CN101838325A - 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
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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 gene thereof and application.
Background technology
(Antigen Presentation Protein is that a kind of relative molecular mass is the glycoprotein 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 molecular chaperones, APP can stop protein aggregation in conjunction with not folding polypeptide chain, 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 may be ATP-binding site.Therefore, APP can have the ATPase activity in conjunction with ATP.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 energy 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 enters APC by acceptor, has avoided behind the endocytosis phagolysosome that the integrity of APP has been safeguarded in the degraded of APP.In sum, APP can be used as a kind of immunostimulant raising antigen immune activity.
In recent years, characteristic and the application of extensive concern APP both at home and abroad.Srivastava etc. have founded the method for extracting the APP peptide complex from tumour cell 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 chemical induction 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, studies show that at the external APP peptide complex that successfully made up, APP is the very important mate molecule of TLRs, in the normal function performance of scavenger cell, play the part of important role.Meng Songdong etc. (2002) discover APP as molecular chaperones, 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 gene and application.
Albumen provided by the present invention (APP-N) from porcine kidney cell line PK-15 cell, participates in antigen presentation, has the immunocompetent function of promotion, for following (a) 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 gene earlier, carry out biology again and express and to obtain.The encoding gene of APP-N in above-mentioned (b) can be by 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.
Described 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 described 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; Described prokaryotic cell prokaryocyte can be intestinal bacteria, as E.coli DH5 α, E.coli TB1 or E.coli BL21 etc.; Described 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.
Described recombinant expression vector specifically can be following A) or B):
A) described gene is inserted the recombinant plasmid that the multiple clone site of pBV220 obtains;
B) described gene is inserted the recombinant plasmid that the multiple clone site of pPICZ α A obtains.
Described reorganization bacterium specifically can be following C) or D):
C) with A) described recombinant plasmid imports the reorganization bacterium that bacillus coli DH 5 alpha obtains;
D) with B) described recombinant plasmid imports the reorganization bacterium that yeast strain SMD1168H obtains.
The total length of described gene of increasing or its any segmental primer are to also belonging to protection scope of the present invention.
The present invention also provides two kinds to prepare described proteic method: (1) is with C) described reorganization bacterium, induce 5～7 hours (inducing 6 hours for preferred 42 ℃), obtain described albumen for 40～43 ℃; (2) with D) described 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).
Described 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 described albumen.Immunostimulant spy of the present invention is applicable to pig fully.
Described 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 described 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 (as viruses such as PRRSV/ porcine reproductive and respiratory syndrome virus, FMDV/ foot and mouth disease virus, CSFV/ Pestivirus suis), 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), to improve swinery antigen immune activity; Immunostimulant provided by the invention can also be injected respectively with antigenic substance.Albumen of the present invention can efficiently express in intestinal bacteria, 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, improve the antigen immune activity, strengthen humoral immunization and the cellular immunization of swinery, reach 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: precipitation.
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 if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.% among the following embodiment if no special instructions, 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 finished 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, extracts the total RNA of pig from porcine kidney cell line PK-15 cell (available from China Veterinery Drug Inspection Office).
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 the primer that designs.The PCR reaction conditions is as follows: 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 company limited, 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 gene 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 production 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 with insert behind restriction enzyme BamHI and the 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 and centrifugal 10 minutes of bacterium liquid 10000g after inducing, 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 is (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 precipitation respectively and carry out the SDS-PAGE electrophoresis.(swimming lane M is molecular weight protein Marker to DH5 α-APP-N/pBV220 detected result as shown in Figure 2, swimming lane 1 is a supernatant, swimming lane 2 is precipitation), go up the target protein band that all occurs in the cleer and peaceful precipitation about 45KD, all there is not the target protein band in the last cleer and peaceful precipitation of DH5 α-pBV220.
Before will inducing respectively and centrifugal 10 minutes of bacterium liquid 10000g after inducing, 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, constant current with every square centimeter of membrane area * 0.65mA was changeed film 1.5～2 hours, 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% is settled to 1000ml), adding resists with one of confining liquid preparation, hatches under the room temperature 3 hours, reclaims an anti-back and preserves.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.(swimming lane M is molecular weight protein Marker to the western blot detected result of DH5 α-APP-N/pBV220 as shown in Figure 3, 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 precipitation of DH5 α-pBV220.Above result shows that APP-N correctly expresses in intestinal bacteria.
3, the fermentative production 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 production 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 (available from the general Bioisystech Co., Ltd that flies in Shanghai after with restriction enzyme Xho I and Xba I double digestion, V19520) between XhoI and the XbaI enzyme cutting site, electricity 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 tryptone, 2% glucose, the 1M sorbyl alcohol, 2% agar) flat board, cultivated 2-3 days for 42 ℃, until growing single bacterium colony, after identifying, PCR obtains positive reorganization bacterium.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 (contains 1% yeast extract, 2% yeast culture 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; Change the BMMY substratum again into and (contain 1% yeast extract, 2% yeast culture tryptone, 100mM potassiumphosphate, 1.34%YNB, 4 * 10
-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, constant current with every square centimeter of membrane area * 0.65mA was changeed film 1.5-2 hour, 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% is settled to 1000ml), adding with confining liquid preparation one anti-(with step 12 in identical one anti-), hatched under the room temperature 3 hours, reclaim an anti-back and preserve.Film is put into TBST solution to decolour, the HRP mark two anti-rear decolorings that adding is prepared with confining liquid, 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 is (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 production 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, be 4.8 with supernatant liquor with the vinegar acid for adjusting pH value; (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 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; 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: with Tris-Cl damping fluid (20mM, pH8.0) as initial liquid, with NaCl concentration is the Tris-Cl damping fluid (20mM of 1M, 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 described 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 described 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 gene 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 '.
With inserting between the BamHI and XhoI restriction enzyme site of carrier pBV220 behind pcr amplification product usefulness restriction enzyme BamHI and the 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 adjuvant I in contrast, is used for the experiment of embodiment 3,4,5.
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 that small white mouse is used in mean body weight and the test 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 penicillin 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 group SI=1.2; SI is the highest for the PRRS-APP group, is nearly 4 times of PRRS group, illustrate that APP-N can obviously strengthen the cell response level of PRRS vaccine, and effect is better than 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 penicillin 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 group SFP=105; The IFN-γ level of PRRS-APP group approximately is 3～4 times of independent immune PRRS group.Illustrate that APP-N can obviously strengthen the cell response level of PRRS vaccine, and effect is better than contrasting adjuvant I.
Two, detect humoral immune reaction
(1) use in serum-virus and 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 sees Table 2.Illustrate 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-APP group | ??1∶8 |
| The PRRS-I group | ??1∶4 |
| The PRRS group | ??1∶2 |
(2) measure the PRRSV antibody horizontal with elisa technique
Separation of serum, carry out ELISA reaction assay PRRSV specific antibody level: usefulness PRRSV virus (50ng/ hole) (available from China Veterinery Drug Inspection Office, JXA1) wrap by 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; Respectively in conjunction with goat-anti mouse HRP-IgG (Santa cruz company, catalog number: SC-2005), HRP-IgG
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 citric acid 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 citric acid 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 results are shown in 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, illustrates 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-APP group | ??1∶25000 | ??1∶2.0×10 8 | ??1∶2.5×10 8 |
| The PRRS-I group | ??1∶25000 | ??1∶2.0×10 8 | ??1∶2.0×10 8 |
| The PRRS group | ??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 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 sees 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 physiological saline 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: physiological saline (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 see Table 5, and appraisal result sees 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 |
| The scoring index | Symptom | Mark |
| 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 the results are shown in Table 7 referring to embodiment 4.
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.As seen 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 intramuscular injection 1ml PRRSV virus (available from China Veterinery Drug Inspection Office, JXA1), detects the protection ratio of swinery body temperature, vaccine and immunostimulant, the results are shown in Table 8.
Table 8 swinery attack poison back body temperature (℃) statistics
"-" represents dead.
As seen, the protection ratio of PRRS-APP group is 100%, and 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 gene 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 (10)
1. protein is following (a) or protein (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.
2. coding claim 1 described proteic gene.
3. gene according to claim 2 is characterized in that: described gene is 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.
4. the recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 2 or 3 described genes.
5. recombinant expression vector as claimed in claim 4 or reorganization bacterium is characterized in that:
Described recombinant expression vector is following A) or B):
A) claim 2 or 3 described genes are inserted the recombinant plasmid that the multiple clone site of pBV220 obtains;
B) claim 2 or 3 described genes are inserted the recombinant plasmid that the multiple clone site of pPICZ α A obtains;
Described reorganization bacterium is following C) or D):
C) with the A of claim 5) described recombinant plasmid imports the reorganization bacterium that bacillus coli DH 5 alpha obtains;
D) with the B of claim 5) described recombinant plasmid imports the reorganization bacterium that yeast strain SMD1168H obtains.
6. prepare the described proteic method of claim 1, be following (1) or (2):
(1) with the C of claim 5) described reorganization bacterium, induced 5～7 hours for 40～43 ℃, induced 6 hours, and obtained described albumen for preferred 42 ℃;
(2) with the D of claim 5) described reorganization bacterium adds methyl alcohol and carries out abduction delivering, add methyl alcohol final concentration be 0.1-1.0% (volumn concentration), be preferably 0.5% (volumn concentration).
7. the application of the described albumen of claim 1 in the preparation immunostimulant.
8. immunostimulant, its activeconstituents is the described albumen of claim 1.
9. a vaccine that is used for porcine reproductive and respiratory syndrome comprises the described albumen of claim 1.
10. the described albumen of claim 1 is used for the application of the vaccine of porcine reproductive and respiratory syndrome in preparation.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586371A (en) * | 2012-02-17 | 2012-07-18 | 中国科学院微生物研究所 | Method for producing swine Gp96 protein by using Pichia pastoris and special medium |
| CN112226408A (en) * | 2020-09-30 | 2021-01-15 | 西北农林科技大学 | Method for screening and identifying specific antigenic peptide of swine pathogen or exogenous protein |
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 |
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| 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 |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102586371A (en) * | 2012-02-17 | 2012-07-18 | 中国科学院微生物研究所 | Method for producing swine Gp96 protein by using Pichia pastoris and special medium |
| 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 |
| CN112226408A (en) * | 2020-09-30 | 2021-01-15 | 西北农林科技大学 | Method for screening and identifying specific antigenic peptide of swine pathogen or exogenous protein |
| 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 |
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| CN101838325B (en) | 2012-02-01 |
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