CN102274496A - 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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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 O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines of producing by gene engineering method and its production and use.
Background technology
Domestic breed scale constantly enlarges, and will become animal vaccine industry sustainable growth power source.Meat, the eggs output of China all are listed as the No. 1 in the world at present, are that world's livestock products are produced first big country.The annual animal husbandry output value of China broke through 1 trillion yuan high point first in 2004, and the proportion that the animal husbandry output value accounts for the general production value of agriculture is progressively improving.Because epidemic situation is continuous in the world wide, foot and mouth disease, bird flu, bovine spongiform encephalopathy etc. have proposed challenge with control work for the animal epidemic control of countries in the world.(Foot and mouth disease FMD) is a kind of acute, hot, the height contagious disease of cloven-hoofed animal in the animal husbandry (pig, cattle, sheep and camel) to foot and mouth disease.Up to now, the popular on a large scale of foot and mouth disease all once broken out in countries in the world except that North America, Australia.The popular of this disease once caused enormous economic loss to Germany (1937-1938), Europe (1951-1952), Turkey (1964-1965), Britain (1967-1968), Austria (1973), France (1974), Korea S countries such as (2002).
The clinical diagnosis characteristics of foot and mouth disease are the skin generation vesicle of oral mucosa, hoof and breast and fester, and are caused by foot and mouth disease virus, and for animal husbandry, the raising of pig, cattle and sheep and the trade of livestock products are 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 toxin expelling amount of a sick cattle can infect 1,000,000 cattle, and the sick Ungula Sus domestica of 1 gram portion vesicle micromicro makes 100,000 pig infection morbidities.Be easy to propagate, propagate rapidly, popular wide, fertility performance descends, the expenses for prevention and control height, and sickness rate 100%, the general 1-2% of poultry mortality rate that grows up, but young stock is up to 50%, even 100%.Often several provinces, several continents even the whole world take place simultaneously.Directly contact and indirect contact transmission all can, mainly propagate through digestive tract, also can be through respiratory infectious, the often interior at one time morbidity of cattle, sheep, pig, have the ability that infects multiple animal and have multiple antigen form, the popular saltatory that is is propagated, and all can take place throughout the year, and is just once popular every 1-2 or 3-5.In recent years, pig has the trend of expansion.The first level replication of FMDV is pharyngeal, infects contiguous lymph node then and enters blood flow, and then be diffused in various organs and the tissue.Clinical symptoms appearred in most cases, zoogenetic infection 2-14 days.Seldom occur death behind the more old zoogenetic infection FMDV, but animal reproduction ability, growth and health are subjected to very big influence.International Office of Epizootics classifies FMDV as the category-A deadly infectious disease.FMDV can only butcher with collective after each outburst and destroy the domestic animal that catches an illness by fire with trouble without offspring in case generation is difficult to control.Because foot and mouth disease is propagated rapidly, is difficult to control, remedial measure is few, is called as " the No.1 killer " of animal husbandry, is crushing blow for animal husbandry.According to OIE's regulation, in case the foot and mouth disease epidemic situation occurs, country concerned will lose the epidemic-stricken area qualification of non-foot and mouth disease automatically.
Foot and mouth disease virus has significant antigen multiformity, and the FMDV that finds has O, A, C, SAT1, SAT2, SAT3 (being South Africa 1,2,3 types) and 7 serotypes of Asia1 (Asia 1 type) at present.Every kind of serotype can also continue to be divided into multiple hypotype.China was the popular district of O type foot and mouth disease originally.The surrounding countries of northwest and the Northeast are O type and the popular district of Asia I type foot and mouth disease.Contain normal chain single stranded RNA polar, that form by 8500 nucleotide in the FMDV granule.The cyst membrane of virion comprises four kinds of structural protein VP1 (molecular weight 34000), VP2 (molecular weight 30000), VP3 (molecular weight 26000), the VP4 (molecular weight 13500) that holds single stranded RNA.Every kind of 60 molecule of four kinds of structural protein constitutes an icosahedral virion.Icosahedral summit is 141-160 zone and 200-213 zone by the immunologic determinants zone of structural protein VP1.
For FMD, vaccinate is the effective means of preventing foot and mouth disease at present.The problem of existing vaccine and existence thereof has:
1, attenuated vaccine and inactivated vaccine:
Traditional attenuated vaccine and inactivated vaccine are to utilize a large amount of zooblasts of cultivating, and infect foot and mouth disease virus then, and through cultivation, isolated viral, the reuse chemical reagent is prepared from after making inactivation of virus.France Merieux company is maximum deactivation FMDV production of vaccine producer.But preparation deactivation FMDV needs the environment of the isolation sealing of special strictness to produce, with leakage-preventing and pollute surrounding, the security requirement height, this potential risk limits this vaccine must be in remote environment production; In addition, the storage of vaccine transportation is preserved and is needed cold chain, increases cost; The change of tiring of deactivation FMDV vaccine is bigger, and vaccine also exists again strong and remaining vestige live virus to cause the danger of disease popularity and large-scale outbreak; In addition, the quality of vaccine also influences the emulsion secretion of milch cow through regular meeting; Use the FMDV vaccine of deactivation that though anaphylactic shock or stupor seldom take place, in case serious consequence takes place just to be enough to cause; At last, first immunisation takes 2 and uses secondary in thoughtful 2 months, later on every injection in 6 months once.The immunoprophylaxis workload is very big.
2, polypeptide vaccine:
Nineteen eighty-two is developed the polypeptide Seedling of synthetic.Synthetic polypeptide vaccine can overcome the shortcoming of conventional vaccine, is considered to the ultimate vaccine of zoonosis prevention usefulness very early.Use escherichia coli only to need 20 hours as the host cell fermented incubation time, period ratio is produced the inactivated vaccine much shorter with zooblast, recombinant vaccine does not pollute environment, there is not the danger of leakage, as safe as a house, and the vaccine of producing does not need cold preservation in storage, transportation and use, it is stable to tire.The research of polypeptide vaccine focuses mostly at the single immunologic determinants of the B of VP1 coat protein cell over more than 20 year, the immunologic determinants that further studies show that structural protein VP1 is 141-160 zone and 200-213 zone, in the polypeptide fragment in these two zones and the antibody capable that carrier protein produces 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; The biological engineering journal, 2009,25 (4) 514-519).Yet because of neutralizing antibody produces lessly, the immanoprotection action that is risen behind the synthetic polypeptide vaccine immune animal does not have the ideal so that people imagined originally, value even denied.In inducing the immunifacient process of body, single in and epitope be far from being enough.And present FMDV vaccine no matter be deactivation FMDV vaccine or polypeptide vaccine, all can not be differentiated immunized animal and infection animal by serology experiment, and immunized animal is obscured and can be caused very big influence to the market import and export with infection animal.Therefore, prepare immunizing potency improve and also can be used for differentiating immunized animal and the genetic engineering multivalent foot-and-mouth disease vaccine of infection animal be our constantly target of pursuit.
Studies have shown that except the B cellular immunization determinant of VP1 in recent years needs the T cellular immunization bunch synergism of VP4, can obtain good immune neutralizing antibody protection effect.
The Asia1 recombinant vaccine of units such as Fudan University development contains T-helper and VP1 immunologic determinants vaccine has the protection effect, can produce neutralizing antibody.
The synthetic polypeptide vaccine of U.S. UBI company has the B cellular immunization determinant polypeptide of T-cell Helper polypeptide and VP1, has strengthened the effect (about 10 times) of neutralizing antibody greatly, has clear and definite immune protective effect.Have good safety: acomia heat symptom-complex shape, do not have that irritated reaction takes place, the injection site does not have the malabsorption phenomenon, to health status and the not influence of gestation of in-pig, to farrowing achievement also not influence.
Need a large amount of, the cheap polypeptide class vaccine of producing at present.But it is very expensive that chemosynthesis surpasses 20 amino acid whose peptide chains.Therefore, need the development biotechnology by genetic engineering mass production polypeptide.Yet practical application also distance is far.According to the nearly 20 years research reports to polypeptide, except that insulin, genetic engineering had advantage unlike chemosynthesis.Basic reason is that engineered theoretical developments and practical application still remain to be developed, moreover polypeptide is degraded rapidly in vivo and is unfavorable for the generation of inducing of corresponding antibodies.Should utilize technique for gene engineering to prepare the FMDV polypeptide vaccine.The same with other gene engineering polypeptide products, producing needs to solve a series of upstream design and downstream process problem.Although it is a lot of reports are arranged, still far away from using.
Summary of the invention
The objective of the invention is to overcome the defective of prior art, a kind of New O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines are provided.A kind of O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines of the present invention, its genetic engineering bacterium is the O/Asia I, foot and mouth disease virus FMDV coat protein has four kinds to be VP1, VP2, VP3, VP4, O/Asia I genetic engineering bacterium is that the DNA sequence of 15 amino acid fragments of the T-cell helper of hoof-and-mouth disease poison strain coding VP4 connects the DNA sequence that the immunologic determinants zone of immunogenicity is arranged in the O type VP1 albumen and connects the DNA sequence in the immunologic determinants zone that immunogenicity is arranged in the Asia I type VP1 albumen as 1 repeated fragment, (the nucleotide collating sequence of 1 repeated fragment is Fig. 1), O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines contain 2
N-1The coded albumen of nucleotide sequence shown in the individual placed in-line SEQ ID, wherein n is the integer of 1-5.
Another purpose of the present invention provides the construction method of described foot-and-mouth disease vaccine.
O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines of the present invention contain placed in-line gene and express with the Lac plasmid that carries in the escherichia coli (E.Coli) in its genetic engineering bacterium.Described vaccine also comprises pharmaceutically acceptable carrier, adjuvant and/or immunological adjuvant.
On the other hand, the present invention also provides the O/Asia I engineering strain that can express described O/Asia I foot and mouth disease virus two bivalent gene engineered polypeptide vaccines.
This O/Asia I engineering strain, its plasmid that carries contains 2 respectively
N-1Nucleotide sequence shown in the individual placed in-line SEQ ID, wherein n is the integer of 1-5.
The invention provides purposes and the purposes during distinguishing infection animal and immunized animal of described O/Asia I foot-and-mouth disease vaccine in preventing and treating the foot and mouth disease disease.
The present invention also provides a kind of cloven-hoofed domestic animal O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccine preparation methoies, comprises the preparation of gene recombinaton, recombination fusion protein, it is characterized in that:
(1) is synthetic O/Asia I genetic engineering bacterium, with the gene of 15 amino acid fragments of the T-cell helper of the method composite coding VP4 of chemosynthesis connect the DNA sequence that immunogenic immunologic determinants zone is arranged in the O type VP1 albumen connect in the Asia I type VP1 albumen immunogenic immunologic determinants zone is arranged DNA sequence as 1 O/Asia I repeated fragment, connect 8 times;
(2) be synthetic O/Asia I genetic engineering bacterium, with 8 repeated fragments insertion Lac plasmid vectors of O/Asia I;
(3) change above-mentioned recombiant plasmid over to escherichia coli and express, obtain O/Asia I bacterial strain;
(4) place nutritious LB culture medium to ferment O/Asia I bacterial strain, 37 ℃ of fermentation temperatures, fermentation time is 12-24 hour, and adding the ampicillin in the fermentation liquid, to make its final concentration be 50 μ g/ml, and IPTG, concentration is 0.5mM, centrifugal acquisition thalline after the fermentation ends, through cell pulverization, renaturing inclusion bodies, the separation and purification of fusion rotein forms O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines with adjuvant homogenate.
The present invention comprises the genetic engineering research of polypeptide and set up the design that the upstream efficiently expresses and the downstream process of intensive new and high technology, set up high yield, simplified technology cheaply.O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines are one of serial polypeptide drugs, can cheap mass production.With escherichia coli is that the engineering bacterium fermentation incubation time only needs 12 hours, and recombinant vaccine does not pollute environment, does not have the danger of revealing.The vaccine of producing need not cold chain in storage, transportation and use.We select O type, two kinds of serotypes of Asia I type according to the epidemic situation of some area generations of China and the foot and mouth disease virus serotype of contiguous country's report of bordering on, and can prevent and treat O type and two kinds of serotype foot and mouth disease of Asia I type effectively, are suitable for for China.Our genetic engineering foot and mouth disease virus vaccine also distinguishable immunized animal except that the immunizing potency height reaches infection animal.
Use vaccine of the present invention to have the following advantages: 1) safety is good, and the present invention is a gene engineering product, is not inactivated vaccine, does not have the danger because of trace live virus leakage causing illness outbreak.In laboratory animal, with the vaccine of higher dosage mice is carried out subcutaneous injection, in the long observation phase, the healthy survival of laboratory animal.
2) vaccine of the present invention is applicable to engineered method large-scale production, has reduced cost.
3) utilize vaccine of the present invention can distinguish infection animal and immune animal.At occurring in nature, infect the animal of A type foot and mouth disease, can produce A type foot-and-mouth disease antibody in its body; Infect the animal of O type foot and mouth disease, can produce O type foot-and-mouth disease antibody in its body; Infect the animal of Asia I type foot and mouth disease, can produce Asia I type foot-and-mouth disease antibody in its body.Vaccine of the present invention has O type and two kinds of foot and mouth disease virus VP1 of Asia I type immunologic determinants simultaneously, after the inoculation, produce the antibody of resisting O-type and two kinds of foot and mouth disease of anti-Asia I type in the animal body, can distinguish infection animal and immune animal by this, avoid causing confusion to the animal import and export.
4) vaccine of the present invention contains O type and two kinds of foot and mouth disease virus VP1 of Asia I type immunologic determinants, the T-Helper polypeptide that contains VP4 again, strengthened the effect of neutralizing antibody greatly, 8 repeated fragments are expressed, the molecular weight of polypeptide is big, half-life in vivo prolongs, and its time that produces neutralizing antibody also prolongs greatly.
5) vaccine of the present invention is applicable to China O type and two kinds of popular districts of foot and mouth disease virus of Asia I type.
Description of drawings
Fig. 1 is the structure SEQ ID in the expression vector of O/Asia I genetic engineering bacterium;
Fig. 2 is the building process sketch map of plasmid F8, and diagram is how with the process of genetic fragment series connection 8 times;
Fig. 3 contains O/Asia I genetic engineering bacterium (E.Coli) the growth collection of illustrative plates during the fermentation of Lac carrier (12hr, fermentation tank: Switzerland is than Europe, 100L).
The specific embodiment
Embodiment one:
The present invention is according to the aminoacid sequence of the T-cell helper of the VP1 immunologic determinants of O and two types of FMDV of Asia I and VP4, design synthetic DNA genetic fragment.Its genetic engineering bacterium is the O/Asia I.O/Asia I genetic engineering bacterium is that the DNA sequence of 15 amino acid fragments of the T-cell helper of hoof-and-mouth disease poison strain coding VP4 connects DNA sequence that the DNA sequence that the immunologic determinants zone of immunogenicity is arranged in the O type VP1 albumen connects the immunologic determinants zone that immunogenicity is arranged in the Asia I type VP1 albumen as 1 repeated fragment, and the nucleotide collating sequence of 1 repeated fragment is referring to Fig. 1.
New O of the present invention/Asia I foot and mouth disease virus two bivalent gene engineered polypeptide vaccines, it contains 2
N-1Nucleotide sequence encoded polypeptide shown in the individual placed in-line SEQ ID, wherein n is the integer of 1-5.The DNA sequence of 15 amino acid fragments that SEQ ID contains the T-cell helper of hoof-and-mouth disease poison strain coding VP4 connects the DNA sequence that the immunologic determinants zone of immunogenicity is arranged in the A type VP1 albumen connects the immunologic determinants zone of immunogenicity in the Asia I type VP1 albumen dna encoding sequence, O type that its forward of can encoding is expressed and the antigenic determinant of Asia I foot and mouth disease virus, its expression product can stimulate the antibody that produces resisting O-type and Asia I foot and mouth disease virus.
Preferably, vaccine of the present invention contains 2
N-1Individual placed in-line SEQ ID encoded polypeptide, wherein, n is the integer of 2-4.
Most preferred, vaccine of the present invention contains 2
N-1Individual placed in-line SEQ ID encoded polypeptide, wherein, n is 4.
Be to improve bioavailability and enhance immunity effect, vaccine of the present invention can also contain pharmaceutically acceptable carrier, adjuvant and or material such as immunological adjuvant.
Immunological adjuvant be a kind of can enhance immunity the material of reaction, it can mix with antigen and uses, and helps the deposition of injection mass or compiles, and can also strengthen antibody response.
The material that can be used as immunological adjuvant has: 1) microorganism and product thereof, and as mycobacteria, coryne bacterium parvum, bordetella pertussis and the extract lipopolysaccharide of locating left Lan Shi negative bacillus, from extract muramyldipeptide of mycobacteria etc.2) polynucleotide is as poly, polyadenylic acid, poly glutaminol etc.3) freund adjuvant (Freund ' sadjuvant), comprise incomplete freund adjuvant (, adding the Water-In-Oil antigen Emulsion that emulsifying agent (lanoline or Tween 80) is made again) and complete freund adjuvant (in Freund, adding mycobacteria such as bacillus calmette-guerin vaccine) with antigen aqueous solution and oil preparation (paraffin oil or vegetable oil) mixed in equal amounts.4) inorganic matter is 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 escherichia coli heat-labile toxin (LT).2) attenuation derivant or the variant of CT and LT.3) the endogenous immunoregulatory factor of people is as IL-2, IL-12, GM-GSF.4) hormone.5) lipopeptid.6) saponin, saponin derivant QS-21.7) contain the synthetic oligonucleotide fragment (CpG ODN) of CpG motif.8) derivant of lipoid A is as lipopolysaccharide derivant monophosphoryl lipid A (MPL).9) derivant of muramyldipeptide (MDP).
In addition, some delivery system with intrinsic immunostimulatory activity also can be used as immunological adjuvant and is used 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.Adjuvant can be used with the polypeptide that proper dosage and the present invention have an immunogenicity, forms vaccine of the present invention.
Preferably, vaccine of the present invention contains incomplete freund adjuvant or aluminium hydroxide, and is preferred, and vaccine of the present invention contains incomplete freund adjuvant as immunostimulant.Not exclusively the ratio of lanoline and paraffin oil is slightly different with changes of seasons in the freund adjuvant, and the ratio of lanoline and paraffin oil is about 3: 7 as, spring, summer, autumn, and winter, the ratio of the two was about 1.5: 8.5.
The preparation method of O/Asia I foot and mouth disease virus two bivalent gene engineered polypeptide vaccines of the present invention comprises 2
N-1Nucleotide sequence shown in the individual placed in-line SEQ ID can directly synthesize, and also can synthesize several dna fragmentations earlier, connects the DNA sequence of back generation shown in SEQ ID.For the convenience of subsequent operation, when the design dna fragment, can be with proper restriction site calling sequence two ends, so that target sequence is cloned into suitable carriers.
In a preferred embodiment of the invention, carrier for synthetic O/Asia I genetic engineering bacterium, the DNA sequence of 15 amino acid fragments that the present invention designs the T-cell helper of synthetic hoof-and-mouth disease poison strain coding VP4 connects the DNA sequence that the immunologic determinants zone of immunogenicity is arranged in the O type VP1 albumen and connects the DNA sequence in the immunologic determinants zone that immunogenicity is arranged in the Asia I type VP1 albumen as a repeated fragment, connect generation by 10 dna fragmentations, each repeated fragment F1 contains nucleotide sequence shown in the SEQ ID and suitable restricted enzyme point of contact.Utilize engineered method that F1 is cloned into carrier, then, the restricted enzyme of the sticky end by having mutual coupling behind the enzyme action is cloned into carrier with F2 (containing 2 placed in-line F1 sequences), and the polypeptide after the expression contains 2 repeated fragments (VP4+O type VP1+Asia I type VP1).According to similar method, can successively F4, F8, F16 be cloned into carrier, they contain 4,8,16 placed in-line F1 sequences successively, after expressing in suitable expression system, the polypeptide that obtains contains 4,8,16 placed in-line repeated fragments (VP4+O type VP1+Asia I type VP1) respectively.
In preparation method of the present invention, the expression system that is used for expression of polypeptides can be a prokaryotic expression system, can also be eukaryotic expression system.Expression system comprises proper host cell, and can duplicate the also plasmid or the carrier of stable existence in host cell.
The example that can be used as host cell includes but not limited to: bacterial cell, as escherichia coli, streptococcus, Salmonella typhimurium etc.; Eukaryotic cell is as yeast etc.
Operable carrier can comprise chromosome source, non-chromosome is the source and synthetic DNA sequence.Make up deutero-carrier as: phage DNA, baculovirus, bacterial plasmid, yeast plasmid and by plasmid, phage and viral DNA.
Preferably, in prokaryotic expression system, express polypeptide of the present invention.Preferred, polypeptide of the present invention can be cloned into high efficiency expression vector (as commercially available expression vector) 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 that carries contains 2 respectively
N-1Nucleotide sequence shown in the individual placed in-line SEQ ID, wherein n is the integer of 1-5.
Preferably, the plasmid that carries of described O/Asia I genetic engineering bacterium contains 2
N-1Nucleotide sequence shown in the individual placed in-line SEQ ID,, wherein n is the integer of 2-4.
Preferred, the plasmid that described O/Asia I genetic engineering bacterium carries contains 2
N-1Nucleotide sequence shown in the individual placed in-line SEQ ID, wherein n is 4.
The purposes of O/Asia I foot and mouth disease virus two bivalent gene engineered polypeptide vaccines of the present invention in prevention and treatment foot and mouth disease.Can give animal with vaccine injection of the present invention, stimulate the antibody that produces specific resisting O-type and two kinds of foot and mouth disease of anti-Asia I type in the animal body, so vaccine of the present invention can be used to distinguish immunity inoculation animal and infection animal.
Table 1 is the measurement result of antigen antibody reaction, antibody provides by academy of agricultural sciences, Shanghai City animal and veterinary institute for O type and A type foot and mouth disease standard serum and O type foot and mouth disease treatment back Ox blood serum, and antigen be the O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines of the present invention that prepare voluntarily.
Table 1: antigen antibody reaction
Embodiment two:
1: the construction expression plasmid
Genetic engineering bacterium is an O/Asia I genetic engineering bacterium.
Carrier for synthetic 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 the DNA sequence that the immunologic determinants zone of immunogenicity is arranged in the O type VP1 albumen connect the DNA sequence in the immunologic determinants zone that immunogenicity is arranged in the Asia I type VP1 albumen as 1 repeated fragment F1, connect generation by 10 dna fragmentations, each repeated fragment F1 contains nucleotide sequence shown in Figure 1 and suitable restricted enzyme point of contact.Utilize engineered method that F1 is cloned into Lac promoter expression plasmid, then according to the method for Fig. 2, can successively F2, F4, F8 be cloned into Lac promoter expression plasmid, they contain 2,4,8 placed in-line F1 sequences (VP4+O type VP1+Asia I type VP1) successively.
The clone of the F8 gene of 2:O/Asia I genetic engineering bacterium in expressing charge material plastochondria Puc18
Plasmid Puc18 is cloned into gene F8 in the Puc18 plasmid according to a conventional method through after restricted enzyme BamH I/Sal I double digestion, obtains expression plasmid O/Asia I.Changing expression plasmid over to escherichia coli, 37 ℃ of cultivations, is that the IPTG of 0.2-0.5mmol/L induces with the final concentration.
3: fermentation
(the 1000ml seed culture fluid contains peptone 10g to the 250ml seed culture fluid, yeast extract 5g, the phosphate buffer 20ml of 0.02mol/L pH7.0) places the 1000ml conical flask, sterilized 20 minutes for 120 ℃, the cooling back adds 20% glucose solution 5ml.The bacterial strain of 1ml cryopreservation in glycerol added above-mentioned solution, and it is 50 μ g/ml that ammonification benzyl XiLin (Ampicillin) makes its final concentration, and 37 ℃, shaking table is cultivated 12-14 hour (150rpm) kind daughter bacteria as amplification culture.
The 1000ml seed culture fluid contains peptone 20g, yeast extract 10g, and the phosphate buffer 20ml of 0.2mol/L, pH7.0,120 ℃ of sterilizations 20 minutes, to make its final concentration be 50 μ g/ml in ammonification benzyl XiLin (Ampicillin) after being cooled to 37 ℃.Add seed culture fluid 20ml, add 20% glucose solution 5ml again, and trace elements of Ca Cl2, NiNO3, CoCl3, MgSO4, each 1mg of FeCl3, keep the listed condition (fermentation tank: 100L that ferments; Temperature: 37 ℃; Mixing speed: 700rpm; Ventilation: 80L/min; PH7.0-7.5).Certain hour is respectively got the 1ml fermentation liquid and is placed 2 plastic centrifuge tubes at interval, and the centrifugal 10min of 8000rpm removes supernatant, and taking by weighing thalline weight is weight in wet base (g/L).As shown in Figure 3, after 8-12 hour, bacterial concentration reaches peak value in fermentation.
After the fermentation, the centrifugal 30min of 4000rpm collects thalline.
Thalline is suspended in the ratio of 1000g/3L in the solution of potassium phosphate pH7.0 of the EDTA, the 20mmol/L that contain 1% sodium chloride, 1mmol/L, add the 1g lysozyme in suspension, stirring at room 1 hour is with smudge cells, thallus suspension liquid is abandoned supernatant in the centrifugal 30min of 10000rpm.
The urea liquid that above-mentioned precipitation is added 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 after the renaturation, with the centrifugal 30min of 10000rpm, get precipitation, make homogenate with sterilized water and form O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines, and be standby.
The detection of 4:O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines
1), reagent
O type and A type foot and mouth disease standard serum antibody gratuitously provide by academy of agricultural sciences, Shanghai City animal and veterinary institute.
2) sample
The O/Asia I genetic engineering bacterium that contains Puc18 after fermentation, centrifugal collection thalline, smudge cells use the 8M urea extraction, centrifugal collecting precipitation afterwards, it is standby that 1mg adds the 1ml sterilized water, supernatant is got 1ml.Add O type foot and mouth disease standard serum at precipitation solution, shake up, produce precipitation, the solution muddiness adds O type foot and mouth disease standard serum, shakes up, and does not produce precipitation, and solution is limpid; Add O type and A type foot and mouth disease standard serum at supernatant solution, shake up, no precipitation, solution is limpid.
The result: O/Asia I fusion rotein and O type foot and mouth disease standard serum produce antigen antibody reaction, and do not contain fusion rotein in the supernatant, so O type foot and mouth disease standard serum is not reacted.
The antigen antibody reaction of 5:O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines
Utilize protein in solution, antigen-antibody can react and produce the mensuration that precipitation is carried out vaccine immunity of the present invention source property.
1) antigen: test article is the albumen that obtains behind the 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 academy of agricultural sciences, Shanghai City animal and veterinary institute.
3) antigen antibody reaction: add O type foot-and-mouth disease antibody and A type foot-and-mouth disease antibody respectively in the O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines.According to the result of table 1, show that O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines and O type antibody have immunoprecipitation, do not have immunological cross-reaction with A type antibody.
The safety experiment (mouse experiment) of 6:O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines
20 of male mouse of kunming are about every body weight 20g, available from Chinese Academy of Sciences's Shanghai animal center.Be divided into two groups, one group is O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines, and another group is matched group.
The O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccine antigen proteins of getting the 1mg purification suspend with the 1ml sterilized water, and matched group injection 1ml sterilized water carries out lumbar injection to mice and observed 24 hours.
Survival rate
O/AsiaⅠ 10/10
Matched group 10/10
Sequence table
Organization?Applicant
----------------------
＜110〉OrganizationName: Wu Xiaoyan; Zhao Hong; Sun Yukun
Street: triumphant return 21 floor Building B, No. one building of No. 3500 HuaYuan Buildings, road
City: Shanghai
Country: China
PostalCode：200030
Application?Project
-------------------
＜120〉Title: a kind of O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines and preparation method and purposes
<130>AppFileReference：PCNWX1001462
<140>CurrentAppNumber：
<141>CurrentFilingDate：_____-___-___
Sequence
--------
＜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 (20)
1. O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines, wherein: genetic engineering bacterium is the O/Asia I, foot and mouth disease virus FMDV coat protein has four kinds to be VP1, VP2, VP3, VP4, O/Asia I genetic engineering bacterium is that the DNA sequence of 15 amino acid fragments of the T-cell helper of hoof-and-mouth disease poison strain coding VP4 connects DNA sequence that the DNA sequence that the immunologic determinants zone of immunogenicity is arranged in the O type VP1 albumen connects the immunologic determinants zone that immunogenicity is arranged in the Asia I type VP1 albumen as 1 repeated fragment, and O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines contain 2
N-1The coded albumen of nucleotide sequence shown in the individual placed in-line SEQ ID, wherein n is the integer of 1-5.
2. O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines as claimed in claim 1, wherein: O/Asia I genetic engineering bacterium contains 2
N-1The coded albumen of nucleotide sequence shown in the individual placed in-line SEQ ID, wherein n is the integer of 2-4.
3. O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines as claimed in claim 2, wherein: O/Asia I genetic engineering bacterium contains 2
N-1The coded albumen of nucleotide sequence shown in the individual placed in-line SEQ ID, wherein n is 4.
4. O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines as claimed in claim 3, wherein: O/Asia I genetic engineering bacterium contains 8 coded albumen of nucleotide sequence shown in the placed in-line SEQ ID, and its molecular weight is respectively 62505.5.
5. O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines as claimed in claim 3, wherein: contain placed in-line gene in the O/Asia I genetic engineering bacterium and express with the Lac plasmid that carries in the escherichia coli.
6. as each described O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines of claim 1-5, wherein: described vaccine also comprises pharmaceutically acceptable carrier, adjuvant and/or immunological adjuvant.
7. O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines as claimed in claim 6, wherein said immunological adjuvant is a freund 's incomplete adjuvant.
8. O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines as claimed in claim 6, wherein said immunological adjuvant is Al (OH) 3.
9. O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines as claimed in claim 6, wherein said immunological adjuvant is γ-polyglutamic acid ploy-γ-glutamic acid.
10. the preparation method of the described O/Asia I of claim 1 type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines comprises 2 of O/Asia I genetic engineering bacterium
N-1The step that nucleotide sequence shown in the individual placed in-line SEQ ID is cloned into carrier and expresses in expression system, wherein n is the integer of 1-5.
11. the preparation method of O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines as claimed in claim 10 comprises 2 of O/Asia I genetic engineering bacterium
N-1The step that nucleotide sequence shown in the individual placed in-line SEQ ID is cloned into carrier and expresses in expression system, wherein n is the integer of 2-4.
12. the preparation method of O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines as claimed in claim 11 comprises 2 of O/Asia I genetic engineering bacterium
N-1The step that nucleotide sequence shown in the individual placed in-line SEQ ID is cloned into carrier and expresses in expression system, wherein n is 4.
13. as the preparation method of each described O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines of claim 10-12, wherein said nucleotide sequence is expressed in prokaryotic expression system.
14. as the preparation method of each described O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines of claim 10-12, wherein said nucleotide sequence is expressed in eukaryotic expression system.
15. the O/Asia I engineering strain of the described O/Asia I of claim 1 type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines, its plasmid that carries contains 2 respectively
N-1Nucleotide sequence shown in the individual placed in-line SEQ ID, wherein n is the integer of 1-5.
16. a kind of O/Asia I engineering strain of O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines as claimed in claim 15, its plasmid that carries contains 2
N-1Nucleotide sequence shown in the individual placed in-line SEQ ID, wherein n is the integer of 2-4.
17. a kind of O/Asia I engineering strain of O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines as claimed in claim 16, its plasmid that carries contains 2
N-1Nucleotide sequence shown in the individual placed in-line SEQ ID, wherein n is 4.
18. the described O/Asia I of claim 1 type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines are used for prevention and treatment O type and Asia I foot and mouth disease.
19. the described O/Asia I of claim 1 type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines are used to distinguish infection animal and immunized animal.
20. the preparation method of cloven-hoofed domestic animal O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines comprises the preparation of gene recombinaton, recombination fusion protein, it is characterized in that:
(1) is synthetic O/Asia I genetic engineering bacterium, connect DNA sequence that the DNA sequence that the immunologic determinants zone of immunogenicity is arranged in the O type VP1 albumen connects the immunologic determinants zone that immunogenicity is arranged in the Asia I type VP1 albumen as 1 O/Asia I repeated fragment with the DNA sequence of 15 amino acid fragments of the T-cell helper of the method composite coding VP4 of chemosynthesis, connect 8 times;
(2) be synthetic O/Asia I genetic engineering bacterium, with 8 repeated fragments insertion Lac plasmid vectors of O/Asia I;
(3) change above-mentioned recombiant plasmid over to escherichia coli and express, obtain O/Asia I engineering strain;
(4) place nutritious LB culture medium to ferment O/Asia I engineering strain, 37 ℃ of fermentation temperatures, fermentation time is 12-24 hour, and adding the ampicillin in the fermentation liquid, to make its final concentration be 50 μ g/ml, and IPTG, concentration is 0.5mM, centrifugal acquisition thalline after the fermentation ends, through cell pulverization, renaturing inclusion bodies, the separation and purification of fusion rotein forms O/Asia I type foot and mouth disease virus two bivalent gene engineered polypeptide vaccines with adjuvant homogenate.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201010199712.2A CN102274496B (en) | 2010-06-12 | 2010-06-12 | O/Asia I type foot and mouth disease virus bivalent genetic engineering polypeptide vaccine, its preparation method and its purpose |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102614507A (en) * | 2012-02-17 | 2012-08-01 | 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 |
| KR20150002908A (en) * | 2013-06-26 | 2015-01-08 | 대한민국(관리부서 : 농림축산식품부 농림축산검역본부) | Foot and mouth disease virus expressing P1-protective antigen of Asia1 type, IV genotype and the manufacturing method |
| CN105367659A (en) * | 2015-11-18 | 2016-03-02 | 李昕阳 | Preparation method and application of urease activity inhibition yolk antibody |
| CN105821011A (en) * | 2015-01-07 | 2016-08-03 | 普莱柯生物工程股份有限公司 | Anti-type-O foot-and-mouth disease vaccine composition, preparation and application thereof |
| 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 |
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| WO1999066954A1 (en) * | 1998-06-20 | 1999-12-29 | United Biomedical Inc. | Synthetic peptide vaccines for foot-and-mouth disease |
| CN1470285A (en) * | 2003-06-13 | 2004-01-28 | 复旦大学 | Polypeptide vaccine of anti Asiatic I virus of foot-and-mouth disease and its preparing method |
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| WO1999066954A1 (en) * | 1998-06-20 | 1999-12-29 | United Biomedical Inc. | Synthetic peptide vaccines for foot-and-mouth disease |
| CN1470285A (en) * | 2003-06-13 | 2004-01-28 | 复旦大学 | Polypeptide vaccine of anti Asiatic I virus of foot-and-mouth disease and its preparing method |
| CN1589901A (en) * | 2003-09-03 | 2005-03-09 | 上海华谊生物技术有限公司 | Foot and mouth disease bivalent polypeptide vaccine and its preparation method and use |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102614507A (en) * | 2012-02-17 | 2012-08-01 | 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 |
| 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 |
| KR20150002908A (en) * | 2013-06-26 | 2015-01-08 | 대한민국(관리부서 : 농림축산식품부 농림축산검역본부) | Foot and mouth disease virus expressing P1-protective antigen of Asia1 type, IV genotype and the manufacturing method |
| 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 |
| CN105821011A (en) * | 2015-01-07 | 2016-08-03 | 普莱柯生物工程股份有限公司 | Anti-type-O foot-and-mouth disease vaccine composition, preparation and application thereof |
| CN105367659A (en) * | 2015-11-18 | 2016-03-02 | 李昕阳 | Preparation method and application of urease activity inhibition yolk antibody |
| 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 |
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| CN102274496B (en) | 2015-05-06 |
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