CN102274496B - O/Asia I type foot and mouth disease virus bivalent genetic engineering polypeptide vaccine, its preparation method and its purpose - Google Patents
O/Asia I type foot and mouth disease virus bivalent genetic engineering polypeptide vaccine, its preparation method and its purpose Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/582—Recycling of unreacted starting or intermediate materials
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to an O/Asia I type foot and mouth disease virus bivalent genetic engineering polypeptide vaccine, its preparation method and its purpose. The method comprises the following steps: selecting two serotypes of O type and Asia I type, taking B cell determinant 15 amino acid fragments of VP1 and T-cell helper of VP4, performing a series connection, cloning without containing carrier protein, constructing O/Asia I gene engineering bacteria. An antigen protein product can be obtained after passing through the processes of high density fermenting, cell disrupting, inclusion body renaturating, fusion protein separating, and is homogenized with an adjuvant to form the O/Asia I type foot and mouth disease virus bivalent genetic engineering polypeptide vaccine. The vaccine of the present invention contains 2<n-1> polypeptide connected in series which is coded by a nucleic acid sequence shown in SEQ ID, wherein, n is an integer of 1-5. The invention has the advantages of good security and high immune efficacy, and can be used once in half year for immunization; and is suitable for large scale production and convenient preservation and transportation; and is capable of effectively preventing and controlling two serotypes foot and mouth disease of O type and Asia I type which is useful in our country; foot and mouth disease virus non-structural protein 3A.B. will not generate, so that the infective animals can be differentiated easily.
Description
Technical field
The present invention relates to a kind of novel foot-and-mouth disease vaccine, particularly a kind of bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two produced by gene engineering method and its production and use.
Background technology
Domestic cultivation scale constantly expands, and will become animal vaccine industry sustainable growth power source.Meat, the eggs output of current China all arrange No. 1 in the world, are that world's livestock products produce the first big country.Within 2004, the annual animal husbandry output value of China breaks through 1 trillion yuan high point first, and the proportion that the animal husbandry output value accounts for the general production value of agriculture is progressively improving.Because epidemic situation in world wide is continuous, foot and mouth disease, bird flu, bovine spongiform encephalopathy etc., propose challenge to the animal epidemic prevention and controlling work of countries in the world.Foot and mouth disease (Foot and mouth disease, FMD) is that the one of artiodactyls in animal husbandry (pig, cattle, sheep and camel) is acute, hot, high degree in contact sexually transmitted disease.Up to now, the popular on a large scale of foot and mouth disease, except North America, Australia, was all once broken out in countries in the world.The popular of this disease once caused huge economic loss to Germany (1937-1938), Europe (1951-1952), Turkey (1964-1965), Britain (1967-1968), Austria (1973), (1974), Korea S (2002) etc. country of France.
The clinical diagnosis feature of foot and mouth disease is the skin generation vesicle of oral mucosa, hoof and breast and festers, and is caused by foot and mouth disease virus, for animal husbandry, pig, cattle and the raising of sheep and the trade of livestock products very harmful.Foot and mouth disease virus (Foot and mouth disease virus, FMDV) belong to Pironavirus, many to pig, cattle and the sheep route of infection, virulence is strong, the shedding virus of a sick cattle can infect 1,000,000 cattle, and 1 gram of sick Ungula Sus domestica portion vesicle micromicro makes 100,000 pig infection morbidities.Be easy to propagate, propagate rapidly, popular wide, fertility performance declines, and expenses for prevention and control is high, sickness rate 100%, and grow up the general 1-2% of poultry mortality rate, but young stock is up to 50%, and even 100%.Often several province, a few continent even whole world occurs simultaneously.Direct contact and indirect contact transmission, main through transmission, also can through respiratory infectious, often cattle, sheep, pig interior morbidity at one time, there is the ability of infection many animals and there is plurality of antigens form, the popular propagation in saltatory, all can occur throughout the year, just once popular every 1-2 or 3-5.In recent years, pig has the trend of expansion.Elementary the copying of FMDV is pharyngeal, then infects contiguous lymph node and enters blood flow, and then being diffused in various Organ and tissue.In most cases, within zoogenetic infection 2-14 days, there is clinical symptoms.Seldom occur death after more old zoogenetic infection FMDV, but the reproductive performance of animal, growth and health are greatly affected.FMDV is classified as category-A deadly infectious disease by International Office of Epizootics.FMDV, once occur, is difficult to control, and can only butcher and destroy the domestic animal that catches an illness by fire with trouble without offspring with collective after each outburst.Because foot and mouth disease propagation is rapid, be difficult to control, remedial measure is few, being called as " number one killer " of animal husbandry, is crushing blow for animal husbandry.According to OIE's regulation, once there is foot and mouth disease epidemic situation, country concerned will lose non-foot and mouth disease epidemic-stricken area qualification automatically.
Foot and mouth disease virus has significant antigen diversity, and the FMDV found at present has O, A, C, SAT1, SAT2, SAT3 (i.e. South Africa 1,2,3 type) and Asia1 (Type Asia 1) 7 serotypes.Often kind of serotype can also continue to be divided into multiple hypotype.China was O type foot and mouth disease Endemic Area originally.The surrounding countries of northwest and the Northeast are O type and Asia I type foot and mouth disease Endemic Area.Single stranded RNA that contain a positive strand polarity in FMDV granule, that be made up of 8500 nucleotide.The cyst membrane of virion comprises the four kinds of Structural protein VP1 (molecular weight 34000), VP2 (molecular weight 30000), VP3 (molecular weight 26000), the VP4 (molecular weight 13500) that hold single stranded RNA.Four kinds of structural protein often plant 60 molecular compositions icosahedral virion.Icosahedral summit is 141-160 region and 200-213 region by the immunologic determinants region of Structural protein VP1.
For FMD, vaccinate is the most effective means of preventing foot and mouth disease at present.Existing vaccine and Problems existing thereof have:
1, attenuated vaccine and inactivated vaccine:
Traditional attenuated vaccine and inactivated vaccine utilize mass propgation zooblast, then infects foot and mouth disease virus, through cultivation, isolated viral, then is prepared from after making inactivation of virus with chemical reagent.Merieux company of France is maximum deactivation FMDV production of vaccine producer.But the environment that preparation deactivation FMDV needs special isolation strictly to close is produced, with leakage-preventing and pollute surrounding, security requirement is high, and this potential danger limits this vaccine must at remote production of environment; In addition, the storage transport preservation of vaccine needs cold chain, increases cost; The variation of tiring of deactivation FMDV vaccine is comparatively large, and vaccine also exists again the danger that strong and remaining vestige live virus causes disease popularity and large-scale outbreak; In addition, the quality of vaccine also often can affect the milk expression of milch cow; Although use the FMDV vaccine of deactivation seldom anaphylactic shock or stupor to occur, be just enough to cause serious consequence once generation; Finally, first immunisation takes 2 with secondary in thoughtful 2 months, once every injection in 6 months later.Immunoprophylaxis workload is very large.
2, polypeptide vaccine:
Nineteen eighty-two develops the polypeptide Seedling of synthetic.Improvement on synthesis vaccine can overcome the shortcoming of conventional vaccine, is considered to the ultimate vaccine of zoonosis prevention very early.Escherichia coli are used only to need 20 hours as host cell fermented incubation time, period ratio zooblast produces inactivated vaccine much shorter, recombinant vaccine does not pollute environment, there is no the danger revealed, very safe, and the vaccine produced does not need cold preservation, titer plateaus in storage, transport and use procedure.Over more than 20 year, the research of polypeptide vaccine focuses mostly at the single immunologic determinants of the B cell of VP1 coat protein, further research shows that the immunologic determinants of Structural protein VP1 is 141-160 region and 200-213 region, in the antibody capable that polypeptide fragment and the carrier protein in these two regions produce after chemistry connects and virion (Nature, 1982,298:30-33; J.Virology, 2000,74:4902-4907; J.Virology, 2001,75:3164-3174; J.Virology, 2003,77:8633-8640; Vaccine, 2002,20:2603-2610; Biological engineering journal, 2009,25 (4) 514-519).But produce less because of neutralizing antibody, after improvement on synthesis vaccine immunity animal the immanoprotection action that rises do not have so ideal that people imagined originally, value is even denied.In the immunifacient process of induction body, single Neutralization and crystallization is far from being enough.And current FMDV vaccine, no matter be deactivation FMDV vaccine or polypeptide vaccine, does not differentiate immunized animal and infection animal by Serologic test, and immunized animal and infection animal are obscured to import and export to market and are caused extreme influence.Therefore, prepare immunizing potency to improve and the genetic engineering multivalent foot-and-mouth disease vaccine that can be used for differentiating immunized animal and infection animal is the target of our continuous pursuit.
Research in recent years proves except the B cell immunologic determinants of VP1, needs the T cell immunity bunch synergism of VP4, can obtain good immune neutralizing antibody protected effect.
The Asia1 recombinant vaccine of the unit developments such as Fudan University has protected effect containing T-helper and VP1 immunologic determinants vaccine, can produce neutralizing antibody.
The polypeptide vaccine of UBI company of U.S. synthesis has the B cell immunologic determinants polypeptide of T-cell Helper polypeptide and VP1, greatly strengthen the effect (about 10 times) of neutralizing antibody, has clear and definite immune protective effect.There is good safety: without heating paresthesia, without anaphylaxis generation, injection site without malabsorption phenomenon, on the health status of in-pig and gestation not impact, also do not affect farrowing achievement.
Current needs are a large amount of, cheap produces many peptide vaccines.But chemosynthesis is very expensive more than 20 amino acid whose peptide chains.Therefore, development biotechnology is needed to produce polypeptide in a large number by genetic engineering.But practical application is also apart from far.According to the nearly 20 years research reports to polypeptide, except insulin, genetic engineering had advantage unlike chemosynthesis.Basic reason is engineered theoretical developments and practical application still awaits development, moreover polypeptide is degraded rapidly in vivo and is unfavorable for that the induction of corresponding antibodies produces.Technique for gene engineering should be utilized to prepare FMDV polypeptide vaccine.The same with other gene engineering polypeptide products, production need solve a series of upstream design and downstream process problem.Although there is a lot of report, away from application still.
Summary of the invention
The object of the invention is to the defect overcoming prior art, provide a kind of New O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccine.The bivalent gene engineered polypeptide vaccine of a kind of O/Asia I type foot and mouth disease virus two of the present invention, its genetic engineering bacterium is O/Asia I, foot and mouth disease virus FMDV coat protein has four kinds of i.e. VP1, VP2, VP3, VP4, O/Asia I genetic engineering bacterium has the DNA sequence in the immunologic determinants region of immunogenicity to connect in Asia I type VP1 albumen in the DNA sequence connection O type VP1 albumen of 15 amino acid fragments of the T-cell helper of hoof-and-mouth disease poison strain coding VP4 to have the DNA sequence in the immunologic determinants region of immunogenicity as 1 repeated fragment, (the nucleotide arrangement sequence of 1 repeated fragment is Fig. 1), the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two contains 2
n-1the albumen coded by nucleotide sequence shown in SEQ ID of individual series connection, wherein n is the integer of 1-5.
Another object of the present invention is to provide the construction method of described foot-and-mouth disease vaccine.
The bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two of the present invention, the gene containing series connection in its genetic engineering bacterium is expressed with the Lac plasmid carried in escherichia coli (E.Coli).Described vaccine also comprises pharmaceutically acceptable carrier, adjuvant and/or immunological adjuvant.
On the other hand, present invention also offers O/Asia I engineering strain can expressing the bivalent gene engineered polypeptide vaccine of described O/Asia I foot and mouth disease virus two.
This O/Asia I engineering strain, its plasmid carried is respectively containing 2
n-1the nucleotide sequence shown in SEQ ID of individual series connection, wherein n is the integer of 1-5.
The invention provides described O/Asia I foot-and-mouth disease vaccine in the purposes of preventing and treating in foot and mouth disease disease and the purposes in differentiation infection animal and immunized animal.
Present invention also offers the bivalent gene engineered polypeptide vaccine preparation method of a kind of cloven-hoofed domestic animal O/Asia I type foot and mouth disease virus two, comprise the preparation of gene recombinaton, recombination fusion protein, it is characterized in that:
(1) be synthesis O/Asia I genetic engineering bacterium, connect in O type VP1 albumen to have in the DNA sequence in immunogenic immunologic determinants region connection Asia I type VP1 albumen with the gene of 15 amino acid fragments of the T-cell helper of the method composite coding VP4 of chemosynthesis and have the DNA sequence in immunogenic immunologic determinants region as 1 O/Asia I repeated fragment, connect 8 times;
(2) be synthesis O/Asia I genetic engineering bacterium, 8 of O/Asia I repeated fragments are inserted Lac plasmid vector;
(3) above-mentioned recombiant plasmid is proceeded to escherichia coli to express, obtain O/Asia I bacterial strain;
(4) O/Asia I bacterial strain is placed in nutritious LB culture medium to ferment, fermentation temperature 37 DEG C, fermentation time is 12-24 hour, adds ampicillin and make its final concentration be 50 μ g/ml in fermentation liquid, and IPTG, concentration is 0.5mM, centrifugal acquisition thalline after fermentation ends, through cell pulverization, renaturing inclusion bodies, the separation and purification of fusion rotein, forms the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two with adjuvant homogenate.
The genetic engineering research of the present invention to polypeptide comprises the downstream process of design and the intensive new and high technology establishing upstream high expression, establishes the Simplified flowsheet of high yield, low cost.The bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two is one of serial polypeptide drugs, can cheap a large amount of production.Be that engineering bacterium fermentation incubation time only needs 12 hours with escherichia coli, recombinant vaccine does not pollute environment, does not have the danger revealed.The vaccine produced storage, transport and use procedure in without the need to cold chain.The hoof-and-mouth disease serotypes that we report according to the epidemic situation of some area generations of China and contiguous country of bordering on, selects O type, Asia I type two kinds of serotypes, effectively can prevent and treat O type and Asia I type two kinds of serotype foot and mouth disease, be suitable for for China.Our genetic engineering foot and mouth disease virus vaccine also distinguishable immunized animal and infection animal except immunizing potency height.
Use vaccine of the present invention to have the following advantages: 1) safety is good, the present invention is gene engineering product, is not inactivated vaccine, there is not the danger causing illness outbreak because trace live virus reveals.In laboratory animal, carry out subcutaneous injection with the vaccine on mouse of higher dosage, within the longer observation phase, the survival of laboratory animal health.
2) vaccine of the present invention is applicable to engineered method large-scale production, reduces cost.
3) vaccine of the present invention is utilized can to distinguish infection animal and immune animal.At occurring in nature, infect the animal of A type foot and mouth disease, A type foot-and-mouth disease antibody in its body, can be produced; Infect the animal of O type foot and mouth disease, O type foot-and-mouth disease antibody in its body, can be produced; Infect the animal of Asia I type foot and mouth disease, Asia I type foot-and-mouth disease antibody in its body, can be produced.Vaccine of the present invention has O type and Asia I type two kinds of FMDV VP1 immunologic determinants simultaneously, after inoculation, produce the antibody of resisting O-type and anti-Asia I type two kinds of foot and mouth disease in animal body, infection animal and immune animal can be distinguished by this, avoid importing and exporting to animal causing confusion.
4) vaccine of the present invention contains O type and Asia I type two kinds of FMDV VP1 immunologic determinants, T-Helper polypeptide again containing VP4, greatly strengthen the effect of neutralizing antibody, 8 repeated fragments are expressed, the molecular weight of polypeptide is large, Increased Plasma Half-life in vivo, its time producing neutralizing antibody also extends greatly.
5) vaccine of the present invention is applicable to China O type and Asia I type two kinds of foot and mouth disease virus Endemic Areas.
Accompanying drawing explanation
Fig. 1 is the structure SEQ ID in the expression vector of O/Asia I genetic engineering bacterium;
Fig. 2 is the building process schematic diagram of plasmid F8, and how genetic fragment connects the process of 8 times by diagram;
Fig. 3 be containing Lac carrier O/Asia I genetic engineering bacterium (E.Coli) growth collection of illustrative plates during the fermentation (12hr, fermentation tank: Switzerland than Europe, 100L).
Detailed description of the invention
Embodiment one:
The present invention according to the aminoacid sequence of the T-cell helper of the VP1 immunologic determinants of O and Asia I liang of type FMDV and VP4, design and synthesis DNA genetic fragment.Its genetic engineering bacterium is O/Asia I.O/Asia I genetic engineering bacterium has the DNA sequence in the immunologic determinants region of immunogenicity to connect in Asia I type VP1 albumen in the DNA sequence connection O type VP1 albumen of 15 amino acid fragments of the T-cell helper of hoof-and-mouth disease poison strain coding VP4 to have the DNA sequence in the immunologic determinants region of immunogenicity as 1 repeated fragment, and the nucleotide arrangement sequence of 1 repeated fragment is see Fig. 1.
The bivalent gene engineered polypeptide vaccine of New O/Asia I foot and mouth disease virus two of the present invention, it contains 2
n-1the polypeptide coded by nucleotide sequence shown in SEQ ID of individual series connection, wherein n is the integer of 1-5.The DNA sequence that SEQ ID contains 15 amino acid fragments of the T-cell helper of hoof-and-mouth disease poison strain coding VP4 connects in A type VP1 albumen has the DNA sequence in the immunologic determinants region of immunogenicity to connect the DNA encoding sequence having the immunologic determinants region of immunogenicity in Asia I type VP1 albumen, the antigenic determinant of the O type that its forward of can encoding is expressed and Asia I foot and mouth disease virus, its expression product can stimulate the antibody producing resisting O-type and Asia I foot and mouth disease virus.
Preferably, vaccine of the present invention contains 2
n-1the polypeptide coded by SEQ ID of individual series connection, wherein, n is the integer of 2-4.
Most preferred, vaccine of the present invention contains 2
n-1the polypeptide coded by SEQ ID of individual series connection, wherein, n is 4.
For improving bioavailability strengthen immune effect, vaccine of the present invention can also containing pharmaceutically acceptable carrier, adjuvant and or the material such as immunological adjuvant.
Immunological adjuvant is that one can strengthen immunoreactive material, and it can be used in combination with antigen, contributes to the deposition of injection mass or collect, and can also strengthen antibody response.
The material that can be used as immunological adjuvant has: 1) microorganism and product thereof, as mycobacteria, coryne bacterium parvum, bordetella pertussis and the extract lipopolysaccharide locating left Lan Shi negative bacillus, from the extract muramyldipeptide etc. of mycobacteria.2) polynucleotide, as poly, polyadenylic acid, poly glutaminol etc.3) freund adjuvant (Freund ' sadjuvant), comprise incomplete freund adjuvant (by antigen aqueous solution and oil preparation (paraffin oil or vegetable oil) mixed in equal amounts, then adding the Water-In-Oil antigen Emulsion that emulsifying agent (lanoline or Tween 80) makes) and Split completely (adding mycobacteria as bacillus calmette-guerin vaccine in Freund's incomplete adjuvant).4) inorganic matter, as Alumen and aluminium hydroxide etc.
In recent years, find that following material also can be used as immunological adjuvant, comprising: 1) bacteriotoxin, as cholera toxin (CT) and E.coli LT (LT).2) attenuated derivative of CT and LT or variant.3) the endogenous immunoregulatory factor of people, as IL-2, IL-12, GM-GSF.4) hormone.5) lipopeptid.6) saponin, saponin deriviatives QS-21.7) oligonucleotide fragment (CpG ODN) of the synthesis containing CpG motif.8) derivant of lipoid A, as lipopolysaccharide derivant Monophosphoryl lipid A (MPL).9) derivant of muramyldipeptide (MDP).
In addition, some delivery system with inherent immunostimulatory activity also can be used as immunological adjuvant for vaccine construction, and these delivery systems include but not limited to: liposome, Emulsion, spiral zooid (cochleate), virion, micropartical and immunostimulating complex (ISCOMs).
Above-mentioned various types of immunological adjuvant all can be used for the present invention.The polypeptide that adjuvant can have an immunogenicity with suitable dosage and the present invention with the use of, form vaccine of the present invention.
Preferably, vaccine of the present invention contains incomplete freund adjuvant or aluminium hydroxide, preferred, and vaccine of the present invention contains incomplete freund adjuvant as immunostimulant.In incomplete freund adjuvant, the ratio of lanoline and paraffin oil is slightly different with the change in season, e.g., the spring, the summer, autumn lanoline and the ratio of paraffin oil be about 3: 7, the ratio in winter both is about 1.5: 8.5.
The preparation method of the bivalent gene engineered polypeptide vaccine of O/Asia I foot and mouth disease virus two of the present invention, comprises 2
n-1the nucleotide sequence shown in SEQ ID of individual series connection, can directly synthesize, and also can first synthesize several DNA fragmentations, produces the DNA sequence as shown in SEQ ID after connecting.In order to the convenience of subsequent operation, when design dna fragment, can by suitable restriction enzyme site calling sequence two ends, target sequence is cloned into suitable carrier.
In a preferred embodiment of the invention, for the carrier of synthesis O/Asia I genetic engineering bacterium, the DNA sequence of 15 amino acid fragments of the T-cell helper of design and synthesis hoof-and-mouth disease poison strain coding VP4 of the present invention connects in O type VP1 albumen to have in the DNA sequence in the immunologic determinants region of immunogenicity connection Asia I type VP1 albumen has the DNA sequence in the immunologic determinants region of immunogenicity as a repeated fragment, connected by 10 DNA fragmentations and produce, each repeated fragment F1 contains the nucleotide sequence shown in SEQ ID and suitable restricted enzyme point of contact.Utilize engineered method that F1 is cloned into carrier, then, by having the restricted enzyme of the sticky end of coupling mutually after enzyme action, F2 (the F1 sequences containing 2 series connection) is cloned into carrier, and the polypeptide after expression contains 2 repeated fragments (VP4+O type VP1+Asia I type VP1).According to similar method, successively F4, F8, F16 can be cloned into carrier, the F1 sequence that they are connected containing 4,8,16 successively, after expressing in suitable expression system, the repeated fragment (VP4+O type VP1+Asia I type VP1) of the polypeptide obtained respectively containing 4,8,16 series connection.
In the preparation process in accordance with the present invention, the expression system for expression of polypeptides can be prokaryotic expression system, can also be eukaryotic expression system.Expression system comprises suitable host cell, and can copy in host cell and the plasmid of stable existence or carrier.
Can include but not limited to as the example of host cell: bacterial cell, as escherichia coli, streptococcus, Salmonella typhimurium etc.; Eukaryotic cell, as yeast etc.
Operable carrier can comprise chromosomal origin, non-chromosome source and synthesis DNA sequence.As: phage DNA, baculovirus, bacterial plasmid, yeast plasmid and the carrier derived by the combination of plasmid, phage and viral DNA.
Preferably, in prokaryotic expression system, polypeptide of the present invention is expressed.Preferred, polypeptide of the present invention can be cloned into high efficiency expression vector (expression vector as commercially available) and carry out amalgamation and expression with carrier protein.
The invention still further relates to a kind of O/Asia I engineering strain, its plasmid carried is respectively containing 2
n-1the nucleotide sequence shown in SEQ ID of individual series connection, wherein n is the integer of 1-5.
Preferably, the plasmid that described O/Asia I genetic engineering bacterium carries contains 2
n-1the nucleotide sequence shown in SEQ ID of individual series connection, wherein n is the integer of 2-4.
Preferred, the plasmid that described O/Asia I genetic engineering bacterium carries contains 2
n-1the nucleotide sequence shown in SEQ ID of individual series connection, wherein n is 4.
The purposes of the bivalent gene engineered polypeptide vaccine of O/Asia I foot and mouth disease virus two of the present invention in prevention and therapy foot and mouth disease.By vaccine injection of the present invention to animal, the antibody of specific resisting O-type and anti-Asia I type two kinds of foot and mouth disease in stimulating animal body, can be produced, therefore vaccine of the present invention may be used for distinguishing immunity inoculation animal and infection animal.
Table 1 is the measurement result of antigen antibody reaction, antibody provides by Shanghai City academy of agricultural sciences animal and veterinary institute for Ox blood serum after O type and A type foot and mouth disease standard serum and the treatment of O type foot and mouth disease, and antigen is the bivalent gene engineered polypeptide vaccine of O/Asia of the present invention I type foot and mouth disease virus two prepared voluntarily.
Table 1: antigen antibody reaction
Embodiment two:
1: construction expression plasmid
Genetic engineering bacterium is O/Asia I genetic engineering bacterium.
For the carrier of synthesis O/Asia I genetic engineering bacterium, the DNA sequence of 15 amino acid fragments of the T-cell helper of hoof-and-mouth disease poison strain coding VP4 is connected in O type VP1 albumen have in the DNA sequence in the immunologic determinants region of immunogenicity connection Asia I type VP1 albumen and have the DNA sequence in the immunologic determinants region of immunogenicity as 1 repeated fragment F1, connected by 10 DNA fragmentations and produce, each repeated fragment F1 contains the nucleotide sequence shown in Fig. 1 and suitable restricted enzyme point of contact.Engineered method is utilized F1 to be cloned into Lac promoter expression plasmid, then according to the method for Fig. 2, F2, F4, F8 can be cloned into Lac promoter expression plasmid successively, the F1 sequence (VP4+O type VP1+Asia I type VP1) that they are connected containing 2,4,8 successively.
The F8 gene of 2:O/Asia I genetic engineering bacterium is expressing the clone in charge material plastochondria Puc18
Gene F8, after restricted enzyme BamH I/Sal I double digestion, is cloned in Puc18 plasmid, obtains expression plasmid O/Asia I by plasmid Puc18 according to a conventional method.Expression plasmid is proceeded to escherichia coli, 37 DEG C of cultivations, is that the IPTG of 0.2-0.5mmol/L induces with final concentration.
3: fermentation
(1000ml seed culture fluid contains peptone 10g to 250ml seed culture fluid, phosphate buffer 20ml, the pH7.0 of yeast extract 5g, 0.02mol/L) be placed in 1000ml conical flask, 120 DEG C of sterilizings 20 minutes, add the glucose solution 5ml of 20% after cooling.The bacterial strain of 1ml cryopreservation in glycerol is added above-mentioned solution, adds ampicillin (Ampicillin) and make its final concentration be 50 μ g/ml, 37 DEG C, 12-14 hour (150rpm) kind daughter bacteria as amplification culture cultivated by shaking table.
1000ml seed culture fluid contains peptone 20g, the phosphate buffer 20ml of yeast extract 10g, 0.2mol/L, pH7.0, and 120 DEG C of sterilizings 20 minutes, add ampicillin (Ampicillin) and make its final concentration be 50 μ g/ml after being cooled to 37 DEG C.Add seed culture fluid 20ml, then add the glucose solution 5ml of 20%, and trace elements of Ca Cl2, each 1mg of NiNO3, CoCl3, MgSO4, FeCl3, maintain listed condition and carry out (the fermentation tank: 100L that ferments; Temperature: 37 DEG C; Mixing speed: 700rpm; Ventilation: 80L/min; PH7.0-7.5).Separated in time is respectively got 1ml fermentation liquid and is placed in 2 plastic centrifuge tubes, the centrifugal 10min of 8000rpm, and removing supernatant, taking thalline weight is weight in wet base (g/L).As shown in Figure 3, in fermentation after 8-12 hour, bacterial concentration reaches peak value.
After fermentation, the centrifugal 30min of 4000rpm collects thalline.
Thalline is suspended in the solution containing the potassium phosphate pH7.0 of the sodium chloride of 1%, EDTA, 20mmol/L of 1mmol/L with the ratio of 1000g/3L, in suspension, add 1g lysozyme, stirring at room temperature 1 hour, with smudge cells, thallus suspension liquid, in the centrifugal 30min of 10000rpm, abandons supernatant.
Above-mentioned precipitation is added the urea liquid of 8mol/L with the ratio of 250g/L.Stirring, extracting are spent the night, and the centrifugal 30min of 20000rpm, gets supernatant, have precipitation to produce, with the centrifugal 30min of 10000rpm, get precipitation after renaturation, make homogenate form the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two with sterilized water, for subsequent use.
The detection of the bivalent gene engineered polypeptide vaccine of 4:O/Asia I type foot and mouth disease virus two
1), reagent
O type and A type foot and mouth disease standard serum antibody gratuitously provide by Shanghai City academy of agricultural sciences animal and veterinary institute.
2) sample
Containing Puc18 O/Asia I genetic engineering bacterium through fermentation after, collected by centrifugation thalline, smudge cells, uses 8M urea extraction, collected after centrifugation precipitate, it is for subsequent use that 1mg adds 1ml sterilized water, and supernatant gets 1ml.Add O type foot and mouth disease standard serum at precipitation solution, shake up, produce precipitation, solutions turbid, add O type foot and mouth disease standard serum, shake up, do not produce precipitation, solution is limpid; Add O type and A type foot and mouth disease standard serum at supernatant solution, shake up, without precipitation, solution is limpid.
Result: O/Asia I fusion rotein and O type foot and mouth disease standard serum produce antigen antibody reaction, and not containing fusion rotein in supernatant, therefore O type foot and mouth disease standard serum is not reacted.
The antigen antibody reaction of the bivalent gene engineered polypeptide vaccine of 5:O/Asia I type foot and mouth disease virus two
Utilize protein in the solution, antigen-antibody can react and produce the mensuration that precipitation carries out vaccine immunity source of the present invention property.
1) antigen: test article is the albumen obtained after O/Asia I genetic engineering bacterium expression and purification of the present invention.
2) antibody: O type and A type foot and mouth disease standard serum antibody gratuitously provide by Shanghai City academy of agricultural sciences animal and veterinary institute.
3) O type foot-and-mouth disease antibody and A type foot-and-mouth disease antibody is added respectively in the bivalent gene engineered polypeptide vaccine of antigen antibody reaction: O/Asia I type foot and mouth disease virus two.According to the result of table 1, show that the O/Asia I type bivalent gene engineered polypeptide vaccine of foot and mouth disease virus two and O type antibody have immunoprecipitation, with A type antibody without immunological cross-reaction.
The safety experiment (mouse experiment) of the bivalent gene engineered polypeptide vaccine of 6:O/Asia I type foot and mouth disease virus two
Male mouse of kunming 20, every about body weight 20g, purchased from Chinese Academy of Sciences's Shanghai animal center.Be divided into two groups, one group is the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two, and another group is matched group.
The bivalent gene engineered polypeptide vaccine antigen protein 1ml sterilized water of O/Asia I type foot and mouth disease virus two getting 1mg purification suspends, and matched group injection 1ml sterilized water, carries out lumbar injection to mice and carry out observation 24 hours.
Survival rate
O/AsiaⅠ 10/10
Matched group 10/10
Sequence table
Organization Applicant
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<110>OrganizationName: Wu Xiaoyan; Zhao Hong; Sun Yukun
Street: floor Building B, HuaYuan Building No. one building 21, No. 3500, Triumph Road
City: Shanghai
Country: China
PostalCode：200030
Application Project
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<120>Title: a kind of O/Asia I type bivalent gene engineered polypeptide vaccine of foot and mouth disease virus two and preparation method and purposes
<130>AppFileReference：PCNWX1001462
<140>CurrentAppNumber：
<141>CurrentFilingDate：_____-___-___
Sequence
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<213>OrganismName:O/Asia I foot and mouth disease virus
<400>PreSequenceString：
gaattccaga tctatcatca acaactacta catgcagcag taccaggata gccttaaggt 60
ctagatagta gttgttgatg atgtacgtcg tcatggtcct atcgatggat ggtgatacca 120
gcaccgatga tgttcgcggt gatctgcagt acctaccact atggtcgtgg ctactacaag 180
cgccactaga cgtcgttctg gcgcagaaag cggaacgcac cggtgaagaa agcacccgcc 240
aagaccgcgt ctttcgcctt gcgtggccac ttctttcgtg ggcgcgcggt gatttcagcg 300
cgctggcgca gcgcctgagc cgccgcctgg cgccactaaa gtcgcgcgac cgcgtcgcgg 360
actcggcggc ggacccggga tcctagaacg ttaagcctag gatcttgcaa 410
<212>Type：DNA
<211>Length：410
SequenceName:O/Asia I FMDV polypeptide vaccine
SequenceDescription：
Claims (16)
1. the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two, wherein: the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two contains 2
n-1the albumen coded by nucleotide sequence shown in SEQ ID of individual series connection, wherein n is the integer of 1-5, the nucleotide sequence shown in SEQ ID:
Gaattccagatctatcatcaacaactactacatgcagcagtaccaggatagcatggatggtgataccagcaccgatgatgttcgcggtgatctgcaggttctggcgcagaaagcggaacgcaccggtgaagaaagcacccgccgcggtgatttcagcgcgctggcgcagcgcctgagccgccgcctgccgggatcctagaacgtt。
2. the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two as claimed in claim 1, wherein: O/Asia I gene engineering polypeptide vaccine contains 2
n-1the albumen coded by nucleotide sequence shown in SEQ ID of individual series connection, wherein n is the integer of 2-4.
3. the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two as claimed in claim 2, wherein: O/Asia I gene engineering polypeptide vaccine contains 2
n-1the albumen coded by nucleotide sequence shown in SEQ ID of individual series connection, wherein n is 4.
4. the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two as claimed in claim 3, wherein: the gene containing series connection in O/Asia I genetic engineering bacterium is expressed with the Lac plasmid carried in escherichia coli.
5. the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two as described in any one of claim 1-4, wherein: described vaccine also comprises pharmaceutically acceptable carrier, adjuvant and/or immunological adjuvant.
6. the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two as claimed in claim 5, wherein said immunological adjuvant is freund 's incomplete adjuvant.
7. the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two as claimed in claim 5, wherein said immunological adjuvant is Al (OH)
3.
8. the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two as claimed in claim 5, wherein said immunological adjuvant is γ-polyglutamic acid ploy-γ-glutamic acid.
9. prepare a method for the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two according to claim 1, comprise 2 of O/Asia I genetic engineering bacterium
n-1the nucleotide sequence shown in SEQ ID of individual series connection is cloned into carrier and in expression system, carries out the step expressed, and wherein n is the integer of 1-5.
10. the preparation method of the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two as claimed in claim 9, comprises 2 of O/Asia I genetic engineering bacterium
n-1the nucleotide sequence shown in SEQ ID of individual series connection is cloned into carrier and in expression system, carries out the step expressed, and wherein n is the integer of 2-4.
11. methods preparing the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two as claimed in claim 10, comprise 2 of O/Asia I genetic engineering bacterium
n-1the nucleotide sequence shown in SEQ ID of individual series connection is cloned into carrier and in expression system, carries out the step expressed, and wherein n is 4.
12. methods preparing the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two as described in any one of claim 9-11, wherein said nucleotide sequence is expressed in prokaryotic expression system.
13. methods preparing the bivalent gene engineered polypeptide vaccine of O/Asia I type foot and mouth disease virus two as described in any one of claim 9-11, wherein said nucleotide sequence is expressed in eukaryotic expression system.
14. 1 kinds of O/Asia I engineering strains, its plasmid carried is respectively containing 2
n-1the nucleotide sequence shown in SEQ ID of individual series connection, wherein n is the integer of 1-5, the nucleotide sequence shown in SEQ ID:
Gaattccagatctatcatcaacaactactacatgcagcagtaccaggatagcatggatggtgataccagcaccgatgatgttcgcggtgatctgcaggttctggcgcagaaagcggaacgcaccggtgaagaaagcacccgccgcggtgatttcagcgcgctggcgcagcgcctgagccgccgcctgccgggatcctagaacgtt。
15. O/Asia I engineering strains as claimed in claim 14, its plasmid carried contains 2
n-1the nucleotide sequence shown in SEQ ID of individual series connection, wherein n is the integer of 2-4, the nucleotide sequence shown in SEQ ID:
Gaattccagatctatcatcaacaactactacatgcagcagtaccaggatagcatggatggtgataccagcaccgatgatgttcgcggtgatctgcaggttctggcgcagaaagcggaacgcaccggtgaagaaagcacccgccgcggtgatttcagcgcgctggcgcagcgcctgagccgccgcctgccgggatcctagaacgtt。
16. O/Asia I engineering strains as claimed in claim 15, its plasmid carried contains 2
n-1nucleotide sequence shown in the SEQID of individual series connection, wherein n is the nucleotide sequence shown in 4, SEQ ID:
Gaattccagatctatcatcaacaactactacatgcagcagtaccaggatagcatggatggtgataccagcaccgatgatgttcgcggtgatctgcaggttctggcgcagaaagcggaacgcaccggtgaagaaagcacccgccgcggtgatttcagcgcgctggcgcagcgcctgagccgccgcctgccgggatcctagaacgtt。
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| CN102614507B (en) * | 2012-02-17 | 2013-12-11 | 中国农业科学院兰州兽医研究所 | Type O foot-and-mouth disease virus molecular marker vaccine and preparation method thereof |
| KR101629345B1 (en) * | 2013-06-26 | 2016-06-15 | 대한민국 | Foot and mouth disease virus expressing P1-protective antigen of Asia1 type, IV genotype and the manufacturing method |
| CN105821011B (en) * | 2015-01-07 | 2020-10-30 | 普莱柯生物工程股份有限公司 | Vaccine composition for resisting O-type foot-and-mouth disease and preparation and application thereof |
| CN105367659A (en) * | 2015-11-18 | 2016-03-02 | 李昕阳 | A kind of preparation method and application of egg yolk antibody inhibiting urease activity |
| RU2650768C1 (en) * | 2016-10-14 | 2018-04-17 | Федеральное государственное бюджетное учреждение "Федеральный центр охраны здоровья животных" (ФГБУ "ВНИИЗЖ") | Strain o no_2212/prymorsky/2014 of aphtae epizooticae foot and mouth disease virus of o type for the control of the antigenic and immunogenic activity of foot-mouth disease vaccines and for the manufacture of biologic drugs for diagnostics and specific prevention of foot and mouth disease of o type |
| RU2682876C1 (en) * | 2017-12-18 | 2019-03-22 | Федеральное государственное бюджетное учреждение "Федеральный центр охраны здоровья животных" (ФГБУ "ВНИИЗЖ") | Inactivated emulsion vaccine for o-type aphthous fever |
| CN116439195A (en) * | 2023-03-09 | 2023-07-18 | 中国农业科学院兰州兽医研究所 | A kind of construction method and application of foot-and-mouth disease virus mouse infection model |
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