CN1730101A - O type foot-and-mouth disease virus poly-gene duplication defect type adenovirus active carrier vaccine and preparation method - Google Patents
O type foot-and-mouth disease virus poly-gene duplication defect type adenovirus active carrier vaccine and preparation method Download PDFInfo
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Abstract
本发明涉及采用基因克隆技术制备一种畜用口蹄疫病毒活载体免疫疫苗及其制备方法。本发明的疫苗及其制备方法是携带有可以表达O型口蹄疫病毒全衣壳蛋白的O型口蹄疫病毒的结构蛋白基因P1和2A、3C蛋白酶基因，以及真核表达元件形成的目的基因表达盒的疫苗和这种疫苗的制备方法。The invention relates to the preparation of a foot-and-mouth disease virus live vector immunization vaccine for livestock by adopting gene cloning technology and a preparation method thereof. The vaccine of the present invention and the preparation method thereof carry the structural protein gene P1 and 2A, 3C protease genes of the O-type foot-and-mouth disease virus that can express the O-type foot-and-mouth disease virus whole capsid protein, and the objective gene expression cassette formed by the eukaryotic expression element. Vaccines and methods for the preparation of such vaccines.
Description
Technical field
The present invention relates to adopt gene clone technology to prepare a breeding stock with foot and mouth disease virus live vector immune vaccine and preparation method thereof, is a kind of O type foot and mouth disease virus immune formulation and preparation method more exactly.
Background technology
Foot and mouth disease virus (Foot-and-Mouth Disease Virus is designated hereinafter simply as FMDV) is the cause of disease that causes the artiodactyl foot and mouth disease, and this disease is a kind of height contagious disease, can cause worldwide being very popular.Animal is produced and products thereof international trade bring and have a strong impact on, thereby countries in the world all take much count of prevention and control to this disease.
The harm of foot and mouth disease is huge, has a far reaching influence, and the international trade of domestic animal and products thereof also is divided into two big camps because of foot and mouth disease, and the national and no epidemic disease of epidemic disease country is arranged.There is epidemic disease country to continue to suffer the tremendous economic loss that the outlet restriction is brought.China will expend billions of units for the anti-system of foot and mouth disease every year, and the direct and consequential damage that is caused is inestimable especially, is necessary to strengthen the research dynamics of Prevention Technique.
Aspect vaccine research, the national active research new generation vaccine of no epidemic disease is to reduce the risk that application traditional vaccine and eqpidemic disease are imported into.Epidemic disease country is arranged also in the research of actively developing the foot and mouth disease new generation vaccine,,, be used for the prevention of foot and mouth disease as a kind of alternate, safe and reliable vaccine so that after following foot and mouth disease is progressively controlled and eliminated.
Traditional inactivated vaccine is made antigen with the totivirus of cell culture after the purification deactivation, thereby has good immunogenicity, produces the good humoral immunity behind the immune animal, and is attended by cellullar immunologic response, has good protective action.Shortcoming is, the poor heat stability of inactivated vaccine requires cryopreservation, and immune duration is short, and antiviral spectrum is limited, and the danger of the persistent infection of causing is arranged, and can not distinguish vaccine injected animal and natural infected animal, has thoroughly the do not loose probability of poison of inactivation of virus simultaneously.In view of the problem that inactivated vaccine exists, the foot and mouth disease new generation vaccine has just become research focus.
Chinese invention patent application 01111121.6 discloses with the viral RNA reverse transcription and has formed the proteic full DNA series of VP1, the VP1 gene is inserted in the plant-bioreactor again, finally forms vaccine.Thisly express main immune section in the foot and mouth disease virus structural protein by plant-bioreactor; host animal had certain immunization; but the epitope that this subunit vaccine can be showed is very limited; and differ greatly with the antigenic structure of natural viral capsid, be not enough to stimulate animal body to produce counteracting toxic substances protective effect completely.
Chinese invention patent application 0210370109 discloses a kind of DNA that has the antigenic peptide section in the foot and mouth disease virus that adopts and has been connected with beta-galactosidase gene, constitutes fusion gene, expresses and merges lactose protein, and purified fusion protein prepares polypeptide vaccine with it.It is that the epitope that can show is limited that the polypeptide vaccine of foot and mouth disease and viral sub-units vaccine all have a shortcoming, and is linear epitope, lacks comformational epitope.And this class vaccine stimulates immunifacient response mechanism and live vector vaccine variant.Live vector vaccine stimulates body to produce the humoral immunization and the cell immune response of system by at the cell inner expression virus antigen as a kind of endogenous antigen, can produce immanoprotection action completely.
The report that in the gene clone technology field, does not still have the development of O type foot and mouth disease virus recombinant adenovirus live vector vaccine so far.
Summary of the invention
It is the genes of interest source with O type foot and mouth disease virus that task of the present invention provides a kind of, is built into the novel live vector vaccine of expressing the hoof-and-mouth disease virus capsid protein, and the preparation method that this vaccine is provided.
Vaccine of the present invention is to carry the structural protein gene P1 of the O type foot and mouth disease virus that can express the full capsid protein of foot and mouth disease virus and 2A, 3C protease gene, and the destination gene expression box that forms of eukaryotic expression element.
Its eukaryotic expression element poly (A) sequence that is CMV promoter and SV40 in an embodiment of the present invention.
The genes of interest of used in an embodiment of the present invention O type foot and mouth disease virus comes from the China99 strain, this strain belongs to the Pan Asia virus groups in the O type foot and mouth disease virus, the propagation that 1999～2001 world's foot and mouth disease are very popular by this group virus causes, pathogenic and the immunogenicity of this strain is all very strong, it is the seedling strain of traditional inactivated vaccine, adopt the gene order of this group virus to make up live vector vaccine, aspect antigenicity, have more representative.
Preparation method of the present invention is
(1) adopt primer sequence to be
| Numbering | Sequence | Length (base) |
| P1K(+) P1B(-) 3CB(+) 3CH(-) | 5’-AGGGGTACCACCATGGGCAACACTGGAAGCATTATCAAC-3’ 5’-GATGGATCCGACATGTCCTCCTGCATCTGGTTG-3’ 5’-GATGGATCCAGGACCGGTGAAGAAAC-3’ 5’-CTCAAGCTTCTACTCGTGGTGTGGTTCGGGATC-3’ | 39 33 26 33 |
(2) be the purpose template with O type foot-and-mouth disease virus gene group reorganization positive plasmid, by the polymerase chain reaction, amplification obtains P1-2A and 3C protease gene,
(3) P1-2A and the 3C protease gene that obtain are cloned in the adenovirus shuttle plasmid, form the destination gene expression box; After this recombinant shuttle plasmid linearize, transform the competence bacteria (BJ5183) that has the adenovirus skeleton carrier, obtain having the recombinant adenovirus plasmid of destination gene expression box.
(4) with after the linearize of recombinant adenovirus plasmid enzyme action, with liposome transfection human embryonic kidney cell (HEK293), obtain just for recombinant adenovirus,
(5) just for the recombinant adenovirus amplification culture that goes down to posterity on the human embryonic kidney cell, preparation and purification get product.
In the preparation of industrialization process of reality, just the amplification culture for recombinant adenovirus can adopt rolling bottle and microcarrier suspension culture method to produce virus, and adopts the ultrafiltration and concentration purified virus.
The present invention adopts the carrier of adenovirus as exogenous gene.The adenovirus kind is a lot, is a kind of distrand DNA virus of not having cyst membrane, and the genome size is about 30-40kb.Because adenovirus is safer, the clinical symptoms after adenovirus infection is also smaller.It is historical for many years with the adenovirus to be that gene therapy vector has had, facts have proved be do not have cause a disease, teratogenesis and carcinogenesis.On the other hand, the adenovirus carrier vaccine recombinant viral vector makes up relatively easy, and the host cell scope of adenovirus is wider, can infect a lot of types division or resting stage cell, the efficient that enters cell is very high, therefore in cell culture, can reach very high titre, the approach of multiple administration is provided, as by muscle, nasal cavity or orally carry out immunity.
Vaccine of the present invention; adopt the recombinant replication-defective type recombinant adenovirus of foot and mouth disease virus structural protein P1 and non-structural protein 2A and 3C coding region; can effectively expressing behind this recombinant adenovirus immune swine and whole capsid antigen compositions of submission foot and mouth disease virus, stimulate the pig body to produce the excellent protective immunoreation.Secondly, because this immune formulation does not have the 3D and the 3AB section of foot-and-mouth disease virus gene group, thereby help differentiating foot-and-mouth disease vaccine immune animal and natural infected animal.
The detailed Description Of The Invention and the specific embodiment
The present invention obtains structural protein P1-2A and the 3C protease gene of China/99 strain O type foot and mouth disease virus by polymerase chain reaction (PCR) amplification, the applied molecular biology technology is cloned into it in adenovirus shuttle plasmid (pShuttle-CMV) again, this shuttle plasmid has POLY (A) tail of giant cell tumor virus (CMV) utmost point early promoter and simian virus 40 (SV40), forms the destination gene expression box therefrom.With recombinant shuttle plasmid restricted enzyme Pme I enzyme action, transform the recombination bacillus coli BJ5183 competence bacteria that has adenovirus skeleton carrier pAdEasy-1 then, by the method for homologous recombination in the antibacterial, obtain having the recombinant adenovirus plasmid of destination gene expression box.
With recombinant adenovirus plasmid after the linearize of restricted enzyme Pac I enzyme action, with liposome transfection human embryonic kidney cell (HEK293), produce the duplicate deficit type recombinant adenovirus of high titre in HEK293 cell inner packing, through fluorescent antibody, evidences such as RT-PCR, PCR, this recombinant adenovirus can be stable carries and expresses genes of interest.
Amplification culture is just for virus on the HEK293 cell, and by the method for difference collecting cell and supernatant, a large amount of preparations and purification obtain recombinant adenovirus.With phosphate buffer dissolved cell precipitation,, centrifugally obtain containing viral supernatant by 37 ℃/-70 ℃ multigelations 3 times; Virus in the culture supernatant concentrates by the ultrafilter membrane purification, and the virus titer that obtains in this way can reach 1～5 * 10
9TCID
50/ ml.
Its concrete way is:
1. design of primers
Design two pairs of primers be used to increase P1-2A and 3C gene respectively, designed specific restriction enzyme site respectively, be used for segmental connection at the two ends of primer.Primer sequence is as follows:
| Numbering | Sequence | Length (base) |
| P1K(+) P1B(-) 3CB(+) 3CH(-) | 5’-AGGGGTACCACCATGGGCAACACTGGAAGCATTATCAAC-3’ 5’-GATGGATCCGACATGTCCTCCTGCATCTGGTTG-3’ 5’-GATGGATCCAGGACCGGTGAAGAAAC-3’ 5’-CTCAAGCTTCTACTCGTGGTGTGGTTCGGGATC-3’ | 39 33 26 33 |
2.PCR amplification
The O type foot and mouth disease virus China/99 pnca gene group recombiant plasmid of preserving with inventor's laboratory is the template of amplifying target genes.With P1K (+) and P1B (-) is primer amplification P1-2A fragment.With 3CB (+) and 3CH (-) is primer amplification 3C fragment.
Reaction system is 100 μ L, that is: 10 * buffer, 10 μ L, dNTP (2.5mmol/L) 8 μ L, MgCl
2(25mmol/L) 6 μ L, forward primer 1 μ L, downstream primer 1 μ L, template 1 μ L, aquesterilisa 71.5 μ L add the high-fidelity Taq of Biolabs company enzyme (10U/ μ L) 0.5 μ L and increase.
The rearmounted PCR of centrifugal mixing increases in the instrument automatically, and 94 ℃ of pre-degeneration 4min carry out following circulation: 94 ℃ of 1min, and 55 ℃ of 1min, 72 ℃ of 2min, after 30 circulations, 72 ℃ are extended 7min.Sample 5～8 μ L on the PCR product, (80～100V), if the amplified production clip size promptly can be used for follow-up test consistent with its intended purposes clip size, and the P1-2A clip size is 2200bp, and 3C is long to be 750bp to carry out electrophoresis in agarose gel.
3. the purification of target DNA fragment reclaims
Earlier the PCR product is carried out in 0.8% agarose gel about electrophoresis (80V) 40min.Under long wave ultraviolet light, observe the electrophoresis situation, after target DNA fragment and assorted band separate fully, contain the segmental gel piece of purpose (less than 300 μ L) and place the 1.5mL centrifuge tube with cleaning scalpel (through flame disinfection) cutting-out.
Subsequent step reclaims the test kit description by precious biotech firm dna fragmentation to carry out.Concrete steps are:
1. add 300 μ L sol solutionses in every 100mg agarose gel, behind 65 ℃ of abundant colloidal sols, add binding buffer liquid 150 μ L, mixing.
2. the glue that melts is added on the centrifugal adsorption column 3600r/min, centrifugal 30s.
3. wash post repeatedly 2 times with cleaning mixture, last centrifugal 1min.
4. adsorption column is put into new collecting pipe, added the elution buffer of 30 μ L, room temperature effect 5min, the centrifugal 1min of 12000r/min.
4. target DNA fragment and pGEM-T Easy carrier is connected
Because employed Taq enzyme adds an adenine (A) at the last product end of giving of polymerization process, and two ends of pGEM-T Easy carrier respectively have an outstanding thymus pyrimidine (T), therefore both can prevent the recirculation of plasmid, can form cohesive end again and connect, make joint efficiency greatly improve.Linked system is 10 μ L, that is: 2 * Buffer, 5 μ L, and T4 DNA Ligase 1 μ L, pGEM-T Easy carrier (Promega company product) (10U/ μ L) 1 μ L, target DNA 3 μ L, 4 ℃ of connections are spent the night or more than 16 ℃ of incubation 4h.
5. the preparation of competent cell (under strict aseptic condition, carrying out)
1. draw in the LB culture medium of bacillus coli DH 5 alpha bacterium liquid (being stored in-70 ℃ of refrigerators) 5 μ L to 3mL, 37 ℃ of 220r/min shakes are cultivated about 16h.
2. draw the above-mentioned bacterium liquid of 500 μ L to the triangular flask that fills 50mL LB (preheating), 37 ℃ are cultured to exponential phase (about 2.5～3h).
3. culture changes in the 50mL centrifuge tube, ice bath 10～15min.
4. 4 ℃ of centrifugal 8min of 4000r/min.
5. abandon supernatant, centrifuge tube is inverted in the absorbent paper, the culture medium control is done.
6. the 0.1mol/L CaCl that adds the 5mL pre-cooling earlier
2The resuspended precipitation of solution (filtration sterilization) adds the CaCl of 15mL pre-cooling again
2Solution, ice bath 45min.
7. 4 ℃ of centrifugal 8min of 4000r/min; Abandon supernatant, be inverted the dried residual liquid of centrifugal management and control (2min), use the CaCl of the 0.1mol/L of 2mL pre-cooling
2The resuspended precipitation of solution is sub-packed in the 1.5mLEppendorf pipe with every pipe 200 μ L, and 4 ℃ are spent the night.
6. the conversion of competent cell
1. add 5 μ L or 10 μ L coupled reaction things in every pipe competent cell, use rifle head mixing gently, ice bath 45min.
2. with centrifuge tube careful be transferred to heat shock 90s in 42 ℃ of water-baths.
3. place ice bath to cool off 3min rapidly, every then pipe adding is preheated to 37 ℃ LB culture medium 800 μ L, and 1h are cultivated in 37 ℃ of 220r/min concussions makes cell recovery.
4. 4 ℃ of centrifugal 2min of 4000r/min abandon the part supernatant, stay the resuspended precipitation in the 200 μ L left and right sides.
5. in cell suspension, add 16 μ L X-gal (50mg/mL) and 4 μ L IPTG (200mg/mL),, coat in advance to 37 ℃ LB agar plate (containing ampicillin 60 μ g/mL) 37 ℃ of incubated overnight with rifle head mixing gently.
7. choose speckle
In the superclean bench with sterilization toothpick single white colony of picking from the flat board, be inoculated in and contain in the 3mLLB culture medium chain bottle of (containing 60 μ g/mL ampicillin), choose a locus coeruleus simultaneously and make negative control, 37 ℃ of 220r/min shakes are cultivated 12～16min.
8. the extraction of plasmid and evaluation
Alkaline lysis extracts plasmid DNA in a small amount.Operating procedure is as follows:
1. use aseptic toothpick from the single bacterium colony of the dull and stereotyped picking white of LB, be inoculated in the 3mL LB culture fluid (ampicillin 100 μ g/mL) 37 ℃ of joltings (230r/min), 12～16h.
2. the aseptic 1.5mL of pipetting bacterium liquid is put in the centrifuge tube, and the centrifugal 3min of 12000r/min abandons supernatant, is inverted the dried residual liquid of control.
3. every pipe adds solution I (the 50mmol/L glucose of 120 μ L pre-coolings; 25mmol/L TrisCl (pH8.0); 10mmol/L EDTA (pH8.0)), suspend with the vortice vibration.
4. solution II (the 0.2mol/L NaOH (facing with preceding) that adds the new preparation of 240 μ L with 10mol/L NaOH dilution; 1%SDS (diluting)) with 10%SDS, gentle 3～4 mixings of centrifuge tube of being inverted, ice bath is placed 5min then.
5. solution III (the 5mol/L potassium acetate 60mL that adds 180 μ L pre-coolings; Glacial acetic acid 11.5mL; Deionized water 28.5mL), put upside down mixed several times, ice bath is placed 5min.
6. the centrifugal 8min of 12000r/min is transferred to supernatant in another centrifuge tube, adds the saturated phenol vibration of 500 μ L Tris extracting.
7. the centrifugal 1min of 12000r/min shifts in the new pipe of upper strata water to, uses the saturated phenol of Tris: chloroform: isoamyl alcohol (25: 24: 1) vibration extracting.
8. the centrifugal 1min of 12000r/min, upper water is added to the dehydrated alcohol of 2 times of volumes, puts more than-20 ℃ of precipitation 0.5h 12000r/min, centrifugal 10min.
9. precipitate with 70% ice-cold washing with alcohol, the centrifugal residual ethanol of removing is put natural drying at room temperature.
10. contain TE (pH8.0) dissolving of Pancreatic RNase (20 μ g/mL) with 30 μ L, room temperature is placed 0.5h, electrophoresis observation.
The evaluation of positive colony:
1. electrophoresis is identified: the plasmid that is extracted is carried out electrophoresis, compare with negative plasmid (locus coeruleus), selecting may positive plasmid.
2. enzyme action is identified: carry out enzyme action with EcoR I.Reaction system is 20 μ L systems, that is: recombiant plasmid 15 μ L, and 10 * Buffer, 2 μ L, EcoR I 1 μ L, sterilized water 2 μ L put in 37 ℃ of incubators, take out behind the 2h, and enzyme action product, molecular weight Marker are carried out electrophoresis relatively.
3. PCR identifies: to get 1 μ L after the positive 10 times of dilutions of plasmid of enzyme action and electrophoresis is template, add 2 μ L, 10 * PCR buffer, 2 μ L dNTP (2.5mmol/L) mixture, 0.5 μ L TaqDNA polymerase (5U/ μ L), 0.2 μ L forward primer (201), 0.2 μ L downstream primer (203), 1.2 μ LMgCl
2(25mmol/L), moisturizing to 20 μ L carries out PCR and identifies, uses 100bp DNA ladder Marker as reference.
A large amount of propagation identify that correct positive plasmid is used for following experiment.
9. purpose fragment and pShuttle-CMV shuttle plasmid is connected
Use restricted enzyme Kpn I and Hind III double digestion pShuttle-CMV shuttle plasmid (Stratagene company product) respectively, reclaim big fragment; Identify the correct segmental reorganization of band P1-2A pGEM-T plasmid with Kpn I and BamH I double digestion, reclaim the 2200bp fragment; With BamH I and the segmental reorganization of Hind III double digestion band 3C pGEM-T plasmid, reclaim the 750bp fragment.With three fragments that the external connection of T4 dna ligase (Biolabs company product) is reclaimed, linked system is as follows: 10 * Buffer2 μ L, and T4DNA ligase (400U/ μ L) 1 μ L reclaims fragment by descending 3,5, the 9 μ L that are respectively, and 4 ℃ of connections are spent the night.
10. the evaluation of the recombinant shuttle plasmid of band foot and mouth disease virus P1-2AX3C genes of interest
Get above-mentioned connection product and transform DH5 α competent cell (method is the same), transformant is grown on the culture plate of kalamycin resistance, the bacterium colony of that resistance of screening card release.Identify (method is the same) by choosing speckle, extraction plasmid, PCR and enzyme action, identify positive reorganization bacterium.Successfully construct the recombinant shuttle plasmid of band destination gene expression box thus, extract plasmid and be provided with down using.
11, homologous recombination method produces the recombinant adenovirus plasmid that carries the destination gene expression box in the antibacterial
Recombinant shuttle plasmid after restricted enzyme Pme I (Biolabs company product) linearisation and alkaline phosphatase treatment with adenovirus skeleton carrier adenoeasy vector (Stratagene company product) cotransformation escherichia coli BJ5183 competence bacteria (Stratagene company product).Perhaps earlier adenoeasy vector is transformed BJ5183, the Adeno-BJ5183 competence bacteria of adenovirus skeleton carrier is carried in preparation, and the recombinant shuttle plasmid with Pme I linearize transforms the Adeno-BJ5183 competence bacteria again.Block that resistance screening, plasmid electrophoresis and Pac I enzyme action identify recombinant adenovirus plasmid, to identify that male recombinant adenovirus plasmid transforms XL-10 competence bacteria (Stratagene company product), with a large amount of propagation recombiant plasmid, adopt β-ME reagent (Stratagene product) to transform, by specification carries out.Positive recombinant adenovirus plasmid called after pAdcmv-p12 * 3c, and send precious biological (Dalian) company conclusive evidence that checks order.
The sequence of measuring is as follows:
ATGGGCAACACTGGAAGCATTATCAACAATTCCTACATGCAGCAGTAC
CAGAACTCCATGGACACGCAACTTGGTGATAACGCTATTAGCGGAGGCTCC
AACGAGGGGTCCACGGACACCACCTCCACCCACACAACCAACACTCAGA
ACAATGACTGGTTTTCAAAGCTGGCCAGTTCCGCTTTTAGCGGTCTTTTCG
GCGCTCTTCTCGCCGACAAGAAAACCGAGGAGACCACTCTTCTCGAGGAC
CGCATCCTCACTACCCGCAACGGACACACGACCTCGACAACCCAGTCGAG
CGTTGGAGTCACTTACGGGTACGCAACAGCTGAGGACTTTGTGAGCGGAC
CAAACACATCTGGGCTTGAGACCAGGGTTGTGCAGGCAGAGCGGTTCTTC
AAAACCCACTTGTTCGACTGGGTCACCGGTGACCCGTTCGGACGGTGCTA
CCTGCTGGAACTCCCAACTGACCACAAAGGTGTCTACGGCAGCCTGACTG
ACTCTTATGCTTACATGAGAAACGGTTGGGATGTTGAGGTCACTGCAGTGG
GAAATCAGTTCAACGGAGGATGTCTGTTGGTGGCCATGGTGCCAGAACTT
TGCTCTATTGACAAGAGAGAGCTGTACCAGCTCACGCTCTTTCCCCACCAG
TTCATCAACCCCCGGACGAACATGACGGCGCACATCACTGTGCCCTTTGTT
GGTGTCAACCGCTACGACCAGTACAAGGTACACAAACCTTGGACCCTCGT
GGTTATGGTTGTGGCCCCGCTGACTGTCAACACCGAAGGTGCCCCACAGA
TCAAGGTCTATGCCAACATCGCCCCTACCAACGTGCACGTTGCGGGTGAGT
TCCCTTCTAAGGAAGGGATCTTCCCCGTGGCATGTAGCGACGGTTACGGTG
GTCTGGTGACCACTGACCCAAAGACGGCTGACCCCGCCTACGGGAAAGTG
TTCAATCCACCTCGCAACATGTTGCCGGGGCGGTTCACCAACTTCCTTGAT
GTGGCTGAGGCGTGCCCTACGTTTCTGCACTTTGAGGGTGACGTGCCGTAC
GTGACCACAAAGACGGACTCAGACAGGGTGCTCGCCCAGTTTGACTTGTC
TCTGGCAGCAAAGCACATGTCAAACACCTTCCTGGCAGGTCTCGCCCAGT
ACTACACACAGTACAGCGGCACCATCAACCTGCACTTCATGTTCACAGGA
CCCACTGACGCGAAAGCGCGTTACATGATTGCATACGCCCCCCCTGGCATG
GAGCCGCCCAAAACACCTGAGGCGGCCGCTCACTGCATTCATGCGGAGTG
GGACACAGGGTTGAATTCAAAATTCACATTTTCAATCCCTTACCTTTCGGC
GGCTGATTACGCGTACACCGCGTCTGACGCTGCGGAGACCACAAATGTAC
AGGGATGGGTCTGCCTGTTTCAAATTACACACGGGAAGGCTGACGGCGAC
GCACTGGTCGTTCTAGCTAGCGCCGGTAAGGACTTTGAGCTGCGTCTGCCA
GTTGACGCTCGCACGCAGACCACCTCCACAGGTGAGTCGGCTGACCCCGT
GACTGCCACTGTTGAGAACTACGGTGGTGAGACACAGGTCCAGAGACGC
CAACACACGGATGTCTCGTTCATATTAGACAGATTTGTGAAAGTAACACCA
AAAGACCAAATTAATGTGTTGGACCTGATGCAAACCCCTGCACACACTTTG
GTAGGCGCGCTCCTCCGTACTGCCACCTACTACTTCGCAGATCTAGAAGTG
GCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGCGCCCG
AGACAGCGTTGGACAACACCACCAATCCAACGGCTTACCACAAGGCACCG
CTCACCCGGCTTGCACTGCCTTACACGGCACCACACCGTGTCTTGGCTACT
GTTTACAACGGGAACTGCAAGTATGACGAGAGCCCCGTGACCAATGTGAG
AGGTGACCTGCAAGTGTTGGCCCAGAAGGCGGCAAGAACGCTGCCTACCT
CCTTCAATTACGGTGCCGTCAAAGCCACTCGGGTGACTGAACTGCTTTACC
GCATGAAGAGGGCCGAAACATACTGCCCCCGGCCTCTTTTGGCTATTCACC
CGAGCGAAGCTAGACACAAACAAAAGATTGTGGCGCCTGTGAAACAGCT
TTTGAACTTTGACCTGCTCAAGTTGGCAGGAGACGTCGAGTCCAACCCTG
GGCCCTTCTTCTCTGACGTCAGGTCAAATCTTTCCAAGTTGGTTGAAACCA
TCAACCAGATGCAGGAGGACATGTCGGATCCAGGACCGGTGAAGAAACCT
GTCGCTTTGAAAGTGAAAGCAAAGAATTTGATTGTCACTGAGAGTGGTGC
TCCCCCGACTGACTTGCAAAAGATGGTCATGGGTAACACCAAGCCTGTTG
AGCTCATCCTCGACGGGAAGACGGTGGCCATCTGCTGCGCCACCGGAGTG
TTTGGTACTGCCTACCTTGTTCCTCGTCATCTTTTCGCAGAGAAGTATGACA
AGATCATGTTGGACGGCAGAGCCATGACAGACAGTGACTACAGAGTGTTT
GAGTTTGAGATTAAAGTGAAAGGACAGGACATGCTCTCAGACGCCGCGCT
CATGGTGCTTCACCGTGGGAATCGCGTGCGGGACATCACGAAGCACTTCC
GTGATGTGGCAAGAATGAAGAAAGGCACCCCCGTCGTCGGCGTGATCAAC
AACGCTGATGTTGGGAGACTGATCTTCTCTGGTGAGGCCCTTACCTACAAG
GACATTGTAGTGTGCATGGACGGAGACACCATGCCCGGTCTCTTCGCCTAC
AAAGCCGCCACCAAGGCGGGTTACTGTGGAGGAGCCGTTCTTGCAAAGG
ACGGAGCCGAGACTTTCATCGTCGGCACTCACTCCGCAGGCGGCAATGGA
GTTGGATACTGCTCATGCGTTTCCAGGTCTATGCTGCTTAAAATGAAGGCG
CACATCGATCCCGAACCACACCACGAGTAG
12, transfection HEK293 cell produces recombinant adenovirus
Extract recombinant adenovirus plasmid with a small amount of plasmid extraction kit (precious biological product), get 20 μ g PacI enzyme action linearize, with the 70% ethanol precipitation enzyme action product that spends the night, 12000r/min, centrifugal 10min, sterile working, remove supernatant, dry up DNA, reuse 20 μ L sterilization ultra-pure water dissolution precipitation, standby.With liposome 2000 transfection reagents (Lipofectamine 2000, the Invitrogen product) transfection HEK293 cell, transfection method carries out with reference to the transfection reagent description.
Specific as follows: preceding 24 hours of transfection, the HEK293 cell is assigned to the 60mm culture dish, treating that cell is long carries out transfection completely the time to about 80%.Use OPTI-MEM culture medium (Invitrogen product) dilution DNA and the liposome of serum-free earlier, the DNA of 2 μ g and the liposome of 10 μ L are diluted to respectively among the OPTI-MEM of 50 μ L, behind the two mixing, room temperature effect 20min, slowly be added drop-wise to then on 24 hours the cell monolayer of growth, put in 37 ℃ of constant incubators that contain 5% carbon dioxide and cultivated 4-8 hour.Afterwards, add the culture fluid continuation cultivation that equivalent contains 20% hyclone.Every day observation of cell pathological changes situation.7～10d after the transfection, centrifugal collection transfectional cell, resuspended with PBS, 37 ℃/-70 ℃ multigelations 4 times, centrifugal, get supernatant and infect 293 cells that grow up to monolayer again.Infect back 3～5d collecting cell repeated transmission, when tangible cytopathy occurring, carry out the mensuration of virus titer and the detection of expression product.
The evaluation of recombinant virus:
(1) indirect fluorescent antibody test testing goal expression of gene
Get the coverslip that 24h after the transfection covers with cell,, wash with PBS after putting drying at room temperature with the fixing 30min of 4 ℃ in acetone; Add the anti-FMDV positive serum of rabbit, put 37 ℃ of effect 1h in the wet box; Wash 4 times with the PBST that contains 0.05% tween 20, wash 1 time with PBS then, each 10min; Add fluorescein-labeled goat anti-rabbit igg, put 37 ℃ of effect 1h in the wet box; After the same washing, the glycerol mounting is put under the fluorescence microscope and is observed.As a result, 24h gets the cell film flying and carries out indirect fluorescent antibody dyeing after the transfection, and as seen transfectional cell has antigen presentation.
(2) RT-PCR detects the generation of recombinant virus
Get 24h cell behind the 4th generation viral infection, extract test kit with QIAGEN RNA and extract cell total rna, carry out RT-PCR and detect.Result: RT-PCR expands the fragment that the expection size.The illustration purpose gene has transcript and expression in cell.
(3) recombinant virus stability and malicious valency are measured
Collect 4,6,8 and 10 generations virus supernatant, 20 μ L, adding E.C. 3.4.21.64, to get 2 μ L behind 56 ℃ of effect 1h be that template is carried out pcr amplification, to detect the stability of recombinant virus.Get the 8th generation virus liquid and be diluted to 10 for 10 times
-8, get 10
-4～10
-8Each 1mL to 6 porocyte culture plate of virus dilution liquid carries out viral level to be measured, and carries out according to a conventional method.The result: get 4,6,8 and 10 generation viral DNA be that template pcr amplification genes of interest all expands the fragment that the expection size.Illustrate recombinant virus 10 generations with interior be stable.Get the 8th generation virus carry out malicious valency and measure, virus titer is 10
9PFU/mL.
(4) electron microscopic observation of recombinant adenovirus
24h behind the 8th generation viral infection, cytopathy is complete, and centrifugal collecting cell adds the PBS re-suspended cell ,-70 ℃/37 ℃ multigelations 3 times, centrifuging and taking supernatant 20 μ L, the phosphotungstic acid negative staining is put and is observed morphology of virus under the ultramicroscope.The result: electron microscopic observation recombinant virus diameter is about 80nm, is regular hexagon, and size shape is similar to adenovirus.Illustrate that the cytopathy that is produced is caused by recombinant adenovirus.
13, a large amount of cultivations and the purifying process of recombinant adenovirus
With aforementioned that institute's virus is passed through to amplify step by step, obtain product.Can cultivate in rolling bottle HEK293 is cell adapted, with the rotating speed that per minute 8-9 changes, cell attachment is functional, and cellular morphology is normal, and growth is very fast.Generally minute after cover with in 2 days, connect poison with the virus quantity of 20MOI (infestation index promptly infects the viral unit of each cell), treat that pathological changes receives poison in (cell detachment) back fully.The centrifugal 20min of 5000r/min collects sick cell and supernatant, and cell precipitation is resuspended with the PBS buffer, multigelation 3 times, and the centrifugal 10min of 12000r/min collects supernatant and is viral liquid, is further purified; Cell precipitation is resuspended with first centrifugal supernatant, puts 4 ℃ and spends the night and soak poison, and is centrifugal again, abandons cell precipitation, and viral supernatant gives over to and is further purified.The PELLICON ultrafilter membrane (Millipore company product) that the viral supernatant of collecting is passed through 300K further concentrates and purified virus, removes some small molecular proteins and exchange buffering liquid.Virus by concentrated and purification can be used for vaccine production.Be used for animal immune after the purified virus packing.
During this vaccine of suitability for industrialized production, need to use rolling bottle and microcarrier suspension culture method to produce virus.The purification of virus can adopt the ultrafiltration and concentration purification.
The immunoprotection test
(1) Cavia porcellus immunization experiment
Experimental animal is the Cavia porcellus of body weight 300～400g, totally 24, is equally divided into 7 groups.One group is the contrasts of 293 cells, every injection 0.5mL 293 cell suspension; Two groups is Ad5 wild-type adenovirus immune group, and every injection 0.5mL Ad5 wild-type adenovirus (is about 1～5 * 10
8Individual TCID
50); Three groups and four groups are respectively adeno-p12a * 3c 1 * 10
8With 2 * 10
8Individual TCID
50The virus quantity immune group; Immunization route is the hindlimb muscle injection.
After immunity the 25th day, adapt to poison with the homotype Cavia porcellus and attack every 10,000 Cavia porcellus 50 3nfective dose (GID
50), the hind leg foot palm part is subcutaneous injects 0.1mL respectively with Intradermal, only injects a sole.Every day twice behind the counteracting toxic substances, observe the Cavia porcellus incidence, and record.Criterion: blister appears in former injection position, and the Secondary cases blister appears in all the other tripodia ends, is judged to 100% protection.
The result is as follows: counteracting toxic substances is after 48 hours, 1 and 2 group of Cavia porcellus begins morbidity, beginning to be mainly former position occurs red and swollen, heating paresthesia, then blister occurs, except that the 3rd and the 4th group, other two groups former and Secondary cases blister all occur after 72 hours, 3 groups and 4 groups all have four equal ends of ball portion of a Cavia porcellus blister to occur, only slightly enlargement; There is Cavia porcellus (the only ball portion appearance after of slight Secondary cases blister to occur except that 3 groups, two front foots to the are still normal in the time of 10 days) the Secondary cases blister appears in all the other equal ends, and the blister area at former position also is significantly less than all the other each groups, substantially, rehabilitation (no redness and heating paresthesia during by the 10th day, the blister incrustation, ulcer surface disappears substantially).The immune protective rate of immune group Cavia porcellus is respectively 83.3% and 100% as a result, and matched group is not all protected.Explanation thus, recombinant adenovirus has good immanoprotection action to Cavia porcellus.See Table 1.
Table 1 Cavia porcellus counteracting toxic substances protection test result
| Group | 1 | 2 | 3 | 4 |
| Former vesicating | 6/6 | 6/6 | 5/6 | 5/6 |
| The secondary blister | 6×6 | 6×6 | 1/6 | 0/6 |
| Protective rate | 0 | 0 | 83.3％ | 100％ |
(2) recombinant adenovirus is to the immunoprotection experiment of pig
Test is respectful white product boar with pig, about 4～5 monthly ages, and body weight 30～40kg, totally 8, purchase in the Wuwei Prefecture, Gansu Province, be healthy non-foot-and-mouth disease vaccine immune swine, detect the foot and mouth disease virus antibody titer all less than 4 with liquid phase blocking-up ELISA method before the test.Test pig is divided into two groups at random, 4 every group: the A group is the counteracting toxic substances matched group, and isolated rearing is not immune; The B group is recombinant adenovirus immune group, every pig basal part of the ear intramuscular inoculation 1 * 10
9Individual TCID
50Virus quantity.
Preceding and immunity back blood sampling in the 7th, 14,21, the 28 day 5ml respectively at immunity, separation of serum is blocked ELISA detection specificity antibody with liquid phase.Block the ELISA test kit mutually with Lanzhou veterinary institute foot and mouth disease virus antibody liquid and detect all porcine blood serum antibody, carry out according to the test kit operating instruction.
Inject 20 pig infective doses with every pig of type seedling poison virulent strain through musculi colli with O, carry out counteracting toxic substances, observe pathological changes situation and record by the sky.
The result:
(1) liquid phase blocking-up ELISA antibody horizontal sees Table 2.
Table 2 test pig liquid phase blocking-up ELISA antibody horizontal
| Group | Overbit | First week | Second week | The 3rd week | Around |
| A group counteracting toxic substances matched group | 1 # | ＜4 | ＜4 | ＜4 | ＜4 |
| 2 # | ＜4 | ＜4 | ＜4 | ＜4 | |
| 3 # | ＜4 | ＜4 | ＜4 | ＜4 | |
| 4 # | ＜4 | ＜4 | ＜4 | ＜4 | |
| B group recombinant virus immune group | 1 # | ＜4 | ＜4 | ＜4 | 11 |
| 2 # | ＜4 | ＜4 | 11 | 11 | |
| 3 # | ＜4 | 11 | 22 | 22 | |
| 4 # | ＜4 | 11 | 11 | 8 |
According to the criterion of test kit, antibody horizontal＜4 are negative, and antibody horizontal 〉=8 are judged to the positive, and antibody horizontal 〉=32 o'clock have the protection more than 95%.The antibody horizontal of No. 3 and No. 4 pigs of B group raise when second week, and antibody horizontal reaches the highest when the 3rd week, No. 1 pig antibody horizontal after immunity around the side positive, illustrate that the immunoreation of this pig generation is slower.
(2) protection test of pig counteracting toxic substances the results are shown in Table 3.
Table 3 counteracting toxic substances protection test result
| Project | Group and numbering | |||||||
| A-1 | A-2 | A-3 | A-4 | B-1 | B-2 | B-3 | B-4 | |
| 72h behind the counteracting toxic substances | ++++ | ++++ | ++++ | ++++ | - | - | - | - |
| Behind the counteracting toxic substances 8 days | ++++ | ++++ | ++++ | ++++ | ++ | - | - | - |
| Behind the counteracting toxic substances 11 days | ++++ | ++++ | ++++ | ++++ | +++ | - | - | - |
| Protective rate (%) | 0 | 75％ | ||||||
Annotate: ++ ++ show whole clinical symptoms; +++hoof is not exclusively fallen ill; ++ 1 hoof morbidity is arranged;-expression is morbidity not
A group contrast pig all began morbidity in second day behind counteracting toxic substances, temperature of pig body rising, lethargy, inappetence, and hoof is walked lamely bitterly, has blister to occur at coronet and frog portion.Serious to morbidity in the 4th day, pig does not play coffin edge debacle, blister erosion with crouching.This group presentation of results counteracting toxic substances seedling seed poison is a kind of virulent strain, has very strong pathogenic effects, reaches the purpose of experimental control.Walking lamely appears in No. 1 pig of immune group behind counteracting toxic substances the 8th day, right back hoof, and the hoof edge has blister to occur, and also fall ill at the 9th day left back hoof, but the symptom of this pig does not have the serious of A group, and the course of disease postpones.Clinical symptoms does not appear during 3 pigs to 11 of all the other of immune group day yet.The description of test recombinant adenovirus has good immanoprotection action to pig, and the test protective rate can reach 75%, proves that the present invention can become a kind of novel gene engineering recombinant virus live vector vaccine of alternative foot and mouth disease inactivated vaccine.
Claims (5)
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