CN1129454C - DNA vaccine for foot-and -mouth disease - Google Patents
DNA vaccine for foot-and -mouth disease Download PDFInfo
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- CN1129454C CN1129454C CN01132249A CN01132249A CN1129454C CN 1129454 C CN1129454 C CN 1129454C CN 01132249 A CN01132249 A CN 01132249A CN 01132249 A CN01132249 A CN 01132249A CN 1129454 C CN1129454 C CN 1129454C
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Abstract
The present invention relates to the technical field of medicines, provides a foot-and-mouth disease DNA vaccine which adopts the synthetic epitope gene SG of swine endemic O-type foot-and-mouth diseases as antigen genes and uses pVAXI as expression vectors. The SG sequence is as follows: ATGGTCACCA GTGACTCATT CGGACGTTGC GGTGGAGGTG GCTCTATCCA AAAGGGCGGT GGAGGATCCC GCTTCGAGGG TGGCGGTGGA TCCGGCGAAA CACAGATCCA GAGGCGCCAA CACACGGACG TCTCGTTCAT CATGGACAGA TTTGTGAAGG TGACACCGCA AAACCAATT AACATTTTGG ACCTCAGCA GATTCCATCA CACACTGGGG GTGGAGGCTC GAGTAAGTAC TCCGCAGGTG GTAGGGCAG ACGGGGCGAC CTAGAGCCTC TCGCGGCGA GGTCGCCGCT CAGCTTCTA CTTCTTTCAA CTTTGGTGCA ATTGGTGGCG GAGGCTCGAG TAGACACAA CAGAAAATTGTGGCTCCGGT GAAACAGACT TTGGGAGGCG GTGGGAGCTC CAGGTACAAC AGAAATACTG TGCCCAACTT GAGAGGTGAC CTTCAGGTGT TGGCTCAAAA GGTGGACGG AGCTGCCTG GGAGCTCCTA A.
Description
Technical field: the present invention relates to the medical biotechnology field, is a kind of DNA vaccine for foot-and-mouth disease.
Background technology: foot and mouth disease is that a kind of distribution is wide, serious harm artiodactyl beast, threatens human health, and animal husbandry production and Livestock Product Trade are influenced great disease, is listed in the category-A deadly infectious disease.Foot-and-mouth disease vaccine is the important means of prevention foot and mouth disease; though the foot and mouth disease inactivated vaccine of using has the effect that excellent prevention homotype foot and mouth disease infects at present; but can not effectively prevent the infection of multiple different serotypes foot and mouth disease; and the immunity persistent period is short, causes protecting effect not satisfactory.Dna vaccination is the new generation vaccine that rises in recent years, has antibody titer and holds time long and advantage such as cross protection is effective.Dna vaccination also has following outstanding advantage in addition: (1) can bring out body fluid and the comprehensive immunne response of cell, plays preventive effect; (2) immunological adjuvant CpG sequence can be integrated in the dna vaccination, improve the immunne response level of body and need not to add in addition adjuvant; (3) simple, with low cost, good stability of production technology and storing are convenient.
The mechanism of action of dna vaccination is: eukaryon expression plasmid is gone in synthetic foot-and-mouth disease antigen gene recombinaton, be injected in muscular tissue, make it in the myocyte, express foot-and-mouth disease epitope,, can activate intravital immunity system and cell immune system through the angtigen presentation process.Activated immunity system can produce special antibody, eliminates the virus that is free in the blood, the invasion of prevention foot and mouth disease; And activated cell immune system can produce cellulotoxic lymphocyte (CTL), attacks infected cells, eliminates the foot and mouth disease virus of hiding.
Summary of the invention: the present invention adopts in the O type foot and mouth disease virus and antigen epitope genes makes up DNA vaccine for foot-and-mouth disease.
In the foot and mouth disease virus and epitope be to stimulate body to produce the important protective antigen composition of neutralizing antibody, be positioned at the foot and mouth disease virus capsid.Of the present invention is the distinctive O type of pig foot and mouth disease virus, and the neutralizing epitope gene is designed and synthesized, and is inserted into carrier for expression of eukaryon pVAX1 again, is built into DNA vaccine for foot-and-mouth disease pVAX-SG.
The present invention is pVAX1 (available from Invitrogen company, sequence and physical map are seen the catalogue of the said firm) in order to the basic framework that makes up carrier for expression of eukaryon; In building process and the preparation process, the used host bacterium of plasmid amplification is DH5 α (available from a GIBCO company); The exogenous gene that inserts is: in the distinctive O type of the pig hoof-and-mouth disease virus capsid protein and antigen epitope genes, its structrual description is as follows:
(1) O type foot and mouth disease in and antigen epitope genes
A) sequence signature
Length: 501bp
Type: nucleic acid
Chain number: two strands
Geometry: linearity
B) molecule type: DNA
C) source: gene is synthetic.
D) sequence description: SG
ATGGTCACCA?GTGACTCATT?CGGACGTTGC?GGTGGAGGTG?GCTCTATCCA?AAAGGGCGGT?GGAGGATCCC
GCTTCGAGGG?TGGCGGTGGA?TCCGgCGAAA?CACAGATCCA?GAGGCGCCAA?CACACGGACG?TCTCGTTCAT
CATGGACAGA?TTTGTGAAGG?TGACACCGCA?AAACCAAATT?AACATTTTGG?ACCTCATGCA?GATTCCATCA
CACACTGGGG?GTGGAGGCTC?GAGTAAGTAC?TCCGCAGGTG?GTATGGGCAG?ACGGGGCGAC?CTAGAGCCTC
TCGCGGCGAG?GGTCGCCGCT?CAGCTTCCTA?CTTCTTTCAA?CTTTGGTGCA?ATTGGTGGCG?GAGGCTCGAG
TAGACACAAA?CAGAAAATTG?TGGCACCGGT?GAAACAGACT?TTGGGAGGCG?GTGGGAGCTC?CAGGTACAAC
AGAAATGCTG?TGCCCAACTT?GAGAGGTGAC?CTTCAGGTGT?TGGCTCAAAA?GGTGGCACGG?ACGCTGCCTG
GGAGCTCCTA?A
The epitope gene of foot and mouth disease adopts the synthetic mode of the full gene of PCR to carry out, the roughly flow process of this method is: synthesizing single-stranded and design corresponding PCR primer according to the gene order of design, be template then with the single stranded DNA, the corresponding genetic fragment of pcr amplification gets final product the extension amplification outcome order-checking again.Concrete grammar and reaction system thereof are referring to " molecular cloning " (Science Press published in 1992) related Sections.
Description of drawings:
Fig. 1 contains the plasmid map of SG antigen gene
The structure flow chart of Fig. 2 dna vaccination pVAX-SG
Fig. 3 dna vaccination pVAX-SG is at the intravital expression curve of BALB/c mouse
Fig. 4 dna vaccination pVAX-SG brings out the time graph of mice serum antibody titer
The specific embodiment:, preparation method of the present invention and effect are described in detail below in conjunction with accompanying drawing.
One, the structure of dna vaccination pVAX-SG, structure and preparation:
The structure of this dna vaccination is with carrier for expression of eukaryon---PVAX1 is the recombinant plasmid dna molecule of skeleton, and general structure is seen Fig. 1.Total length 3.5kb (kilobase to) except that containing elements such as pMB1 ori plasmid replication initiation site, kalamycin resistance gene Kanamycin, has also comprised a complete eukaryotic transcription translation unit.The eukaryotic transcription translation unit contains eukaryotic transcription translates necessary element: the polyadenylic acid BGH pA of eucaryon cytomegalovirus promoter Pcmv, respective coding gene SG and 3 ' end.On the whole, but this vaccine is the recombinant plasmid dna that can express SG antigen and activated lymphocyte in eukaryotic cell.
1, makes up engineering bacteria DH5 α (pVAX-SG) (see figure 2)
To O type foot and mouth disease synthetic gene SG EcoR I/Xba I double digestion, electrophoresis reclaims the purpose fragment (containing restriction enzyme site) of 519bp.With EcoR I/Xba I double digestion carrier pVAX1, electrophoresis reclaims the big fragment of carrier, and above two fragments are connected into the pVAX-SG plasmid simultaneously.Use CaCl
2Method transformed into escherichia coli DH5 α, screening obtains positive recombinant, promptly obtains the engineering bacteria DH5 α (pVAX-SG) that contains recombiant plasmid pVAX-SG.(concrete reaction system and experimental program are referring to aforementioned " molecular cloning " related content)
2, preparation dna vaccination pVAX-SG
Dna vaccination of the present invention prepares with above-mentioned engineered strain DH5 α (pVAX-SG).The cultivation amplification reaches to make with extra care can carry out (seeing aforementioned " molecular cloning " related content for details) by antibacterial culturing, plasmid amplification and the purification process of routine.
Training method is generally liquid culture.Available air shaking table shaken cultivation during a small amount of the amplification; When producing, industrialness need use the capable high density suspension of bacterial fermentation jar aerobic culture.Cultivation source in the culture medium there is not special requirement, the general LB fluid medium of using, as the need High Density Cultivation, then can select for use 2 * YT (contain 1.6% tryptone, 1% yeast extract and 0.5% sodium chloride, pH7.0) or the SOC fluid medium (contain 2% tryptone, 0.5% yeast extract, 0.05% sodium chloride, 20mmol/L glucose, 2.5mmol/L KCl, 10mmol/L MgCl
2, pH7.0).Also can add in case of necessity moving, plant, the mineral wet goods is as defoamer.Cultivation temperature is generally 37 ℃, the setting that is decided by culture medium, training method and ventilation condition opportunity of incubation time and collection antibacterial.
Separation and purification plasmid DNA from the engineering bacteria after the amplification can adopt some conventional methods.For example available detergent SDS cracking thalline utilizes plasmid DNA and chromosomal DNA to become the difference of renaturation feature, by to the degeneration of DNA and the processing of renaturation, plasmid DNA and chromosomal DNA is separated; Utilize plasmid DNA and impurity to carry out further purification again, and remove endotoxin in the difference of aspects such as dissolubility, ions binding power, absorption affinity.Specifically, after being present in the plasmid DNA process desalting processing in the renaturation solution, available anion exchange resin (as DEAESepharose) exchanges, use TE (containing 10mmol/L Tris and 1mmol/L EDTA, pH 8.0) buffer solution elution then, can make highly purified plasmid DNA (specifically seeing embodiment).Through PBS dilution packing, can obtain the pure product of transparent clarifying dna vaccination of the present invention.This product physicochemical property is very stable, can preserve down and transportation in room temperature, before the use, gets final product with the water for injection dissolving.
Two, the Function detection of dna vaccination pVAX-SG:
1, vivoexpression of this dna vaccination and detection, be by the electroporation rotaring redyeing COS 7 cell with constructed dna vaccination, harvesting after 72 hours, detect the antigenic expression of SG in culture supernatant and the cell breakage liquid with indirect elisa method, the result shows expression, illustrates that dna vaccination of the present invention really can express in mammalian cell.(seeing the experiment 1 of back for details)
2, expression in vivo of this dna vaccination and detection, be the pure product immune mouse of dna vaccination that will obtain after, detect antigen levels (see figure 3) in the serum with the ELISA method.After the result shows this dna vaccination immune animal, can express SG antigen.(seeing the experiment 2 of back for details)
3, this dna vaccination brings out the detection that body produces the humoral immunoresponse(HI) level, be the pure product immune mouse of dna vaccination that will obtain after, detect the level of anti-foot-and-mouth disease antibody in the serum with the ELISA method.The dna vaccination group can be kept the antibody titer of higher level in for a long time as a result, Zu inactivated vaccine (available from Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences) antibody titer then descended from March in contrast, subunit vaccine group antibody titer is than dna vaccination height, the (see figure 4) but antibody titer promptly descended since March.(seeing the experiment 3 of back for details)
The present invention can bring out the immune Response of Mice reaction effectively, illustrates that its foot and mouth disease virus infection to animal has certain immanoprotection action; And production technology is simple, and is with low cost, and physicochemical property is stablized, is convenient to store and transportation.
Use engineering bacteria of the present invention to prepare the embodiment of DNA vaccine for foot-and-mouth disease:
From-80 ℃ of refrigerators, take out frozen engineered strain---DH5 α (pVAX-SG), after room temperature is melted, dip in inoculating loop and to take a morsel the streak inoculation of bacterium liquid on 1.5% LB agar plate (containing kanamycin 50 μ g/ml), after 37 ℃ of overnight incubation, get monoclonal be inoculated in 50ml LB fluid medium (contain 1% tryptone, 0.5% yeast extract and 1% sodium chloride, kanamycin 50 μ g/ml, pH7.0) in, 37 ℃ of shaken cultivation 40 hours are as seed liquor.In 1: 20 ratio seed liquor is inoculated in (on the basis of aforementioned composition, other contains kanamycin 50 μ g/ml) in 1 liter of SOC fluid medium, cultivated 25 hours in 37 ℃ of violent joltings; Adding chloromycetin to final concentration is 170 μ g/ml, continues to cultivate 16 hours in 37 ℃ of violent joltings.With bacterium liquid in 4 ℃ with 6000rpm centrifugal 10 minutes; Remove supernatant, thalline is resuspended in the solution I (containing 50mmol/L glucose, 25mmol/LTris.Cl, 10mmol/L EDTA pH8.0) of the alkaline lysis of 30ml and adds lysozyme 40mg, add 60ml solution II (0.2mol/L NaOH and 1%SDS), slowly put upside down mixing 5 times, room temperature was placed 5 minutes.The solution III (containing potassium ion 3mol/L, acetate 5mol/L) that adds the pre-cooling of 45ml ice is slowly put upside down mixing for several times, ice bath 10 minutes; In 4 ℃ with 10000r/m centrifugal 10 minutes, get supernatant, cross 500ml G25 post desalination after, last 20ml DEAE Sepharose anion-exchange column, treat in conjunction with adsorb finish after, press salt ion (Cl
-) (0～1mol/L) progressive linear gradient elution method is slowly cleaned anion-exchange column with the PBS (pH 8.0) and the sodium chloride solution of 10 column volumes to concentration, collects the eluent of 260nm place absworption peak, obtains the crude product of dna vaccination from low to high.Be further purified then, add the dehydrated alcohol precipitation of 2 times of volumes, centrifugal 10 minutes of 4 ℃ of 12000rpm remove supernatant, behind 70% ethanol rinsing airing, are dissolved in PBS (pH 8.0) solution, promptly get the pure product of dna vaccination of the present invention; With ultraviolet spectrophotometry quantitatively after, make finished product by every part 200 μ g/ml packing again.
Prophylactic immunization scheme of the present invention is: get final product with finished product 1ml intramuscular injection, dosage is every piglet 200 μ g.Supplementary immunization once can obtain to react than strong immune response after 10 days.As then effect is better with the subunit vaccine therapeutic alliance.
Test the vivoexpression and the detection of 1 dna vaccination
Method with electroporation changes plasmid over to the COS7 cell, and the perforation condition is: electric capacity 960 μ F, and voltage is 250V, plasmid 40 μ g, the COS7 cell concentration is 1 * 10
7/ ml, the time is 16.7
Establish the contrast of empty carrier pVAX1 simultaneously, harvesting after 72 hours.Detect the expression of SG antigen in COS7 cell conditioned medium and cell pyrolysis liquid with indirect elisa method, cells and supernatant and cell pyrolysis liquid are wrapped respectively by 96 hole elisa plates, one anti-is mouse anti foot and mouth disease serum (making according to the conventional method immune mouse with the foot-and-mouth disease vaccine available from Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences), and two anti-ly are sheep anti mouse-HRP (available from magnificent biological engineering company limited).After the colour developing, positive greater than 1.8 with P/N ratio, SG antigen has obtained expression in the COS7 cell as a result, and culture supernatant and cell pyrolysis liquid are all positive.Illustrate that this dna vaccination can express in the COS7 cell.
Intravital antigen levels detects test after testing 2 immunity inoculations
40 of the female BALB/c mouse in 4～6 ages in week are divided into 4 groups at random, and 10 every group, the 1st group of immunity dna vaccination of the present invention (every inoculation 200 μ g), the 2nd, 3 group is PBS or pVAX1 negative control.DNA 200 μ g are injected to 1 group of mice through tibialis anterior, and 2 groups of mices then inoculate equal-volume PBS or pVAX1.Got blood through eye socket on the 2nd, 4,6,8,10 day before injection injection on the same day and after the injection, detect DNA behind the separation of serum in the lump in the expression of serum situation.
Detect the level of SG in the serum with indirect elisa method, 1: 10 dilution back of mice serum is wrapped by 96 hole elisa plates, one anti-is the anti-foot and mouth disease serum of pig (available from Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences), and two anti-ly are the anti-pig-HRP of rabbit (available from magnificent biological engineering company limited).Negative control is the blank mice serum of vaccinate not.After the colour developing, detect the hole is detected in the hole respectively with blank mice OD with 1,2,3 group
450Ratio P/N＞1.5 are positive.The results are shown in Figure 3.
As can be seen from the figure, vaccine detected antigen levels in the mice body is the highest at the 2nd day, descends gradually later on, during by the 10th day, is the weak positive.
Test 3 dna vaccinations and bring out the detection that body produces the humoral immunoresponse(HI) level
Be holding time of comparison dna vaccine pVAX-SG humoral immunoresponse(HI), BALB/c mouse is divided into 5 groups at random, every group 10, the 1st group is the dna vaccination group, every injected in mice pVAX-SG vaccine 200 μ g, the 2nd group is inactivated vaccine group (available from Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences), every mouse subcutaneous injection inactivated vaccine 10 μ l, the 3rd group is the subunit vaccine group, every injected in mice protein 10 μ g (subunit vaccine preparation method: the strain that contains fusion expression plasmid GST-SG, the GST purified fusion protein is used in ultrasonication after IPTG induces, and is dissolved in PBS.), the 4th group is the PBS matched group.Per 2 weeks get blood 1 time in two months after the same day of injecting and injection, got blood once in every month after 2 months, eye socket is got blood.Observation to 1 year.Detect the antibody horizontal of anti-foot and mouth disease in the serum with indirect elisa method, GST-SG with purification wraps by elisa plate, after adding serum to be checked, the anti-mice IgG two of rabbit anti-(available from magnificent biological engineering company limited) that adds the HRP labelling again, under the 450nm wavelength, measure optical density value after the chromogenic reaction, positive with P/N greater than 2.
The dna vaccination group can be kept the antibody titer of higher level in for a long time as a result, and the inactivated vaccine antibody titer then obviously descended from March; Subunit vaccine group antibody titer is than dna vaccination height, but antibody titer promptly descended since March.(see figure 4)
Above experimental result can prove that dna vaccination pVAX-SG of the present invention can bring out the immune Response of Mice reaction effectively, illustrates that its foot and mouth disease virus infection to animal has certain immanoprotection action; And production technology is simple, and is with low cost, and physicochemical property is stablized, is convenient to store and transportation.
The antigen gene SG nucleotide sequence of DNA vaccine for foot-and-mouth disease pVAX-SG is as follows:
ATGGTCACCA?GTGACTCATT?CGGACGTTGC?GGTGGAGGTG?GCTCTATCCA?AAAGGGCGGT?GGAGGATCCC
GCTTCGAGGG?TGGCGGTGGA?TCCGGCGAAA?CACAGATCCA?GAGGCGCCAA?CACACGGACG?TCTCGTTCAT
CATGGACAGA?TTTGTGAAGG?TGACACCGCA?AAACCAAATT?AACATTTTGG?ACCTCATGCA?GATTGGATCA
CACACTGGGG?GTGGAGGCTC?GAGTAAGTAC?TCCGCAGGTG?GTATGGGCAG?ACGGGGCGAC?CTAGAGCCTC
TCGCGGCGAG?GGTCGCCGCT?CAGCTTCCTA?CTTCTTTCAA?CTTTGGTGCA?ATTGGTGGGG?GAGGCTCGAG
TAGACACAAA?CAGAAAATTG?TGGCACCGGT?GAAACAGACT?TTGGGAGGCG?GTGGGAGCTC?CAGGTACAAC
AGAAATGCTG?TGCCCAACTT?GAGAGGTGAC?CTTCAGGTGT?TGGCTCAAAA?GGTGGCACGG?ACGCTGCCTG
GGAGCTCCTA?A
Claims (2)
1. DNA vaccine for foot-and-mouth disease; Comprise carrier for expression of eukaryon and antigen gene SG; SGO,：ATGGTCACCA GTGACTCATT CGGACGTTGC GGTGGAGGTG GCTCTATCCA AAAGGGCGGTGGAGGATCCC GCTTCGAGGG TGGCGGTGGA TCCGGCGAAA CACAGATCCA GAGGCGCCAACACACGGACG TCTCGTTCAT CATGGACAGA TTTGTGAAGG TGACACCGCA AAACCAAATTAACATTTTGG ACCTCATGCA GATTCCATCA CACACTGGGG GTGGAGGCTC GAGTAAGTACTCCGCAGGTG GTATGGGCAG ACGGGGCGAC CTAGAGCCTC TCGCGGCGAG GGTCGCCGCTCAGCTTCCTA CTTCTTTCAA CTTTGGTGCA ATTGGTGGCG GAGGCTCGAG TAGACACAAACAGAAAATTG TGGCACCGGT GAAACAGACT TTGGGAGGCG GTGGGAGCTC CAGGTACAACAGAAATGCTG TGCCCAACTT GAGAGGTGAC CTTCAGGTGT TGGCTCAAAA GGTGGCACGGACGCTGCCTG GGAGCTCCTA A。
2. by the described DNA vaccine for foot-and-mouth disease of claim 1, it is characterized in that said carrier for expression of eukaryon is pVAX1.
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| Application Number | Priority Date | Filing Date | Title |
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| CN01132249A CN1129454C (en) | 2001-11-20 | 2001-11-20 | DNA vaccine for foot-and -mouth disease |
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|---|---|---|---|
| CN01132249A CN1129454C (en) | 2001-11-20 | 2001-11-20 | DNA vaccine for foot-and -mouth disease |
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| CN1129454C true CN1129454C (en) | 2003-12-03 |
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| Title |
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| 科学通报,46(6) 2001-03-31 李光金等.CPG DNA 增强口蹄疫病毒DNA疫苗诱发豚鼠产生的免疫应答 * |
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