CN102586161A - Mycobacterium smegmatis IL-17A and preparation method thereof - Google Patents
Mycobacterium smegmatis IL-17A and preparation method thereof Download PDFInfo
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- CN102586161A CN102586161A CN2011100091647A CN201110009164A CN102586161A CN 102586161 A CN102586161 A CN 102586161A CN 2011100091647 A CN2011100091647 A CN 2011100091647A CN 201110009164 A CN201110009164 A CN 201110009164A CN 102586161 A CN102586161 A CN 102586161A
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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
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 is claimed the non-specific immunity proliferant agent again; Be a kind of and antigen simultaneously or be injected in advance body, can the 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 the antigen physical behavior; Prolong the antigenic action time, strengthen the effect of helper cell, the division that stimulates 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.
Aluminium salt adjuvant is first adjuvant that is approved for the people, and major function is a slow releasing function, also possesses the activation to immunocyte simultaneously, but can cause the local inflammation reaction after aluminium salt adjuvant and the injection of some mixed antigen, even forms granuloma.Freund's adjuvant is a kind of adjuvant that contains the mycobacterium composition, has stronger immune-enhancing effect, is that adjuvant is used in most widely used test, because the intensive side reaction of freund's adjuvant injection site, so this adjuvant only is used for the immune Research of experiment purpose at present.
Cytokine as immunologic adjuvant in clinical application; Recombinant bacillus Calmette-Guerin vaccines such as IL-2, IFN-γ have been successfully applied to the treatment research of superficial bladder cancer; Like the disclosed a kind of secretion of tumour necrosis factor-alpha recombinant bacillus Calmette-Guerin vaccine of 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 IFN-betaser strain of BCG vaccine, when increasing immune effect, can also reduce the BCG consumption, reduces toxic side effect.But the poky characteristic of BCG is brought inconvenience for the transgenic process.
M. smegmatics (Mycobacterium smegmatis) is to have similar antigenic mycobacterium with BCG, also is the cellular immunization adjuvant of using always, compares with BCG, and M. smegmatics has the characteristics fast, non-virulent of growing, and its range of application is more extensive.The at present existing various kinds of cell factor obtains expressing in M. smegmatics, is applied to bladder cancer like the recombinant Mycobacterium smegmatis of expressing TNF-α, and obtains enhancing immunity response effect (Int. J. Cancer, 2004,112 (4): 653-660) preferably; Like the disclosed recombinant Mycobacterium smegmatis of patent CN1339583A, contain bacillus tuberculosis typus humanus's heat shock protein 70 promotor 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 white plaque; The recombinant Mycobacterium smegmatis of and for example expressing IL-2 and GLS can strengthen immunne response (Vaccine, 2007,25 (4): 638-648) that mouse anti pulmonary tuberculosis bacterium infects.But the recombinant Mycobacterium smegmatis of expressing IL-17A (interleukin-17 A, Interleukin 17A) does not see that as yet report is arranged.
Summary of the invention
The invention provides recombinant Mycobacterium smegmatis (Mycobacterium smegmatis) IL-17A of a kind of expression IL-17A (SEQUENCE No.4); Made up the recombinant Mycobacterium smegmatis of a kind of IL-17A of expression through shuttle expression carrier; And prepare this recombinant Mycobacterium smegmatis vaccine; Make recombiant vaccine stimulate inherent immunity and acquired immunity responsibility 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 the IL-17A gene is expressed in said M. smegmatics IL-17A.
M. smegmatics preparation method according to the invention is following:
Preferably, introduce at the IL-17A upstream region of gene at the upstream primer that is designed
BamH I restriction enzyme site; The downstream primer that is designed is introduced in IL-17A gene downstream
HinD III restriction enzyme site.
Wherein, said 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, GGATCC (underscore part) does in the said upstream primer
BamH I restriction enzyme site; AAGCTT in the said downstream primer (underscore part) does
HinD III restriction enzyme site.
Wherein, said carrier is preferably bacillus coli-mycobacteria shuttle expression carrier (SEQUENCE No.3).
The present invention also provides said M. smegmatics IL-17A application in medicine, vaccine or the immunological adjuvant of the non-specific anti-infectious immunity of preparation enhancing.
The present invention also provides said M. smegmatics IL-17A application in medicine, vaccine or the immunological adjuvant of the immunity of preparing property.
Wherein, be preferably application, the especially application in preparation treatment and prevention in children allergic asthma medicine in the asthma air flue damage medicine that preparation prevention and control cause because of allergic asthma or respiratory tract infection.And be preferably further that (mycoplasma pneumoniae MP) infects application in the medicine of the inflammation damnification that causes because of mycoplasma pneumoniae in preparation prevention and control.
In sum, the invention provides the M. smegmatics IL-17A and preparation method thereof of the reorganization of a kind of IL-17A of expression, and said M. smegmatics IL-17A is provided the application in the preparation medicine.
IL-17A is a kind of important factor of regulating inherent immunity and acquired immunity, participates in the defence and the scavenging process of pathogenic infection through promoting number of ways such as chemokine, inflammatory cytokine expression; And after waking immunization up, have great importance aspect the T cell memory effect.Therefore the mycobacterium vaccine of construction expression IL-17A helps to strengthen the non-specific anti-infection ability that vaccine inoculation excites, and helps prophylaxis of microbial to infect the acute aggravation of asthma that causes; And exogenous IL-17A mainly shows as when low dosage and suppresses eosinophil and raise and suppress the Th2 reaction, and experimentation on animals shows that exogenous IL-17A dosage increases the weight of asthma mouse neutrophil leucocyte hardly and raises when being lower than 2.5 μ g/kg; And IL-17A to suppress ability that TNF-α induces RANTES to express be that it activates TNF-α and induces 6 times of IL-6, IL-8 excretory, so the mycobacterium vaccine that low dosage is expressed IL-17A has remarkable advantages 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 living bacterium and non-virulent bacterium, and the better application prospect is arranged.
M. smegmatics IL-17A according to the invention has the dual function of M. smegmatics and IL-17A; Make the mycobacterium after the reorganization promote that the non-specific anti-infectious immunity and the effect of the air flue immune inflammation damage that suppresses the sensibiligen mediation are strengthened significantly, especially in the asthma air flue damage that prevention and control allergic asthma or respiratory tract infection cause, can play important effect.
Description of drawings
Fig. 1 is M. smegmatics and M. smegmatics IL-17A growth curve chart of the present invention;
Fig. 2 identifies the expression of results figure of IL-17A in M. smegmatics IL-17A of the present invention for WB;
Fig. 3 is that the pMFA41-mIL-17a recombinant plasmid is at r
M.smegmatisThe PCR that expresses identifies and enzyme is cut qualification result.
Embodiment
The invention provides the M. smegmatics bacterial strain IL-17A of a kind of express recombinant interleukin-17 A, be with existing M. smegmatics difference: the bacillus coli-mycobacteria shuttle expression carrier of expressing the IL-17A gene is expressed in M. smegmatics.
Its preparation method is following:
M. smegmatics IL-17A according to the 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, through concrete embodiment M. smegmatics IL-17A of the present invention and preparation method thereof are specifically introduced, and understand the present invention with better, but following embodiment do not limit the scope of the invention.
Experiment material: mouse,
M.smegmatisMc
2155 (upright university department of microbiology mycobacterium researchs department, Colorado), pMD
TM18-T carrier (TaKaRa company), pET28a carrier (Novagen company), intestinal bacteria
E.coli, TOP10 (market is buied), pMFA41 carrier (self-control).
1.1. the preparation of mouse boosting cell and cultivation
6 ages in week, the spleen of female BALB/c mouse was put into substratum, and in 200 order stainless steel cells sieve, ground pressure gently, the preparation cell suspension.
Obtained cell suspension moves in the centrifuge tube, centrifugal three times of the washing of 1640 substratum, 1000r/min; Abandon supernatant, add erythrocyte cracked liquid and carry out cracking, add the 1640 substratum mixing that vibrates gently then, the centrifugal 10min of refrigerated centrifuge horizontal rotor (1000r/min).
Abandon supernatant, add 10% foetal calf serum and two anti-(penicillium mould 100U/ml, Streptomycin sulphate 100U/ml) process 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 prepares cDNA then.Wherein the cDNA preparation method is following: RNA places cooled on ice immediately 65 ℃ of following preparatory sex change 5 minutes; Carry out reverse transcription reaction then, the reverse transcription reaction system is following:
Design primer amplification mouse IL-17A, primer is following:
IL-17a gene PCR product isolation and purification: the PCR product carries out 1.2% agarose gel electrophoresis, downcuts the sepharose piece contain target DNA under the uv lamp in about 438bp place; Centrifuge tube is added the gel front and back 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 ℃ are heated to blob of viscose and melt fully; The Buffer DE-B that adds 1.5 times of gel volumes, mixing; Shift solution to the DNA adsorption column, centrifugal 30 seconds of 5000rpm; Centrifugate is poured adsorption column, spinning again into; Adsorption column is transferred in the sterilization 1.5ml centrifuge tube, adds Eluent 25 ~ 30 μ l in the film centre of adsorption column, 65 ℃ of incubations 2 minutes, centrifugal eluted dna calculates the concentration that reclaims each dna fragmentation of back by 60% recovery.
For can easily with IL-17A purify and with the reorganization of shuttle expression carrier, can carry out following construction of recombinant plasmid step:
1.3. reorganization pMD
TMThe structure of 18-T-IL-17A plasmid
PMD
TM18-T is connected with the PCR product of IL-17A.Reaction system is following:
1.4. the structure of recombinant expression plasmid pET28a-IL-17A
Extracting pMD
TM18-T-IL-17A plasmid, double digestion prepare the IL-17A gene fragment; The double digestion reaction system is:
PET28a adds in the TOP10 competence bacterial cell, places 30min on ice, and 42 ℃ were heated 90 seconds, placed on ice.37 ℃ of cultivations of LB substratum; Extracting, double digestion pET28a plasmid prepares the big fragment of pET28a carrier, and the double digestion reaction system is:
The big fragment of pET28a that double digestion is obtained is connected 2 hours for 22 ℃ with the IL-17A gene fragment, makes recombinant plasmid pET28a-IL-17A, and the ligation system is following:
The structure of
1.5.pMFA41-IL-17A recombinant vectors
37 ℃ of double digestion pET28a-IL-17A plasmids obtain IL-17A, and the double digestion reaction system is following:
pMFA41 plasmid adds in the TOP10 competent cell; Place 30min on ice; 42 ℃ were heated 90 seconds, placed on ice; Add 37 ℃ of cultivations of LB substratum; Extracting pMFA41 plasmid then.
The pMFA41 plasmid is carried out 37 ℃ of double digestions make the big fragment of pMFA41 carrier, the double digestion reaction system is following:
PMFA41 is connected 2 hours with IL-17A under 22 ℃ of temperature, the ligation system is following:
With M. smegmatics (
Mycobacterium smegmatis) mc
2155 bacterial classifications are example, cultivate 3 ~ 5 days for 37 ℃ in the LBG nutrient agar, scrape and get single colony inoculation to LBG substratum 200rpm shaking culture to the logarithmic growth cycle, reclaim thalline, and the preparation competent cell.
Get competent cell and add pMFA41-IL-17A; Place 10min on ice; Be transferred in the electric shock cup, be provided with electric conversion instrument (Gene Pulser Xcell Electioporation System, Bio-Rad) parameter is 2.5kv, 25 μ F, 1000 Ω; Add the LBG substratum after the discharge immediately, 37 ℃ of vibration incubations made the antibiotic resistance gene of bacteria resuscitation and expression plasmid coding in 4 hours.
The competent cell that transforms is coated on the LBG nutrient agar that contains KNA, is inverted plate and cultivated 3 ~ 5 days for 37 ℃, obtain M. smegmatics IL-17A (Mycobacterium smegmatis-IL-17A).
3.1.PCR identify
Select the full well-grown bacterium colony of form, adopt the primer in the step 1 to carry out the PCR reaction; The PCR response procedures: 95 ℃, 5min-(94 ℃, 30s-52 ℃, 30s-72 ℃, 1min) * 30 times-72 ℃ extend 10min, and 1.2% agarose gel electrophoresis detects amplification.The PCR reaction system is following:
The 3rd swimming lane can find out that the present invention has obtained the gene amplification product that conforms to the expection size among the figure, and promptly the IL-17A gene correctly is cloned into said M. smegmatics.
3.2.pMFA41-IL-17A plasmid transforms the influence to Mycobacterium smegmatis-IL-17A growth curve
Distinguish picking Mycobacterium smegmatis, Mycobacterium smegmatis-IL-17A colony inoculation in the LBG substratum, 37 ℃ of slow speed of revolution shaking culture, timing sampling is measured the OD600 value, draws growth curve, and is as shown in Figure 1.
Among Fig. 1, Mycobacterium smegmatis-IL-17A of the present invention is slightly slower than the wild-type bacterium speed of growth, behind inoculation 14h, gets into logarithmic phase, and can keep logarithmic growth more than 10 hours.
3.3.Western-Bolt (WB) identify the expression of IL-17A in Mycobacterium smegmatis-IL-17A
(1) Mycobacterium smegmatis-IL-17A tropina preparation: the bacterium liquid after getting 2 ml and inducing is put in the Eppendorf tube; Centrifugal 5 min of 12000 rpm collect bacterial sediment; After TBS washs 2 times, be resuspended among the TBS of 150 μ l, ultrasonic disruption cell to solution is clarified in the ice bath.In whole cell ultrasonic degradation liquid, add 4 times of diluted protein sample-loading buffers of 50 ml.95 ℃ of thermally denature 5 min are after 12% SDS-polyacrylamide gel electrophoresis protein isolates sample.
(2) SDS-PAGE electrophoresis.
(3) semidrying is changeed film: it is the same to change membrane method, is summarized as follows: make gel, pvdf membrane, filter paper sandwich structure, be flat in the half-dried transfer device (Bio-Rad), it is 15 V that voltage is set, and constant voltage mode changes film 20 min.
(3) antibody incubation: seal and to wash membrane method the same; Be summarized as follows: the pvdf membrane after will sealing is one anti-with rat anti-mouse IL-17A antibody (R&D system), is diluted in sample diluting liquid (5% skim-milk by 1:4000; 1 ' TBS-T20); Pvdf membrane immerses 4 ℃ of incubated overnight in the antibody liquid, abandons an anti-solution, washes film 5 times with the TBS-T of 1 times of dilution; The goat anti-mouse igg (Santa Cruz Biotechnology Inc.) that adds the HRP mark that dilutes by 1:10000 is hatched 1h for 37 ℃, abandons two anti-solution, and the TBS-T of 1 times of dilution washes film 5 times.
(4) luminous and development: get 300 μ l ECL substrate A and substrate B component mixing respectively and be placed on the preservative film, PVDF transfer film front is covered on it, drag pvdf membrane gently, make it fully and substrate reactions.Vertically clamp one jiao of pvdf membrane with tweezers, the drip-dry substrate solution is wrapped to face up with another preservative film and is placed the dark sheet of X-ray folder.Get an X-ray sheet in the darkroom and place on the film, the sheet that closes folder is according to fluorescence intensity; 1 min that makes public takes out film and drops in the developing solution, observe under the red light development band clear after; Press from both sides out film and drop in the stop bath, rock washing gently, the flushing with clean water film; Dry the ventilation, and scanner is gathered image.
The Western-Bolt qualification result is 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, positive band appears in about 20kDa place, and the standard control strain does not have positive band herein, show the recombinant Mycobacterium smegmatis successful expression can with antibodies specific bonded target protein.
Can find out that through above-mentioned qualification result the method for the invention has been prepared purpose 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 the RAW264.7 cell of vitro culture respectively with M. smegmatics (M.smegmatis), M. smegmatics IL-17A; Detect macrophage inflammatory protein (macrophage inflammatory protein, MIP)-1 expression of α, MIP-2 β, INF-γ and IL-4 in the culture supernatant with ELISA (enzyme-linked immuno sorbent assay) method after 72 hours; Expression with Defensin β 2, Eotaxin-1, MIP-1 α, MIP-2 β, IL-6, IL-10 and IL-23mRNA in the real-time fluorescence quantitative PCR detection culturing cell.
The result: M. smegmatics and M. smegmatics IL-17A all can promote Defensin β 2, MIP-1 α, MIP-2 β, INF-γ, IL-4 inhibition Eotaxin-1 in the RAW264.7 of vitro culture cell, to express, and M. smegmatics IL-17A of the present invention adjusting immunne response ability improves than 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. immunity security of M. smegmatics IL-17A nasal mucosa and immunogenicity
M. smegmatics IL-17A rhinovaccination immunity BALB/c mouse, M. smegmatics, IL-17A albumen dynamic observe reaction of inoculation and detect the body weight growth pattern as contrast; Carry out mycobacterium CFU in lung tissue and the homogenation of spleen tissue (colony-forming unit) counting and lung tissue morphologic observation, assessment M. smegmatics viability and inoculation security; The ELISA method detects the expression of IL-17A, INF-γ and IL-4 in lung tissue homogenate and the serum, and assessing it stimulates the nonspecific immune response ability.
Result: after expressing IL-17A mycobacterium vivo and vitro viability is not obviously influenced; Lung tissue pathology and pathogen culture result show; M. smegmatics IL-17A of the present invention rhinovaccination is a kind of safe immunization ways, can not cause the damage of lung tissue pathology; The effect that the M. smegmatics IL-17A of the present invention of rhinovaccination simultaneously induces IL-17A and IFN-γ to express is better than M. smegmatics, regulates holding time than the remarkable enhancing of IL-17A of immunne response effect.
4.3. inoculating anti-MP, M. smegmatics IL-17A infects the effect that asthma increases the weight of that causes
Laboratory animal is divided into 9 groups at random, accepts the different treatment intervention respectively: the M.smegmatis+OVA winding receives M.smegmatis inoculation and simulated infection; The M.smegmatis+OVA+MP winding is infected by M.smegmatis inoculation and MP; The M.smegmatis+OVA+IL-17A+MP winding is infected by M.smegmatis inoculation IL-17A immunity and MP; M. smegmatics IL-17A+OVA winding receives 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; The PBS+MP winding is infected by simulation inoculation and MP; The IL-17A+MP winding is infected by IL-17A immunity and MP; M. smegmatics IL-17 A+MP winding is infected by M. smegmatics IL-17 A inoculation and MP.All OVA treatment group all accept OVA abdominal injection sensitization and the aerosol suction excites.The immunohistochemical methods of the detection of IL-17A and lung tissue pathology IL-17A detected in increasing BALF and cells and supernatant, all the other indexs detections and evaluation method were all the same.
The result: the present invention expresses IL-17A 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.Compare M. smegmatics IL-17 A of the present invention suppresses the Eotaxin-1 expression, induces the ability of the allergic immune reaction of Th1 type immunne response and downward modulation IgE mediation all to obtain reinforcement with M.smegmatist.
Therefore M. smegmatics IL-17A of the present invention has the non-specific anti-infectious immunity of enhancing, regulates function such as immune inflammation reaction, can be used for preparing the asthma air flue damage medicine that prevention and control cause because of allergic asthma or respiratory tract infection; Especially to present BCG vaccination to the inapt defective of childhood asthma provide protection, M. smegmatics IL-17A of the present invention can play a significant role in the asthma air flue damage aggravation that prevention and control allergic asthma or respiratory tract infection cause.
In the foregoing of the present invention, said 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.
More than specific embodiment of the present invention is described in detail, wherein NM working method and means are to be understood that to adopting present technique field ordinary method and means to operate.The foregoing description is just as example simultaneously, and the present invention is not restricted to the specific embodiment of above description.To those skilled in the art, any equivalent modifications that the present invention is carried out with substitute also all among category of the present invention.Therefore, not breaking away from impartial conversion and the modification of being done under the spirit and scope of the present 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
<211> 30
<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
<400> 3
Gly Cys Gly Gly Cys Thr Ala Cys Ala Gly Thr Gly Ala Ala Gly Gly
1 5 10 15
Cys Ala Gly Cys Ala Gly Cys Gly Ala Thr Cys Ala Thr Cys Cys Cys
20 25 30
Thr Cys Ala Ala Ala Gly Cys Thr Cys Ala Gly Cys Gly Thr Gly Thr
35 40 45
Cys Cys Ala Ala Ala Cys Ala Cys Thr Gly Ala Gly Gly Cys Cys Ala
50 55 60
Ala Gly Gly Ala Cys Thr Thr Cys Cys Thr Cys Cys Ala Gly Ala Ala
65 70 75 80
Thr Gly Thr Gly Ala Ala Gly Gly Thr Cys Ala Ala Cys Cys Thr Cys
85 90 95
Ala Ala Ala Gly Thr Cys Thr Thr Thr Ala Ala Cys Thr Cys Cys Cys
100 105 110
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
260 265 270
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
290 295 300
Ala Gly Ala Thr Cys Cys Thr Gly Gly Thr Cys Cys Thr Gly Ala Ala
305 310 315 320
Gly Ala Gly Gly Gly Ala Gly Cys Cys Thr Gly Ala Gly Ala Gly Cys
325 330 335
Thr Gly Cys Cys Cys Cys Thr Thr Cys Ala Cys Thr Thr Thr Cys Ala
340 345 350
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
atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta 120
tgagccatat tcaacgggaa acgtcttgct cgaggccgcg attaaattcc aacatggatg 180
ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct 240
atcgcttgta tgggaagccc catgcgccag agttgtttct gaaacatggc aaaggtagcg 300
ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc 360
ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga 420
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 (10)
- A M. smegmatics ( Mycobacterium smegmatis) IL-17A, it is characterized in that, express the bacillus coli-mycobacteria shuttle expression carrier of IL-17A gene and in M. smegmatics IL-17A, express; This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC NO.4446.
- A M. smegmatics as claimed in claim 1 ( Mycobacterium smegmatis) preparation method of IL-17A, it is characterized in that step is following:Step 1 is according to IL-17 A gene order design of amplification primers, through PCR clone IL-17 A maturation protein encoding sox fragment; Said IL-17 A gene fragment clone to the bacillus coli-mycobacteria shuttle expression carrier, is obtained recombinant vectors;Step 2, electricity transform said recombinant vectors to M. smegmatics, obtain said M. smegmatics IL-17A.
- 3. preparation method according to claim 2 is characterized in that, in the said design primer process, the upstream primer that is designed is introduced at the IL-17A upstream region of gene BamH I restriction enzyme site; The downstream primer that is designed is introduced in IL-17A gene downstream HinD III restriction enzyme site.
- 4. preparation method according to claim 3 is characterized in that amplimer is following described in the step 1:Upstream primer: 5 '-CAG GGATCCGCGGCTACAGTGAAGGC-3 ';Downstream primer: 5 '-AGT AAGCTTTTAGGCTGCCTGGCGGACAAT-3 ';GGATCC does in the wherein said upstream primer BamH I restriction enzyme site; AAGCTT does in the said downstream primer HinD III restriction enzyme site.
- 5. preparation method according to claim 2 is characterized in that, electric conversion process described in the step 2, the shock parameters of electric conversion instrument are voltage 2.5kv, electric capacity 25 μ F, resistance 1000 Ω.
- 6. preparation method according to claim 2 is characterized in that, said electricity transforms after product and on the LBG of kantlex nutrient agar, screens and cultivate.
- A M. smegmatics as claimed in claim 1 ( Mycobacterium smegmatis) application of IL-17A in medicine, vaccine or the immunological adjuvant of preparation non-specific anti-infectious immunity of enhancing or the immunity of preparing property.
- 8. application according to claim 7 is characterized in that, is applied to prepare the asthma air flue damage medicine that prevention and control cause because of the medicine or the respiratory tract infection of allergic asthma.
- 9. application according to claim 8 is characterized in that, the respiratory inflammation damage of said asthma air flue damage for causing because of mycoplasma pneumoniae infection.
- 10. application according to claim 7 is characterized in that, said medicine is the medicine of prevention and control children allergic asthma.
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| CN106520816A (en) * | 2016-11-03 | 2017-03-22 | 扬州大学 | Function and purpose of ppe27 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106456745A (en) * | 2014-02-11 | 2017-02-22 | 北京艾棣维欣生物技术有限公司 | Vaccines with interleukin-17 as an adjuvant |
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| CN106520816B (en) * | 2016-11-03 | 2020-02-07 | 扬州大学 | Function and use of ppe27 |
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