CN101838325B - Antigen-presenting protein for swines and encoding gene and application thereof - Google Patents
Antigen-presenting protein for swines and encoding gene and application thereof Download PDFInfo
- Publication number
- CN101838325B CN101838325B CN2010101405986A CN201010140598A CN101838325B CN 101838325 B CN101838325 B CN 101838325B CN 2010101405986 A CN2010101405986 A CN 2010101405986A CN 201010140598 A CN201010140598 A CN 201010140598A CN 101838325 B CN101838325 B CN 101838325B
- Authority
- CN
- China
- Prior art keywords
- app
- protein
- sequence
- glu
- antigen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Abstract
The invention discloses an antigen-presenting protein for swines and an encoding gene and application thereof. The protein (APP-N) of the invention is a protein of the following (a) or (b): (a) the protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table; and (b) the protein which is prepared by carrying out substitution and/or deletion and/or addition of one or more amino acid residues on the amino acid sequence of the sequence 1, has a function of promoting immune activity and is derived from the sequence 1. The protein of the invention can be efficiently expressed. The expression amount reaches over 50 percent of the total protein of the strain. The antigen-presenting protein of the invention has the advantages of simple operation, short period, low cost and high specificity. The APP-N can carry out combined immunization together with antigen or vector vaccines of various viruses, improves antigen immune activity, strengthens humoral immunity and cellular immunity of the swinery, fulfills the aims of eliminating viruses and curing diseases, is very suitable for prevention of porcine virus diseases and is easy to be popularized and applied on a large scale.
Description
Technical field
The present invention relates to a kind of antigen-presenting protein for swines and encoding sox thereof and application.
Background technology
(Antigen Presentation Protein is that a kind of relative molecular mass is the gp of 94～96kDa APP) to antigen presentation albumen, is heat shock protein(HSP) (heat shock protein, HSP) a member in the family.Not only be expressed in all cells of living, and be eukaryotic cell and the conservative and abundant class protein of prokaryotic cell prokaryocyte camber, it extensively is present in the endocytoplasmic reticulum.As Chaperones Molecular, APP can combine not folding polypeptied chain, stops protein aggregation, assists protein folding, stretching, extension, assembling and transhipment, suppresses the secretion of misfolded proteins matter.Under the normal circumstances, APP is the persistence low expression level in cell, but stressed condition such as heat-shocked, heavy metal poisoning, glucose shortage, bacterium and virus infection and cytodifferentiation etc. all can induce it to express to increase.
The C-end of APP contains endoplasmic reticulum signal for locating KDEL (Lys-Asp-Glu-Leu) sequence; The latter has constituted a feedback signal from the golgi body to the endoplasmic reticulum; Its N-terminal has a signal sequence, near the N-end region 2 Walker boxes is arranged, and possibly be ATP-binding site.Therefore, APP can combine ATP, has the ATPase activity.The peptide of 400～2000Da does not have obvious aminoacid sequence specificity, in conjunction with occurring in the endoplasmic reticulum with the non-covalent combination of about 1: 1 ratio in its ability and the cell.Its APP-peptide complex also can be in external generation, and this mixture quite stable is inferred according to APP-peptide combination model; APP peptide binding pocket is made up of 200 amino-acid residues approximately; Be a ditch of forming by the α spiral, be positioned on the surface of reverse β lamella formation that the peptide part promptly is positioned at this ditch.Peptide binding pocket and APP dimerization structural domain adjoin in this model, and pointing out this structural domain and peptide to be passed to the MHC-I quasi-molecule with the high stability of APP-peptide complex, to carry out angtigen presentation relevant.APP bonded antigen peptide can be given the MHC-I quasi-molecule by the crf receptor CD91 submission on antigen presenting cell (APC) surface, and the two-way interaction activates the atpase activity of APP, and the ATP hydrolysis provides energy that peptide is transferred on the MHC-I quasi-molecule from APP.APP gets into APC through acceptor, has avoided behind the endocytosis phagolysosome that the integrity of APP has been safeguarded in the degraded of APP.In sum, it is active that APP can be used as a kind of immunostimulant raising antigen immune.
In recent years, characteristic and the application of extensive concern APP both at home and abroad.Srivastava etc. have founded the method for from tumour cell, extracting the APP peptide complex the earliest; From 1g tumor tissues or cell, can the purify mixture of about 15～30 μ g; Therefore can prepare APP-antigenic peptide complexes vaccine with patient self tumour, can produce antigen-specific immune responses.The APP-peptide complex in autologous tumor source has been used for the research of mouse spontaneous tumor chemically induced immunotherapy of tumors at present, and has set up mouse model.Thomas etc. (1996) with vesicular stomatitis virus (VSV) model at the external APP peptide complex that confirmed to the lymphocytic activation of CD8+T.Subsequently, research is presented at the external APP peptide complex that successfully made up, and APP is the very important mate molecule of TLRs, in the normal function performance of scavenger cell, plays the part of important role.Meng Songdong etc. (2002) discover APP as Chaperones Molecular, can specific combination hepatitis B virus (HBV) epitope peptide, and the bonded polypeptide intersected to be pass MHCI quasi-molecule, thereby activate virus-specific CD8+T cell (CTL).Subsequently, (2006) achievement in research such as Tian Bo is converted into patent of invention " immunological adjuvant and the application in antiviral vaccine or medication preparation thereof the " (patent No.: ZL 200410069192.8; Granted publication CN 100467063C; The applying date: on July 7th, 2004).
Summary of the invention
The purpose of this invention is to provide a kind of antigen-presenting protein for swines (the active albumen of enhancing immunity) and encoding sox and application.
Albumen provided by the present invention (APP-N) from porcine kidney cell line PK-15 cell, is participated in antigen presentation, has the immunocompetent function of promotion, for (a) as follows or (b):
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have promote immunocompetent function by sequence 1 deutero-protein.
APP-N is made up of 334 amino-acid residues.
In order to make the APP-N in (a) be convenient to purifying, proteinic N-terminal or C-terminal that can the aminoacid sequence shown in the sequence 1 is formed in by sequence table connect label as shown in table 1.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tagII | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned (b) but in the APP-N synthetic, also can synthesize its encoding sox earlier, carry out biology again and express and to obtain.The encoding sox of APP-N in above-mentioned (b) can be through the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 2; And/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
The above-mentioned proteic gene (APP-N) of encoding also belongs to protection scope of the present invention.
Said gene can be following 1) or 2) or 3) dna molecular:
1) dna molecular shown in the sequence 2 in the sequence table;
2) under stringent condition with 1) the dna sequence dna hybridization that limits and have the dna molecular that promotes immunocompetent function;
3) with 1) or 2) dna sequence dna that limits has 90% above homology, and have the dna molecular of the immunocompetent function of promotion.
Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
The dna molecular shown in the sequence 2 is made up of 1002 Nucleotide in the sequence table.
The recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain above arbitrary said gene all belong to protection scope of the present invention.
When making up recombinant expression vector, the carrier that sets out can adopt pBV220, pPICZ α A, pET carrier series, pGEX, pQE carrier series, pPIC9K or pPICZ etc.When making up the reorganization bacterium, but host cell can be selected the protokaryon or the eukaryotic cell of arbitrary expression alien gene for use; Said prokaryotic cell prokaryocyte can be intestinal bacteria, like E.coli DH5 α, E.coli TB1 or E.coli BL21 etc.; Said eukaryotic cell can be yeast cell, and mammalian cell or vegetable cell etc. comprise yeast SMD1168H, yeast GS115, yeast X-33, COS-7, CHO etc.
Said recombinant expression vector specifically can be following A) or B):
A) said gene is inserted the recombinant plasmid that the MCS of pBV220 obtains;
B) said gene is inserted the recombinant plasmid that the MCS of pPICZ α A obtains.
Said reorganization bacterium specifically can be following C) or D):
C) with A) said recombinant plasmid imports the reorganization bacterium that bacillus coli DH 5 alpha obtains;
D) with B) said recombinant plasmid imports the reorganization bacterium that yeast strain SMD1168H obtains.
Increase total length or its any segmental primer of said gene to also belonging to protection scope of the present invention.
The present invention also provides two kinds to prepare said proteic method: (1) is with C) said reorganization bacterium, induce 5～7 hours (inducing 6 hours for preferred 42 ℃), obtain said albumen for 40～43 ℃; (2) with D) said reorganization bacterium, add methyl alcohol and carry out abduction delivering, add methyl alcohol final concentration can be 0.1-1.0% (volumn concentration), be preferably 0.5% (volumn concentration).
Said albumen can be applicable to prepare immunostimulant, especially for the immunostimulant of preparation pig.
The present invention also protects a kind of immunostimulant, and its activeconstituents is said albumen.Immunostimulant spy of the present invention is applicable to pig fully.
Said albumen can be applicable to prepare the vaccine that is used for porcine reproductive and respiratory syndrome.
The present invention also protects a kind of vaccine that is used for porcine reproductive and respiratory syndrome, comprises said albumen.
With APP-N of the present invention and commercially available PRRS vaccine combined immunization pig, can promote pig generation specific humoral immunity (antibody generation) and cell immune response (ctl response) to reply.Albumen provided by the present invention can be used as immunostimulant and uses, and strengthens CTL level and antibody horizontal that epitope peptide and attenuation or inactivated vaccine produce, improves body to external antigenic immunne response.With attenuation or the inactivated vaccine combined immunization of albumen of the present invention with virus (like viruses such as PRRSV/ porcine reproductive and respiratory syndrome virus, FMDV/ foot and mouth disease virus, CSFV/ CSFVs); Closely octuple improves the immunocompetence of vaccine, reaches the purpose of removing virus, disease preventing and treating.Immunostimulant provided by the invention can with the direct blended mode of antigenic substance; Adopt subcutaneous multiple spot or modes such as intradermal injection or intramuscular injection to carry out immunity (immunizing dose is 0.05mg/Kg, 0.5mg/Kg or 5mg/Kg), active to improve the swinery antigen immune; Immunostimulant provided by the invention can also be injected respectively with antigenic substance.Albumen of the present invention can efficiently express in intestinal bacteria, and expression amount reaches more than 50% of bacterial protein, it is simple to operate, the cycle is short, with low cost, specificity good; APP-N can with the antigen or the carrier bacterin combined immunization of multiple virus; It is active to improve antigen immune, strengthens humoral immunization and the cellular immunization of swinery, reaches the purpose of removing virus, treatment disease; Be highly suitable for the prevention of virus diseases of pigs, be easy to apply on a large scale.
Description of drawings
Fig. 1 induces the SDS-PAGE detected result of front and back supernatant for DH5 α-APP-N/pBV220; M: molecular weight protein Marker; 1: before inducing; 2: after inducing.
Fig. 2 is supernatant and a sedimentary SDS-PAGE detected result behind DH5 α-APP-N/pBV220 abduction delivering; M: molecular weight protein Marker; 1: supernatant; 2: deposition.
Fig. 3 is the western blot detected result of DH5 α-APP-N/pBV220; M: molecular weight protein Marker; 1: before inducing; 2: after inducing.
Fig. 4 is the western blot detected result of SMD-APP-N/pPICZ α A; M: molecular weight protein Marker; 1: before inducing; 2: after inducing.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.% among the following embodiment like no specified otherwise, is the quality percentage composition.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.All primers synthesize and examining order is accomplished by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
The commercially available vaccine of PRRS: available from Qilu Animal Health Products Co., Ltd., veterinary drug new word: (2005) 080011054; Vaccine is that (every ml viral level is>=2 * 10 for the packing of 50ml/ bottle
7.0TCID
50).
1, the extraction of the total RNA of pig
RNA according to invitrogen company extracts test kit, from porcine kidney cell line PK-15 cell (available from China Veterinery Drug Inspection Office), extracts the total RNA of pig.
2, the reverse transcription of APP goal gene and PCR
RT-PCR test kit according to invitrogen company carries out reverse transcription, obtains the cDNA of APP-mRNA.With reference to pig app gene sequence (GenBank number: DQ306708), design following primer:
Upstream primer: 5 '-GAGGATGAAGTGGATGTGG-3 ';
Downstream primer: 5 '-GTATTCATCATCTTCTACTTCCTTTG-3 '.
With cDNA is template, carries out pcr amplification with designed primer.The PCR reaction conditions is following: 94 ℃ 4 minutes; 94 ℃ 50 seconds, 55 ℃ 50 seconds, 72 ℃ 1 minute; Totally 30 circulations; 72 ℃ 10 minutes.Obtain being about the PCR product of 1Kb.PCR product subclone is gone into the pMD18-T carrier, and (available from precious biotechnology ltd, D102A), (available from Beijing health is the century bio tech ltd to transformed into escherichia coli DH5 α, and CW0808), the picking positive monoclonal carries out gene sequencing.Sequencing result shows that the PCR product is shown in the sequence 2 of sequence table.
Protein shown in the gene coded sequence 1 shown in the sequence 2.With the protein called after APP-N shown in the sequence 1, with the encoding sox name APP-N of APP-N, with the above-mentioned pMD18-T carrier called after pMD18-APP that contains APP-N that obtains.
The expression of embodiment 2, APP-N and the purifying of expression product thereof
One, expression and the fermentative prodn of APP-N in intestinal bacteria
1, the structure of coli expression carrier APP-N/pBV220
Give birth to the APP-N shown in the worker company composition sequence 2 by Shanghai, then with following primer to carrying out pcr amplification, introducing EcoRI and BamHI enzyme are cut recognition site:
APP-F-E：5’-CATGAATTC
ATGGAGGATGAAGTGGATGTGG-3’；
APP-R-B：5’-CATGGATCC
TTAGTATTCATCATCTTCTACTTCCTTTG-3’。
With pcr amplification product insert after with restriction enzyme BamHI and EcoRI double digestion carrier pBV220 (available from Shanghai Jierui Biology Engineering Co., Ltd, between BamHI GV0302) and the EcoRI restriction enzyme site, transformed into escherichia coli DH5 α.Carry out PCR and enzyme and cut the positive reorganization of evaluation back acquisition bacterium; The positive bacterium of recombinating is extracted plasmid and checks order, and the result shows and obtains purpose plasmid APP-N/pBV220 (APP-N shown in the sequence 2 of insertion sequence table between the BamHI of pBV220 and EcoRI restriction enzyme site).The bacillus coli DH 5 alpha called after DH5 α-APP-N/pBV220 that will contain APP-N/pBV220.
With pBV220 transformed into escherichia coli DH5 α, method is the same, and bacterium DH5 α-pBV220 (contrast) obtains recombinating.
2, the expression of APP-N in intestinal bacteria
(or DH5 α-pBV220), (bacterium liquid OD is cultivated in 30 ℃, 200 rev/mins (rotation radius is 13mm) joltings with DH5 α-APP-N/pBV220
600To 1.0), under 42 ℃, induced 6 hours.
Before will inducing respectively with induce after centrifugal 10 minutes of bacterium liquid 10000g, it is resuspended through PBS to collect thalline, carrying out ultrasonic bacteria breaking, 12000 rev/mins centrifugal 15 minutes, get supernatant and carry out the SDS-PAGE electrophoresis.DH5 α-APP-N/pBV220 detected result (swimming lane M is molecular weight protein Marker, and swimming lane 1 is for before inducing, and swimming lane 2 is for inducing the back) as shown in Figure 1.Do not have protein band before the bacterium liquid of DH5 α-APP-N/pBV220 is induced, occur the target protein band about 45KD after inducing, all do not have the target protein band before and after the bacterium liquid of DH5 α-pBV220 is induced.
With centrifugal 10 minutes of the bacterium liquid 10000g after inducing, it was resuspended through PBS to collect thalline, carrying out ultrasonic bacteria breaking, 12000 rev/mins centrifugal 15 minutes, get cleer and peaceful deposition respectively and carry out the SDS-PAGE electrophoresis.DH5 α-APP-N/pBV220 detected result is as shown in Figure 2, and (swimming lane M is molecular weight protein Marker; Swimming lane 1 is a supernatant; Swimming lane 2 is deposition), in the cleer and peaceful deposition target protein band about 45KD appears all upward, all there is not the target protein band in the last cleer and peaceful deposition of DH5 α-pBV220.
Before will inducing respectively with induce after centrifugal 10 minutes of bacterium liquid 10000g, it is resuspended through PBS to collect thalline, carrying out ultrasonic bacteria breaking; 12000 rev/mins centrifugal 15 minutes, get supernatant and carry out the 12%SDS-PAGE electrophoresis detection, changeed film 1.5～2 hours with the constant current of every square centimeter of membrane area * 0.65mA; Put into 5% skimmed milk with TBST configuration; Discard confining liquid (the 5g skimmed milk is dissolved in 100ml) after the decolouring, add TBST (NaCl 25mM, Tris 100Mm, Tween-20 0.2% are settled to 1000ml), adding resists with one of confining liquid preparation; Hatched under the room temperature 3 hours, and reclaimed an anti-back and preserve.Film is put/gone in the TBST solution and decolour, add HRP mark two anti-rear decolorings, add TBST and wash 3 rear decolorings of film, develop, photographic fixing is developed a film, compressing tablet with the confining liquid preparation.The western blot detected result of DH5 α-APP-N/pBV220 is as shown in Figure 3, and (swimming lane M is molecular weight protein Marker; Swimming lane 1 is for before inducing; Swimming lane 2 is for inducing the back), show to have produced target protein really after inducing all do not have the target protein signal in the last cleer and peaceful deposition of DH5 α-pBV220.Above result shows that APP-N correctly expresses in intestinal bacteria.
3, the fermentative prodn of APP-N
DH5 α-APP-N/pBV220 is inoculated in the 100mlLB liquid nutrient medium (containing the 100mg/ml penbritin), and 37 ℃ of shake-flask culture 12 hours are as primary seed solution; With 1% (volume ratio) inoculum size primary seed solution is transferred in the fermentor tank of the LB liquid nutrient medium that contains 6L again, be cultured to OD
600To 1.0, be rapidly heated to 42 ℃, induced 6 hours.
Two, expression and the fermentative prodn of APP-N in yeast
1, the structure of Yeast expression carrier APP/pPICZ α A
By the APP-N shown in Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's composition sequence 2, then with following primer to carrying out pcr amplification, introduce XhoI and XbaI enzyme cutting recognition site:
APP-F：5’-CATCTCGAGGAGGATGAAGTGGATGTGG-3’；
APP-R：5’-CATTCTAGAGTATTCATCATCTTCTACTTCCTTTG-3’。
Pcr amplification product is inserted pPICZ α A after with restriction enzyme Xho I and Xba I double digestion, and (available from the general Bioisystech Co., Ltd that flies in Shanghai, between XhoI V19520) and the XbaI enzyme cutting site, electric transformed yeast bacterial strain SMD1168H is (available from U.S. invitrogen company; C18400), transformant is laid on contains zeocin (available from U.S. invitrogen company, C19840) YPDS of 100 μ g/mL (contains 1% yeast extract; 2% yeast culture is used tryptone, 2% glucose, 1M sorbyl alcohol; 2% agar) flat board; Cultivated 2-3 days for 42 ℃,, after PCR identifies, obtain positive reorganization bacterium until growing single bacterium colony.The positive bacterium of recombinating is extracted plasmid and checks order, and the result shows and obtains purpose plasmid APP-N/pPICZ α A (APP-N shown in the sequence 2 of insertion sequence table between the XhoI of pPICZ α A and XbaI enzyme cutting site).The yeast strain SMD1168H called after SMD-APP-N/pPICZ α A that will contain APP-N/pPICZ α A.
With pPICZ α A transformed yeast bacterial strain SMD1168H, bacterium SMD-pPICZ α A (contrast) obtains recombinating.
2, the expression of APP-N in yeast
SMD-APP-N/pPICZ α A (or SMD-pPICZ α A) is inoculated in BMGY earlier, and (contain 1% yeast extract, 2% yeast culture is used tryptone, 100mM potassiumphosphate, 1.34%YNB, 4 * 10
-5Vitamin H, 1% glycerine) in the substratum, cultivate 18h-20h, treat OD for 42 ℃
600When value was 2-6,1500g collected thalline in centrifugal 5 minutes; (contain 1% yeast extract, 2% yeast culture is used tryptone, 100mM potassiumphosphate, 1.34%YNB, 4 * 10 to change the BMMY substratum again into
-5Vitamin H, 0.5% volumn concentration methyl alcohol), continue under the same conditions to cultivate to make its OD
600Value reaches 1.0; Adding final concentration in per then 24 hours is the methanol induction expression of 0.5% (volumn concentration), collects a sample, and induces 48 hours for 30 ℃ in per 24 hours.After cultivate finishing, to tunning 10000g centrifugal 10 minutes, to get supernatant and carry out the SDS-PAGE electrophoresis detection, the target protein band about 45KD appears.Before and after inducing, the tunning of SMD-pPICZ α A all do not have the target protein band.
Centrifugal 10 minutes of bacterium liquid 10000g before and after will inducing respectively, it is resuspended through PBS to collect thalline, carrying out ultrasonic bacteria breaking; 12000 rev/mins centrifugal 15 minutes, get supernatant and carry out the SDS-PAGE electrophoresis, changeed film 1.5-2 hour with the constant current of every square centimeter of membrane area * 0.65mA; Put into 5% skimmed milk of TBST configuration; Discard confining liquid (the 5g skimmed milk is dissolved in 100ml) after the decolouring, add TBST (NaCl 25mM, Tris 100Mm, Tween-200.2% are settled to 1000ml), add with the confining liquid preparation one anti-(with step 12 in identical one anti-); Hatched under the room temperature 3 hours, and reclaimed an anti-back and preserve.Film is put into TBST solution to decolour; Adding is with the HRP mark two anti-rear decolorings of confining liquid preparation; Add/go into TBST and wash 3 rear decolorings of film, develop, photographic fixing is developed a film, compressing tablet, the western blot detected result of SMD-APP-N/pPICZ α A (swimming lane M molecular weight protein Marker as shown in Figure 4; Swimming lane 1 is for before inducing, and swimming lane 2 is for inducing the back).Show and produced target protein really after inducing.Before and after inducing, the tunning of SMD-pPICZ α A all do not have the target protein signal.
Above experimental result shows that APP-N correctly expresses in yeast.
3, the fermentative prodn of APP-N
Fermentor tank is the Biostat B. of B.Braun company, and capacity is 5L.After choosing bacterium, inoculation, fermentation culture, in 8 hours, methanol concentration is increased to 70%, coinduction was expressed 5 days, can obtain APP-N.
Three, the purifying of APP-N
1, get DH5 α-APP-N/pBV220 fermented liquid that the 5L step 1 obtains, centrifugal 10 minutes of 10000g collects thalline.
2, TE damping fluid washing, carrying out ultrasonic bacteria breaking, PBS washs inclusion body, 6M guanidine hydrochloride dissolution inclusion body, PBS renaturation.
3, Guanidinium hydrochloride, centrifuging and taking supernatant are removed in dialysis.
4, supernatant being used the vinegar acid for adjusting pH value is 4.8; (chromatography column is Capto Q, available from GE Healthcare to cross the anion-exchange chromatography post; The anion-exchange chromatography column filling is efficient agarose and DEXTRAN 500.000 mixture; Column length is that the internal diameter of 10cm, pillar is 0.77cm), collect the target elution peak; (molecular sieve is SuperDex 75, available from GE Healthcare to cross the Sephacry1 molecular sieve chromatography; Sephacry1 sieve chromatography column filling is the crosslinked Epicholorohydrin of DEXTRAN 500.000; Column length is that the internal diameter of 50cm, pillar is 1.5cm), collect the target elution peak; Obtain the APP-N solution of 100mL purifying after the filtration sterilization, be used for the experiment of embodiment 3,4,5 as the APP adjuvant.
Anion-exchange chromatography comprises the steps: that (20mM pH8.0) as initial liquid, is the Tris-Cl damping fluid (20mM of 1M with NaCl concentration with the Tris-Cl damping fluid; PH8.0) as stop buffer; Carry out linear gradient elution, elution time is 1 hour, and NaCl concentration gradient rate of change is constant in the said elution process; Each 1ml elutriant of collecting, collection NaCl concentration are the solution that the Tris-Cl damping fluid of 0.25M is eluted;
It is that (20mM pH7.5) carries out, and the time of carrying out is 1 hour, the said NaCl constant concentration in the process (begin to collect elutriant at the 16ml place, collect 5ml altogether) of carrying out for the Tris-Cl damping fluid of 150mM that sieve chromatography comprises the steps: with NaCl concentration.
Four, the preparation of contrast adjuvant I
Patent " immunological adjuvant and the application in antiviral vaccine or medication preparation thereof the " (patent No.: ZL200410069192.8; Granted publication CN 100467063C; The applying date: on July 7th, 2004), disclose a kind of albumen (Hgp96N holds 22-355) and seen Instructions Page 9 the 2nd to 5 row.This albumen and albumen provided by the invention have higher homology, so as the contrast of APP-N of the present invention.
This reference protein is the protein shown in the sequence 3 of sequence table, and its encoding sox is the dna molecular shown in the sequence 4 of sequence table.
By the dna molecular shown in the sequence 4 of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's composition sequence table, then with following primer to carrying out pcr amplification, introduce BamHI and XhoI enzyme and cut recognition site:
Upstream primer: 5 '-CTGGATCCGACGATGAAGTTGATGTGGATG-3 ';
Downstream primer: 5 '-GCACTCGAGTCATTCATCTTCTTCTACTTC-3 '.
Pcr amplification product is inserted between the BamHI and XhoI restriction enzyme site of carrier pBV220 after with restriction enzyme BamHI and XhoI double digestion, obtain recombinant plasmid.Use the recombinant plasmid transformed bacillus coli DH 5 alpha, obtain the bacterium of recombinating.The reorganization bacterium is adopted method abduction delivering and the purifying identical with DH5 α-APP-N/pBV220, and the solution that obtains is used for the experiment of embodiment 3,4,5 as contrast adjuvant I.
The security animal experiment of embodiment 3, APP-N
1, imitates inspection with small white mouse
With 10 of the healthy mices of body weight 14～18g, each abdominal injection (but also subcutaneous injection) 0.2ml APP-N observed 7.At viewing duration, if untoward reactions such as the tic that appearance causes because of the APP-N injection, death, it is defective declaring this batch APP-N; If untoward reactions such as the tic that the non-APP-N factor of appearance causes, death, it is invalid to declare this check, Ying Chongjian, if heavily do not examine, it is defective then to declare this batch APP-N.
2, imitate inspection with cavy
With 10 of the healthy guinea pigs of body weight 350～400g, each abdominal injection (but also subcutaneous injection) 1ml APP-N observed 7.At viewing duration, if untoward reactions such as the tic that appearance causes because of the APP-N injection, death, it is defective declaring this batch APP-N; If untoward reactions such as the tic that the non-APP-N factor of appearance causes, death, it is invalid to declare this check, Ying Chongjian, if heavily do not examine, it is defective then to declare this batch APP-N.
The security verification result of small white mouse and cavy shows that the APP-N that embodiment 2 prepares is safe, does not cause any untoward reaction.
The test of embodiment 4, APP-N adjuvant and the commercially available vaccine immunity small white mouse of PRRS
Select for use about body weight 30g, at random small white mouse of sex (kind is the BalB/c mouse, grows institute's Experimental Animal Center available from Chinese Academy of Sciences's heredity) carries out immunity for experimental animal.According to the mean body weight and the mean body weight of test with small white mouse of pig, (the 50ml/ bottle, every ml viral level is>=2 * 10 to calculate the commercially available vaccine of PRRS
7.0TCID
50) act on the dosage of mouse.According to the conversion result, draw the single-dose dosage of 50uL as every mouse from the commercially available vaccine of PRRS.
Test is divided into four groups (every group of 10 small white mouses):
First group (APP group): inject with the APP-N adjuvant, single immunization dosage is 100uL/;
Second group (PRRS-APP group): with APP-N adjuvant and the commercially available vaccine immunity of PRRS, the single injection dosage of APP-N adjuvant is 50uL/, and the single immunization dosage of the commercially available vaccine of PRRS is 50uL/;
The 3rd group (PRRS-I group): with contrast adjuvant I and the commercially available vaccine immunity of PRRS, the single injection dosage of contrast adjuvant I is 50uL/, and the single immunization dosage of the commercially available vaccine of PRRS is 50uL/;
The 4th group (PRRS group): with the commercially available vaccine immunity of PRRS, single immunization dosage is 100uL/;
Immune programme for children is to carry out immunity after swinery is observed a week.The immunity back was carried out cellular immunization and humoral immunization experimental analysis on the 7th day.
One, detects cell immune response
(1) measures the lymphopoiesis level with MTT dyeing
Separation and results splenocyte carry out lymphocyte proliferation assay: splenocyte is suspended from the PRIM-1640 substratum that contains 10mM HEPES, microbiotic (each 100U/ml of penicillium mould and Streptomycin sulphate) and 10% (volumn concentration) foetal calf serum (FBS); After cell counting, divide equally to 96 orifice plates (2 * 10
6Individual cells/well), in containing 5%CO
237 ℃ of cell culture incubators in cultivate; The PRRSV virus (available from China Veterinery Drug Inspection Office) that adds deactivation after 24 hours is as stimulator, simultaneously with the stimulator of Marc-145 cell (available from China Veterinery Drug Inspection Office) as negative control.Cultivate and add MTT (20uL/ hole) after 72 hours, cultivate and add DMSO (150uL/ hole) after 4 hours again, mixing was measured the 570nm light absorption value in 10 minutes, calculated stimulating factor SI.SI=OD570 value (sample aperture-blank well)/OD570 value (negative hole-blank well).The result shows: APP group SI=1.0, PRRS-APP group SI=3.8, PRRS-I group SI=3.0, PRRS organize SI=1.2; SI is the highest for the PRRS-APP group, is nearly 4 times of PRRS group, explain that APP-N can obviously strengthen the cell response level of PRRS vaccine, and effect is superior to contrasting adjuvant I.
(2) measure IFN-γ level with the ELISPOT method
Separation and results splenocyte carry out the ELISPOT experiment: splenocyte is suspended from the PRIM-1640 substratum that contains 10mMHEPES, microbiotic (each 100U/ml of penicillium mould and Streptomycin sulphate) and 10% (volumn concentration) foetal calf serum (FBS); After cell counting, divide equally to 6 orifice plates (5 * 10
8Individual cells/well), in containing 5%CO
237 ℃ of cell culture incubators in cultivate; The PRRSV virus (available from China Veterinery Drug Inspection Office) that adds deactivation after 24 hours is as stimulator, simultaneously with the stimulator of Marc-145 cell as negative control.APP group SFP=10, PRRS-APP group SFP=350, PRRS-I group SFP=330, PRRS organize SFP=105; The IFN-γ level of PRRS-APP group approximately is 3～4 times of independent immune PRRS group.Explain that APP-N can obviously strengthen the cell response level of PRRS vaccine, and effect is superior to contrasting adjuvant I.
Two, detect humoral immune reaction
(1) with in serum-virus with the measuring immunity neutralizing antibody level that produces
Separation of serum carries out mouse serum-virus neutralization experiment: trysinization Marc-145 cell, divide equally to 96 orifice plates, and the 100uL/ hole, and guarantee that cell count is (2～8) * 10
5Cell count/hole, CO
2Cell culture incubator is cultivated 16～24h; 56 ℃ of water-bath inactivated serums 30 minutes, (available from China Veterinery Drug Inspection Office, DMEM JXA1) (2%FBS) adopts different volumes than mixing (serum diluted 2 with containing 100TCID50PRRSV virus
1Doubly, 2
2Doubly, 2
3Doubly, 2
4Doubly), behind the mixing, hatch 1h for 37 ℃; Cell culture medium in reject 96 orifice plates adds serum-virus mixture (100 μ L/ hole), places CO
2In the cell culture incubator, absorption 1h; The reject supernatant is washed 2 times with aseptic PBS, blot the debris in each hole after, add DMEM (2%FBS), place CO
2Cell culture incubator was cultivated 3～5 days; Observation of cell pathology CPE; Calculate NAT (tiring), detected result is seen table 2.Explain that APP-N has tangible enhancement to immunogenicity of antigens.
The detected result of table 2 NAT (tiring)
| Group | NAT (tiring) |
| The APP group | -- |
| The PRRS- |
1∶8 |
| The PRRS- |
1∶4 |
| The |
1∶2 |
(2) measure the PRRSV antibody horizontal with elisa technique
Separation of serum, carry out ELISA reaction assay PRRSV specific antibody level: with PRRSV virus (50ng/ hole) (available from China Veterinery Drug Inspection Office, JXA1) encapsulate 96 orifice plates, 4 ℃ spend the night (12 hours), PBST washes 3 times; With 37 ℃ of sealings of the confining liquid that contains 4%BSA 2 hours, PBST washed 6 times; 10 times of doubling dilution serum were hatched 1 hour for 37 ℃, and PBST washes 6 times; Combine goat-anti mouse HRP-IgG (Santa cruz company, catalog number: SC-2005), HRP-IgG respectively
1(Santa cruz company, catalog number: sc-2060) and HRP-IgG2a (Santacruz company, catalog number: sc-2061), hatched 1 hour for 37 ℃, PBST washes 6 times; Add equal-volume blended A/B liquid 100uL/ hole (A liquid formula: 0.2M Na
2HPO
451.4ml, 0.1M Hydrocerol A 48.6ml, 30%H
2O
267 μ l, adding distil water be to 100mL, pH value 5.0～5.4; The B liquid formula: TMB 2HCl 50mg, 0.1M Hydrocerol A 5ml, 0.1M EDTA 0.5ml, adding distil water is to 100ml), develop the color after 5 minutes, add the stop buffer termination reaction; Measure the 450nm light absorption value, calculate and respectively organize antibody titer (sample well and negative control hole A450nm ratio are antibody titer greater than 2.1 highly diluted multiples).According to 10 times of doubling dilutions, set amount level (10
4).Again according to 5 times and 2 times of doubling dilutions definite accurately tire (method is the same).The result sees table 3.The result shows IgG antibody, the IgG of PRRS-APP group
1Antibody and IgG
2aAntibody horizontal all is higher than other groups far away, explains that APP can obviously improve the PRRS specific antibody level.
Table 3 is respectively organized antibody titer quantitative analysis table
| Group | Antibody horizontal (IgG 2a) | Antibody horizontal (IgG) | Antibody horizontal (IgG 1) |
| The APP group | -- | -- | -- |
| The PRRS- |
1∶25000 | 1∶2.0×10 8 | 1∶2.5×10 8 |
| The PRRS- |
1∶25000 | 1∶2.0×10 8 | 1∶2.0×10 8 |
| The |
1∶7500 | 1∶0.5×10 8 | 1∶0.5×10 8 |
Embodiment 5, APP-N adjuvant and the clinical trial of commercially available PRRS vaccine immunity swinery
One, grouping administration
Selecting 20 clinical identification for use is that antigen/antibody jack to jack adapter property 40 age in days pigs (kind is a landrace, available from experimentation on animals base, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences Changping) carry out immunity for experimental animal.Test is divided into four groups, every group of 5 pigs, and the immune situation of each group is seen table 4.The experiment carried out 50 days, immune programme for children be immunity once, immunization ways is for to be dissolved in intramuscular injection in the saline water with sample.(the 50ml/ bottle, every ml viral level is>=2 * 10 with every part of commercially available vaccine of PRRS
7.0TCID
50) being divided into 25 parts, every part of 2ml is as the single-dose dosage of every pig.
Table 4 pair swinery immunity grouping information slip
| Group | 0 day | 7 days |
| Negative control group: saline water (single immunization dosage is 4ml/) | Observe | 4ml |
| PRRS-APP group: the commercially available vaccine of PRRS (single immunization dosage is 2ml/) APP adjuvant (single immunization dosage is 2ml/) | Observe | 4ml |
| PRRS-I group: the commercially available vaccine of PRRS (single immunization dosage is 2ml/) contrast adjuvant I (single immunization dosage is 2ml/) | Observe | 4ml |
| PRRS group: the commercially available vaccine of PRRS (single immunization dosage is 4ml/) | Observe | 4ml |
Two, clinical symptom scoring
Every day, the viewing test animal was added up scoring to clinical symptom.Clinicing symptom observation project: the mental status, have or not appetite, whether vomit, whether have secretory product, ight soil shape, search for food and death condition, whether scleroma, redness and cyanosis, weightening finish situation etc. are arranged.The clinical symptom standards of grading: only give a mark for pig according to following table, mark is high more, and body condition is good more, full marks 15 minutes, and standards of grading are seen table 5, appraisal result is seen table 6.Scoring time and frequency: experimental animal was marked once weekly since first day.
Table 5 clinical symptom standards of grading
| The scoring index | Symptom | Mark |
| The mental status | Stupor (unconscious) dispirited (consciously but debility) spirit (consciously and have vigor) | 0 |
| Have or not appetite | Do not take food, no appetite can be taken food but the honey stomach of being off one's feed | 0 |
| Whether vomit | There is the vomiting phenomenon not vomit | 0 |
| Whether secretory product is arranged | There is secretory product not have secretory product | 0 |
| Ight soil form etc. | Ight soil is invisible, and bloody stool ight soil have loose bowels water sample or stiff ight soil consecutive are nodositas, and smooth in appearance is moistening | 0 |
| The situation of searching for food | Do not search for food and normally search for food | 0 |
| Whether scleroma, redness and cyanosis are arranged | Scleroma, redness and cyanosis are arranged, very serious a small amount of red and swollen, the nothing scleroma cyanosis of situation, situation does not seriously have scleroma, redness and cyanosis and takes place | 0 |
| The weightening finish situation | Do not have weightening finish, body weight is seriously become thin does not have weightening finish, the body weight weight increase that maintains an equal level | 0 |
| The anus temperature | The anus temperature is too high or too low be difficult to detect<=38 ℃ low slightly or>=39.5 ℃ high slightly 38 ℃～39.5 ℃ | 0 |
Table 6 swinery clinical symptom scoring statistics
According to clinical observation, the result shows on PRRS-APP group, PRRS-I group, PRRS group and the negative control group clinical symptom scoring statistics and exists difference.
Three, amynologic index
Respectively at precaval vein blood sampling in 14,21 and 28 days before the immunity, after the immunity, detect the following amynologic index in the pig blood: IgG, NAT, IL-4 and IFN-γ, detection method is referring to embodiment 4, and the result sees table 7.
Respectively organize pig blood IFN-γ horizontal analysis table after table 7 immunity
Show that according to ELISA result IFN-γ, IgG, IL-4 and NAT level exist difference in the PRRS-APP group, obviously exceed other three groups.It is thus clear that the APP adjuvant can significantly strengthen the immune efficacy of the commercially available vaccine of PRRS.Though the APP adjuvant is very approaching with the aminoacid sequence of contrast adjuvant, be much higher than contrast adjuvant I as the effectiveness of immunostimulant.
Four, attack the poison experiment
Carry out challenge test 28 days 5 pigs in to every group, attacking malicious mode is that (available from China Veterinery Drug Inspection Office, JXA1), detect the protection ratio of swinery body temperature, vaccine and immunostimulant, the result sees table 8 to intramuscular injection 1ml PRRSV virus.
Table 8 swinery attack poison back body temperature (℃) statistics
"-" represents dead.
It is thus clear that the protection ratio of PRRS-APP group is 100%, the protection ratio of PRRS-I group is 80%, and the protection ratio of PRRS group is 80%, and the negative control group swinery is all dead.
Above data show that all APP can be used as immunostimulant, significantly increase the immune effect of vaccine.APP adjuvant and existing vaccine are used as novel vaccine, can effectively prevent the generation of pig " blue otopathy ", swinery is had protection.
Sequence table
< 110>Institute of Microorganism, Academia Sinica
< 120>antigen-presenting protein for swines and encoding sox thereof and application
<130>CCGNARY102197
<160>4
<210>1
<211>334
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>1
Glu Asp Glu Val Asp Val Asp Gly Thr Val Glu Glu Asp Leu Gly Lys
1 5 10 15
Ser Arg Glu Gly Ser Arg Thr Asp Asp Glu Val Val Gln Arg Glu Glu
20 25 30
Glu Ala Ile Gln Leu Asp Gly Leu Asn Ala Ser Gln Ile Arg Glu Leu
35 40 45
Arg Glu Lys Ser Glu Lys Phe Ala Phe Gln Ala Glu Val Asn Arg Met
50 55 60
Met Lys Leu Ile Ile Asn Ser Leu Tyr Lys Asn Lys Glu Ile Phe Leu
65 70 75 80
Arg Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu Asp Lys Ile Arg Leu
85 90 95
Ile Ser Leu Thr Asp Glu Asn Ala Leu Ala Gly Asn Glu Glu Leu Thr
100 105 110
Val Lys Ile Lys Cys Asp Lys Glu Lys Asn Leu Leu His Val Thr Asp
115 120 125
Thr Gly Val Gly Met Thr Arg Glu Glu Leu Val Lys Asn Leu Gly Thr
130 135 140
Ile Ala Lys Ser Gly Thr Ser Glu Phe Leu Asn Lys Met Thr Glu Ala
145 150 155 160
Gln Glu Asp Gly Gln Ser Thr Ser Glu Leu Ile Gly Gln Phe Gly Val
165 170 175
Gly Phe Tyr Ser Ala Phe Leu Val Ala Asp Lys Val Ile Val Thr Ser
180 185 190
Lys His Asn Asn Asp Thr Gln His Ile Trp Glu Ser Asp Ser Asn Glu
195 200 205
Phe Ser Val Ile Ala Asp Pro Arg Gly Asn Thr Leu Gly Arg Gly Thr
210 215 220
Thr Ile Thr Leu Val Leu Lys Glu Glu Ala Ser Asp Tyr Leu Glu Leu
225 230 235 240
Asp Thr Ile Lys Asn Leu Val Lys Lys Tyr Ser Gln Phe Ile Asn Phe
245 250 255
Pro Ile Tyr Val Trp Ser Ser Lys Thr Glu Thr Val Glu Glu Pro Met
260 265 270
Glu Glu Glu Glu Ala Ala Lys Glu Glu Lys Glu Glu Ser Asp Asp Glu
275 280 285
Ala Ala Val Glu Glu Glu Glu Glu Glu Glu Lys Lys Pro Lys Thr Lys
290 295 300
Lys Val Glu Lys Thr Val Trp Asp Trp Glu Leu Met Asn Asp Ile Lys
305 310 315 320
Pro Ile Trp Gln Arg Pro Ser Lys Glu Val Glu Asp Asp Glu
325 330
<210>2
<211>1002
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>2
gaggatgaag tggatgtgga tgggacagtg gaagaagatc tgggtaaaag tagagaaggt 60
tccaggacag atgatgaagt agtacagaga gaggaagaag ctattcaatt ggatggatta 120
aatgcatccc aaataagaga acttagagag aaatcagaaa aatttgcctt ccaagctgaa 180
gttaacagaa tgatgaaact tatcatcaat tcattatata aaaataaaga gattttccta 240
agagaactga tttcaaatgc ttctgatgct ttggataaga taagactaat atcactgact 300
gatgaaaatg ctcttgctgg aaatgaggag ttaacggtca aaatcaagtg tgacaaggag 360
aagaacctgc tccatgtcac agacactggt gtgggaatga cccgggaaga gttggttaaa 420
aaccttggta ccatagccaa atctgggaca agcgagtttt taaacaaaat gacggaggca 480
caagaagatg gccagtcaac ttcggaactg attggccagt tcggtgttgg cttctattct 540
gccttccttg tagcagataa agttattgtc acgtcaaaac acaacaatga cacccagcac 600
atctgggagt ccgactccaa tgaattttct gtaattgctg accccagagg aaacacctta 660
ggacggggaa cgacaattac ccttgtttta aaagaagaag catctgatta ccttgaattg 720
gatacaatta aaaatctcgt gaaaaaatat tcacagttca taaactttcc tatttatgta 780
tggagcagca agactgaaac tgtcgaggaa cctatggaag aagaagaagc agcaaaagaa 840
gaaaaagagg aatctgatga tgaagctgca gtagaagaag aagaagaaga agaaaagaaa 900
ccaaaaacta aaaaagttga aaaaactgtc tgggactggg aacttatgaa tgatatcaaa 960
ccaatatggc agagaccatc aaaggaagta gaagatgatg aa 1002
<210>3
<211>333
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>3
Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu Glu Asp Leu Gly Lys
1 5 10 15
Ser Arg Glu Gly Ser Arg Thr Asp Asp Glu Val Val Gln Arg Glu Glu
20 25 30
Glu Ala Ile Gln Leu Asp Gly Leu Asn Ala Ser Gln Ile Arg Glu Leu
35 40 45
Arg Glu Lys Ser Glu Lys Phe Ala Phe Gln Ala Glu Val Asn Arg Met
50 55 60
Met Lys Leu Ile Ile Asn Ser Leu Tyr Lys Asn Lys Glu Ile Phe Leu
65 70 75 80
Arg Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu Asp Lys Ile Arg Leu
85 90 95
Ile Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly Asn Glu Glu Leu Thr
100 105 110
Val Lys Ile Lys Cys Asp Lys Glu Lys Asn Leu Leu His Val Thr Asp
115 120 125
Thr Gly Val Gly Met Thr Arg Glu Glu Leu Val Lys Asn Leu Gly Thr
130 135 140
Ile Ala Lys Ser Gly Thr Ser Glu Phe Leu Asn Lys Met Thr Glu Ala
145 150 155 160
Gln Glu Asp Gly Gln Ser Thr Ser Glu Leu Ile Gly Gln Phe Gly Val
165 170 175
Gly Phe Tyr Ser Glu Phe Leu Val Ala Asp Lys Val Ile Val Thr Ser
180 185 190
Lys His Asn Asn Asp Thr Gln His Ile Trp Glu Ser Asp Ser Asn Glu
195 200 205
Phe Ser Val Ile Ala Asp Pro Arg Gly Asn Thr Leu Gly Arg Gly Thr
210 215 220
Thr Ile Thr Leu Val Leu Lys Glu Glu Ala Ser Asp Tyr Leu Glu Leu
225 230 235 240
Asp Thr Ile Lys Asn Leu Val Lys Lys Tyr Ser Gln Phe Ile Asn Phe
245 250 255
Pro Ile Tyr Val Trp Ser Ser Lys Thr Glu Thr Val Glu Glu Pro Met
260 265 270
Glu Glu Glu Glu Ala Ala Lys Glu Glu Lys Glu Glu Ser Asp Asp Glu
275 280 285
Ala Ala Val Glu Glu Glu Glu Glu Glu Lys Lys Pro Lys Thr Lys Lys
290 295 300
Val Glu Lys Thr Val Trp Asp Trp Glu Leu Met Asn Asp Ile Lys Pro
305 310 315 320
Ile Trp Gln Arg Pro Ser Lys Glu Val Glu Glu Asp Glu
325 330
<210>4
<211>999
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>4
gacgatgaag ttgatgtgga tggtacagta gaagaggatc tgggtaaaag tagagaagga 60
tcaaggacgg atgatgaagt agtacagaga gaggaagaag ctattcagtt ggatggatta 120
aatgcatcac aaataagaga acttagagag aagtcggaaa agtttgcctt ccaagccgaa 180
gttaacagaa tgatgaaact tatcatcaat tcattgtata aaaataaaga gattttcctg 240
agagaactga tttcaaatgc ttctgatgct ttagataaga taaggctaat atcactgact 300
gatgaaaatg ctctttctgg aaatgaggaa ctaacagtca aaattaagtg tgataaggag 360
aagaacctgc tgcatgtcac agacaccggt gtaggaatga ccagagaaga gttggttaaa 420
aaccttggta ccatagccaa atctgggaca agcgagtttt taaacaaaat gactgaagca 480
caggaagatg gccagtcaac ttctgaattg attggccagt ttggtgtcgg tttctattcc 540
gaattccttg tagcagataa ggttattgtc acttcaaaac acaacaacga tacccagcac 600
atctgggagt ctgactccaa tgaattttct gtaattgctg acccaagagg aaacactcta 660
ggacggggaa cgacaattac ccttgtctta aaagaagaag catctgatta ccttgaattg 720
gatacaatta aaaatctcgt caaaaaatat tcacagttca taaactttcc tatttatgta 780
tggagcagca agactgaaac tgttgaggag cccatggagg aagaagaagc agccaaagaa 840
gagaaagaag aatctgatga tgaagctgca gtagaggaag aagaagaaga aaagaaacca 900
aagactaaaa aagttgaaaa aactgtctgg gactgggaac ttatgaatga tatcaaacca 960
atatggcaga gaccatcaaa agaagtagaa gaagatgaa 999
Claims (12)
1. protein, the protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1.
2. coding claim 1 said proteic gene.
3. gene according to claim 2 is characterized in that: said gene is the dna molecular shown in the sequence 2 in the sequence table.
4. the recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 2 or 3 said genes.
5. recombinant expression vector as claimed in claim 4 or reorganization bacterium is characterized in that:
Said recombinant expression vector is following A) or B):
A) claim 2 or 3 said genes are inserted the recombinant plasmid that the MCS of pBV220 obtains;
B) claim 2 or 3 said genes are inserted the recombinant plasmid that the MCS of pPICZ α A obtains;
Said reorganization bacterium is following C) or D):
C) with the A of claim 5) said recombinant plasmid imports the reorganization bacterium that bacillus coli DH 5 alpha obtains;
D) with the B of claim 5) said recombinant plasmid imports the reorganization bacterium that yeast strain SMD1168H obtains.
6. preparation claim 1 said proteic method is (1) or (2) as follows:
(1) with the C of claim 5) said reorganization bacterium, induced 5～7 hours, and obtained said albumen for 40～43 ℃;
(2) with the D of claim 5) said reorganization bacterium adds methyl alcohol and carries out abduction delivering, add methyl alcohol final concentration be 0.1-1.0% (volumn concentration).
7. method as claimed in claim 6 is characterized in that: in said (1), induced 6 hours, and obtained said albumen for 42 ℃.
8. method as claimed in claim 6 is characterized in that: in said (2), add methyl alcohol final concentration be 0.5% (volumn concentration).
9. the application of the said albumen of claim 1 in the preparation immunostimulant.
10. immunostimulant, its activeconstituents is the said albumen of claim 1.
11. a vaccine that is used for porcine reproductive and respiratory syndrome comprises the said albumen of claim 1.
12. the said albumen of claim 1 is used for the application of the vaccine of porcine reproductive and respiratory syndrome in preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101405986A CN101838325B (en) | 2010-04-02 | 2010-04-02 | Antigen-presenting protein for swines and encoding gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101405986A CN101838325B (en) | 2010-04-02 | 2010-04-02 | Antigen-presenting protein for swines and encoding gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101838325A CN101838325A (en) | 2010-09-22 |
| CN101838325B true CN101838325B (en) | 2012-02-01 |
Family
ID=42742025
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010101405986A Expired - Fee Related CN101838325B (en) | 2010-04-02 | 2010-04-02 | Antigen-presenting protein for swines and encoding gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101838325B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586371B (en) * | 2012-02-17 | 2014-01-01 | 中国科学院微生物研究所 | Method for producing swine Gp96 protein by using Pichia pastoris and special medium |
| CN112226408B (en) * | 2020-09-30 | 2024-04-02 | 西北农林科技大学 | Method for screening and identifying swine pathogen or exogenous protein specific antigen peptide |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993021951A1 (en) * | 1992-04-27 | 1993-11-11 | Michigan State University | Method for producing a bacterial vaccine and novel vaccines produced thereby |
-
2010
- 2010-04-02 CN CN2010101405986A patent/CN101838325B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993021951A1 (en) * | 1992-04-27 | 1993-11-11 | Michigan State University | Method for producing a bacterial vaccine and novel vaccines produced thereby |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101838325A (en) | 2010-09-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103172749B (en) | Preparation of African swine fever protein engineering vaccine | |
| CN103386128B (en) | Tuberculosis subunit vaccine containing unite adjuvant | |
| CN101905018A (en) | Recombinant fusion protein vaccine and attenuated live vector vaccine for treating and preventing helicobacter pylori (Hp) infection | |
| CN107098974A (en) | A kind of fusion protein and its application | |
| CN113512096A (en) | Weever rhabdovirus recombinant G2 protein and application thereof | |
| CN102816246B (en) | Human cytomegalo virus immunogen fusion protein as well as preparation method and usage thereof | |
| CN100391969C (en) | Helicobacter Pylori urease B subunit Th epitope peptide, its coding DNA, vaccine and uses | |
| CN104292339A (en) | Recombinant protein containing SARS virus RBD antigen and baculovirus displaying RBD protein | |
| CN109467606A (en) | A kind of escherichia coli enterotoxin STa-LTB-STb fusion protein and its encoding gene and application | |
| CN108273054A (en) | Swine foot-and-mouth disease virus is O-shaped, A type Fc polypeptide bivalent vaccines and its preparation method and application | |
| CN106279431B (en) | A kind of pig circular ring virus subunit inactivated vaccine | |
| CN102796200B (en) | Hybrid peptide bursin adjuvant and preparation method and application thereof | |
| CN113862284A (en) | Gene for coding recombinant avian influenza virus HA protein, virus-like particle, vaccine, preparation and application | |
| CN101838325B (en) | Antigen-presenting protein for swines and encoding gene and application thereof | |
| CN101376887B (en) | Preparation and use of swine foot-and-mouth disease recombinant immune composite peptide | |
| CN102533629B (en) | Preparation method of avian encephalomyelitis virus VP1 protein subunit vaccine | |
| CN102134278A (en) | Preparation of subunit vaccine for mucosal immune in circovirus genetic engineering and application thereof | |
| CN101070347B (en) | Bird-flu H5N1 novel mucous membrane immunization vaccine and its use | |
| CN103352047B (en) | Trichinella Spiralis larva ES antigen gene vaccine and preparation method | |
| CN106397602B (en) | A kind of reinforced chicken Marek's disease protein engineering vaccine of molecule adjuvant | |
| CN1864746B (en) | Gene engineered poly-valence subunit vaccine of pylorus Hp AhpC-NapA and preparation method thereof | |
| CN103509815A (en) | Preparation method of recombinant panda IL-2 immune adjuvant | |
| CN113603793A (en) | Novel coronavirus recombinant S protein, recombinant plasmid, recombinant bacterium and application for preparing exosome drug or exosome vaccine | |
| CN103547286B (en) | A kind of composition of DNA vaccine for preventing and treat hepatitis B | |
| CN105481953A (en) | Target cell specificity fusion protein serving as porcine reproductive and respiratory syndrome viral vaccine antigen and vaccine composition |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20100922 Assignee: Beijing HFK Bioscience Co., Ltd. Assignor: Institute of Microbiology, Chinese Academy of Sciences Contract record no.: 2013990000052 Denomination of invention: Antigen-presenting protein for swines and encoding gene and application thereof Granted publication date: 20120201 License type: Exclusive License Record date: 20130207 |
|
| LICC | Enforcement, change and cancellation of record of contracts on the licence for exploitation of a patent or utility model | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120201 Termination date: 20210402 |