CN101838319B - Asia 1-type FMDV compound multiepitope - Google Patents
Asia 1-type FMDV compound multiepitope Download PDFInfo
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Abstract
The invention provides an Asia 1-type FMDV compound multiepitope which adopts an Asia 1-type FMDV capsid protein VP1 antigen activity area as the basic skeleton, and comprises the epitope of Asia 1/JL/05 plant VP1, the epitope of the IND 63/72 plant VP1, the epitope of YNBS/58 plant VP1, cholera toxin B subunit and helper T lymphocyte epitope. The compound multiepitope can be used for preparing Asia 1-type FMDV subunit vaccine, nucleic acid vaccine, synthetic peptide vaccine, live vector vaccine and a plurality of genetic engineering vaccines.
Description
Technical field
The present invention relates to a kind of Asia 1 type FMDV composite multi-epitope; Particularly relating to a kind of is the Asia 1 type FMDV composite multi-epitope (albumen) of skeleton with Asia 1 type FMDV capsid protein VP1 antigenic activity zone; The invention still further relates to the polynucleotide of this composite multi-epitope of coding (albumen), and use the constructed recombinant vaccine of this composite multi-epitope.
Background technology
Foot and mouth disease (Foot-and-Mouth Disease; FMD) be that (Foot-and-Mouth Disease Virus, what FMDV) cause serves as a kind of acute, hot, the height contagious disease that infects object with multiple artiodactyls such as ox, pig, sheep by foot and mouth disease virus.This disease have the diffusion of propagating rapidly, host range is wide, sickness rate is high, popular characteristics such as extremely wide, therefore is decided to be by OIE (OIE) and must reports one of transmissible disease, China also classifies it as type of transmissible disease.At present, the foot and mouth disease virus of having found has seven serotypes (O, A, C, Asia 1 and SAT 1, SAT 2, SAT 3), and more than 80 kind of hypotype, do not have the cross immunity protection between serotype, and virus morphs very easily.Since the 17th century was on the books, breaking out each time all of foot and mouth disease can bring enormous economic loss and serious political fallout to country, like extensive popular the breaking out that 1967-1968 Britain has experienced a foot and mouth disease, surpassed 430 000 livestocks and catched and killed; Calendar year 2001, foot and mouth disease broke out in Britain on a large scale, spread to continental European countrieses such as France, Ireland, Holland afterwards, this time only the British agriculture loss just above 3,100,000,000.China in March, 2005 reported first Asia 1 type FMD takes place in Hong Kong, subsequently,, reported that Asia 1 type FMD has taken place in the China's Mainland in April, 2005.By in September, 2006, in state-owned 15 Asia 1 type FMD has taken place more than the area.
Take the policy of catching and killing different with developed country, most of developing countries come the prevention and control should disease with the method for general immunity, and China then takes planned immunization and catches and kills the method that combines.The widely used FMD vaccine of China mainly is a deactivation vaccine at present; Although it occupies critical role in the FMD control; But in production and use, still there are potential safety hazards such as virus spreads, deactivation is not thorough; Therefore, development safety, new generation vaccine efficiently are very important for China's prevention and control foot and mouth disease.
The research of making a general survey of aftosa vaccine is historical, has mainly experienced and has updated and perfect four-stage: animal tissues's deactivation vaccine (20～forties of 20th century), attenuated live vaccine (50～eighties of 20th century), cell culture and virus deactivation vaccine (using after the seventies in 20th century), new generation vaccine (after the eighties in 20th century so far).In recent years, along with development of molecular biology, and the proposition of new theory of immunity, make the research of new generation vaccine become focus.See that from security, high efficiency, broad spectrum, economy and operability new generation vaccine all exists the incomparable advantage of traditional vaccine, the also final FMD of elimination of control that appears as of new generation vaccine has brought new hope.At present, the foot and mouth disease new generation vaccine mainly comprises: synthetic peptide vaccine, protein carrier vaccine, gene-deleted vaccine, can raise vaccine, live vector vaccine, subunit vaccine, nucleic acid vaccine etc.
Polyepitope vaccines (multi-epitope vaccine) is a kind of as new generation vaccine; The aminoacid sequence that is based on epitope prepares; Can carry the vaccine of a plurality of target antigen associated epitope and complementary epi-position simultaneously; Also claiming the cocktail type vaccine, is the vaccine design thinking of development in recent years a kind of uniqueness of getting up.In general; Immune system recognition, submission antigen only need tens amino acid; And only need several amino acid with antibodies; Therefore polyepitope vaccines can comprise a large amount of antigenic informations according to design demand; Can give fluent replies effectively susceptible poison (IV), human immunodeficiency virus (HIV), hepatitis C virus (HCV), foot and mouth disease viruses (FMDV) etc. have no cross protection between a plurality of serotypes, type or the cross protection rate is low, variability is high pathogenic micro-organism, thereby reduce due to illness substance variation and the vaccine immunity failure that causes.In addition, because polyepitope vaccines can be connected various epitopes, therefore the quantity of autotelic increase type epi-position strengthening having special advantages aspect the cellular immunization.
Reported first such as nineteen eighty-two Bittle succeed with the FMDV small peptide immune animal of chemosynthesis, opened the prelude of FMDV polyepitope vaccines development.From then on, Bittle, research worker such as Morgand, Zamorano etc. have both at home and abroad carried out a large amount of research to FMDV epi-position and epiposition vaccine, for the development of FMDV epiposition vaccine has been gained most valuable experience.Research at present shows that FMDV capsid protein VP1 major part is exposed to the virus particle surface, is the staple of decision virus antigenicity.Its 141st～160 and 200～213 amino acids residues are major antigen districts of FMDV, can produce neutralizing antibody by induced animal, also are antigenic hypervariable regions simultaneously.With the full gene of VP1 or its important epitope is that the based gene engineered vaccine is to study maximum FMD new generation vaccines now.
Research confirms that antigen realizes through epitope that to the stimulation of body immune system therefore the research to epitope has become one of main means of screening candidate vaccine at present.But because epitope quantity is huge,, waste time and energy, be difficult to filter out advantage epi-position effectively through traditional method screening and evaluation epi-position.Along with development of molecular biology; The quantity of nucleotide sequence, protein sequence and structural information increases gradually; And set up associated databases for your guidance; For the foundation of immune information biology (Immuno-bioinformatics) lays the foundation, making applying biological information science software predict epi-position becomes possibility.Through 20 years of development, had software, DB and the webserver of various epitopes prediction at present.As be used for " BIMAS (http://www-bimas.cit.nih.gov) ", " SYFPEITHI (http://www.syfpeithi.de) ", " Predict (http://sdmc.lit.org.sg:/predict-demo) ", " EPIPREDICY (the http://www.epipredict.de) " etc. of CTL functional epitope prediction; Be used for " SYFPEITHI ", " Bcepred (http://www.imtech.res.in/raghava/bcepred/bcepred_submission.htm l) ", " BepiPred (http://www.cbs.dtu.dk/services/BepiPred/) ", " ABcpred (the http://www.imtech.res.in/raghava/abcpred) " of B cell epitope prediction etc.; " mhc2pred (http://www.imtech.res.in/raghava/mhc2pred/) " MHCPred (http://the www.jenner.ac.uk/MHCPred/) " Propred (http://www.imtech.res.in/raghava/propred/) " that is used for the prediction of Th epi-position.Applied Forecasting Methodology mainly comprises: simple motif method (simple motify), artificial neural network (ANNs), hidden Markov model (HMMs), molecular model method (spatial structure based method), frequency analysis and quantitative matrix method (frequency matrix), support vector machine method (SVMs) etc.
Summary of the invention
The composite multi-epitope that the purpose of this invention is to provide many strain epi-positions of a kind of Asia of comprising 1 type FMDV, and the dna vaccination of this composite multi-epitope of encoding.
For achieving the above object; The present invention at first provides a kind of Asia 1 type FMDV composite multi-epitope (CTE-Asia); It is to be basic skeleton with Asia 1 type FMDV capsid protein VP1 antigenic activity zone; Include the epitope of Asia 1/JL/05 strain VP1, the epitope of IND 63/72 strain VP1 and the epitope of YNBS/58 strain VP1, and choleratoxin B subunit (CTB) and helper T cell epi-position (Th).
Wherein, the epitope of said Asia 1/JL/05 strain VP1 is:
RLHTDVAFVLDRFVKLTQPKS，
TTYGKESSRRGDLAALARRVNNRLP, and
LLALDTTQDRRKQKIIAPEKQTLNFD。
Wherein, the epitope of the VP1 of said IND 63/72 strain is:
KKPITRLALPYTAPHR, and
TYGTQPTRRGDLAVLAQRVSNRLPTSFNY。
Wherein, the epitope of said YNBS/58 strain VP1 is:
KTTYGEESTRRGDFAALAQRLSRRL, and
KADTITELLIR。
Connect through flexible amino acid Linker between above-mentioned each epitope sequences.
The present invention also provides the albumen that has above-mentioned composite multi-epitope with a kind of, of the present invention one preferred embodiment in, the proteic aminoacid sequence of this composite multi-epitope is shown in SEQ IDNo.2.Those skilled in the art can not influence under its active prerequisite according to this sequence, replace, disappearance and/or add one or several amino acid, obtain said proteic mutant nucleotide sequence.For example the AAA with the 249-251 position replaces with GPGPG, perhaps with the GGGGS disappearance of 132-136 position, perhaps adds GP at the 221st.Therefore, composite multi-epitope albumen of the present invention also comprises aminoacid sequence shown in the SEQ ID No.2 through replacing, replace and/or increasing one or several amino acid, has the protein of being derived and being obtained by SEQ ID No.2 of same function.
The present invention also comprises above-mentioned composite multi-epitope of coding or proteic gene.In embodiments of the present invention, encode the nucleotide sequence of this composite multi-epitope protein gene shown in SEQ ID No.1.Should be understood that the degeneracy of considering codon and the preferences of different plant species codon, those skilled in the art can use as required and be fit to the codon that specific species are expressed.
Can said gene of the present invention be operably connected with expression vector, obtain to express the proteic recombinant expression vector of composite multi-epitope of the present invention.
Composite multi-epitope albumen of the present invention is to have good immunogenicity, can make the animal body immunity system produce the antibody to FMDV, thereby FMDV is produced prevention or therapeutic action.And then the present invention provides a kind of composite multi-epitope vaccine, and it comprises above-mentioned composite multi-epitope albumen and acceptable carrier.
The present invention also provides a kind of dna vaccination, its above-mentioned composite multi-epitope albumen of encoding.Those skilled in the art can design corresponding nucleotide sequence according to composite multi-epitope, and the transcript and expression system that this nucleotide sequence can pass through organism in vivo produces this composite multi-epitope albumen.
Technique scheme has following advantage: on the basis of the epitope that is closely related through detailed understanding Asia 1 type FMDV genome structure and with immunity; In conjunction with the specialized network server (CTL Pred:http: //www.imtech.res.in/cgibin/ctlpred/ctlpred.pl, Bce Pred:http: //www.imtech.res.in/raghava/bcepred and Harvard:http: //the bio.dfci.harvard.edu/Tools/antigenic.pl epi-position predicts the outcome, and selected 2 antigenic activity zones and 2 the antigenic activity zones of YNBS/58 strain VP1 of VP1 of 3 antigenic activity zones, IND 63/72 strain of Asia 1/JL/05 strain VP1.Through flexible micromolecule polypeptide Linker, these antigenic activity zones are combined the synthetic Asia 1 type FMDV multi-epitope gene (Epi-Asia) that obtains of applied chemistry.Utilize molecular biology method, the choleratoxin B subunit that end is had Linker links to each other with Asia 1 type FMDV multi-epitope with the helper T cell epi-position, obtains Asia 1 type FMDV composite multi-epitope gene (CTE-Asia).With this Asia 1 type FMDV composite multi-epitope; Utilize molecular biology software DNAStar and Insight II to carry out the prediction of secondary structure and tertiary structure; In the proof composite multi-epitope neighboar lists interdigit secondary structure influence each other lessly, and have antigens with higher property; Have a plurality of epi-positions to show in molecular surface simultaneously outward, particularly composite multi-epitope Epi-Asia part explains that the composite multi-epitope of design meets theory demand, is expected to become immunogen preferably.This composite multi-epitope can be used for the preparation of several genes engineered vaccines such as Asia 1 type FMDV subunit vaccine, nucleic acid vaccine, synthetic peptide vaccine, live vector vaccine.
Description of drawings
Fig. 1 is an embodiment of the invention cloning vector pBluescript II KS-Epi-Asia structure iron;
Fig. 2 is an embodiment of the invention cloning vector pBluescript II KS-CTE-Asia structure iron;
Fig. 3 is Nucleotide and the aminoacid sequence and the note of embodiment of the invention Asia 1 type FMDV composite multi-epitope (CTE-Asia).
Embodiment
Below in conjunction with accompanying drawing and embodiment, specific embodiments of the invention is described in further detail.Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
The present invention fully utilizes different subject knowledges, technology such as molecular biology, Molecular Virology, molecular immunology, information biology; Select and recombinant gene in conjunction with information biology simulation, molecular designing, epi-position; Experience, lesson and newest research results to pathogenic agent FMDV that causes FMD and FMD research field; Take all factors into consideration characteristic, genome structure and the characteristics of FMDV and the problem that is closely related with immunity, on molecular level, made up a kind of NEW TYPE OF COMPOSITE multi-epitope (CTE-Asia) to Asia 1 type FMD.The design of this composite multi-epitope meets theory demand, is expected to become immunogen preferably.Can be used for the preparation of several genes engineered vaccines such as Asia 1 type FMDV subunit vaccine, nucleic acid vaccine, synthetic peptide vaccine, live vector vaccine.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.The screening of being connected of the recovery of the extraction of the preparation of competent escherichia coli cell and conversion, plasmid and digestion with restriction enzyme, dna fragmentation, linear DNA fragment, recombinant plasmid and evaluation etc. are translated " molecular cloning experiment guide " second edition related Sections with reference to Jin Dongyan, Li Mengfeng etc. and are carried out among the embodiment.
The structure of embodiment 1 Asia 1 type FMDV composite multi-epitope
On the basis of the epitope that is closely related through detailed understanding Asia 1 type FMDV genome structure and with immunity; Select the epidemic isolates of Chinese Asia 1 type FMDV; In conjunction with the specialized network server (CTL Pred:http: //www.imtech.res.in/cgibin/ctlpred/ctlpred.pl, Bce Pred:http: //www.imtech.res.in/raghaVa/bcepred and Harvard:http: //bio.dfci.harvard.edu/Tools/antigenic.pl) epi-position predicts the outcome and adjusts, and makes the antigenic activity zone that obtains be in the common factor district of a plurality of epi-positions as far as possible.The antigenic activity that connects selection with the flexible amino acid of small molecules is regional, forms one section new artificial multi-epitope sequence.Carry out full gene chemosynthesis.Utilize molecular biology method, the choleratoxin B subunit that end is had Linker links to each other with Asia 1 type FMDV multi-epitope with the helper T cell epi-position, obtains Asia 1 type FMDV composite multi-epitope gene (CTE-Asia).
Selected antigenic activity zone is as shown in the table:
| Sequence number | The position | Sequence | The |
| 1 | VP1 26-46 | RLHTDVAFVLDRFVKLTQPKS | Asia 1/JL/05 |
| 2 | VP1 133-157 | TTYGKESSRRGDLAALARRVNNRLP | Asia 1/JL/05 |
| 3 | VP1 188-213 | LLALDTTQDRRKQKIIAPEKQTLNFD | Asia 1/JL/05 |
| 4 | VP1 107-122 | KKPITRLALPYTAPHR | IND 63/72 |
| 5 | VP1 133-161 | TYGTQPTRRGDLAVLAQRVSNRLPTS FNY | IND 63/72 |
| 6 | VP1 133-157 | KTTYGEESTRRGDFAALAQRLSRRL | YNBS/58 |
| 7 | VP1 166-187 | VKADTITELLIR | YNBS/58 |
1, Asia 1 type FMDV multi-epitope gene is synthetic
By JaRa biotechnology (Shanghai) Co., Ltd., adopt full gene synthetic method to carry out artificial chemosynthesis, and be cloned in the pBluescript II KS carrier, called after pBluescriptII KS-Epi-Asia, its plasmid map is as shown in Figure 1.
2, the structure of Asia 1 type FMDV composite multi-epitope gene
With BamHI and EcoR I double digestion plasmid pBluescript II KS-CTB-Th2 behind agarose gel electrophoresis; Reclaim purpose fragment CTB-Th2; Under 16 ℃, be connected among the plasmid pBluescript II KS-Epi-Asia with EcoR I and Bgl II double digestion; Utilizing BamHI and Bgl II is isocaudarner, obtains plasmid pBluescript II KS-CTB-Th2-Epi-Asia, i.e. pBluescript II KS-CTE-Asia.
The Asia that obtains 1 type FMDV composite multi-epitope gene clone plasmid collection of illustrative plates as shown in Figure 2.
3, the secondary structure analysis of Asia 1 type FMDV composite multi-epitope
Utilize molecular biology software DNAStar that Asia 1 type FMDV composite multi-epitope is carried out secondary structure analysis; The result shows: most Linker form flexible region and corner between two peptide sections or epi-position; Many epi-positions all have the possibility that appears at molecular surface simultaneously, and composite multi-epitope has antigens with higher property.
4, the prediction of Asia 1 type FMDV composite multi-epitope tertiary structure
Utilize molecular biology software I nsight II that Asia 1 type FMDV composite multi-epitope is carried out the tertiary structure prediction, the result shows: have a plurality of epi-positions to show in molecular surface outward, meet the design requirements of multi-epitope.
5, Asia 1 expression of type FMDV composite multi-epitope gene in pichia spp
(1) structure of yeast expression vector
With Not I and EcoR I double digestion plasmid pBluescript II KS-CTE-Asia and yeast expression vector pPIC9K, reclaim the purpose fragment, connect down at 16 ℃, obtain expression plasmid pPIC9K-CTE-Asia.Utilize alkaline lysis to prepare pPIC9K-CTE-Asia in a large number, be stored in-70 ℃.
(2) the expression vector electricity transforms and gets in the pichia spp
Pichia spp GS115 is inoculated in the fresh YPD substratum of 200mL, and 30 ℃ of incubated overnight are to OD
600Between 1.3～1.5; 4 ℃, the centrifugal 10min collection of 3000r/min bacterium are used ice-cold sorbyl alcohol (1mol/L) the washing bacterial sediment of ice-cold deionized water of 200mL and 10mL respectively, and be with the resuspended deposition of 400 μ L sorbyl alcohols, subsequent use.
After getting linearizing recombinant plasmid 10 μ L of Bgl II (containing DNA 5～20 μ g approximately) and the above-mentioned competent cell mixing of 80 μ L, change in the electric revolving cup and shock by electricity.Condition is: voltage 1500V, time 5ms.After adding the ice-cold resuspended thalline of sorbyl alcohol of 1mL, coat on the MD flat board.
(3) screening of pichia spp positive colony
Be inoculated in respectively on MD and the MM nutrient agar with toothpick picking bacterium colony to be checked, cultivate 2～4d for 30 ℃, choose MD substratum well-grown, and the MM substratum poor growth or the bacterium colony of not growing.
Extract above-mentioned pastoris genomic dna with glass bead method, carry out PCR with Auele Specific Primer and yeast universal primer respectively and identify.
(4) abduction delivering of pichia spp positive colony
With identifying that correct pichia spp bacterium colony is inoculated among the YPD, cultivate 16～18h, get 1mL and be inoculated in the 50mL BMGY substratum, for 30 ℃ as thalline OD
600Be 2～6 o'clock, centrifugal collection thalline, BMMY is resuspended with the 25mL inducing culture, cultivates 48h for 28 ℃, and it is 1.0% that every separated 24h adds 100% methyl alcohol to final concentration.After the 48h, centrifugal recovery supernatant is stored in-70 ℃.
(5) SDS-PAGE and the Western blot that express target protein in the supernatant detect
Get the yeast expression supernatant and utilize SDS-PAGE to detect, anti-with anti-Asia 1 type FMDV serum of rabbit and the anti-Toxins,exo-, cholera serum of rabbit respectively simultaneously as one, target protein is carried out the protein immunoblot analysis.
The result shows: Asia 1 type FMDV composite multi-epitope gene can be expressed in eucaryon host pichia spp GS115, and expression product can be discerned by Asia 1 type FMDV serum and the anti-Toxins,exo-, cholera serum of rabbit, has antigenic activity.
6, the mouse immune of Asia 1 type FMDV composite multi-epitope subunit vaccine experiment
(1) mouse immune program
Get 15 of female BALB/c mouses in age in 6-8 week, immunity twice altogether, 21d at interval, each immune subunit vaccine 40 μ g/ only take the abdominal injection mode, and head exempts to mix with Freund's complete adjuvant, and inferior exempting from mixed with Freund's incomplete adjuvant.Respectively at 0d, 7d, 14d, 21d, 31d tail vein blood, carry out serum ELISA antibody analysis.Two exempt from back 10d, get mouse spleen, and preparation SPL suspension carries out lymphopoiesis conversion test and IFN γ-ELISPOT and detects.
(2) immune serum ELISA antibody test
Adopt indirect ELISA method to measure specific IgG antibodies level in the immune serum.Its method is: with the Asia 1 type FMDV of coating buffer dilution formalin-inactivated, and 100 μ L/ holes, 4 ℃ are spent the night and encapsulate elisa plate; Next day, wash plate 3 times with washings, each 3min, last drains on filter paper, adds confining liquid 100 μ L/ holes, 37 ℃ of sealing 1h; After washing plate, add serum to be checked 100 μ L/ holes (100 times of PBS dilutions), hatch 1h for 37 ℃; After washing plate, the anti-mouse IgG antibody (PBS dilution) that adds horseradish peroxidase-labeled is hatched 1h for 37 ℃; After washing plate, add 100 μ L/ hole substrate solutions, put dark place 5～15min after, add 50 μ L/ holes and stop to dissolve; Detect the OD value under the 492nm.
The result shows: Asia 1 type FMDV composite multi-epitope can stimulate the mouse body to produce the specific antibody to Asia 1 type FMDV.
(3) preparation of immune mouse SPL suspension
Aseptic condition takes out spleen down, with slide glass mouse spleen is ground, and cell is resuspended in the 4mL mouse lymphocyte parting liquid; With nylon wire (200 order) cell suspension is filtered in the 10mL centrifuge tube, adherent adding 1mL serum-free RPMI-1640 is in the centrifugal 15min of 1500r/min; Draw buffy coat in new centrifuge tube; The RPMI-1640 that adds 4mL 5% serum, mixing, the centrifugal 15min of 1500r/min; Abandoning supernatant, the resuspended SPL of RPMI-1640 of adding 1mL 10% serum.Count with blood counting chamber, adjustment lymphocyte suspension concentration is 1 * 10
7Individual cell/mL.
(4) mouse spleen lymphocyte transforms proliferation experiment
Get 96 porocyte culture plates, the lymphocyte 50 μ L/ holes that add dilution (contain 2 * 10
4Individual); Each sample comprises nonspecific stimulation (ConA), differential stimulus (FMDV) and non-stimulated (RPMI-1640); Corresponding FMDV (1MOI) and the nutrient solution that adds 50 μ L ConA (final concentration is 5 μ g/mL), deactivation, the various stimulations of sample are all established 3 multiple holes.After culture plate placed cell culture incubator 60h, every hole added 10 μ L WST-1 solution and continues to cultivate 1-4h.Measure the 450nm light absorption value, calculate SI (SI).
SI (SI)=stimulation hole OD value/control wells OD value
The result shows: after the specific anti primary stimuli, mouse spleen lymphocyte shows specific conversion propagation phenomenon.
(5) mouse spleen lymphocyte IFN-γ ELISPOT detects
Reaching section according to Beijing is that the Quick Spot of Bioisystech Co., Ltd mouse IFN-γ ELISPOT encapsulates the test kit working instructions in advance and carries out.1. encapsulate the plate activation in advance: every hole adds the RPMI-1640 substratum of 200 μ L, and room temperature leaves standstill 10min, topples over substratum.2. add lymphocyte: the arrangement according to the experiment card adds sample cell, and every hole 100 μ L (contain 1 * 10
5Individual cell).If positive control wells and negative control wells.3. add stimulator: positive control wells adds 100 μ L ConA (final concentration is 5 μ g/mL); Negative control wells adds the RPMI-1640 of 100 μ L, 10% serum; Sample well adds 100 μ L differential stimulus things.4. cultivate: be put in and cultivate 36h in the cell culture incubator.5. lysing cell: discard liquid in the hole, add the deionized water 200 μ L/ holes of precooling, 4 ℃ of refrigerators leave standstill 10min.6. wash plate: discard liquid in the hole, with 1 * Washing buffer washing 7 times, each 30s.For the last time, on filter paper paper, buckle and do.7. detect antibody incubation: every hole adds the biotin labeling antibody of 100 μ L dilution, hatches 1h for 37 ℃.8. avidin is hatched: wash plate, add the good enzyme mark avidin of 100 μ L/ holes dilution, hatch 1h for 37 ℃.9. colour developing: wash plate, add the AEC colour developing liquid 100 μ L/ holes that prepare, the about 25min of room temperature lucifuge colour developing.It is suitable when big or small to treat that spot grows into, with deionized water wash 2 times, and color development stopping.Do the tiny globule at the filter paper arsis, take off the resist of bottom, be placed on the airy place, film is dried naturally.10. count: ELISPOT plate spot counting.
The result shows: after the specific anti primary stimuli, mouse spleen lymphocyte shows specific IFN-γ secreting phenomenon.Transforming proliferation experiment in conjunction with mouse spleen lymphocyte shows: Asia 1 type FMDV composite multi-epitope can stimulate the mouse body to produce the specific cellular immunity to Asia 1 type FMDV.
Above-mentioned experiment shows; In the new A sia 1 type FMDV composite multi-epitope that the present invention designed neighboar lists interdigit secondary structure influence each other less; And has antigens with higher property; There are a plurality of epi-positions to show simultaneously outward, meet the design requirements of multi-epitope, be expected to become immunogen preferably in molecular surface.Simultaneously, Asia 1 type FMDV composite multi-epitope gene can be expressed in eucaryon host pichia spp GS115, and expression product can be discerned by Asia 1 type FMDV serum and the anti-Toxins,exo-, cholera serum of rabbit, has antigenic activity.The mouse immune experiment shows, is that subunit vaccine not only can stimulate the mouse body to produce specific humoral immunity with Asia 1 type FMDV composite multi-epitope, can also produce specific cellular immunity simultaneously.
The new A sia 1 type FMDV composite multi-epitope that the present invention designed has antigens with higher property; Can be used for the preparation of several genes engineered vaccines such as Asia 1 type FMDV subunit vaccine, nucleic acid vaccine, synthetic peptide vaccine, live vector vaccine, have bright development prospect.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from know-why of the present invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Sequence table
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< 120>a kind of Asia 1 type FMDV composite multi-epitope
<130>KHP10112700.2
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<170>PatentIn version 3.5
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gga tcc atg att aaa tta aaa ttt ggt gtt ttt ttt aca gtt tta cta 48
Gly Ser Met Ile Lys Leu Lys Phe Gly Val Phe Phe Thr Val Leu Leu
1 5 10 15
tct tca gca tat gca cat gga aca cct caa aat att act gat ttg tgt 96
Ser Ser Ala Tyr Ala His Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys
20 25 30
gca gaa tac cac aac aca caa ata tat aca cta aat gat aag ata ttt 144
Ala Glu Tyr His Asn Thr Gln Ile Tyr Thr Leu Asn Asp Lys Ile Phe
35 40 45
tcg tat aca gaa tct cta gct gga aaa aga gag atg gct atc att act 192
Ser Tyr Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr
50 55 60
ttt aag aat ggt gca att ttt caa gta gaa gta cca ggt agt caa cat 240
Phe Lys Asn Gly Ala Ile Phe Gln Val Glu Val Pro Gly Ser Gln His
65 70 75 80
ata gat tca caa aaa aaa gcg att gaa agg atg aag gat acc ctg agg 288
Ile Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg
85 90 95
att gca tat ctt act gaa gct aaa gtc gaa aag tta tgt gta tgg aat 336
Ile Ala Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn
100 105 110
aat aaa acg cct cat gcg att gcc gca att agt atg gca aat ggt gga 384
Asn Lys Thr Pro His Ala Ile Ala Ala Ile Ser Met Ala Asn Gly Gly
115 120 125
ggc ggt tca ggc gga ggt ggc tct ggc ggt ggc gga tcg gaa ttg atg 432
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Met
130 135 140
gca gct att gag ttc ttt gag ggc atg gtc cac gac tcc atc aaa gcc 480
Ala Ala Ile Glu Phe Phe Glu Gly Met Val His Asp Ser Ile Lys Ala
145 150 155 160
gtt ctt gcc aaa gac gga gct gac act ttc atc gtc ggc act ggc aat 528
Val Leu Ala Lys Asp Gly Ala Asp Thr Phe Ile Val Gly Thr Gly Asn
165 170 175
act ggc agc ata ata aac aac tac tac atg cag cag tat caa aac ggt 576
Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln Asn Gly
180 185 190
cca gga cca gga tct cgt ctt cat act gat gtt gct ttc gtt ttg gac 624
Pro Gly Pro Gly Ser Arg Leu His Thr Asp Val Ala Phe Val Leu Asp
195 200 205
aga ttc gtt aaa ttg acc caa cct aag tct ggt cct ggt cct ggt aca 672
Arg Phe Val Lys Leu Thr Gln Pro Lys Ser Gly Pro Gly Pro Gly Thr
210 215 220
act tac gga aaa gaa tct tca aga cgt ggt gat ctt gct gct ctt gca 720
Thr Tyr Gly Lys Glu Ser Ser Arg Arg Gly Asp Leu Ala Ala Leu Ala
225 230 235 240
aga aga gtt aac aac aga ttg cct gct gct gct ttg ttg gct ctt gac 768
Arg Arg Val Asn Asn Arg Leu Pro Ala Ala Ala Leu Leu Ala Leu Asp
245 250 255
acc aca caa gac aga aga aaa caa aag atc atc gca cct gag aaa caa 816
Thr Thr Gln Asp Arg Arg Lys Gln Lys Ile Ile Ala Pro Glu Lys Gln
260 265 270
act ttg aac ttt gat ggt cct ggt cct ggt aag aaa cct atc acc cgt 864
Thr Leu Asn Phe Asp Gly Pro Gly Pro Gly Lys Lys Pro Ile Thr Arg
275 280 285
ttg gct ttg cct tac acc gct cct cat cgt gct gct gct act tac ggt 912
Leu Ala Leu Pro Tyr Thr Ala Pro His Arg Ala Ala Ala Thr Tyr Gly
290 295 300
aca caa cct act aga cgt ggt gac ctt gct gtt ctt gca caa aga gta 960
Thr Gln Pro Thr Arg Arg Gly Asp Leu Ala Val Leu Ala Gln Arg Val
305 310 315 320
tct aac aga ttg cct acc tct ttc aac tac ggt cct ggt cct ggt aag 1008
Ser Asn Arg Leu Pro Thr Ser Phe Asn Tyr Gly Pro Gly Pro Gly Lys
325 330 335
aca acc tac ggt gaa gag tca aca aga aga ggt gac ttt gca gct ttg 1056
Thr Thr Tyr Gly Glu Glu Ser Thr Arg Arg Gly Asp Phe Ala Ala Leu
340 345 350
gct caa aga ttg tca aga aga ttg ggt cct ggt cct ggt gtt aag gct 1104
Ala Gln Arg Leu Ser Arg Arg Leu Gly Pro Gly Pro Gly Val Lys Ala
355 360 365
gac gcc atc acc gag ttg ttg atc aga ggt cct ggt cct ggt act agt 1152
Asp Thr Ile Thr Glu Leu Leu Ile Arg Gly Pro Gly Pro Gly Thr Ser
370 375 380
<210>2
<211>384
<212>PRT
< 213>artificial sequence
<400>2
Gly Ser Met Ile Lys Leu Lys Phe Gly Val Phe Phe Thr Val Leu Leu
1 5 10 15
Ser Ser Ala Tyr Ala His Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys
20 25 30
Ala Glu Tyr His Asn Thr Gln Ile Tyr Thr Leu Asn Asp Lys Ile Phe
35 40 45
Ser Tyr Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr
50 55 60
Phe Lys Asn Gly Ala Ile Phe Gln Val Glu Val Pro Gly Ser Gln His
65 70 75 80
Ile Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg
85 90 95
Ile Ala Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn
100 105 110
Asn Lys Thr Pro His Ala Ile Ala Ala Ile Ser Met Ala Asn Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Met
130 135 140
Ala Ala Ile Glu Phe Phe Glu Gly Met Val His Asp Ser Ile Lys Ala
145 150 155 160
Val Leu Ala Lys Asp Gly Ala Asp Thr Phe Ile Val Gly Thr Gly Asn
165 170 175
Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln Asn Gly
180 185 190
Pro Gly Pro Gly Ser Arg Leu His Thr Asp Val Ala Phe Val Leu Asp
195 200 205
Arg Phe Val Lys Leu Thr Gln Pro Lys Ser Gly Pro Gly Pro Gly Thr
210 215 220
Thr Tyr Gly Lys Glu Ser Ser Arg Arg Gly Asp Leu Ala Ala Leu Ala
225 230 235 240
Arg Arg Val Asn Asn Arg Leu Pro Ala Ala Ala Leu Leu Ala Leu Asp
245 250 255
Thr Thr Gln Asp Arg Arg Lys Gln Lys Ile Ile Ala Pro Glu Lys Gln
260 265 270
Thr Leu Asn Phe Asp Gly Pro Gly Pro Gly Lys Lys Pro Ile Thr Arg
275 280 285
Leu Ala Leu Pro Tyr Thr Ala Pro His Arg Ala Ala Ala Thr Tyr Gly
290 295 300
Thr Gln Pro Thr Arg Arg Gly Asp Leu Ala Val Leu Ala Gln Arg Val
305 310 315 320
Ser Asn Arg Leu Pro Thr Ser Phe Asn Tyr Gly Pro Gly Pro Gly Lys
325 330 335
Thr Thr Tyr Gly Glu Glu Ser Thr Arg Arg Gly Asp Phe Ala Ala Leu
340 345 350
Ala Gln Arg Leu Ser Arg Arg Leu Gly Pro Gly Pro Gly Val Lys Ala
355 360 365
Asp Thr Ile Thr Glu Leu Leu Ile Arg Gly Pro Gly Pro Gly Thr Ser
370 375 380
<210>3
<211>21
<212>PRT
< 213>artificial sequence
<400>3
Arg Leu His Thr Asp Val Ala Phe Val Leu Asp Arg Phe Val Lys Leu
1 5 10 15
Thr Gln Pro Lys Ser
20
<210>4
<211>25
<212>PRT
< 213>artificial sequence
<400>4
Thr Thr Tyr Gly Lys Glu Ser Ser Arg Arg Gly Asp Leu Ala Ala Leu
1 5 10 15
Ala Arg Arg Val Asn Asn Arg Leu Pro
20 25
<210>5
<211>26
<212>PRT
< 213>artificial sequence
<400>5
Leu Leu Ala Leu Asp Thr Thr Gln Asp Arg Arg Lys Gln Lys Ile Ile
1 5 10 15
Ala Pro Glu Lys Gln Thr Leu Asn Phe Asp
20 25
<210>6
<211>16
<212>PRT
< 213>artificial sequence
<400>6
Lys Lys Pro Ile Thr Arg Leu Ala Leu Pro Tyr Thr Ala Pro His Arg
1 5 10 15
<210>7
<211>29
<212>PRT
< 213>artificial sequence
<400>7
Thr Tyr Gly Thr Gln Pro Thr Arg Arg Gly Asp Leu Ala Val Leu Ala
1 5 10 15
Gln Arg Val Ser Asn Arg Leu Pro Thr Ser Phe Asn Tyr
20 25
<210>8
<211>25
<212>PRT
< 213>artificial sequence
<400>8
Lys Thr Thr Tyr Gly Glu Glu Ser Thr Arg Arg Gly Asp Phe Ala Ala
1 5 10 15
Leu Ala Gln Arg Leu Ser Arg Arg Leu
20 25
<210>9
<211>12
<212>PRT
< 213>artificial sequence
<400>9
Val Lys Ala Asp Thr Ile Thr Glu Leu Leu Ile Arg
1 5 10
Claims (6)
1. Asia 1 type FMDV composite multi-epitope, this composite multi-epitope are the epitope, epitope and choleratoxin B subunit and the helper T cell epi-position of YNBS/58 strain VP1 of VP1 of epitope, IND 63/72 strain of Asia1/JL/05 strain VP1;
The epitope of wherein said Asia 1/JL/05 strain VP1 is:
RLHTDVAFVLDRFVKLTQPKS，
TTYGKESSRRGDLAALARRVNNRLP, and
LLALDTTQDRRKQKIIAPEKQTLNFD；
The epitope of the VP1 of said IND 63/72 strain is:
KKPITRLALPYTAPHR, and
TYGTQPTRRGDLAVLAQRVSNRLPTSFNY；
The epitope of said YNBS/58 strain VP1 is:
KTTYGEESTRRGDFAALAQRLSRRL, and
KADTITELLIR。
2. composite multi-epitope as claimed in claim 1 is characterized in that, connects through flexible amino acid joint between said each epitope sequences.
3. composite multi-epitope albumen, said composite multi-epitope albumen are the albumen that aminoacid sequence shown in the SEQ ID No.2 is formed.
4. coding claim 1 or 2 said composite multi-epitopes or the proteic gene of the said composite multi-epitope of claim 3.
5. the recombinant vectors that contains the said gene of claim 4.
6. composite multi-epitope vaccine, it comprises described composite multi-epitope albumen of claim 3 and acceptable carrier.
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| CN201010180733XA CN101838319B (en) | 2010-05-24 | 2010-05-24 | Asia 1-type FMDV compound multiepitope |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201010180733XA CN101838319B (en) | 2010-05-24 | 2010-05-24 | Asia 1-type FMDV compound multiepitope |
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| CN101838319B true CN101838319B (en) | 2012-11-07 |
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| CN102154224A (en) * | 2010-12-14 | 2011-08-17 | 山东农业大学 | Construction of recombinant chicken marek's disease vaccine virus SC9-1 strain and application thereof |
| CN103224548B (en) * | 2013-03-25 | 2015-12-23 | 中国牧工商(集团)总公司 | For the preparation of the polypeptide and its production and use of ox foot and mouth disease ASIA I type peptide vaccine |
| CN104162152A (en) * | 2013-05-16 | 2014-11-26 | 申联生物医药（上海）有限公司 | Swine foot and mouth disease O-type synthetic peptide vaccine and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1730101A (en) * | 2005-04-06 | 2006-02-08 | 中国农业科学院兰州兽医研究所 | O type foot-and-mouth disease virus poly-gene duplication defect type adenovirus active carrier vaccine and preparation method |
| CN1730650A (en) * | 2005-06-30 | 2006-02-08 | 复旦大学 | Foot-and-mouth disease virus resistant recombinant live bacteria and its preparation method |
| CN101130780A (en) * | 2007-08-01 | 2008-02-27 | 中国农业科学院兰州兽医研究所 | Process for preparing A type hoof-and-mouth disease subunit vaccine |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1730101A (en) * | 2005-04-06 | 2006-02-08 | 中国农业科学院兰州兽医研究所 | O type foot-and-mouth disease virus poly-gene duplication defect type adenovirus active carrier vaccine and preparation method |
| CN1730650A (en) * | 2005-06-30 | 2006-02-08 | 复旦大学 | Foot-and-mouth disease virus resistant recombinant live bacteria and its preparation method |
| CN101130780A (en) * | 2007-08-01 | 2008-02-27 | 中国农业科学院兰州兽医研究所 | Process for preparing A type hoof-and-mouth disease subunit vaccine |
Non-Patent Citations (3)
| Title |
|---|
| 胡博等.Asia 1型复合多表位与CTB基因融合蛋白在毕赤酵母中的表达.《中国畜牧兽医学会家畜传染病学分会第七届全国会员代表大会暨第十三次学术研讨会论文集》.2009, * |
| 霍晓伟.亚洲1型口蹄疫核酸疫苗与重组鸡痘疫苗的构建及实验免疫研究.《中国博士学位论文全文数据库 农业科技辑》.2008, * |
| 鲁会军等.Asia 1 型口蹄疫病毒多表位基因的元和表达及ELISA检测方法的建立.《中国兽医科学》.2009,第39卷(第一期), * |
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