CN102586161B

CN102586161B - Mycobacterium smegmatis IL-17A and preparation method thereof

Info

Publication number: CN102586161B
Application number: CN201110009164.7A
Authority: CN
Inventors: 张建华; 吴良霞; 郭盛; 范小勇; 陈凌; 郝春莉
Original assignee: Shanghai Sixth Peoples Hospital
Current assignee: Shanghai Sixth Peoples Hospital
Priority date: 2011-01-17
Filing date: 2011-01-17
Publication date: 2014-08-13
Anticipated expiration: 2031-01-17
Also published as: CN102586161A

Abstract

The invention relates to a mycobacterium smegmatis IL-17A and a preparation method thereof. An escherichia coli-mycobacteria shuttle expression vector for expressing an IL-17A gene is expressed in the mycobacterium smegmatis IL-17A; and a bacterial strain is preserved at Ordinary Microorganism Center of China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC No. 4446. The mycobacterium smegmatis IL-17A has the double effects of mycobacterium smegmatis and IL-17A, so that the effects of the recombined mycobacteria on promoting the nonspecific anti-infections immunity and restraining the allergen mediated airway immune inflammatory damage are remarkably enhanced, and particularly, the recombined mycobacteria can play important roles in preventing and controlling the asthma airway injury caused by allergic asthma or respiratory tract infection.

Description

M. smegmatics IL-17A and preparation method thereof

Technical field

The present invention relates to a kind of M. smegmatics (Mycobacterium smegmatis) bacterial strain and preparation method thereof, relate in particular to recombinant Mycobacterium smegmatis of a kind of IL-17A of expression and preparation method thereof.

Background technology

Immunological adjuvant claims again non-specific immunity proliferant agent, be a kind of and antigen simultaneously or be injected in advance body, can enhancing body immunne response or change the auxiliary goods and materials of type of immune response, it is long-pending that it can increase antigenic surface, make antigen be easy to by macrophage phagocytic, change antigen physical behavior, extend the antigenic action time, strengthen helper cell effect, stimulate the division of primed lymphocyte and plasmacytic generation, improve dripping to become and change antibody and producing type and delayed allergy of the first and secondary immune response antibody of body, and make its enhancing; Therefore, immunological adjuvant is having aspect the prevention of disease and important effect.

Alum adjuvant is first adjuvant that is approved for people, and major function is slow releasing function, also possess the activation to immunocyte, but Alum adjuvant can cause that with after some antigen hybrid injection local inflammation reacts, and even forms granuloma simultaneously.Freund's adjuvant is a kind of adjuvant that contains mycobacterium composition, has stronger immune-enhancing effect, is most widely used test adjuvant, due to the strong side reaction in freund's adjuvant injection site, and therefore this adjuvant immune Research for experiment purpose at present.

Cytokine as immunologic adjuvant in clinical application, the recombinant bacillus Calmette-Guerin vaccines such as IL-2, IFN-γ have been successfully applied to the treatment research of superficial bladder cancer, a kind of secretion of tumour necrosis factor-alpha recombinant bacillus Calmette-Guerin vaccine as disclosed in Chinese patent CN100360668C, and the disclosed secretion human interleukin-2 of Chinese patent CN1710072A recombinant bacillus Calmette-Guerin vaccine, all can effectively prevent and treat superficial bladder tumor; Chinese patent CN100374548C openly also discloses a kind of BCG vaccine strain of recombined interferon, in increasing immune effect, can also reduce BCG consumption, reduces toxic side effect.But the poky characteristic of BCG is that transgenosis process is brought inconvenience.

M. smegmatics (Mycobacterium smegmatis) is to have and the mycobacterium of BCG comm on antigen, also be conventional cellular immunization adjuvant, compare with BCG, M. smegmatics has the feature fast, non-virulent of growing, and its range of application is more extensive.At present existing cytokine profiles is expressed in M. smegmatics, as the recombinant Mycobacterium smegmatis of expressing TNF-α is applied to bladder cancer, and acquisition strengthens immunne response effect (Int. J. Cancer, 2004,112 (4): 653-660) preferably; Recombinant Mycobacterium smegmatis as disclosed in patent CN1339583A, contains bacillus tuberculosis typus humanus's Heat Shock Protein 70 Promoter cDNA, also can be used to treat tumor of bladder; The recombinant Mycobacterium smegmatis (CCTCC NO:M2010097) that patent CN101875913A discloses a kind of mycobacterium tuberculosis Ag 85 B and ESAT-6 fusion rotein can be used for prevention and treatment tuberculosis; The recombinant Mycobacterium smegmatis of and for example expressing IL-2 and GLS can strengthen the immunne response (Vaccine, 2007,25 (4): 638-648) that mouse treating pulmonery tuberculosis bacterium infects.But the recombinant Mycobacterium smegmatis of expressing IL-17A (interleukin-17 A, Interleukin 17A) there is not yet report.

Summary of the invention

The invention provides recombinant Mycobacterium smegmatis (Mycobacterium smegmatis) IL-17A of a kind of IL-17A of expression (SEQUENCE No.4); built the recombinant Mycobacterium smegmatis of a kind of IL-17A of expression by shuttle expression carrier; and prepare this recombinant Mycobacterium smegmatis vaccine; make recombiant vaccine stimulate inherent immunity and Acquired immune response ability to be strengthened, improve immune protection effectiveness.

M. smegmatics IL-17A of the present invention has been preserved in Chinese common micro-organisms culture presevation administrative center, and preserving number is CGMCC NO.4446, preservation date: on December 10th, 2010, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; The bacillus coli-mycobacteria shuttle expression carrier of wherein, expressing IL-17A gene is expressed in described M. smegmatics IL-17A.

M. smegmatics preparation method of the present invention is as follows:

Step 1, according to IL-17A gene order design of amplification primers, obtains IL-17A genetic expression fragment through pcr amplification, then described IL-17A genetic expression fragment is cloned into carrier, obtains recombinant vectors;

Step 2, electricity transforms described recombinant vectors to M. smegmatics, obtains described M. smegmatics IL-17A.

Preferably, introduce at IL-17A upstream region of gene at designed upstream primer bamh I restriction enzyme site; Designed downstream primer is introduced in IL-17A gene downstream hind III restriction enzyme site.

Wherein, described amplimer is preferably by 5 ' end to 3 ' end oligonucleotide sequence:

Upstream primer: CAG gGATCCgCGGCTACAGTGAAGGC (SEQUENCE No.1);

Downstream primer: AGT aAGCTTtTAGGCTGCCTGGCGGACAAT (SEQUENCE No.2).

Wherein, in described upstream primer, GGATCC (underscore part) is bamh I restriction enzyme site; AAGCTT in described downstream primer (underscore part) is hind III restriction enzyme site.

Wherein, described carrier is preferably bacillus coli-mycobacteria shuttle expression carrier (SEQUENCE No.3).

The present invention also provides the application of described M. smegmatics IL-17A in medicine, vaccine or the immunological adjuvant of the non-specific anti-infectious immunity of preparation enhancing.

The present invention also provides the application of described M. smegmatics IL-17A in medicine, vaccine or the immunological adjuvant of the immunity of preparing property.

Wherein, be preferably in preparation prevention and control application, the especially application in preparation treatment and prevention children allergic asthma medicine in the asthma air flue damage medicine causing because of allergic asthma or respiratory tract infection.And more preferably prevent and control the application in the medicine that infects the inflammation damnification causing because of mycoplasma pneumoniae (mycoplasma pneumoniae, MP) in preparation.

In sum, the invention provides M. smegmatics IL-17A of the restructuring of a kind of IL-17A of expression and preparation method thereof, and provide described M. smegmatics IL-17A in the application of preparing in medicine.

IL-17A is a kind of important factor that regulates inherent immunity and acquired immunity, by promoting the number of ways such as chemokine, expression of inflammatory cytokines to participate in defence and the scavenging process of pathogenic infection; And aspect T cell memory effect, have great importance waking up after immunization.Therefore the mycobacterial vaccine of construction expression IL-17A contributes to strengthen the non-specific anti-infection ability that vaccine inoculation excites, the acute aggravation of asthma that contributes to prophylaxis of microbial to infect to cause; And exogenous IL-17A main manifestations in the time of low dosage raises and suppress Th2 reaction for suppressing eosinophil, experimentation on animals increases the weight of hardly mouse asthma neutrophil leucocyte while showing exogenous IL-17A dosage lower than 2.5 μ g/kg and raises; And the ability that IL-17A inhibition TNF-α induces RANTES to express is that it activates 6 times that TNF-α induces IL-6, IL-8 to secrete, therefore the mycobacterial vaccine of low dosage expression IL-17A has obvious advantage in prevention of asthma.

M. smegmatics and mycobacterium tuberculosis (Mycobacterium Tuberculosis) have high homology, can be used as the alternative bacterial classification of antitubercular agent, and M. smegmatics is fast raw bacterium and non-virulent bacterium, has better application prospect.

M. smegmatics IL-17A of the present invention has the dual function of M. smegmatics and IL-17A, the effect that makes mycobacterium after restructuring promote non-specific anti-infectious immunity and to suppress the air flue immune inflammation damage of anaphylactogen mediation is strengthened significantly, especially prevention and control allergic asthma or asthma air flue damage that respiratory tract infection causes in can play important effect.

Brief description of the drawings

Fig. 1 is M. smegmatics and M. smegmatics IL-17A growth curve chart of the present invention;

Fig. 2 is the expression of results figure of WB qualification IL-17A in M. smegmatics IL-17A of the present invention;

Fig. 3 is that pMFA41-mIL-17a recombinant plasmid is at r m.smegmatispCR qualification and the enzyme of expressing are cut qualification result.

Embodiment

The invention provides a kind of M. smegmatics bacterial strain IL-17A that expresses recombinant interleukin-17A, be with existing M. smegmatics difference: the bacillus coli-mycobacteria shuttle expression carrier of expressing IL-17A gene is expressed in M. smegmatics.

Its preparation method is as follows:

Step 1 according to known IL-17A gene order design of amplification primers, and is introduced respectively in upstream and downstream primer bamh I restriction enzyme site and hind III restriction enzyme site; Obtain IL-17A genetic expression fragment through PCR reaction; Described IL-17A genetic expression fragment is cloned into carrier (as bacillus coli-mycobacteria shuttle expression carrier, pMFA41 carrier), obtains recombinant vectors; Wherein, obtaining and purifying of IL-17A gene order can operate according to prior art, and operational condition is introduced in content below, does not repeat them here.

Step 2, M. smegmatics is cultivated and is prepared competent cell, and electricity transforms described recombinant vectors to M. smegmatics, obtains described M. smegmatics IL-17A.

M. smegmatics IL-17A of the present invention has been preserved in Chinese common micro-organisms culture presevation administrative center, and preserving number is CGMCC NO.4446.

Below with mouse interleukin-17-A, bacillus coli-mycobacteria shuttle expression carrier pMFA41 and M. smegmatics m.smegmatismc ²155 bacterial strains are example, by specific embodiment, M. smegmatics IL-17A of the present invention and preparation method thereof are specifically introduced, and better to understand the present invention, but following embodiment does not limit the scope of the invention.

Experiment material: mouse, m.smegmatismc ²155 (department of microbiology mycobacterium researchs department of vertical university, Colorado), pMD ^tM18-T carrier (TaKaRa company), pET28a carrier (Novagen company), intestinal bacteria e.coli, TOP10 (market is buied), pMFA41 carrier (self-control).

step 1, according to IL-17 A gene order design of amplification primers, obtains IL-17 A genetic expression fragment through PCR reaction; And described IL-17 A genetic expression fragment is cloned into carrier, obtain recombinant vectors;

1.1. the preparation of mouse boosting cell and cultivation

6 week age, the spleen of female BALB/c mouse was put into substratum, and ground gently pressure in 200 order stainless steel cell sieves, prepared cell suspension.

Obtained cell suspension moves in centrifuge tube, centrifugal three times of 1640 substratum washings, 1000r/min; Abandon supernatant, add erythrocyte cracked liquid to carry out cracking, then add 1640 substratum to vibrate and mix gently, the centrifugal 10min of refrigerated centrifuge horizontal rotor (1000r/min).

Abandon supernatant, add 10% foetal calf serum and dual anti-(penicillin 100U/ml, Streptomycin sulphate 100U/ml) to make cell suspension, be inoculated in Tissue Culture Plate, add the activation of PHA-P irritation cell, cultivate 24 hours harvested cells.

1.2. the amplification of mouse IL-17A gene

The total RNA of Trizol method extracting mouse boosting cell, then prepares cDNA.Wherein cDNA preparation method is as follows: RNA denaturation 5 minutes at 65 DEG C, is placed in cooled on ice immediately; Then carry out reverse transcription reaction, reverse transcription reaction system is as follows:

reverse transcription reaction condition: 37 DEG C of reaction 15min, then 98 DEG C of reaction 5min, the cDNA obtaining.

Design primer amplification mouse IL-17A, primer is as follows:

wherein upstream primer is introduced in mouse IL-17A upstream bamh I restriction enzyme site (underscore part), downstream primer is introduced in mouse IL-17A downstream hindiII restriction enzyme site (underscore part), carries out PCR reaction, and reaction system is as follows:

pCR reaction conditions: PCR response procedures is 95 DEG C, 5 minutes-(94 DEG C, 30 seconds-52 DEG C, 30 seconds-72 DEG C, 1 minute) × 30 times circulation, 72 DEG C extend 5 minutes, add TaqDNA polymerase extension 10min.

IL-17a gene PCR product separation and purifying: PCR product carries out 1.2% agarose gel electrophoresis, cut the sepharose piece containing target DNA in about 438bp place under ultraviolet lamp; Before and after centrifuge tube is added to gel, weigh respectively, calculated for gel weight is designated as 1 gel volume (GV, 100mg=100 μ l volume), adds the sol solutions Buffer DE-A of 3 times of GV; 65 ~ 75 DEG C are heated to blob of viscose and melt completely; The Buffer DE-B that adds 1.5 times of gel volumes, mixes; Shift solution to DNA adsorption column, centrifugal 30 seconds of 5000rpm; Centrifugate is poured adsorption column, centrifugation again into; Adsorption column is transferred in sterilizing 1.5ml centrifuge tube, adds Eluent 25 ~ 30 μ l in the film centre of adsorption column, 65 DEG C of incubations 2 minutes, centrifugal eluted dna, by 60% rate of recovery calculate reclaim after the concentration of each DNA fragmentation.

For IL-17A can easily be purified and with the restructuring of shuttle expression carrier, can carry out the construction step of following recombinant plasmid:

1.3. pMD recombinates ^tMthe structure of 18-T-IL-17A plasmid

PMD ^tM18-T is connected with the PCR product of IL-17A.Reaction system is as follows:

wherein, solution I refers to commercially available pMD ^tM18-T carrier carries solvent.

1.4. the structure of recombinant expression plasmid pET28a-IL-17A

Extracting pMD ^tM18-T-IL-17A plasmid, double digestion is prepared IL-17A gene fragment; Double digestion reaction system is:

double digestion reaction conditions, 37 DEG C, 2 hours.

PET28a adds in TOP10 competence bacterial cell, places 30min on ice, and 42 DEG C are heated 90 seconds, place on ice.37 DEG C of cultivations of LB substratum; Extracting, double digestion pET28a plasmid is prepared pET28a carrier large fragment, and double digestion reaction system is:

double digestion reaction conditions, 37 DEG C, 2 hours.

The pET28a large fragment that double digestion is obtained is connected 2 hours with 22 DEG C of IL-17A gene fragments, makes recombinant plasmid pET28a-IL-17A, and ligation system is as follows:

1.5.pMFA41-IL-17A the structure of recombinant vectors

37 DEG C of double digestion pET28a-IL-17A plasmids obtain IL-17A, and double digestion reaction system is as follows:

pMFA41 plasmid adds in TOP10 competent cell, places 30min on ice, and 42 DEG C are heated 90 seconds, place on ice; Add 37 DEG C of cultivations of LB substratum; Then extracting pMFA41 plasmid.

PMFA41 plasmid is carried out to 37 DEG C of double digestions and make pMFA41 carrier large fragment, double digestion reaction system is as follows:

wherein, pMFA41 plasmid is with reference to existing document (Fan X Y, Ma H, et al., A novel differential expression system for gene modulation in Mycobacteria, [J]. Plasmid, 2009,61 (1): 39 ~ 46, wherein, Fan X Y for one of present inventor model little brave) build.

PMFA41 is connected 2 hours with IL-17A at 22 DEG C of temperature, and ligation system is as follows:

step 2, electricity transforms described recombinant vectors to M. smegmatics

With M. smegmatics ( mycobacterium smegmatis) mc ²155 bacterial classifications are example, in LBG nutrient agar, cultivate 3 ~ 5 days for 37 DEG C, and scraping list colony inoculation, to LBG substratum 200rpm shaking culture to the logarithmic growth cycle, reclaims thalline, and prepares competent cell.

Get competent cell and add pMFA41-IL-17A, place 10min on ice, be transferred in electric shock cup, electric conversion instrument (Gene Pulser Xcell Electioporation System is set, Bio-Rad) parameter is 2.5kv, 25 μ F, 1000 Ω, after electric discharge, add immediately LBG substratum, 37 DEG C of vibration incubations make the antibiotic resistance gene of bacteria resuscitation expression plasmid coding for 4 hours.

The competent cell of conversion is coated on the LBG nutrient agar that contains KNA, is inverted 37 DEG C of plates and cultivates 3 ~ 5 days, obtain M. smegmatics IL-17A (Mycobacterium smegmatis-IL-17A).

step 3, the qualification of M. smegmatics IL-17A (Mycobacterium smegmatis-IL-17A, M.smegmatis-IL-17A)

3.1.PCR qualification

Select the full well-grown bacterium colony of form, adopt the primer in step 1 to carry out PCR reaction; PCR response procedures: 95 DEG C, 5min-(94 DEG C, 30s-52 DEG C, 30s-72 DEG C, 1min) × 30-72 DEG C of extension 10min, 1.2% agarose gel electrophoresis detects amplification.PCR reaction system is as follows:

as shown in Figure 3, in figure, M1 swimming lane is DL-2000 plus DNA marker to electrophoresis detection result; The 1st swimming lane is blank; The 2nd swimming lane is m.smegmatis-IL-17A bacterium colony PCR; The 3rd swimming lane is recombinant vectors pMFA41-IL-17A; The 4th swimming lane is the BamH I-of recombinant vectors pMFA41-IL-17A hind III enzyme is cut product; M2 swimming lane is DL-250 DNA marker.

In figure, the 3rd swimming lane can find out that the present invention has obtained the gene amplification product conforming to expection size, and IL-17A gene is correctly cloned into described M. smegmatics.

3.2.pMFA41-IL-17A plasmid transforms the impact on Mycobacterium smegmatis-IL-17A growth curve

Distinguish picking Mycobacterium smegmatis, Mycobacterium smegmatis-IL-17A colony inoculation in LBG substratum, 37 DEG C of slow speed of revolution shaking culture, timing sampling is measured OD600 value, draws growth curve, as shown in Figure 1.

In Fig. 1, Mycobacterium smegmatis-IL-17A of the present invention is slightly slower than the wild-type bacterium speed of growth, enters logarithmic phase, and can maintain logarithmic growth more than 10 hours after inoculation 14h.

3.3.Western-Bolt the expression of (WB) qualification IL-17A in Mycobacterium smegmatis-IL-17A

(1) Mycobacterium smegmatis-IL-17A tropina preparation: the bacterium liquid of getting after 2 ml inductions is put in Eppendorf tube, 12000 rpm are centrifugal, and 5 min collect bacterial sediment, after TBS washs 2 times, be resuspended in the TBS of 150 μ l, in ice bath, ultrasonic disruption cell to solution is clarified.In whole cell ultrasonic degradation liquid, add 4 times of diluted protein sample-loading buffers of 50 ml.After 95 DEG C of thermally denature 5 min through 12% SDS-polyacrylamide gel electrophoresis protein isolate sample.

(2) SDS-PAGE electrophoresis.

(3) semidrying transferring film: transferring film method is the same, is summarized as follows: make gel, pvdf membrane, filter paper sandwich structure, be flat in half-dried transfer device (Bio-Rad), it is 15 V that voltage is set, constant voltage mode transferring film 20 min.

(3) antibody incubation: seal and to wash membrane method the same, be summarized as follows: by the pvdf membrane after sealing, taking rat anti-mouse IL-17A antibody (R & D system) as primary antibodie, be diluted in sample diluting liquid (5% skim-milk by 1:4000,1 ' TBS-T20), pvdf membrane immerses 4 DEG C of overnight incubation in antibody liquid, abandons primary antibodie solution, washes film 5 times with the TBS-T of 1 times of dilution; Add by 37 DEG C of the goat anti-mouse iggs (Santa Cruz Biotechnology Inc.) of the HRP mark of 1:10000 dilution and hatch 1h, abandon two anti-solution, the TBS-T of 1 times of dilution washes film 5 times.

(4) luminous and develop: to get respectively 300 μ l ECL substrate A and substrate B component and mix and be placed on preservative film, cover by positive PVDF transfer film that it is upper, drag gently pvdf membrane, make it abundant and substrate reactions.Vertically clamp one jiao of pvdf membrane with tweezers, drip-dry substrate solution, wraps to face up with another preservative film and is placed in the dark sheet folder of X-ray.In darkroom, getting an X-ray is placed on film, the sheet that closes folder, according to fluorescence intensity, exposure 1 min, takes out film and drops in developing solution, under red light, observe development band clear after, press from both sides out film and drop in stop bath, rock gently washing, clear water develops photographic film, dry ventilation, and scanner gathers image.

Western-Bolt qualification result as shown in Figure 2, wherein, band 1 is M. smegmatics cell degradation production (total cell lysate of wild type Mycobacterium smegmatis), and band 2 ~ 5 is M. smegmatics-17A cell degradation production (total cell lysate of wild type Mycobacterium smegmatis-IL-17A).As can be seen from the figure, there is positive band in about 20kDa place, and standard control strain is herein without positive band, the target protein that can be combined with antibodies specific that shown recombinant Mycobacterium smegmatis successful expression.

Can find out by above-mentioned qualification result, the method for the invention has been prepared object product M. smegmatics IL-17A.

step 4, M. smegmatics IL-17A functional verification

4.1. strengthen the non-specific anti-infection ability of RAW264.7 cell

Stimulate respectively the RAW264.7 cell of vitro culture with M. smegmatics (M.smegmatis), M. smegmatics IL-17A, after 72 hours, detect the expression of macrophage inflammatory protein (macrophage inflammatory protein, MIP)-1 α, MIP-2 β, INF-γ and IL-4 in culture supernatant with ELISA (enzyme-linked immuno sorbent assay) method; With the expression of Defensin β 2, Eotaxin-1, MIP-1 α, MIP-2 β, IL-6, IL-10 and IL-23mRNA in real-time fluorescence quantitative PCR detection culturing cell.

Result: M. smegmatics and M. smegmatics IL-17A all can promote Defensin β 2, MIP-1 α, MIP-2 β, INF-γ, IL-4 to suppress the RAW264.7 cells that Eotaxin-1 cultivates in vitro, and M. smegmatics IL-17A of the present invention regulates immunne response ability to improve compared with M. smegmatics.Wherein, M. smegmatics IL-17A group of the present invention is respectively 80:20,2.5:1.5,2.5:1,3:1 with the ratio of M. smegmatics group Defensin β 2, MIP-1 α, MIP-2 β, IL-6 multiple inductivity,

4.2. M. smegmatics IL-17A nasal mucosa immunological safety and immunogenicity

M. smegmatics IL-17A rhinovaccination immunity BALB/c mouse, M. smegmatics, IL-17A albumen in contrast, are dynamically observed reaction of inoculation and are detected body weight growth pattern; Carry out mycobacterium CFU in lung tissue and homogenation of spleen tissue (colony-forming unit) counting and pulmonary morphology and observe, assessment M. smegmatics viability and inoculation security; The expression of IL-17A, INF-γ and IL-4 in the detection lung tissue homogenate of ELISA method and serum, assessing it stimulates nonspecific immune response ability.

Result: after expressing IL-17A, mycobacterium vivo and vitro viability is not had a significant effect, pathologic and pathogen culture result show, M. smegmatics IL-17A of the present invention rhinovaccination is a kind of safe immunization ways, can not cause pathologic damage; The effect that the M. smegmatics IL-17A induction IL-17A of the present invention of rhinovaccination simultaneously and IFN-γ express is better than M. smegmatics, regulates holding time than the remarkable enhancing of IL-17A of immunne response effect.

4.3. M. smegmatics IL-17A inoculates anti-MP infection and causes the effect that asthma increases the weight of

Laboratory animal is divided into 9 groups at random, accepts respectively different treatment intervention: M.smegmatis+OVA group is accepted M.smegmatis inoculation and simulated infection; M.smegmatis+OVA+MP group is accepted M.smegmatis inoculation and MP infects; M.smegmatis+OVA+IL-17A+MP group is accepted M.smegmatis inoculation IL-17A immunity and MP infects; M. smegmatics IL-17A+OVA group is accepted M. smegmatics IL-17A inoculation and simulated infection; M. smegmatics IL-17 A+OVA+MP accepts M. smegmatics IL-17 A inoculation and MP infects; PBS+MP group is accepted simulation inoculation and MP infects; IL-17A+MP group is accepted IL-17A immunity and MP infects; M. smegmatics IL-17 A+MP group is accepted M. smegmatics IL-17 A inoculation and MP infects.All OVA treatment group all accept OVA abdominal injection sensitization and aerosol suction excites.In increasing BALF and cells and supernatant, the detection of IL-17A and the immunohistochemical methods of pathologic IL-17A detect, all the other index detecting and assessing methods are all the same.

Result: the present invention expresses IL-17A and makes recombiant vaccine stimulate the ability of IL-6, IL-23 and Defensin β 2 genetic expressions, and the removing ability of mycoplasma pneumoniae is strengthened.M. smegmatics IL-17 A of the present invention suppresses Eotaxin-1 expression, induces the ability of the allergic immune reaction of Th1 type immunne response and downward IgE mediation all to obtain reinforcement compared with M.smegmatist.

Therefore M. smegmatics IL-17A of the present invention has the non-specific anti-infectious immunity of enhancing, regulates the functions such as immune inflammation reaction, can be used for preparation prevention and control the asthma air flue damage medicine causing because of allergic asthma or respiratory tract infection; Especially for current BCG (Bacille Calmette-Guerin) vaccination to the inapt defect of childhood asthma provide protection, M. smegmatics IL-17A of the present invention can and control in the asthma air flue damage aggravation that allergic asthma or respiratory tract infection cause in prevention and play a significant role.

In foregoing of the present invention, described M.smegmatis and Mycobacterium smegmatis all refer to M. smegmatics; M.smegmatis-IL-17A and Mycobacterium smegmatis-IL-17A all refer to M. smegmatics IL-17A of the present invention.

Above specific embodiments of the invention be have been described in detail, wherein NM working method and means should be understood to adopt the art ordinary method and means to operate.Above-described embodiment is just as example simultaneously, and the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and alternative also all among category of the present invention.Therefore, equalization conversion and the amendment done without departing from the spirit and scope of the invention, all should contain within the scope of the invention.

SEQUENCE

<110> Shanghai City Sixth People's Hospital

<120> M. smegmatics IL-17A and preparation method thereof

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 26

<212> DNA

<213> artificial sequence

<220>

<223> PCR primer

<400> 1

cagggatccg cggctacagt gaaggc 26

<210> 2

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<212> DNA

<213> artificial sequence

<220>

<223> PCR primer

<400> 2

agtaagcttt taggctgcct ggcggacaat 30

<210> 3

<211> 417

<212> PRT

<213> the unknown

<220>

<223> interleukin-17 A

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Gly Cys Gly Gly Cys Thr Ala Cys Ala Gly Thr Gly Ala Ala Gly Gly

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Cys Ala Gly Cys Ala Gly Cys Gly Ala Thr Cys Ala Thr Cys Cys Cys

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Thr Cys Ala Ala Ala Gly Cys Thr Cys Ala Gly Cys Gly Thr Gly Thr

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Cys Cys Ala Ala Ala Cys Ala Cys Thr Gly Ala Gly Gly Cys Cys Ala

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Ala Gly Gly Ala Cys Thr Thr Cys Cys Thr Cys Cys Ala Gly Ala Ala

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Thr Gly Thr Gly Ala Ala Gly Gly Thr Cys Ala Ala Cys Cys Thr Cys

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Ala Ala Ala Gly Thr Cys Thr Thr Thr Ala Ala Cys Thr Cys Cys Cys

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Thr Thr Gly Gly Cys Gly Cys Ala Ala Ala Ala Gly Thr Gly Ala Gly

115 120 125

Cys Thr Cys Cys Ala Gly Ala Ala Gly Gly Cys Cys Cys Thr Cys Ala

130 135 140

Gly Ala Cys Thr Ala Cys Cys Thr Cys Ala Ala Cys Cys Gly Thr Thr

145 150 155 160

Cys Cys Ala Cys Gly Thr Cys Ala Cys Cys Cys Thr Gly Gly Ala Cys

165 170 175

Thr Cys Thr Cys Cys Ala Cys Cys Gly Cys Ala Ala Thr Gly Ala Ala

180 185 190

Gly Ala Cys Cys Cys Thr Gly Ala Thr Ala Gly Ala Thr Ala Thr Cys

195 200 205

Cys Cys Thr Cys Thr Gly Thr Gly Ala Thr Cys Thr Gly Gly Gly Ala

210 215 220

Ala Gly Cys Thr Cys Ala Gly Thr Gly Cys Cys Gly Cys Cys Ala Cys

225 230 235 240

Cys Ala Gly Cys Gly Cys Thr Gly Thr Gly Thr Cys Ala Ala Thr Gly

245 250 255

Cys Gly Gly Ala Gly Gly Gly Ala Ala Ala Gly Cys Thr Gly Gly Ala

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Cys Cys Ala Cys Cys Ala Cys Ala Thr Gly Ala Ala Thr Thr Cys Thr

275 280 285

Gly Thr Thr Cys Thr Cys Ala Thr Cys Cys Ala Gly Cys Ala Ala Gly

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Ala Gly Ala Thr Cys Cys Thr Gly Gly Thr Cys Cys Thr Gly Ala Ala

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Gly Ala Gly Gly Gly Ala Gly Cys Cys Thr Gly Ala Gly Ala Gly Cys

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Thr Gly Cys Cys Cys Cys Thr Thr Cys Ala Cys Thr Thr Thr Cys Ala

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Gly Gly Gly Thr Cys Gly Ala Gly Ala Ala Gly Ala Thr Gly Cys Thr

355 360 365

Gly Gly Thr Gly Gly Gly Thr Gly Thr Gly Gly Gly Cys Thr Gly Cys

370 375 380

Ala Cys Cys Thr Gly Cys Gly Thr Gly Gly Cys Cys Thr Cys Gly Ala

385 390 395 400

Thr Thr Gly Thr Cys Cys Gly Cys Cys Ala Gly Gly Cys Ala Gly Cys

405 410 415

Cys

<210> 4

<211> 4395

<212> DNA

<213> artificial sequence

<220>

<223> pMFA41 shuttle expression carrier

<400> 4

gctagcaaca aagcgacgtt gtgtctcaaa atctctgatg ttacattgca caagataaaa 60

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tgagccatat tcaacgggaa acgtcttgct cgaggccgcg attaaattcc aacatggatg 180

ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct 240

atcgcttgta tgggaagccc catgcgccag agttgtttct gaaacatggc aaaggtagcg 300

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tccccgggaa aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg 480

ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt 540

ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg 600

ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag 660

aaatgcataa tcttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac 720

ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg 780

gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc 840

cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat 900

tgcagtttca tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac 960

actggcagag cattacgctg acttgacggg acggcggctt tgttgaataa atcgaacttt 1020

tgctgagttg aaggatcaga tcacgcatct tcccgacaac gcagaccgtt ccgtggcaaa 1080

gcaaaagttc aaaatcacca actggtccac ctacaacaaa gctctcatca accgtggctc 1140

cctcactttc tggctggatg atggggcgat tcaggcctgg tatgagtcag caacaccttc 1200

ttcacgaggc agacctcact agttccactg agcgtcagac cccgtagaaa agatcaaagg 1260

atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 1320

gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 1380

tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 1440

ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 1500

ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 1560

ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 1620

aacgacctac accgaactga gatacctaca gcgtgagcat tgagaaagcg ccacgcttcc 1680

cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 1740

gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 1800

ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 1860

cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 1920

tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 1980

cgctcgccgc agccgaacga ccgagcgcaa cgcgtgcggc cgcacgcgtg agcccaccag 2040

ctccgtaagt tcgggtgctg tgtggctcgt acccgcgcat tcaggcggca gggggtctaa 2100

cgggtctaag gcggcgtgta cggccgccac agcggctctt agcggcccgg aaacgtcctc 2160

gaaacgacgc atgtgttcct cctggttggt acaggtggtt gggggtgctc ggctgtcgct 2220

ggtgtttcat catcagggct cgacgggaga gcgggggagt gtgcagttgt ggggtggccc 2280

ctcagcgaaa tatctgactt ggagctcgtg tcggaccata caccggtgat taatcgtggt 2340

ctactaccaa gcgtgagcca cgtcgccgac gaatttgagc agctctggct gccgtactgg 2400

ccgctggcaa gcgacgatct gctcgagggg atctaccgcc aaagccgcgc gtcggcccta 2460

ggccgccggt acatcgaggc gaacccaaca gcgctggcaa acctgctggt cgtggacgta 2520

gaccatccag acgcagcgct ccgagcgctc agcgcccggg ggtcccatcc gctgcccaac 2580

gcgatcgtgg gcaatcgcgc caacggccac gcacacgcag tgtgggcact caacgcccct 2640

gttccacgca ccgaatacgc gcggcgtaag ccgctcgcat acatggcggc gtgcgccgaa 2700

ggccttcggc gcgccgtcga tggcgaccgc agttactcag gcctcatgac caaaaacccc 2760

ggccacatcg cctgggaaac ggaatggctc cactcagatc tctacacact cagccacatc 2820

gaggccgagc tcggcgcgaa catgccaccg ccgcgctggc gtcagcagac cacgtacaaa 2880

gcggctccga cgccgctagg gcggaattgc gcactgttcg attccgtcag gttgtgggcc 2940

tatcgtcccg ccctcatgcg gatctacctg ccgacccgga acgtggacgg actcggccgc 3000

gcgatctatg ccgagtgcca cgcgcgaaac gccgaatttc cgtgcaacga cgtgtgtccc 3060

ggaccgctac cggacagcga ggtccgcgcc atcgccaaca gcatttggcg ttggatcaca 3120

accaagtcgc gcatttgggc ggacgggatc gtggtctacg aggccacact cagtgcgcgc 3180

cagtcggcca tctcgcggaa gggcgcagca gcgcgcacgg cggcgagcac agttgcgcgg 3240

cgcgcaaagt ccgcgtcagc catgcatgga ggcattgcta tgagcgacgg ctacagcgac 3300

ggctacagcg acggctacaa ccggcagccg actgtccgca aaaagcggcg cgtgaccgcc 3360

gccgaaggcg ctcgaatcac cggactatcc gaacgccacg tcgtccggct cgtggcgcag 3420

gaacgcagcg agtggctcgc cgagcaggct gcacgccgcg aacgcatccg cgcctatcac 3480

gacgacgagg gccactcttg gccgcaaacg gccaaacatt tcgggctgca tctggacacc 3540

gttaagcgac tcggctatcg ggcgaggaaa gagcgtgcgg cagaacagga agcggctcaa 3600

aaggcccaca acgaagccga caatccaccg ctgttctaac gcaattgggg agcgggtgtc 3660

gcgggggttc cgtggggggt tccgttgcaa cgggtcggac aggtaaaagt cctggtagac 3720

gctagttttc tggtttgggc catgcctgtc tcgttgcgtg tttcgttgcg tccgttttga 3780

ataccagcca gacgagacgg ggttctacga atcttggtcg ataccaagcc atttccgctg 3840

aatatcgtgg agctcaccgc cagaatcggt ggttgtggtg atgtacgtgg cgaactccgt 3900

tgtagtgctt gtggtggcat ccgtggcgcg gccgcggtac cagatcttta aatctagagg 3960

cgggcaccgg gacacaccac taccggttta ccctctacca ccttcctgcc gtgcctccac 4020

tcgcgggact ggctgggaca caagcggcgc gggtgatcgc gcaggccgcc accatgcagg 4080

cccggctcat cggaacatac gaaggctgat ccacccgcca tcccacgatc cagcggcccc 4140

ggggcgatcg ggtcctagca gacgcctgtc acgctagcca aagtcttgac tgattcctct 4200

cctgggagtc atattgtcta gtatgtcctc tataccggac ggatccagct gcagaattcg 4260

aagcttatcg atgtcgacgt agttaactag cgtacgatcg actgccaggc atcaaataaa 4320

acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt ccggccatca 4380

tggccgcggt gatca 4395

Claims

1. M. smegmatics (Mycobacterium smegmatis) IL-17A, is characterized in that, expresses the bacillus coli-mycobacteria shuttle expression carrier of IL-17A gene and expresses in M. smegmatics IL-17A; This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCC NO.4446.

2. a preparation method of M. smegmatics as claimed in claim 1 (Mycobacterium smegmatis) IL-17A, is characterized in that, step is as follows:

Step 1, according to IL-17A gene order design of amplification primers, through PCR clone IL-17A maturation protein encoding gene fragment; Described IL-17A gene fragment clone, to bacillus coli-mycobacteria shuttle expression carrier, is obtained to recombinant vectors; Described amplimer is as follows:

Upstream primer: 5 '-CAGGGATCCGCGGCTACAGTGAAGGC-3 ';

Downstream primer: 5 '-AGTAAGCTTTTAGGCTGCCTGGCGGACAAT-3 ';

In wherein said upstream primer, GGATCC is BamH I restriction enzyme site; In described downstream primer, AAGCTT is Hind III restriction enzyme site; Step 2, electricity transforms described recombinant vectors to M. smegmatics, obtains described M. smegmatics IL-17A, and wherein, the shock parameters of electric conversion instrument is voltage 2.5kv, electric capacity 25 μ F, resistance 1000 Ω.

3. preparation method according to claim 2, is characterized in that, described electricity transforms after product and on the LBG of kantlex nutrient agar, screens and cultivate.

4. a M. smegmatics as claimed in claim 1 (Mycobacterium smegmatis) IL-17A is in preparation prevention with control the application in the medicine of the asthma air flue damage causing because of allergic asthma or respiratory tract infection.

5. application according to claim 4, is characterized in that, the respiratory inflammation damage of described asthma air flue damage for causing because of mycoplasma pneumoniae infection.

6. application according to claim 4, is characterized in that, described medicine is prevention and the medicine of controlling children's allergic asthma.

7. according to the application described in any one in claim 4-6, it is characterized in that, described medicine is vaccine or immunological adjuvant.