CN101724636B - Asial type foot-and-mouth disease virus (FMDV) infectious cDNA and construction method as well as application thereof - Google Patents
Asial type foot-and-mouth disease virus (FMDV) infectious cDNA and construction method as well as application thereof Download PDFInfo
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Abstract
The invention discloses Asial type Jiangsu pedigree foot-and-mouth disease virus (FMDV) infectious cDNA and a construction method as well as application thereof. The infectious cDNA contains a poly (A) sequence and a poly (C) sequence required for ensuring synthesis and transcription of the infectious cDNA; the tail end of the poly (A) sequence has 19-22 A; the poly (C) structure has 14-20 polymerized C; and the 5'end has a T7RNA polymerase promoter sequence, and the 3' end has single restriction enzyme sites. The infectious cDNA and an eukaryotic expression vector for expressing T7RNA polymerase can form a reverse genetic operating system. The infectious cDNA and the reverse genetic operating system thereof can be used for curing the FMDV, and establish the experimental basis for researching the structure and the functions of FMDV genes, researching the molecular pathogenesis of the FMDV, developing attenuated vaccine strains of the FMDV, developing novel FMDV vaccines, and the like.
Description
Technical field
The present invention relates to the infections clone of sub-thread positive chain RNA virus; Relate in particular to Asia1 type foot and mouth disease virus infectious CDNA and construction process thereof, the invention still further relates to the reverse genetic operating system of the Jiangsu pedigree Asia1 type foot and mouth disease virus of forming by this Asia1 type foot and mouth disease virus infectious CDNA and the carrier for expression of eukaryon of expressing t7 rna polymerase; The present invention also further relates to this infectious CDNA through in-vitro transcription and mode the application in rescue foot and mouth disease virus of this reverse genetic operating system through transcribing in the body, belongs to the genetically engineered field.
Background technology
(Foot-and-mouth disease virus FMDV) belongs to the member of Picornaviridae Hostis to foot and mouth disease virus, is the sub-thread positive chain RNA, the about 8.5kb of its geneome RNA total length.Foot and mouth disease is one of serious harm important transmissible disease artiodactylous, and this disease causes that cub is dead, milk yield descends, meat reduces, meat descends, the production performance of animal reduces, and causes enormous economic loss.In addition, owing to the restriction of trade with forbid that the loss that causes is bigger, the direct economic loss that the whole world causes every year thus can reach tens billion of dollars.Asia1 type FMD (Asia1/JS/CHA/05, GenBank accession number: EF149009),, be known as Asia1 type Jiangsu pedigree have been broken out since 2005 owing at first separate in China Jiangsu Province on China Jiangsu, Shandong, Gansu, Beijing, Hebei and other places.Once popular, propagation trend is swift and violent in China cows for this pedigree foot and mouth disease virus, and the loss that causes is very serious.Therefore, actively develop the research of Asia1 type FMD Jiangsu pedigree strain, the prevention and control of China FMD are had great importance.
Reverse genetic operating system is in one of important tool of molecular level research virus.Through viral genome being carried out rite-directed mutagenesis, disappearance, the genomic arbitrary portion of replacement to produce two mutants, whereby on molecular level the structure of research virogene with function, understand molecular basis, host's preferendum that determines virus virulence and the mechanism of striding kind of propagation.Infections clone also can be used for the research of the research of epitope, particularly conformational epitope.Established experiment basis for studying viral attenuation, for the development new generation vaccine provides strong instrument.
There has been report successfully to make up ox source strain 01K strain (Zibert A at present; Maass G; Strebel K, et al.Infectious foot-and-mouth disease viruses derived from acloned full-length cDNA [J] .J Virol, 1990; 64:2467-2473), ox source strain A12 strain (Rieder E; Bunch T, Brown F, et al.Genetically engineeredfoot-and-mouth disease viruses with poly (C) tracts of two nucleotide arevirulent in mice [J] .J Virol; 1993; 67:5139-5145), the strain OH/99 strain of pig source (Guanqing liu, et al.Generation of an infectious cDNA clone of anFMDV strain isolated from swine.Virus Research, 2004; 104:157-164) and rabbitization attenuated vaccine strain ZB/CHA/58 (att) (Chinese patent publication number: CN101225395A, denomination of invention: foot-and-mouth disease virus Asia I infectious cDNA and construction process thereof.) infectious CDNA of foot and mouth disease virus, and on this basis FMDV has been carried out exploratory research.But above-mentioned infections clone and China recent years popular Jiangsu pedigree Asia1 type foot and mouth disease virus have very big genetic divergence; Therefore make up the reverse genetic manipulation platform of the Jiangsu pedigree Asia1 type foot and mouth disease virus that meets China's popularity, the development of carrying out Asia1 type foot and mouth disease virus structure and functional study and new generation vaccine is had great importance.
Summary of the invention
Primary and foremost purpose of the present invention provides the full-length infectious CDNA of a kind of Asia1 type Jiangsu pedigree foot and mouth disease virus, and this full-length infectious CDNA has comprised assurance institute synthetic transcript and had infectious desired poIy (A) sequence and poIy (C) sequence;
Full-length infectious CDNA clones 5 ' the end of said Asia1 type foot and mouth disease virus has the t7 rna polymerase promoter sequence, and 3 ' end has single restriction enzyme site; Wherein, described 3 ' end limit property restriction enzyme site is EcoRV restriction enzyme site or NotI restriction enzyme site; 5 ' the terminal upper reaches have the SphI restriction enzyme site.
The full-length infectious CDNA clones of said Asia1 type foot and mouth disease virus is except promotor, and outside poIy (A) sequence and poIy (C) sequence, remaining nucleotide sequence and foot-and-mouth disease virus gene group native sequences differ and be no more than 6 nucleotide sequences;
PoIy (C) structure of the full-length infectious CDNA clones of said Asia1 type foot and mouth disease virus has 14-20 polymerization C, and poIy (A) tail has 19-22 A (more preferably having 19 A); PoIy (C) structure and flanking sequence thereof are directly to increase through RT-PCR to obtain, and utilize two restriction enzyme enzyme recognition sites that exist naturally in the FMDV genome, and the fragment that comprises poIy (C) structure that obtains is imported in the full-length cDNA.
The full-length infectious CDNA of said Asia1 type foot and mouth disease virus is a carrier with pBluescriptSK (+);
Further preferred, the full-length infectious CDNA of said Asia1 type foot and mouth disease virus comprises the sequence shown in the SEQ ID NO:1.
A kind of method that makes up the full-length infectious CDNA of above-mentioned Asia1 type Jiangsu pedigree foot and mouth disease virus comprises:
(1) is template with Asia1 type Jiangsu pedigree foot and mouth disease virus RNA, uses 5 ' end sequence fragment of 5 ' RACE method amplification gene group;
(2) adopt RT-PCR to obtain 6 fragments of foot-and-mouth disease virus gene group each several part, comprise poIy (C) structure and flanking sequence thereof;
(3) adopt digestion with restriction enzyme clone's method assembling to be cloned in the plasmid vector institute's amplification PCR fragment, full length cDNA clone 5 ' end has the t7 rna polymerase promoter sequence, and 3 ' end has single restriction enzyme site;
(4) each fragment of said cDNA is connected, constitute the plasmid vector that has the genome full-length cDNA; Purifying and recovering comprises the plasmid of viral genome full-length cDNA, promptly gets.
Wherein, step (1) primer sequence of using is shown in SEQ ID NO:15, the SEQ ID NO:16; The primer sequence of using is shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, the SEQ ID NO:14 in the step (2);
Plasmid vector described in the step (3) is pBluescript II SK (+) carrier;
Another object of the present invention provides a kind of Asia1 type foot and mouth disease virus reverse genetic operating system, and this Asia1 type foot and mouth disease virus reverse genetic operating system is made up of with the carrier for expression of eukaryon of expressing t7 rna polymerase the full-length infectious CDNA of above-mentioned Asia1 type Jiangsu pedigree foot and mouth disease virus.
The carrier for expression of eukaryon pT7RNAP of described expression t7 rna polymerase; Its construction process comprises: with the t7 rna polymerase gene clone in carrier for expression of eukaryon pCDNA3.1; And in 5 of this gene ' end introducing Kozak sequence, to guarantee efficiently expressing of t7 rna polymerase;
Simultaneously; Whether expression vector pT7RNAP expresses pT7RNAP and whether this enzyme has transcriptional activity in order to detect; Having made up is the detection carrier pT7-IRES-EGFP of detection signal with the green fluorescent protein; This carrier is to utilize the IRES of FMDV can mediate the characteristic of non-cap dependency expression alien gene, IRES and the EGFP of FMDV is cloned among the prokaryotic expression carrier PET-28a in order, and makes it to be controlled by under the IRES element of foot and mouth disease virus;
Above-mentioned two recombinant plasmid cotransfection BHK-21 cells, cultivate 20～48h, observation can be seen typical green fluorescence under ultraviolet microscope, shows that the pT7RNAP carrier can express t7 rna polymerase and have transcriptional activity.
Another object of the present invention is that the full-length infectious CDNA with constructed Asia1 type Jiangsu pedigree foot and mouth disease virus is used to save foot and mouth disease virus, comprising: the mode transfection BHK-21 cell that the full-length infectious CDNA of said Asia1 type Jiangsu pedigree foot and mouth disease virus is adopted in-vitro transcription; Or with the full-length infectious CDNA of said Asia1 type Jiangsu pedigree foot and mouth disease virus and carrier for expression of eukaryon cotransfection (transcribing in the body) the BHK-21 cell of expressing t7 rna polymerase with the rescue foot and mouth disease virus;
Evidence, the not human relations of full-length infectious CDNA that utilize the constructed Asia1 type Jiangsu pedigree foot and mouth disease virus of the present invention are to adopt in-vitro transcription or adopt in the body mode of transcribing (with the carrier for expression of eukaryon cotransfection of expressing t7 rna polymerase) all can successful rescue go out foot and mouth disease virus.
The place that foot and mouth disease virus infectious CDNA of the present invention is different from domestic and international foot and mouth disease virus infectious CDNA mainly contain following some:
(1) infectious CDNA of the U.S. is that material development comes out with A type foot and mouth disease virus, and the infectious CDNA of Germany is that the representative strains 01K with European epidemic isolates is raw-material, separates from the ox body.The strain that the present invention selected for use is China's popular Asia1 type Jiangsu pedigree aphthovirus Asial/YS/CHA/05 strain; Separation is from the ox body; The epidemic characteristic that has reflected China's foot and mouth disease; Therefore foot and mouth disease virus infectious CDNA of the present invention has more specific aim to the research of China's foot and mouth disease, and the prevention and control of China's foot and mouth disease are had positive meaning.
(2) strategy of development infectious CDNA is different:
The A strain infectious CDNA of the U.S. is poly (the C) (Cn=2,6,16,25 with the synthetic a series of length of manual method; 35); And artificial add the restriction enzyme enzyme recognition site at poly (C) two ends; And in genome full-length cDNA fragment downstream, add corresponding restriction endonuclease recognition site, thus poly (C) is imported in the full length cDNA sequence, this strategy can cause the change of base and introduce the Nucleotide of non-genomic group in full-length cDNA.
The 01K strain infectious CDNA of Germany is the enzymatic activity that utilizes terminal enzyme (DNA), adds poly (C) structure to full-length cDNA, and this method can not artificially be controlled, poor operability, and also needs change the true sequence of full-length cDNA.
The present invention directly increases through RT-PCR and obtains poly (C) and flanking sequence thereof; Utilize the restriction enzyme enzyme recognition site that exists naturally in the FMDV genome; Poly (C) structure is imported in the full-length cDNA, need not change genomic true sequence, workable.
(3) poly that is comprised (A) tail sequence length is different: the A type infectious CDNA of the U.S. contains 15 A, and the 01K strain infectious CDNA of Germany contains 90 A.Infectious CDNA of the present invention contains poly (A) structure of 19 these length of A, can guarantee the needs of viral infection.
(4) the reverse genetic manipulation platform of setting up foot and mouth disease virus both at home and abroad all is to use the method rescue virus of in-vitro transcription, and the present invention has set up the dual system of transcribing in the body of rescue foot and mouth disease virus with in-vitro transcription.
At present, judge that the constructed foot and mouth disease virus infectious CDNA key that whether meets the requirements will satisfy following some requirement:
(1) viral genome will be guaranteed its verity in the molecular cloning process: thus the structure that just might influence whole genome because of the variation of a base in the ORFs causes whole total length plasmid not possess infectivity; In addition, because the characteristics of RNA viruses self, its genome is also unstable;
(2) to guarantee that constructed infectious CDNA contains sufficiently long poIy (C) sequence and poIy (A) tail structure;
(3) 3 ' ends have suitable restriction enzyme site;
Full-length infectious CDNA of the present invention is except promotor, poIy (A) sequence and poIy (C) sequence; Remaining nucleotide sequence and foot-and-mouth disease virus gene group native sequences differ and are no more than 6 nucleotide sequences; PoIy (C) structure and flanking sequence thereof are directly to increase through RT-PCR to obtain, so infectious CDNA of the present invention its verity in clone's process is high; In addition, the poIy of full-length infectious CDNA of the present invention (A) tail has 19-22 A; Said poIy (C) structure has 14-20 polymerization C, contains sufficiently long poIy (C) sequence and poIy (A) tail structure; 3 ' end of said infectious CDNA has EcoRV restriction enzyme site or NotI restriction enzyme site.
The successful structure of infectious CDNA of the present invention will provide a strong gene engineering service platform for the research foot and mouth disease, can be convenient for people to the virus genomic operation of foot and mouth disease.The concrete application mainly contains the following aspects:
(1) structure and the function of understanding gene;
(2) pathogenesis and genomic of understanding virus duplicates, expression and regulation mechanism;
The interaction of (3) research virus and host cell;
The B cell epitope of (4) research virus is conformation type B cell epitope particularly;
(5) prepare the attenuated vaccine strain that the virulence reversion can not take place through reverse genetic operating system;
(6) research and develop novel aftosa vaccine;
Asia1 type foot and mouth disease virus infectious CDNA of the present invention; Can be on molecular level through sudden change, insert, foot and mouth disease virus is studied in disappearance and replacement duplicates, the structure of the molecule determinative of virulence and attenuation mechanism, molecule pathogenesis, gene product is with function, the change of host's preferendum and stride kind of a propagation; And the research and development of new generation vaccines such as attenuated live vaccine, epiposition vaccine, nucleic acid vaccine, marker vaccine, be the extremely valuable instrument of further studying foot and mouth disease virus and anti-system foot and mouth disease.
Description of drawings
Figure 1A sia1/YS/CHA/05 strain FMDV genome splices tactful synoptic diagram.
The structural representation of Fig. 2 pET-28a (+) carrier.
Fig. 3 pBluescript II SK (+).
Fig. 4 pcDNA3.1 (+).
The enzyme of the full genome cDNA of Fig. 5 is cut evaluation figure: A:1kb Ladder; B:Sph1 and Not1 double digestion are identified; C:1kb Ladder; The D:EcoR1 single endonuclease digestion is identified.
The RNA electrophorogram of Fig. 6 in-vitro transcription.
The normal BHK-21 cell of BHK-21 cell (B) of Fig. 7 (A) rescue virus infection.
The immune electron microscopy (bar=100nm) of Fig. 8 foot and mouth disease virus particle.
Fig. 9 saves evaluation (A) the lane1:100bp DNA Ladder of viruses molecule label; Lane2: parent's poison PCR product P stI enzyme is cut evaluation; Lane3: rescue virus PCR product P stI enzyme is cut the sequential analysis checking of the viral PstI of sequential analysis (C) the rescue site elimination in evaluation (B) parent strain genome PstI site district.
The immunofluorescence that infects the BHK-21 cell of saving Figure 10 detects the normal BHK-21 cell contrast of BHK-21 cell (B) parental virus infected B HK-21 cell (C) of (A) rescue virus infection.
Figure 11 saves the one step growth of virus and parental virus.
48h EGFP expression behind the expression of Figure 12 t7 rna polymerase and active detection (A) pT7RNAP and the pT7-IRES-EGFP carrier corotation BHK-21 cell; (B) pEGFP-N1 carrier transfection BHK-21 cell is as positive control; (C) pT7-IRES-EGFP carrier transfection BHK-21 cell is as negative control.
Figure 13 pT7RNAP carrier and pT7-IRES-EGFP carrier synoptic diagram.
Embodiment
One, test materials, relational term and method
In the present invention; Described term " reverse genetic manipulation " refers to: for classical genetics; Be on the basis that obtains foot and mouth disease virus (Asia1/YS/CHA/05 strain) genome full sequence,, let it assemble out and have bioactive virus particle by forming sequential build total length viral genome; For further foot and mouth disease virus-virus gene being carried out necessary processing and modification; Like rite-directed mutagenesis, gene insertion/disappearance, gene substitution etc., thereby study genomic structure and function, and these modifications possibly there is the content of aspects such as which kind of influence to establish experiment basis to phenotype, the proterties of virus.
Asia1 type hoof-and-mouth disease poison strain according to the invention refers to the Asia1/YS/CHA/05 strain.
Used reference among the present invention: (1) Wang Haiwei, Tu Yabin, Zhou Guohui, Yang Decheng, Zhang Yake, in power, the foundation of Asia1 type foot and mouth disease virus infections clone.China's Preventive Veterinary Medicine newspaper, 2008 the 12nd phases; (2) Yang Decheng, Tu Yabin, Wang Haiwei, Zhou Guohui, in power, the foundation of O type foot and mouth disease virus Pan Asia pedigree infections clone, Chinese Preventive Veterinary Medicine newspaper, 2009 the 1st phases; (3) Zibert A, Maass G, Strebel K, etal.Infectious foot-and-mouth disease viruses derived from a cloned full-length cDNA.J Virol, 1990,64:2467-2473; (4) Rieder E; Bunch T, Brown F, et al.Genetically engineered foot-and-mouth disease viruseswith poly (C) tracts of two nucleotide are virulent in mice.J Virol; 1993,67:5139-5145.
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, the condition described in " molecular cloning: lab guide " (New York:Cold Spring HarborLaboratory Press, 2001) is carried out.
In an embodiment of the present invention, (depositary institution: Chinese common micro-organisms culture presevation administrative center preserves the plasmid pASi that contains aphthovirus Asial/YS/CHA/05 strain complete genome group of use; Preservation address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date: on October 08th, 2008; Its microbial preservation number is: CGMCC 2689; Its called after of classifying: ETEC (Escherichia coli)); (depositary institution: Chinese common micro-organisms culture presevation administrative center preserves the eukaryotic expression plasmid pT7RNAP of T7 RNA polymerase; Preservation address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date: on October 08th, 2008; Its microbial preservation number is: CGMCC 2688, its called after of classifying: ETEC (Escherichia coli)); (depositary institution: Chinese common micro-organisms culture presevation administrative center preserves to detect the vector plasmid pT7-IRES-EGFP of T7 rna polymerase activity; Preservation address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date: on October 08th, 2008; Its mikrobe preserves and number is: CGMCC 2690; Its called after of classifying: ETEC (Escherichia coli)).
In an embodiment of the present invention, the plasmid of use and bacterial strain: pBluescript II SK (+) carrier is available from Stratagene company, and DH5 α competent cell, BHK-21 cell are preserved by this laboratory.
In an embodiment of the present invention, other reagent of use: PrimeSTAR archaeal dna polymerase, various restriction enzyme, dNTPs, DNA Ladder Marker, 5 ' FULL-RACEKit are available from TaKaRa company; SEAP, T4 dna ligase New EnglandBiolabs company; TRIzol is available from GibcoBRL company, and in-vitro transcription adopts the RiboMAX of Promega company
TMLarge Scale RNA Production Systems-T7 test kit.Transfection reagent adopts Effectene
the Transfection Reagent transfection reagent box of QIAGEN company, and sheep anti mouse fluorescence two is anti-available from Sigma company.SDS, agarose, Tryptones, yeast extract, Tris, EDTA, diethylpyrocarbonate reagent such as (DEPC) are the packing of external product domestic corporation; Sodium-chlor, chloroform, Virahol, ethanol, primary isoamyl alcohol, NaCl, MgCl
2, SODIUM PHOSPHATE, MONOBASIC, saturated phenol etc. be homemade AR.
The foundation of embodiment 1 Asia1 type foot and mouth disease virus Jiangsu pedigree reverse genetic operating system
In an embodiment of the present invention, BHK-21 monolayer culture condition: nutrient solution is the DMEM that contains 10% foetal calf serum, 37 ℃ of cultivations in containing the incubator of 5%CO2.(the GenBank accession number: nucleotide sequence homology EF149009) is more than 98% for Asia1 type aphthovirus Asial/YS/CHA/05 strain and Asia1/JS/CHA/05 strain; Belong to identical source; Its genome total length 8193bp comprises 14nt poly (C) and 19nt poly (A).Virus is bred in containing the DMEM nutrient solution of 2% foetal calf serum, results virus when CPE appears in the cell when 80%, and behind the multigelation three times, the centrifugal 10min of 2000 * g (4 ℃), supernatant is in-70 ℃ of preservations.
1.1 design of primers
According to the genome sequencing result of aphthovirus Asial/YS/CHA/05 strain, the design primer is respectively as the primer of pcr amplification;
1.2?5′RACE
With 5 ' FULL-RACE test kit, adopt " going cap method principle ", measure the true sequence of viral genome 5 ' end.In brief, with 5 ' the end cap minor structure that Tobacco Acid Pyrophosphatase (TAP) removes mRNA, use T afterwards
4RNA Ligase is connected to 5 ' of mRNA with 5 ' RACEAdaptor and holds, and uses primer and known array primer partly on 5 ' the RACE Adaptor to carry out reverse transcription and pcr amplification 5 ' end sequence at last, and the primer of employing is seen table 1.
The primer of table 1 FMDV Asia1/YS/CHA/05 pnca gene group pcr amplification
Reverse transcription: extract viral RNA according to TRIzol reagent (Gibco BRL) product description, reaction system is 20 μ l:H
2O 6 μ l, 5x damping fluid 4 μ l, dNTPs (2.5mM) 2 μ l, the reverse transcription primer is RACE2,2 μ l, AMV ThermoScript II (TakaRa) 1 μ l, viral RNA 5 μ l.Reaction conditions is 25 ℃ of 10min, 42 ℃ of 1h, 4 ℃ of 20min.
Pcr amplification: Outer PCR reaction system is 50 μ l:5x damping fluids, 10 μ l, dNTPs (2.5mM) 4 μ l, 5 ' RACE Outer Primer, 2 μ l, RACE2 2 μ l, archaeal dna polymerase 1 μ l, H
2O 26 μ l, cDNA template 5 μ l.
Inner PCR reaction system is 50 μ l:5x damping fluids, 10 μ l, dNTPs (2.5mM) 4 μ l, 5 ' RACE Inner Primer, 2 μ l, RACE1 2 μ l, archaeal dna polymerase 1 μ l, H
2O 26 μ l, cDNA template 5 μ l.
The PCR response procedures: 94 ℃ of sex change 4min at first, carry out 25 circulations by following parameter again: 94 ℃ of 1min, 56 ℃ of 30s, 72 ℃ of 30s extend 10min in 72 ℃ at last, and the PCR product is electrophoretic separation on 1.5% sepharose, and the PCR product is identified in EB dyeing.1.3 the structure of full-length cDNA carries out following test referring to the technological line synoptic diagram that Fig. 1 makes up the foot and mouth disease virus infectious CDNA.
(1) with TRIzol extracting foot-and-mouth disease virus genome RNA from the cell culture of aphthovirus Asial/YS/CHA/05 strain, concrete operations are undertaken by the test kit process specifications.
(2) use the Auele Specific Primer that designed and the round pcr of employing: the Auele Specific Primer that is designed is: S1, S2, A1, A2, B1, B2, C1, C2, D1, D2, F1, F2; Reverse transcription: extract viral RNA according to TRIzol reagent (Gibco BRL) product description, reaction system is 20 μ l:H
2O 6 μ l, 5x damping fluid 4 μ l, dNTPs (2.5mM) 2 μ l, the reverse transcription primer is RACE2,2 μ l, AMV ThermoScript II (TakaRa) 1 μ l, viral RNA 5 μ l.Reaction conditions is 25 ℃ of 10min, 42 ℃ of 1h, 4 ℃ of 20min.CDNA first chain of synthetic gene group RNA.
With PrimeSTAR HS archaeal dna polymerase (Takara, Dalian, China), segmentation amplification S, A, B, C, D, six fragments of F.Its reaction conditions is: the PCR primer is for seeing table 1, and the reaction system of amplified fragments is 50 μ l:5x damping fluids, 10 μ l, dNTPs4 μ l, upstream primer 2 μ l, downstream primer 2 μ l, archaeal dna polymerase 1 μ l, H
2O 26 μ l, cDNA template 5 μ l.
The PCR response procedures: 94 ℃ of sex change 4min at first, carry out 25 circulations by following parameter again: 94 ℃ of 1min, 56 ℃ of 30s, 72 ℃ of 1-3min extend 10min in 72 ℃ at last, and the PCR product is electrophoretic separation on 1% sepharose, and the PCR product is identified in EB dyeing.With PCR product electrophoretic separation purifying (carrying out) according to Axygen company Agarose Gel DNAPurification Kit operation instructions; Then the PCR product behind the purifying is connected in pBluescript II SK (+) carrier (Stratagene company), will connects product again and transform competent escherichia coli cell DH5 α according to the preparation of Inoue method.The positive bacterium colony that picking is possible, amplification cultivation is extracted matter in a small amount with alkaline lysis and is drawn.Identify positive plasmid (Fig. 5) with pcr amplification method and restriction enzyme complete digestion method respectively.
1.4 the structure of pT7RNAP carrier and pT7-IRES-EGFP carrier and active the detection.
Referring to Figure 13 (pT7RNAP carrier and pT7-IRES-EGFP carrier synoptic diagram)
(1) pT7RNAP vector construction
Use round pcr, use high-fidelity Taq enzyme (TakaRa), amplification T7 rna polymerase gene: the PCR primer is T7RNAP (sense/Antisense; Primer sequence is seen table 2), the reaction system (50ul) of amplification t7 rna polymerase gene is: 5x damping fluid 10ul, dNTP 4ul, upstream primer 2ul, downstream primer 2ul, TaqDNA polysaccharase (TakaRa) 1ul, BL21 (DE3) bacterium liquid 1ul, water 30ul.The PCR response procedures: 94 ℃ of sex change 4min at first, carry out 30 circulations by following parameter again: 94 ℃ of 1min, 56 ℃ of 30s, 72 ℃ of 3min extend 10min in 72 ℃ at last, and the PCR product is about 2652bp.The PCR product electrophoretic separation on 1% sepharose that takes a morsel, the PCR product is identified in EB dyeing.With PCR product electrophoretic separation purifying (carrying out) according to Hua Shun company Agarose Gel DNAPurification Kit operation instructions; Then the PCR product behind the purifying is used restriction enzyme BamHI (TakaRa) and EcoRI (TakaRa) double digestion; After glue is received purifying, be connected among the pCDNA3.1 (Invitrogen), will connect product again according to calcium chloride method transformed into escherichia coli JM109 bacterial strain (TakaRa).The positive bacterium colony that picking is possible, amplification cultivation is extracted matter in a small amount with alkaline lysis and is drawn.Use pcr amplification method and restriction enzyme complete digestion method (BamHI (TakaRa) and EcoRI (TakaRa)) to identify positive plasmid respectively, thereby obtain the pT7RNAP carrier for expression of eukaryon.The PCR reaction of following EGFP gene, IRES sequence, clone, authentication method are identical therewith.
2) structure of pT7-IRES-EGFP carrier
Use round pcr, use high-fidelity Taq enzyme (TakaRa) amplification EGFP gene and IRES sequence, the primer is shown in table one.Its reaction conditions is respectively:
The EGFP gene: the PCR primer is EGFP (sense/Antisense; Primer sequence is seen table 2), template is the pEGFP-N1 plasmid purification.The PCR response procedures: 94 ℃ of sex change 4min at first, carry out 30 circulations by following parameter again: 94 ℃ of 1min, 58 ℃ of 30s, 72 ℃ of 1min extend 10min in 72 ℃ at last, and the PCR product is about 705bp.
The IRES sequence: extracting foot and mouth disease virus RNA according to TRIzol reagent (Gibco BRL) product description, use Oligod (T)-15 to carry out reverse transcription then, is template with this reverse transcription product, uses IRES (sense/Antisense; Primer sequence is seen table 2) primer, the PCR response procedures: 94 ℃ of sex change 4min at first, carry out 30 circulations by following parameter again: 94 ℃ of 1min, 58 ℃ of 30s, 72 ℃ of 1min extend 10min in 72 ℃ at last, and the PCR product is about 434bp.
The PCR product electrophoretic separation on 1% sepharose that takes a morsel respectively, the PCR product is identified in EB dyeing.Respectively with PCR product electrophoretic separation purifying (carrying out) according to QIAGEN company Agarose Gel DNA Purification Kit operation instructions; Then earlier with the PCR product EGFP behind the purifying with BamHI and HindIII double digestion and be connected among the carrier PET-28a of same processing, will connect product transformed into escherichia coli DH5a bacterial strain again.The positive bacterium colony that picking is possible, amplification cultivation is extracted matter in a small amount with alkaline lysis and is drawn.Use pcr amplification method and restriction enzyme complete digestion method (BamHI and HindIII) to identify positive plasmid respectively, thereby obtain recombinant vectors pT7-EGFP.And then with use SpeI of the PCR product IRES behind the purifying and BamHI double digestion; Use XbaI and BamHI to handle carrier pT7-EGFP; Because SpeI and XbaI are isocaudarners; Can the IRES sequence be inserted among the carrier pT7-EGFP, same picking positive colony obtains recombinant vectors pT7-IRES-EGFP.
Table 2 makes up pT7RNAP carrier and pT7-IRES-EGFP carrier the primer
(3) pT7RNAP and pT7-IRES-EGFP plasmid are used chemical transfection reagent (QIAGEN) cotransfection BHK-21 cell, detect the expression and the activity of T7 RNA polymerase.The concrete operations step is following:
When the BHK-21 cell grows to the 70%-90% individual layer in 6 orifice plates, wash cell twice, add 1.5ml normal cell nutrient solution with PBS.The pT7-IRES-EGFP carrier of purifying is mixed back transfection BHK-21 cell with plasmid pT7RNAP equal proportion; The Effectene of QIAGEN company is pressed in transfection
Transfection Reagent transfection reagent box specification sheets carries out, and cells transfected is at 5%CO
2Incubator in 37 ℃ of incubation 4h, change substratum with the DMEM that contains 2% foetal calf serum and continue to cultivate, the expression of observation EGFP under the Olympus fluorescent microscope behind the 48h.
Transfection results: with pT7RNAP and pT7-IRES-EGFP plasmid co-transfection BHK-21 cell; Visible fluorescence under fluorescent microscope (Figure 12 A); Fluorescence intensity is close with pEGFP-N1 positive control (Figure 12 B); Show that EGFP is efficiently expressed, and normal BHK-21 cell there is not fluorescence appearance (Figure 12 C).The result shows that the pT7RNAP carrier can efficiently express the t7 rna polymerase with transcriptional activity.
1.5 in-vitro transcription and transfection
Restriction enzyme EcoRV or NotI 5 μ l, the infectious recombinant plasmid 50 μ l of the above-mentioned constructed foot and mouth disease virus that comprises full-length cDNA are in 37 ℃ of water-bath 3h; After the phenol-chloroform extracting; Add 0.1 times of volume 3M sodium-acetate (pH5.2), the absolute ethyl alcohol of 2.5 times of volumes is placed 15min for-70 ℃; The centrifugal 10min of 1200 * g (4 ℃) abandons supernatant.After 70% ethanol, 800 μ l washing and precipitating, the centrifugal 5min of 1200 * g (4 ℃) abandons supernatant again.The seasoning deposition adds the nuclease free water dissolution to suitable volumes, and-20 ℃ of preservations are subsequent use.Get the 0.5mleppendorf pipe, add 5 * T7 RNA polymerase damping fluid, 4 μ l, each 1.5 μ l of rNTPs (100mmolATP, CTP, GTP, UTP), linear DNA template 8 μ l, T7 RNA polymerase mixture 2 μ l, incubation 2.5h in 37 ℃ of water-baths.Per 1 μ g template DNA adds the DNA nucleicacidase RQ1 1U of no RNA enzyme, 37 ℃ of incubation 15min.The saturated phenol of TE-(pH4.5) with 1 times of volume: chloroform: primary isoamyl alcohol (25: 24: 1) extracting, vibration 1min, the centrifugal 2min of 1200 * g (4 ℃), upper water move to 1 new eppendorf pipe mutually, add isopyknic chloroform: primary isoamyl alcohol (24: 1).Vibration 1min, the centrifugal 2min of 1200 * g (4 ℃); Upper water moves to 1 new eppendorf pipe mutually, adds the 3M sodium-acetate (pH5.2) of 0.1 times of volume and the absolute ethyl alcohol of 2.5 times of volumes, mixing, the centrifugal 10min of 1200 * g behind the ice bath 5min (4 ℃); Carefully outwell supernatant, with 70% ethanol washing and precipitating of DEPC water preparation.After the drying, dissolve the RNA sample again with 20 μ l nuclease free water.The RNA of prepared in-vitro transcription is grown up to the BHK-21 cell of 80% individual layer with Effectene
the Transfection Reagent transfection reagent box transfection of QIAGEN company, and operation is undertaken by product description.Be cultured to after the transfection and can be observed cytopathy, results virus, with the centrifugal 10min of cell culture 2000 * g of continuous 3 freeze thawing, it is viral to get supernatant inoculation BHK-21 cell amplification, and continuous passage 7 times.
1.6 transcribe rescue virus in the body
When the BHK-21 cell grows to the 70%-90% individual layer in 6 orifice plates, wash cell twice, add 1.5ml normal cell nutrient solution with PBS.Mixes the BHK-21 of transfection afterwards cell behind the infectious recombinant plasmid purifying of the foot and mouth disease virus that comprises full-length cDNA that the present invention is constructed with plasmid pT7RNAP equal proportion.The Effectene of QIAGEN company is pressed in transfection
TransfectionReagent transfection reagent box specification sheets carries out, and cells transfected is at 5%CO
2Incubator in 37 ℃ of incubation 4h, change substratum with the DMEM that contains 2% foetal calf serum and continue to cultivate, the observation of cell pathology, about about 3 days results virus is inoculated the BHK-21 cell behind the multigelation 3 times once more, can stablize generation CPE up to virus.
1.7 the electron microscopic observation of virus particle
In the centrifugal 30min of 3300 * g (4 ℃), get supernatant with the BHK-21 cell culture of saving virus infection, virus particle is containing NTE damping fluid (NaCl, the 100Mm of 25% sucrose; Tris, 10mM; EDTA, 1mM [pH 7.4]) in 1 * 10
5Behind the centrifugal 2h of g (4 ℃), will precipitate dissolving, add 1% hyper-immune serum with PBS; 37 ℃ of incubation 2h; 4 ℃ are spent the night then, and the centrifugal 20min of 800 * g (4 ℃) uses 100 μ l PBS dissolution precipitations at last; Send Harbin veterinary institute Electron Microscopy Room to do conventional negative staining, under the JEM-1200EX Electronic Speculum, observe virus particle (Fig. 8).
1.8 indirect immunofluorescence
The cell culture supernatant of gathering in the crops after the transfection is inoculated the BHK-21 cell on 96 orifice plates, and with the fixing 20min of the absolute ethyl alcohol of precooling, raffinate exhausts after the rinsing; Add Asia1/YS/CHA/05 monoclonal antibody specific 3E11 (1: 200) 50 μ l/ holes, put wet box 1h, wash 3 times with PBS; Drip sheep anti mouse FITC (1: 200), put wet box and hatch 50min, PBS washes 3 times; Observe down in Olympus fluorescence inverted microscope, with Leica imaging system take pictures (Figure 10).
1.9 the stability of reverse genetic system
Infections clone carries out sequencing to its full genome after in bacillus coli DH 5 alpha, passing for 10 generations continuously, does not have the sudden change or the disappearance of any base, shows that this recombinant plasmid is high stability in bacillus coli DH 5 alpha; In addition, rescue virus passed for 7 generations continuously in the BHK-21 cell, and its pathology time is identical with the time that CPE appears in parental virus after the 4th generation.The result shows that the Asia1 type FMDV reverse genetic system that the present invention sets up is high stability.
Sequence table
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Claims (6)
1. the full-length infectious CDNA of an Asial type Jiangsu pedigree foot and mouth disease virus, it is characterized in that: described full-length infectious CDNA sequence is the sequence shown in the SEQ ID NO:1.
2. an Asial type foot and mouth disease virus reverse genetic operating system is made up of full-length infectious CDNA and the carrier for expression of eukaryon of expressing t7 rna polymerase of the described Asial type of claim 1 Jiangsu pedigree foot and mouth disease virus.
3. according to the described Asial type of claim 2 foot and mouth disease virus reverse genetic operating system; The carrier for expression of eukaryon that it is characterized in that described expression T7 RNA polymerase is pT7RNAP; Its construction process comprises: the T7 rna polymerase gene is cloned among the carrier for expression of eukaryon pCDNA3.1, and introduces the Kozak sequence at 5 of this gene ' end.
4. according to claim 2 or 3 described Asial type foot and mouth disease virus reverse genetic operating systems, it is characterized in that: also comprise and a kind ofly detect whether the carrier for expression of eukaryon of expressing t7 rna polymerase expresses and whether expressed enzyme has the detection expression vector of transcriptional activity.
5. according to the described Asial type of claim 4 foot and mouth disease virus reverse genetic operating system; It is characterized in that: described detection expression vector is pT7-IRES-EGFP; Its construction process comprises: IRES and the EGFP of FMDV are cloned among the prokaryotic expression carrier PET-28a in order; And EGFP is controlled by under the IRES element of foot and mouth disease virus, promptly get.
6. the application of the said full-length infectious CDNA of claim 1 in the rescue foot and mouth disease virus comprises the carrier for expression of eukaryon cotransfection mammalian cell with described full-length infectious CDNA and T7 RNA polymerase, and rescue obtains foot and mouth disease virus.
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| CN108085302B (en) | 2016-11-21 | 2020-02-14 | 中国农业科学院哈尔滨兽医研究所 | Foot-and-mouth disease virus temperature sensitive attenuated strain and construction method and application thereof |
| CN108642068B (en) * | 2018-04-19 | 2022-02-22 | 新疆畜牧科学院兽医研究所（新疆畜牧科学院动物临床医学研究中心） | Whole genome sequence of foot-and-mouth disease virus artificial attenuated strain B, primer and application |
| CN111218474A (en) * | 2018-11-23 | 2020-06-02 | 成都生物制品研究所有限责任公司 | Paramyxovirus rescue system and method |
| CN111303251B (en) * | 2020-02-25 | 2021-07-30 | 中国农业科学院兰州兽医研究所 | Method for in-vitro assembly of foot-and-mouth disease virus-like particles and application thereof |
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