CN101092631B - Modified ORF2 gene of toroidal virus of pig, and application - Google Patents
Modified ORF2 gene of toroidal virus of pig, and application Download PDFInfo
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Abstract
This invention relates to modified ORF2 gene of porcine circovirus type 2 and its application. The modified ORF2 gene is obtained by: replacing the gene coding 41 amino acids at N-termal of natural ORF2 with the gene coding signal peptide of human tissue plasminogen activator, deleting terminator of ORF2 gene, and fusing the gene coding C-terminal of ORF2 with the gene coding the transmembrane peptide of avian influenza virus HA protein. The nucleotide sequence of ORF2 gene is shown in SEQ ID No.1. This invention also relates to eukaryotic plasmid pcDNA-spORF2 Delta41TM, and host E. coli strain DH5alpha/pcDNA-spORF2 Delta 41TM (CCTCC No.M207069). The ORF2 gene and the plasmid can be used for manufacturing the vaccine for preventing post-weaning multi-systematic syndrome.
Description
Technical field
The present invention relates to animal virology and genetic engineering technical field.Relate in particular to a kind of modification of porcine circovirus 2 type ORF2 gene, also relate to immunogenicity evaluation and the application in the Novel DNA vaccine thereof behind this genetic modification.
Background technology
Because the biological characteristics of PCV2 is more special, it breeds titre on cell very low, and do not cause cytopathy, though use D-glucosamine (the .Replication ofporcine circovirusinduction by glucosamine and cell cycle dependence.Arch Virol such as Tischer I that handles that virus titer can raise behind the cell, 1987,96:39-57), make the very fast death of cell but D-glucosamine has cytotoxicity, the difficulty that therefore is used to develop the inactivated vaccine of prevention PCV2 and attenuated vaccine is very big.Based on this, the vaccine (dna vaccination, subunit vaccine etc.) that utilizes gene engineering method to be used to develop PCV2 will become anti-this sick important directions of system.But subunit vaccine will be very limited in clinical application because cost is too high.And dna vaccination is compared with traditional vaccine and is had many superiority: at first be that dna vaccination preparation technology is simple, only need to extract a plasmid and get final product; Next is that the contained antigen of dna vaccination is very pure, does not have proteinic pollution; In addition, dna vaccination can be broken through the interference of body maternal antibody, and no matter whether maternal antibody exists in the body, and dna vaccination can produce corresponding immunne response, this to the generation of weanling pig immunoprotection reaction with keep quite important.Therefore, dna vaccination capable very application prospects of tool aspect the anti-system of PCV2 relative disease.
Porcine circovirus 2 type ORF2 gene is the primary structure protein gene of PCV2, the capsid protein of coding virus, mainly be positioned in the cell caryoplasm, can the oneself be assembled into virus like particle (.Open reading frame 2 ofporcine circovirus type 2encodes a major capsid protein.J Gen Virol such as Nawagitgul P, 2000,81:2282-2287), and (.Production such as McNeilly F such as McNeilly, characterization and applications of monoclonalantibodies to porcine circovirus 2.Arch Virol, 2001,146:909-922) studies confirm that contain in the ORF2 albumen virus that can neutralize in and epitope, therefore be the preferred object gene of design PCV2 new generation vaccine.(.Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type 2 (PCV2) proteins.Vaccine such as Blanchard P such as Blanchard; 2003; 21:4565-4575) utilize the dna vaccination and the subunit vaccine of PCV2ORF1 and ORF2 gene to discover; to be dna vaccination or subunit vaccine to PCV2 infect all has certain immune protective effect, and the vaccine of ORF1 and ORF2 combined immunization can be induced the generation of neutralizing antibody.(.Immunisation against PCV2 structural protein by DNAvaccination of mice.Vaccine such as Kamstrup S such as Kamst, 2004,22:1358-1361) also use the nucleic acid vaccine immunity small white mouse of ORF2 gene, find in head exempts from back 62d mouse body, can detect higher antibody horizontal.But above-mentioned research all finds to express ORF2 proteic dna vaccination inductive neutralizing antibody and cellular immune level is not high.(.The immunogenicityof VP7 such as Andrew M such as Andrew, a rotavirus antigen resident in the endoplasmic reticulum, is enhanced by cell surfaccexpression.J Virol, 1990,64:4776-4783) find during as the proteic immunogenicity of study on the carrier rotavirus VP 7 with vaccinia virus recombinant, in the born of the same parents of wild-type VP7 alignment restrictions its immunogenic performance, and express the level that the modification type VP7 gene that can be anchored to the host cell membrane surface can be induced higher antibody, immunogenicity improves greatly than wild-type.Based on this discovery, we modify the ORF2 gene of pig 2 type PCV-II, promptly according to (the human tissue plasminogen activator of people tissue plasminogen activator, tpA) signal peptide sequence (.C3d enhanced DNAvaccination induced humoral immune response to glycoprotein C of pseudorabies virus.Biochemical and Biophysical Research Communications such as Tong T Z, 2006,347:845-851) and avian influenza virus HA gene stride aminoacid sequence (the .Amino acid sequence requirements of thetransmembrane and cytoplasmic domains of influenza virus hemagglutinin for viable membranefusion.Molecular Biology of the Cell such as Melikyan G B in film district, 1999,10:1821-1836), the ORF2 gene is added signal peptide and the modification of striding the film district, be anchored to surface of cell membrane with the ORF2 genetic expression albumen that reaches after the modification, and compared the immunogenicity of ORF2 gene of modification and unmodified and the immune response of bringing out thereof in the mode of dna vaccination.
Summary of the invention
First purpose of the present invention is that the ORF2 gene of porcine circovirus 2 type is modified, and can be anchored to the host cell membrane surface after it is expressed, and obtains the better ORF2 gene of a kind of immunogenicity.
Second purpose of the present invention provides a kind of dna vaccination of expressing the ORF2 gene of porcine circovirus 2 type modification.
The 3rd purpose of the present invention is to utilize the present invention through transforming and modify the application in preparation pig wean multisystemic wasting syndrome vaccine of ORF2 gene and expression plasmid.
The present invention implements by the following technical programs:
A kind of porcine circovirus 2 type ORF2 gene of modification, its nucleotide sequence is shown in sequence table SEQ ID NO:1.
A kind of porcine circovirus 2 type ORF2 gene of modification is to utilize (the humantissue plasminogen activator of people tissue plasminogen activator, tpA) signal peptide sequence is replaced 41 amino acid of natural ORF2 gene N-end, and disappearance ORF2 gene terminator, merge avian influenza virus HA gene at its C end and stride the film region sequence and obtain.
The dna vaccination of the ORF2 gene that described expression porcine circovirus 2 type is modified is that the BamHI that the ORF2 gene that will modify inserts carrier for expression of eukaryon pcDNA3.1 (+) forms with XhoI site structure, intestinal bacteria (Escherichia coli) DH5 α/pcDNA-spORF2 Δ 41TM that comprises this eucaryon plasmid pcDNA-spORF2 Δ 41TM, deliver Chinese typical culture collection center (CCTCC) preservation on May 16th, 2007, its deposit number is: CCTCC NO:M207069.
The present invention also comprises the porcine circovirus 2 type ORF2 gene and the application of eukaryon expression plasmid in preparation pig wean multisystemic wasting syndrome vaccine of above-mentioned modification.
More detailed scheme is as described in the following step:
One, the structure of the dna vaccination of the ORF2 gene of the modification of PCV2ORF2 gene and expression modification
1, primer design
According to (the human tissue plasminogen activator of people tissue plasminogen activator, be called for short tpA) signal peptide of tpA (.C3d enhanced DNA vaccination induced humoral immune response toglycoprotein C of pseudorabies virus.Biochemical and Biophysical Research Communications such as Tong T Z, 2006,347:845-851) and avian influenza virus HA gene stride aminoacid sequence (the .Amino acidsequence requirements of the transmembrane and cytoplasmic domains of influenza virushemagglutinin for viable membrane fusion.Molecular Biology of the Cell such as Melikyan G B in film district, 1999,10:1821-1836), utilize pcr amplification to splice in conjunction with DNA, clone's method (referring to: J. Sa nurse Brooker etc., Wang Jiaxi etc. translate. molecular cloning experiment guide (third edition), Science Press, version in 2002) the ORF2 gene is added signal peptide and the modification of striding the film district.Method by PCR lacks 41 amino acid that ORF2 gene N holds, and obtains ORF2 Δ 41 genes.On this basis, further remove the terminator of ORF2 Δ 41 genes by PCR method, merge aminoacid sequence (the Melikyan G B that avian influenza virus HA strides the film district at its C end, Deng .Amino acid sequence requirements of the transmembraneand cytoplasmic domains of influenza virus hemagglutinin for viable membrane fusion.MolecularBiology of the Cell, 1999,10:1821-1836), thus obtain the ORF2 gene (spORF2 Δ 41TM) of modification type.SpORF2 Δ 41TM gene is inserted among the carrier for expression of eukaryon pCDNA3.1 (+) construction recombination plasmid pcDNA-spORF2 Δ 41TM.Primer P8 and P17 be used to increase disappearance ORF2 gene whole nuclear localization sequences (promptly lacking 41 amino acid of ORF2 gene N end) and do not contain the ORF2 gene fragment of terminator, the amplified fragments size is 582bp, two ends are designed EcoRV site and Not I site successively.Primer P18 and the P11 avian influenza virus HA that is used to increase strides the aminoacid sequence in film district, and clip size is 126bp, and two ends are designed Not I site and Xho I site successively.Primer PtPAF and PtPAR can directly anneal, form length and be 21 amino acid whose double-stranded tPA sequences of 63bp coding (.C3d enhancedDNA vaccination induced humoral immune response to glycoprotein C of pseudorabies virusBiochemical and Biophysical Research Communications such as Tong T Z, 2006,347:845-851), two ends are designed BglII site and BamH I site successively.Primer P3 and P4 can be used for the increasing complete coding region of ORF2 gene, upstream and downstream primer two ends have been designed HindIII and Sal I site respectively.Above-mentioned primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized.Primer sequence is as follows:
P3：5’-CAAAAGCTTACCATGACGTATCCAAGGAGG-3’
P4：5’-TATGTCGACTCACTTAGGGTTAAGTGG-3’
P8：5’-CATGATATCATGAATGGCATCTTCAACACC-3’
P11：5’-AGGCTCGAGTTAAATGCAAATTCTGCATTG-3’
P17：5’-TATGCGGCCGCCTTAGGGTTAAGTGGGGG-3’
P18：5’-ATAGCGGCCGCAACTTACCAAATACTG-3’
PtPAF：5’-GATCTATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCT
GTGTGGAGCAGTCTTCGTTTCGG-3’
PtPAR：5’-GATCCCAGAACGAAGACTGCTCCACACAGCAGCAGCACACAGCA
GAGCCCTCTCTTCATTGCATCCATA-3’
2, pcr amplification
To contain the plasmid pT-PCV2 (Xi Xin etc. of PCV2 Henan A strain (Genebank accession number AY035820) complete genome group, complete genomic clone of pig II type PCV-II and infectious the evaluation. the microorganism journal, 2004,44:172-176) carry out the amplification of ORF2 gene for template, the amplification condition of ORF2 gene order is: enter circulation after 95.0 ℃ of 5min sex change, loop parameter is: 95.0 ℃ of 1min, 55.0 ℃ of 1min, 72.0 ℃ 1min, 35 circulations are extended 10min for back 72.0 ℃.Plasmid pMD-HA (Zhang Ruihua etc. with the HA gene that contains avian influenza virus H9 hypotype, avian influenza hemagglutinin Prokaryotic Expression and the application in the diagnosis of H9 hypotype thereof. the biotechnology journal, 2005,21:315-319) increase for template, the pcr amplification condition of the aminoacid sequence of striding film district (TM) of HA is: 94 ℃ of 5min; Enter PCR circulation, 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s, carry out 35 circulations after, 72 ℃ are extended 10min.After reaction finishes, detect amplification with 1% agarose gel electrophoresis.The tPA double-stranded DNA can mix by primer PtPAF and PtPAR, obtains in 42 ℃ of 10min that directly anneal behind 94 ℃ of 5min.
3, modify the acquisition of ORF2 gene and the structure of eukaryon expression plasmid thereof
Utilize primer P8, P17 to carry out pcr amplification ORF2 gene, obtain ORF2 Δ 41 gene fragments of disappearance ORF2 gene 41 amino acid of N end and terminator, after reclaiming this gene fragment and using EcoR and Not I double digestion, be inserted between the EcoRV and Not I site of pcDNA3.1 (+) (available from American I nvitrogen company), obtain recombinant plasmid pcORF2 Δ 41.That further utilizes P11, P18 primer amplification HA gene strides film district (TM) sequence, acquisition is striden film district PCR product with behind Not I and the Xho I double digestion, be connected with pcORF2 Δ 41 plasmids that Xho I enzyme is cut with Not I, obtain ORF2 Δ 41 gene C ends and add the ORF2 Δ 41TM gene fragment of striding the film region sequence.The BamHI that the two ends sticky end of the tPA double-stranded DNA that utilizes PtPAF, PtPAR primer directly to anneal to obtain directly inserts the eucaryon plasmid pcDNA-ORF2 Δ 41TM that comprises ORF2 Δ 41TM gene fragment is point, thereby finish the ORF2 gene is added signal peptide and the work of striding the film district, obtain the ORF2 gene (spORF2 Δ 41TM) of modification type and the eukaryon expression plasmid pcDNA-spORF2 Δ 41TM of this modification type gene of expression, it is cut with PCR through enzyme and identifies that the confirmation structure is correct.SpORF2 Δ 41TM gene shows that through order-checking structure is correct, and its nucleotide sequence is shown in SEQ ID NO:1.
Two, the structure of dna vaccination pcORF2 that is used for the expression unmodified ORF2 gene of simultaneous test
To comprise the complete genomic recombinant plasmid pT-PCV of PCV2 Henan A strain ((Genebank accession number AY035820)) is template (Cao Shengbo etc., the full genomic clone and the sequential analysis of the Henan A strain of pig II type PCV-II, the virus journal, 2002,18 (2): 137-141), with P3, P4 is the pcr amplification that primer carries out ORF2 gene complete coding region, and the amplification size is 705bp.The PCR reaction conditions is: 94 ℃ of 5min; Enter the PCR circulation, 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, after 35 circulations, 72 ℃ are extended 10min.After reaction finishes, detect amplification with 1.0% agarose gel electrophoresis.Purifying reclaims the purpose fragment, the amplified production of purifying is behind Hind III, Sal I double digestion, directly be cloned between the Hind III, Sal I site of carrier for expression of eukaryon pcDNA3.1 (+) (available from American I nvitrogen company), obtain to express the reorganization eucaryon plasmid pcORF2 of natural ORF2 gene, cut through Hind III, Sal I and Hind III and Sal I enzyme and to identify with PCR and to confirm to make up correct that order-checking confirms that no base mismatches.
Three, a large amount of preparations of plasmid pcDNA-spORF2 Δ 41TM
(1) picking contains the colony inoculation of plasmid pcDNA-spORF2 Δ 41TM in 75mL LB nutrient solution, 37 ℃ of 300r/min overnight incubation, 6000r/min 5min, the collecting cell precipitation, with 3mL solution I (50mmol/L glucose, 10mmol/L EDTA, 25mmol/L Tris-Cl (pH8.0), according to conventional high-pressure sterilizing method 121 ℃ of sterilizations after 20 minutes rearmounted 4 ℃ of preservation standby) suspension (dispelling or vortex).
(2) add 6mL solution II (0.2mol/L NaOH, 1% sodium lauryl sulphate (SDS), now with the current), ice bath 7-10min.
(3) add 4.5mL solution III (3mol/L potassium acetate, Glacial acetic acid adjust pH to 4.8), ice bath 7-10min.4℃10000r/min 10-15min。
(4) get supernatant, add the Virahol of 0.6 times of volume, mixing ,-20 ℃ of 30min or room temperature 5min.Room temperature, 10000r/min6-15min.
(5) abandon supernatant, with 75% ethanol rinsing once, vacuum is drained or seasoning, adds 1.5mL TE (10.0mmol/LTris-HCl, 1.0mmol/L EDTA) suspension (washing about wall 20min).
(6) add the 5mol/L NH that 1.5ml is the precooling of 1 times of volume ice
4Ac, mixing.4℃10000r/min 10min。
(7) supernatant is transferred to the centrifuge tube of 7mL, adds the Virahol mixing of 1 times of volume (3mL) ,-20 ℃ of effect 30min.12000r/m 15min。
(8) abandon supernatant, with 75% ethanol rinsing once, vacuum is drained or seasoning, adds 500 μ L TE and suspends (washing about wall 20min), and be transferred to the centrifuge tube of 1.5mL.
(9) add an amount of RNase, 37 ℃ of 1h remove RNA.
(10) add 13% polyoxyethylene glycol 8000 (PEG8000) (containing 1.6mol/L NaCl) of 1 times of volume, mixing ,-20 ℃ of 30min (can spend the night).Centrifugal, abandon supernatant, heavy molten with 400 μ L TE.
(11) add equal-volume phenol: imitative: primary isoamyl alcohol extracting 2 times, chloroform: primary isoamyl alcohol extracting 1 time.
(12) add 100 μ l 10mol/L NH
4Ac fully behind the mixing, adds the dehydrated alcohol of 2 times of volumes ,-20 ℃ of precipitation 30min.4 ℃ of centrifugal 5-10min of 12000r/min.
(13) abandon supernatant, with 75% ethanol rinsing once, vacuum is drained or seasoning, adds 50 μ L TE or H
2O is heavy molten, put-20 ℃ standby.
Four, the production technique of pig wean multisystemic wasting syndrome dna vaccination
The main stream of pig wean multisystemic wasting syndrome dna vaccination production technique comprises the conversion of plasmid, a large amount of extractions of plasmid, the mensuration and the dilution of plasmid concentration.
1, the conversion of plasmid
With dna vaccination pcDNA-spORF2 Δ 41TM and pcORF2 (ammonia benzyl resistance) difference transformed into escherichia coli DH5 α competent cell.Concrete operations are: 1) get 100 μ l competent cell suspensions and transfer in the aseptic 1.5ml EP pipe, add 10 μ l and connect product, rotate gently with the mixed content thing, place 30min on ice.2) centrifuge tube was put in the circulator bath that is warmed to 42 ℃ in advance thermal shocking 90 seconds.3) fast centrifuge tube is transferred in the ice bath, made cell cooling 1～2min.4) every pipe adds 400 μ l LB substratum.With water-bath substratum is heated to 37 ℃, centrifuge tube is transferred on 37 ℃ of shaking tables then, incubation 45min makes bacteria resuscitation.For reaching effective conversion, rotating speed should not be above 225 rev/mins during recovery.5) competent cell of getting 100 μ l conversion is transferred on the LB agar plate that contains ammonia benzyl resistance, with an aseptic elbow glass rod cell transformed is uniformly applied to agar plate surface.6) plate is put 37 ℃ of cultivations, be absorbed until liquid, be inverted plate then and cultivate, bacterium colony can appear in 12～16h.
2, a large amount of extractions of plasmid are with the method (this place omits this detailed preparation process) of above-mentioned " two, a large amount of preparations of the plasmid pcDNA-spORF2 Δ 41TM ".
3, the mensuration of plasmid concentration, dilution
Utilize the concentration of the big upgrading grain of spectrophotometric determination, (be called for short PBS, it is as follows to fill a prescription: NaCl 8.0g, KCl 0.2g, NaHPO with phosphate buffered saline buffer
4(12H
2O) 2.9g, KH
2PO
40.2g, pH7.4) it is diluted to 1 μ g/ μ l, promptly can be used for the Balb/c injected in mice.
Five, the immune efficacy check of pig wean multisystemic wasting syndrome dna vaccination
With dna vaccination pcDNA-spORF2 Δ 41TM and pcORF2 (respectively through the back leg intramuscular injection Balb/c mouse in 6 ages in week), 6 every group, every mouse 100 μ l (containing 100 μ g plasmids), immunity is 2 times altogether, at interval 2 weeks; Use the negative control of blank vector plasmid pcDNA3.1 (+) simultaneously as nucleic acid immunization.2,4,6 weeks took a blood sample through tail vein negative pressure after first immunisation, and separation of serum detects neutralizing antibody; Exempt from 6 weeks of back in head, put to death all immune mouses by cervical vertebra, aseptic taking-up spleen, utilize lymphocyte separation medium (available from Tianjin TBD company) to separate splenic lymphocyte, carry out cellular immunization and (comprise that the stimulate proliferation mRNA of exponential sum IFN-γ of splenic lymphocyte transcribes, secretion level detects (Comparison of immuncresponses and protective efficacy of suicidal DNA vaccine and conventional DNA vaccineencoding glycoprotein C of pseudorabies virus in mice.Vaccine.2004 such as Xiao, 22,345-351).The result shows that the dna vaccination pcDNA-spORF2 Δ 41TM of the expression modification ORF2 gene that the present invention makes up induces neutralizing antibody and cell immune response all apparently higher than the dna vaccination pcORF2 of the ORF2 gene of expressing unmodified, has showed the better immunogenicity of ORF2 gene (spORF2 Δ 41TM) that the present invention modifies.
Description of drawings
Sequence table SEQ ID NO:1 is the ORF2 gene order of modifying among the present invention
Fig. 1: the structure iron that has shown the ORF2 gene DNA vaccine pcORF2 of PCV2 Henan A strain (GenBank accession number AY035820) ORF2 gene DNA vaccine pcDNA A-spORF2 Δ 41TM that the expression among the present invention is modified and unmodified
Fig. 2: the enzyme that has shown the ORF2 gene DNA vaccine pcDNA A-spORF2 Δ 41TM that the expression among the present invention is modified is cut the qualification result with PCR
Fig. 3: shown the neutralizing antibody level behind pig wean multisystemic wasting syndrome dna vaccination pcDNA-spORF2 Δ 41TM and the pcORF2 immunity Balb/c mouse
Fig. 4: shown behind pig wean multisystemic wasting syndrome dna vaccination pcDNA-spORF2 Δ 41TM and the pcORF2 immunity Balb/c mouse inductive splenic lymphocyte index that stimulates proliferation
Fig. 5: after having shown pig wean multisystemic wasting syndrome dna vaccination pcDNA-spORF2 Δ 41TM and pcORF2 immunity Balb/c mouse, detect mouse spleen lymphocyte is stimulated the back secretion of gamma-IFN by specific antigen level with the ELISPOT method
Fig. 6: after having shown pig wean multisystemic wasting syndrome dna vaccination pcDNA-spORF2 Δ 41TM and pcORF2 immunity Balb/c mouse, detect mRNA that mouse spleen lymphocyte stimulated back IFN-γ by specific antigen level relatively with fluorescent quantitative RT-PCR method.
Embodiment
The present invention is further illustrated below in conjunction with Figure of description, but be not restriction protection scope of the present invention.
Embodiment 1: the plasmid and the preparation that contains the plasmid of crt gene that contain the gene of the present invention's modification
1, the structure of the eucaryon plasmid pcORF2 of the ORF2 gene of expression unmodified
With pT-PCV is template, p3 and P4 are primer amplification ORF2 gene, reclaim and the purifying amplified production, behind Hind III, Sal I double digestion, directly be cloned into same enzyme and cut between the Hind III, Sal I site of the carrier for expression of eukaryon pcDNA3.1 (+) of processing, obtain recombinant plasmid pcORF2.Its structure is seen Fig. 1.
2, express the structure of the eucaryon plasmid pcDNA-spORF2 Δ 41TM of the ORF2 gene of modifying
Utilize primer P8, P17 to carry out pcr amplification ORF2 gene, obtain ORF2 Δ 41 gene fragments of disappearance ORF2 gene 41 amino acid of N end and terminator, after reclaiming this gene fragment and using EcoR and Not I double digestion, be inserted between the EcoR V and Not I site of pcDNA3.1 (+) (available from American I nvitrogen company), obtain recombinant plasmid pcORF2 Δ 41.That further utilizes P11, P18 primer amplification HA gene strides film district (TM) sequence, acquisition is striden film district PCR product with behind Not I and the Xho I double digestion, be connected with pcORF2 Δ 41 plasmids that Xho I enzyme is cut with Not I, obtain ORF2 Δ 41 gene C ends and add the ORF2 Δ 41TM gene fragment of striding the film region sequence.The BamH I that the two ends sticky end of the tPA double-stranded DNA that utilizes PtPAF, PtPAR primer directly to anneal to obtain directly inserts the eucaryon plasmid pcDNA-ORF2 Δ 41TM that comprises ORF2 Δ 41TM gene fragment is point, thereby finish the ORF2 gene is added signal peptide and the work of striding the film district, obtain the ORF2 gene (spORF2 Δ 41TM) of modification type and the eukaryon expression plasmid pcDNA-spORF2 Δ 41TM of this modification type gene of expression, it is cut with PCR through enzyme and identifies that the confirmation structure is correct.This eukaryon expression plasmid pcDNA-spORF2 Δ 41TM structure is seen Fig. 1, and qualification result is seen Fig. 2.
Embodiment 2: the biological experiment that pig wean multisystemic wasting syndrome dna vaccination of the present invention and control vaccine are renderd a service mouse immune
1) immune programme for children of Balb/c mouse
The Balb/c mouse is divided into 3 groups, be respectively pig wean multisystemic wasting syndrome dna vaccination pcDNA-spORF2 Δ 41TM of the present invention, pcORF2 immune group and blank carrier pcDNA3.1 (+) control group, every group 6, adopt the back leg intramuscular injection, every mouse 100 μ l (containing 100 μ g plasmids), immunity is 2 times altogether, at interval 2 weeks.Exempt from 2,4,6 weeks of back through the blood sampling of tail vein negative pressure at head, separation of serum detects neutralizing antibody.Exempt from 6 weeks of back in head, put to death all immune mouses by cervical vertebra, aseptic taking-up spleen, utilize lymphocyte separation medium (available from Tianjin TBD company) to separate splenic lymphocyte, carry out cellular immunization and (comprise that the stimulate proliferation mRNA of exponential sum IFN-γ of splenic lymphocyte transcribes, secretion level) detects (Comparison of immune responses and protective efficacy of suicidal DNA vaccine andconventional DNA vaccine encoding glycoprotein C of pseudorabies virus in mice.Vaccine.2004 such as Xiao, 22,345-351).
2) neutralizing antibody level detection
Adopt reports such as Wang (.Construction and immunogenicity of recombinant adenovirusexpressing the capsid protein of porcine circovirus 2 (PCV2) in mice.Vaccine such as Wang X, 2006, the neutralizing antibody detection method of PCV2 24:3374-3380), detect the neutralizing antibody level of the anti-PCV2 of specificity in the serum, the result shows that the neutralizing antibody level of not modified dna vaccination pcORF2 immune group is not high, and rise comparatively slowly, and the present invention modifies the neutralizing antibody fast rise of improved dna vaccination pcDNA-spORF2 Δ 41TM immune group, reach higher level, compare significant difference (P＜0.05 with the pcORF2 immune group, t-test), test-results such as Fig. 3.
3) the splenic lymphocyte index that stimulates proliferation detects
Adopt mtt assay measure the stimulation index (SI) of mouse spleen lymphocyte (chief editor such as Shen Guanxin. modern immunological experiment technology. Hubei science tech publishing house, version in 1998).The index that stimulates proliferation of the modified improved dna vaccination pcDNA-spORF2 Δ 41TM immune group of the present invention as a result to be significantly higher than not modified dna vaccination pcORF2 immune group (P＜0.01, t-test).Test-results such as Fig. 4.
4) the ELISPOT method detects splenic lymphocyte excretory IFN-γ level
Employing ELISPOT method (Long Zhenzhou. Medical Immunology (the 2nd edition). Beijing: People's Health Publisher, version in 2000) detect mouse spleen lymphocyte by the ability of specific antigen stimulation back secretion of gamma-IFN.The secretion of gamma-IFN level of the modified improved dna vaccination pcDNA-spORF2 Δ 41TM immune group of the present invention as a result to be significantly higher than not modified dna vaccination pcORF2 immune group (P＜0.01, t-test).Test-results such as Fig. 5.
5) fluorescence relative quantification RT-PCR method detects lymphocyte is stimulated back IFN-γ by specific antigen mRNA level
Employing fluorescence relative quantification RT-PCR method (referring to: Zhang Jingbo etc. the Cell Biology Experiment technology. Chemical Industry Press, version in 2006) detect mouse spleen lymphocyte by the mRNA level of specific antigen stimulation back IFN-γ.Mouse spleen lymphocyte extracts total RNA external after specific antigen (PCV2) stimulates, utilize oligo dT that the mRNA in-vitro transcription is become cDNA.Utilize primer β-actins:5 '-CACTGCCGCATCCTCTTCCTCCC-3 ', β-actinr:5 '-CAATAGTGATGACCTGGCCGT-3 ' amplification mouse house-keeping gene β-actin, and with its confidential reference items as relative quantification RT-PCR; Primer I FN-γ s:5 '-TCAAGTGGCATAGATGTGGAAGAA-3 ', IFN-γ r:5 '-TGGCTCTGCAGGATTTTCATG-3 ', amplification mouse IFN-γ gene.Quantitative fluorescent PCR reaction system (25 μ l) comprising: each 0.5 μ l (10 μ M) of IFN-γ and β-actin upstream and downstream primer,
Green Realtime PCR MasterMix (comprising reaction buffer, dNTP, Mgcl2, SYBR Green, Taq enzyme) (available from ToYoBo company, Shanghai) 12.5 μ l, distilled water 11.0 μ l, cDNA sample 0.5 μ l.Each sample is done three repetitions.The reaction amplification condition is: 50 ℃ of 2min, 94 ℃ of pre-sex change 10min and then 94 ℃ of sex change 15s of 40 round-robin and 60 ℃ of annealing with extend 1min.The variation of the fluorescent signal in the entire reaction course is detected by ABI Prism 7500 real-time fluorescence quantitative PCR instrument (available from U.S. Applied Biosystems company).The mRNA level of the modified improved dna vaccination pcDNA-spORF2 Δ 41TM immune group IFN-γ of the present invention as a result will apparently higher than not modified dna vaccination pcORF2 immune group (P＜0.01, t-test).Test-results such as Fig. 6.
Appendix
Term definition:
| English name | Chinese |
| spORF2Δ41TM | The ORF2 gene of modification type |
| pT-PCV | Recombinant plasmid |
| pcDNA-ORF2Δ41 | Recombinant plasmid |
| pcDNA3.1(+) | Blank plasmid vector |
| pcORF2 | Express the dna vaccination of the ORF2 gene of unmodified |
| English name | Chinese |
| pcDNA-spORF2Δ41TM | The pig wean multisystemic wasting syndrome dna vaccination synonym of the dna vaccination of the ORF2 gene of expression modification type and the present invention's preparation |
Sequence table
＜110〉Hua Zhong Agriculture University
＜120〉a kind of porcine circovirus 2 type ORF2 gene and application of modification
<130>
<141>2007-04-11
<160>1
<170>PatentIn version 3.1
<210>1
<211>822
<212>DNA
＜213〉pig circular ring virus (Porcine circovirus)
<220>
<221>gene
<222>(1)..(822)
<223>
<400>1
atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60
tcgggatcca ctagtccagt gtggtggaat tctgcagata tcatgaatgg catcttcaac 120
acccgcctct cccgcacctt cggatatact gtcaagaaaa ccacagtcag aacgccctcc 180
tgggcggtgg acatgatgag atttaatatt aacgatttcc ttcccccagg agggggctca 240
aaccccctca ctgtgccctt tgaatactac agaataagaa aggttaaggt tgaattctgg 300
ccctgctccc caatcaccca gggtgacagg ggagttggat ccactgctgt tattctagat 360
gataactttg taacaaaggc cacagccctg acttatgatc cctatgtaaa ctactcctcc 420
cgccatacca taacccagcc cttctcctac cactcccggt actttacccc gaaacctgtt 480
cttgattcca ctattgatta cttccaacca aataacaaaa ggaatcagct ttggctgagg 540
ctacaaacct ctgcaaatgt ggaccacgta ggcctcggca ctgcgttcga aaacagtata 600
tacgaccagg actacaatat ccgtgtaacc atgtatgtac aattcagaga atttaatctt 660
aaagaccccc cacttaaccc taaggcggcc gcaacttacc aaatactgtc aatttattca 720
acagtggcga gttccctagc actggcaatc atggtagctg gtctatcttt atggatgtgc 780
tccaatggat cgttacaatg cagaatttgc atttaactcg ag 822
Claims (3)
1. the porcine circovirus 2 type ORF2 gene of a modification, it is characterized in that, utilize the signal peptide sequence of people tissue plasminogen activator (tpA) to replace 41 amino acid that natural ORF2 gene N-holds, and disappearance ORF2 gene terminator, merging avian influenza virus HA gene at its C end strides the film region sequence and obtains, its nucleotide sequence is shown in sequence table SEQ ID NO:1, this modifying factor is included among the eukaryon expression plasmid pcDNA-spORF2 Δ 41TM, the intestinal bacteria Escherichia coli DH5 α/pcDNA-spORF2 Δ 41TM that contains this plasmid, be deposited in Chinese typical culture collection center, preserving number is CCTCC NO:M207069.
2. dna vaccination that comprises the described gene of claim 1.
3. the application of the described gene of claim 1 in preparation pig wean multisystemic wasting syndrome vaccine.
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| CN200710099886XA CN101092631B (en) | 2007-05-31 | 2007-05-31 | Modified ORF2 gene of toroidal virus of pig, and application |
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| CN101092631A CN101092631A (en) | 2007-12-26 |
| CN101092631B true CN101092631B (en) | 2010-04-14 |
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| US20100150959A1 (en) * | 2008-12-15 | 2010-06-17 | Vectogen Pty Ltd. | PCV 2-Based Methods and Compositions for the Treatment of Pigs |
| CN101875941A (en) * | 2010-03-09 | 2010-11-03 | 胡文锋 | Artificially modified and synthesized dORF2art gene and protein encoded by same |
| CN104789514B (en) * | 2015-04-14 | 2018-06-15 | 华中农业大学 | A kind of amicine overexpression DNA vaccination and construction method and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1597956A (en) * | 2003-09-18 | 2005-03-23 | 中国疾病预防控制中心性病艾滋病预防控制中心 | Trans menbrance type and secretory type HIV Gag antigen code gene and AIDS vaccine containing said gene |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1597956A (en) * | 2003-09-18 | 2005-03-23 | 中国疾病预防控制中心性病艾滋病预防控制中心 | Trans menbrance type and secretory type HIV Gag antigen code gene and AIDS vaccine containing said gene |
Non-Patent Citations (2)
| Title |
|---|
| P. Blanchard et al..Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type 2(PCV2) proteins.Vaccine21.2003,21第4565页摘要，第4566页第2.1节、2.4节. * |
| Tiezhu Tong et al..C3d enhanced DNA vaccination induced humoral immune response to glycoprotein C of pseudorabies virus.Biochemical and Biophysical Research Communications347.2006,347第846页右栏第2段. * |
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