltd., carrying out full gene synthesizes, and subclone is to carrier pUC57, obtain pUC57-nArah2(synthetic by the raw work in Shanghai, wherein pUC57 is commercialization plasmid).
Natural gene order (genbank:AY158467.1) before optimizing, as SEQ ID No.1.
Gene order (nArah2) after optimizing, SEQ ID No.2.
Aminoacid sequence (genbank:AAN77576.1) before optimizing, SEQ ID No.3.
Aminoacid sequence after optimizing, SEQ ID No.4.
The structure of embodiment 2 recombinant expression vector pNZ1
The plasmid pUC57-nArah2(of take is synthetic by the raw work in Shanghai) be template, utilize primer Pra1F 5'-CATGCCATGGGTCAACAATGGGAATTACAAG-3' and Pra1R:5'-CGGGGTACCTTAATAACGATCACGACCAC-3' to carry out pcr amplification to nArah2 gene.PCR reaction system (cumulative volume is 50 μ L) is: 10 * KOD plus buffer:5 μ L; DNTP:5 μ L; MgSO4:2 μ L; DNA profiling: 2 μ L; Upstream primer/downstream primer (20 μ M): each 1 μ L; KOD plus:0.5 μ L; ddH
2o:33.5 μ L.Response procedures: 95 ℃ of 30s (sex change), 61 ℃ of 30s(annealing), 68 ℃ of 50s(extend) and, 30 circulations of increasing.1.0% agarose gel electrophoresis detects and reclaims purifying nArah2 gene fragment.Amplified fragments and carrier pNZ8148(Mierau and Kleerebezem.10years of the nisin-controlled gene expression system (NICE) the in Lactococcus lactis.Appl Microbiol Biotechnol.2005 of purifying will be reclaimed, 68 (6): 705-717) after double digestion, carry out ligation, obtain recombinant vectors pNZ8148-nArah2, called after pNZ1, plasmid forms referring to Fig. 1.
The preparation of embodiment 3 Recombinant Lactococcus lactis
Above-mentioned structure plasmid pNZ1 electric shock is transformed to L.lactis NZ9000(Kuipers et al.Quorum sensing-controlled gene expression in lactic acid bacteria.Journal of Biotechnology.1998,64 (1): 15-21) competent cell.Picking list bacterium colony carries out bacterium colony PCR and double digestion and verifies and deliver to Sangon Biotech (Shanghai) Co., Ltd. and carry out sequence verification, by the correct transformant called after L.lactis NZNH of order-checking, will carry empty plasmid bacterial strain L.lactis NZ9000/pNZ8148 called after L.lactis NZ48.
(1) abduction delivering of restructuring L.lactis NZ9000 bacterial strain:
Picking verifies that single bacterium colony access of correct recombinant bacterial strain is containing 10 μ g/ml paraxin (Cm) GM17 substratum (every liter of GM17 culture medium prescriptions: soy peptone 5g, bacto peptone 5g, extractum carnis 5g, yeast powder 2.5g, glucose 5g, β-phospho-glycerol disodium 19g, vitamins C 0.5g, 1mol/L MgSO
41mL), 30 ℃ of standing overnight incubation; By incubated overnight bacterium liquid with the 2% inoculum size GM17(Cm 10 μ g/ml that transfer) in liquid nutrient medium, 30 ℃ are cultured to OD
600be about 0.6, add nisin(Sigma, purchased from melodious bio tech ltd, Shanghai) as inductor, making its final concentration is 50ng/ml, 30 ℃ are continued to cultivate 6 hours.
(2) protein sample preparation:
After induction finishes by bacterium liquid in 4 ℃, the centrifugal 10min of 5,000g, gained bacterial sediment, with after PBS washed twice, is resuspended in PBS.Bacteria suspension is carried out to ultrasonication, 4 ℃, after the centrifugal 30min of 12,000 * g, collect supernatant ,-80 ℃ of preservations are with to be detected; In culture supernatants, add 100%(w/v) trichoroacetic acid(TCA) (TCA) to final concentration is 16%(w/v), place at least 30min on ice, 4 ℃, 12, the centrifugal 30min of 000 * g, the washing with acetone twice of precooling for collecting precipitation, finally will be precipitated and dissolved in 50mM NaOH solution, and-80 ℃ of preservations are with to be detected.
(3) Western blot analyzes expression of recombinant proteins situation:
The protein sample of above-mentioned preparation after the separation of 12%SDS-PAGE gel, 150mA constant current transferring film 50min by protein delivery to pvdf membrane (Millipore, purchased from middle Ke Ruitai bio tech ltd); Transfer film is through every liter of 10 * TBS buffer formulation of TBST(: Tris 2.42g, and NaCl 8g, adjusts PH to 7.6 with HCl; TBST buffer formulation: 10 * TBS 100ml, distilled water 900ml, Tween-20 0.1%) after washing, with TBST(containing 5% skim-milk) room temperature sealing 1.5h; Then transfer film and primary antibodie (1:750 extent of dilution) are in 4 ℃ of night incubation; With anti-(1:1.2 * 10 of HRP mark goat-anti rabbit two
4extent of dilution, purchased from melodious bio tech ltd, Shanghai) and transfer film room temperature condition under hybridize on the backward transfer film of 1h, to drip after nitrite ion (purchased from Kang Wei century bio tech ltd, Beijing) and develop the color under lucifuge condition.Detected result is referring to Fig. 2.As shown in the figure: in the supernatant of negative control bacterial strain and precipitation part (swimming lane 2 and 3), do not detect signal, precipitation part (swimming lane 5) at positive control (swimming lane 1) and bacterial strain L.lactis NZNH all detects about 19kDa fragment (in the same size with the expection of nArah2 albumen), and detects without corresponding signal in the supernatant part (swimming lane 4) of bacterial strain L.lactis NZNH.
Vaccine preparation process is as follows: the abduction delivering step of recombinant bacterial strain as mentioned above.4 ℃ of bacterium liquid after induction is finished, the centrifugal 10min of 5,000g, gained bacterial sediment is with being resuspended in PBS after aseptic PBS washed twice, and making its concentration is 1 * 10
10cFU/ml, the fresh bacteria suspension of gained is for immune animal.
Experimentation on animals flow process is as shown in Figure 3: 40 3-5 week female C3H/HeJ mouse in age (purchased from Shanghai Slac Experimental Animal Co., Ltd.) adaptability are fed after one week and are divided at random 4 groups (10/group), and experimentation on animals divides three phases:
(1) prevention stage: the-14-0 gavage continuous two week every day 2 * 10
9the recombinant bacterial strain of the fresh preparation of CFU;
(2) the sensitization stage: 0th, 1,2,7,14,21 day gavage 12mg Semen arachidis hypogaeae protein crude extract (CPE) and 10 μ g CT;
(3) excitation phase: gavage 30mg CPE excited in the 28th day.
If the 1st group is natural group (Naive group), in whole experimentation, normally feed.The 2nd group is model group (Sham group), at prevention stage gavage PBS, and carries out normal sensitization in the sensitization stage.3rd, 4 groups (called after Vector group and CYT groups respectively) are experimental group, at prevention stage difference gavage recombinant bacterial strain L.lactis NZ48 and L.lactis NZNH, at equal gavage CPE of sensitization stage and CT, carry out sensitization.The mouse of all groups all excites at the 28th day gavage CPE.
Within the 35th day, excite rear 30-40min to collect mice serum sample ,-80 ℃ of preservations are for subsequent detection allergen-specific antibody in serum and mMCP-1 level; Experiment finishes rear execution mouse and aseptic separating mouse spleen is prepared splenocyte suspension, adds 50 μ g/mL peanut crude extracts (CPE) in 37 ℃, 5%CO
2in incubator, cultivate altogether 72h, collect supernatant and be stored in-80 ℃ for detection of cytokine levels.
Experimental result shows: compare with Vector group with Sham group, excite serological specificity IgE and the IgG1 level of rear CYT group mouse to decrease, specific IgG 2a level rising (referring to Fig. 4), the Lactococcus lactis recombiant vaccine that intracellular expression mode is described can suppress Th2 type antibody response to a certain extent, promotes the generation of Th1 type antibody response; In CYT group Mice Body, mastocyte threshing is also suppressed (referring to Fig. 5) simultaneously.Gavage recombinant bacterial strain L.lactis NZNH also can suppress the secretion of Th2 cytokines IL-4 in mouse boosting cell culture supernatant, promotes the generation of Th1 cytokines IFN-γ, IL-12 in splenocyte and improves Th1/Th2 cytokine ratio (referring to table 1).Based on above result, in the present invention, intracellular expression mode vaccine reversible Th2 type immunne response changes to Th1 type immunne response, and peanut allergy is had to certain immunoregulation effect.
Table 1: cytokine levels in spleen cell cultures supernatant
* represent that between experimental group and Sham group, otherness is remarkable, P<0.05
Although patent of the present invention with preferred embodiment openly as above, it is not in order to limit the present invention.Any person skilled in the art, without departing from the spirit and scope of the present invention, can do various changes and modification.Therefore protection scope of the present invention should be with being as the criterion that claims were defined.
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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| CN107653260A (en) * | 2017-11-08 | 2018-02-02 | 中国水产科学研究院珠江水产研究所 | A kind of preparation method and application of Recombinant Lactococcus lactis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102584966A (en) * | 2011-11-14 | 2012-07-18 | 广州医学院第二附属医院 | Recombinant peanut allergen and mutant and preparation method and application of recombinant peanut allergen and mutant |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107653260A (en) * | 2017-11-08 | 2018-02-02 | 中国水产科学研究院珠江水产研究所 | A kind of preparation method and application of Recombinant Lactococcus lactis |
| CN107653260B (en) * | 2017-11-08 | 2020-11-17 | 中国水产科学研究院珠江水产研究所 | Preparation method and application of recombinant lactococcus lactis |
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