CN103614331A - Preparation method and application of recombinant lactococcus lactis capable of displaying peanut allergen on surface - Google Patents
Preparation method and application of recombinant lactococcus lactis capable of displaying peanut allergen on surface Download PDFInfo
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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]
The present invention relates to surface display and express the Recombinant Lactococcus lactis bacterial strain of 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, there is rapid onset, lethality rate is high and
The features 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 > >; KatrinLehmann etc. successfully utilize Recombinant protein expression to have good immunogenic Arah2 albumen (KatrinLehmann, etal.High-yieldexpressioninEscherichiacoli, purification, andcharacterizationofproperlyfoldedmajorpeanutallergenAr ah2.ProteinExpressionand 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.There are some researches show, utilize host cell surface display technique to express exogenous antigen or human cytokines, the effectively immune response of excitating organism protectiveness of prepared Lactococcus lactis mucosal vaccine.The investigators such as Xin are anchored on Lactococcus lactis cell surface by the V2-V4 district fragment of HIV virus envelope protein and carry out presenting and expressing, with this recombinant bacterium oral immunity mouse, can stimulate body to produce specific serum IgG and the ight soil IgA antibody of high-level anti-HIV, make HIV virus load in mouse ovarian reduce by 350 times of (Xin, etal.Immunogenicityandprotectiveefficacyoforallyadminist eredrecombinantLactococcuslactisexpressingsurface-boundH IVEnv.Blood, 2003,102:223-228).Therefore, we utilize Lactococcus lactis (Lactococcuslactis) to be delivery carrier as mucosal vaccine, Peanut Allergen Arah2 is carried out to presenting and expressing at L.lactis cell surface, and constructed Recombinant Lactococcus lactis has broad application prospects 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 that a kind of surface display is expressed Peanut Allergen albumen nArah2, this bacterial strain is to obtain with the recombinant plasmid pNZ3 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.lactisNZ9000.The bacterial strain that after transforming, screening obtains is L.lactisNZCW, this bacterial strain Yi China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, and its deposit number is CGMCCNo.8218.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 to be 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 grappling expression vector plasmid PNZ3, while building recombinant expression plasmid PNZ3, comprise that take the genomic dna of L.lactissubsp.cremorisMG1363 is template, utilizes primer Pca1F:5'-TAGGGTACCTCTGGTGGCTCGACAACCACAATTAC-3' and Pca1R:5'-CCGCAAGCTTTTATTTTATTCGTAGATACTGACCAATT-3'PCR amplification-cwa sequence; Utilize primer Psp1F:5'-CCGGCCATGGTGAAAAAAAAGATTATCTCAG-3' and Pra1 ' R:5'-CGGGGTACCATAACGATCACGACCACCT-3'PCR amplification-SP
usp45the step of-nArah2 fusion sequence, 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 is successfully anchored on L.lactisNZ9000 cell surface and carries out presenting and expressing, and results of animal shows, the adjustable hypersensitive systemic immunity reaction of Recombinant Lactococcus lactis vaccine of surface display phraseology, 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 LactococcuslactisNZCW the present invention relates to, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 18th, 2013, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, Institute of Microorganism, Academia Sinica, deposit number is CGMCCNo.8218;
Lactococcus lactis Lactococcus lactisNZNH, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 18th, 2013, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, Institute of Microorganism, Academia Sinica, deposit number is CGMCCNo.8216.
[accompanying drawing explanation]
The structural representation of Fig. 1 Lactococcus lactis recombinant plasmid PNZ3;
Fig. 2: in recombinant bacterial strain L.lactisNZCW, the Westernblot 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 L.lactisNZ48 bacterial strain, and swimming lane 4-6 is respectively protoplastis, cell walls and the supernatant component of L.lactisNZCW strain culture;
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, * represents that between experimental group and Sham group, otherness is remarkable, P<0.05.
[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 SignalP4.1Server 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, SEQIDNo.1.
Gene order (nArah2) after optimizing, SEQIDNo.2.
Aminoacid sequence (genbank:AAN77576.1) before optimizing, SEQIDNo.3.
Aminoacid sequence after optimizing, SEQIdNo.4.
The structure of embodiment 2 recombinant expression vector pNZ3
Utilize L.lactissubsp.cremorisMG1363(Gasson.Plasmidcomplementsof Streptococcus lactis NCDO712and other lactic streptococci after protoplast-induced curing.Journal of Bacteriology.1983,154 (1): (be openly gene order of Genebank, the cA structural domain in GenBank:U17696.1) is as grappling functional sequence CWA for intrinsic N-acetyl muramidase AcmA gene 1-9).Utilizing the genomic dna of L.lactis subsp.cremorisMG1363 is template, utilizes primer Pca1F:5'-TAGGGTACCTCTGGTGGCTCGACAACCACAATTAC-3' and Pca1R:5'-CCGCAAGCTTTTATTTTATTCGTAGATACTGACCAATT-3'PCR amplification to carry out pcr amplification-cwa sequence.PCR reaction system (cumulative volume is 50 μ L) is: 10 * KODplusbuffer: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; KODplus:0.5 μ L; ddH
2o:33.5 μ L.Response procedures: 95 ℃ of 30s (sex change), 59 ℃ of 30s (annealing), 68 ℃ of 60s (extension), increase and circulate for 30 times.Simultaneously with plasmid pNZ8148-SP
usp45-nArah2, as template, utilizes primer Psp1F:5'-CCGGCCATGGTGAAAAAAAAGATTATCTCAG-3' and Pra1 ' R:5'-CGGGGTACCATAACGATCACGACCACCT-3'PCR amplification to carry out pcr amplification-SP
usp45-nArah2 fusion sequence.PCR reaction system is the same.Response procedures: 95 ℃ of 30s (sex change), 60 ℃ of 30s (annealing), 68 ℃ of 50s (extension), increase and circulate for 30 times.Two fragments-cwa ,-SP of purifying will be reclaimed
usp45-nArah2 and carrier pNZ8148 carry out ligation after endonuclease reaction respectively, obtain plasmid pNZ8148-SP
usp45-nArah2-cwa, called after pNZ3, plasmid forms referring to Fig. 1.
The preparation of embodiment 3 Recombinant Lactococcus lactis
Above-mentioned structure plasmid pNZ3 electric shock is transformed to L.lactisNZ9000(Kuipersetal.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.lactisNZCW of order-checking, will carry empty plasmid bacterial strain L.lactisNZ9000/pNZ8148 called after L.lactisNZ48.
(1) abduction delivering of restructuring L.lactisNZ9000 bacterial strain:
Picking verifies that single bacterium colony access of correct recombinant bacterial strain is containing 10 μ g/ml paraxin (Cm) GM17 trainings
Support in base (every liter of GM17 culture medium prescription: 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/LMgSO41mL) 30 ℃ of standing overnight incubation; By incubated overnight bacterium liquid with the 2% inoculum size GM17(Cm10 μ g/ml that transfers) 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:
Induction finish after by bacterium liquid in 4 ℃, 5, the centrifugal 10min of 000g, adds 100%(w/v in gained supernatant liquor) trichoroacetic acid(TCA) (TCA) to final concentration is 16%(w/v), place at least 30min on ice, 4 ℃, the centrifugal 30min of 12,000 * g, twice of the washing with acetone of collecting precipitation use precooling, finally will be precipitated and dissolved in 50mMNaOH solution ,-80 ℃ of preservations are with to be detected.Bacterial sediment TES(10mMTris-HCl[pH8.0], 1mMEDTA, 25%sucrose) after twice of solution washing, be resuspended in TES(10mg/mL N,O-Diacetylmuramidase, 0.1mg/mLRNase) in, 37 ℃ of temperature are bathed 1h.To obtain 4 ℃ of above-mentioned bacterium liquid, the same culture supernatants for the treatment of process of gained supernatant after the centrifugal 10min of 2500 * g, gained sample is for analysis of cells wall albumen distribution situation; Centrifugal gained precipitation is resuspended in after TES for analyzing protoplastis albumen distribution situation.Gained sample is all stored in-80 ℃ 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: Tris2.42g of TBST(, and NaCl8g, adjusts PH to 7.6 with HCl; TBST buffer formulation: 10 * TBS100ml, distilled water 900ml, Tween-200.1%) 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 after 1h, to dripping on transfer film after nitrite ion (purchased from Kang Wei century bio tech ltd, Beijing), under lucifuge condition, develop the color.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, at positive control (swimming lane 1), detect about 19kDa fragment (in the same size with the expection of nArah2 albumen); Cell walls part (swimming lane 5) at bacterial strain L.lactisNZCW detects about 46kDa fragment, and supposition may be for removing the nArah2-CWA albumen of signal peptide; The protoplastis part (swimming lane 4) of L.lactisNZCW detects about 49kDa fragment, may be SP
usp45-nArah2-CWA precursor protein.
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: 50 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 5 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 gCT; (3) excitation phase: gavage 30mgCPE 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.3-5 group (respectively called after Vector group, CYT group and CWA group) is experimental group, the prevention stage respectively gavage recombinant bacterial strain L.lactis NZ48, L.lactis NZNH( built can intracellular expression nArah2 Recombinant Lactococcus lactis bacterial strain; Deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is CGMCC No.8216) and L.lactis NZCW, 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 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: gavage L.lactis NZNH and L.lactis NZCW all can suppress to excite specific IgE and the generation that promotes specific IgG 2a in rear mice serum, but only the variation of L.lactis NZCW group reaches statistical significance level (P<0.05); Explanation is aspect the humoral immune reaction of regulation system, and the regulating effect of L.lactis NZCW is better than L.lactis NZNH(referring to Fig. 4).Compare with Sham group, in CWA group and CYT group splenocyte, the quantity of Th2 cytokines IL-4 and quantity, the Th1/Th2 cytokine ratio of Th1 cytokines IFN-γ have respectively the trend that reduces and raise, and the intensity of variation of CWA group is obviously greater than CYT group; The regulating effect more superior (referring to table 1) of this explanation L.lactisNZCW aspect adjusting cell immune response.Based on above result; aspect humoral immunization and cellular immunization; surface display type Recombinant Lactococcus lactis all can effectively reverse hypersensitive Th2 type immunne response to the Th1 type immunne response direction transformation of protectiveness as mucosal vaccine, and peanut allergy is had to effective 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 an energy surface display Peanut Allergen nArah2, it is characterized in that, described bacterial strain is LactococcuslactisNZCW, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 18th, 2013, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, Institute of Microorganism, Academia Sinica, deposit number is CGMCCNo.8218.
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 SEQNo.2;
B) utilize optimized gene nArah2 further to build surface display expression vector plasmid PNZ3;
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 PNZ3 in step b), comprise that take the genomic dna of L.lactissubsp.cremorisMG1363 is template, utilizes primer Pca1F:5'-TAGGGTACCTCTGGTGGCTCGACAACCACAATTAC-3' and Pca1R:5'-CCGCAAGCTTTTATTTTATTCGTAGATACTGACCAATT-3'PCR amplification-cwa sequence; Utilize primer Psp1F:5'-CCGGCCATGGTGAAAAAAAAGATTATCTCAG-3' and Pra1 ' R:5'-CGGGGTACCATAACGATCACGACCACCT-3'PCR amplification-SP
usp45the step of-nArah2 fusion sequence.
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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| 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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| CN113278643A (en) * | 2021-05-19 | 2021-08-20 | 中国农业科学院饲料研究所 | Method for constructing genetic engineering bacteria with functions of reducing liver fat accumulation and protecting intestinal barrier and application |
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Application publication date: 20140305 |