CN102250843A - Genetic engineering marked attenuated vaccine strain of porcine reproductive and respiratory syndrome virus and application thereof - Google Patents
Genetic engineering marked attenuated vaccine strain of porcine reproductive and respiratory syndrome virus and application thereof Download PDFInfo
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Abstract
The invention discloses a genetic engineering marked attenuated vaccine strain of a porcine reproductive and respiratory syndrome virus (PRRSV). The attenuated vaccine strain comprises a genomic nucleic acid of a porcine reproductive and respiratory syndrome virus attenuated vaccine strain HuN4-F112; the HuN4-F112 genome includes a mutation in a genetic region for coding an Nsp2 protein, and the mutation is as follows: a nucleotide sequence for coding a Newcastle disease virus NP protein is inserted to a lacking region of a nucleotide sequence for coding 480-532-site amino acid of the Nsp2 protein; or the nucleotide sequence for coding the Newcastle disease virus NP protein is inserted to the lacking region of a nucleotide sequence for coding 508-532-site amino acid of the Nsp2 protein. The invention also discloses an application of the genetic engineering marked attenuated vaccine strain. The genetic engineering marked attenuated vaccine strain of the porcine reproductive and respiratory syndrome virus provided by the invention not only can provide completely safe immune protection to resist high-pathogenicity PRRSV after the porcine is immunized, but also can effectively distinguish the immunized porcine of the porcine reproductive and respiratory syndrome vaccine with the naturally infected porcine of the field virus.
Description
Technical field
The present invention relates to technical field of bioengineering, relate in particular to a kind of high-pathogenicity porcine reproductive and breath syndrome virus genetically engineered mark attenuated vaccine strain and application.
Background technology
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) be one of the transmissible disease of present serious harm pig industry, this disease is by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome Virus, PRRSV) infect and to cause, breeding difficulty that its main clinical characteristics is that farrowing sow is miscarried, premature labor and stillborn foetus etc. are serious and piglet and growing and fattening pigs show symptoms such as tangible dyspnoea.Up to now, PRRS is still most of regional popular in the whole world, is one of main transmissible disease of pig.
Since in May, 2006, China has broken out high-pathogenicity porcine reproductive and the respiration syndrome that is caused by the american type variant, causes very large economy loss for the pig industry of China.Compared with former popular strain, the virulence of current popular high-pathogenicity porcine reproductive and breath syndrome virus obviously strengthens, and 482 and 534～562 in its Nonstructural Protein Nsp2 exist a characteristic 1+29 aminoacid deletion respectively.The Nsp2 albumen of PRRSV is a kind of special Nonstructural Protein, has cysteine protease activity, and there is significantly sudden change in numerous studies have shown that near Nsp2 albumen intermediate zone and the PL2 district, inserts or the deletion phenomenon between each strain.Find that by reverse genetic manipulation Nsp2 plays an important role in the reproduction process of virus.Studies confirm that its virulence strengthens and 30 amino acid of Nsp2 characteristic disappearance of this strain are irrelevant though have, at present this virus virulence enhanced reason still be it be unclear that.
Vaccine immunity remains prevention and controls this sick important means, commercial inactivated vaccine and attenuated vaccine all have use on market at present, comparatively speaking, the effect of attenuated live vaccines is more obvious at the prevention of high-pathogenicity porcine reproductive and respiration syndrome and control, the existing several attenuated vaccines of China at high-pathogenicity porcine reproductive and respiration syndrome, all with virulent strain extremely weak acquisition of the external continuous passage of process on cell, as high-pathogenicity porcine reproductive and respiration syndrome HuN4-F112 attenuated vaccine, these vaccines have been brought into play vital role in the prevention and control of highly pathogenic PRRSV at present.However, existing attenuated vaccine in use still exists can not effectively distinguish natural infection and this major issue of vaccine immunity animal, this brings serious obstruction for more effectively controlling and purify the high PRRSV of causing a disease in the future, therefore is necessary to develop the novel gene engineering mark attenuated vaccine that can reach the differential diagnosis purpose.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of porcine reproductive and respiratory syndrome virus genetically engineered mark attenuated vaccine strain, and this vaccine strain can effectively be distinguished the animal of natural infection street strain and vaccine immunity strain.
In addition, also need to provide a kind of application of said gene engineering mark attenuated vaccine strain.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, a kind of reorganization porcine reproductive and respiratory syndrome virus is provided, the genomic nucleic acids that comprises porcine reproductive and respiratory syndrome virus attenuated vaccine strain HuN4-F112, described HuN4-F112 genome comprises a disappearance or sudden change in the proteic gene region of coding porcine reproductive and respiratory syndrome virus Nsp2
Described disappearance is selected from:
(1) disappearance of the nucleotide sequence of coding Nsp2 albumen 480-532 amino acids;
(2) disappearance of the nucleotide sequence of coding Nsp2 albumen 508-532 amino acids;
Described sudden change is selected from:
(a) insert the proteic nucleotide sequence of coding Newcastle disease virus NP in (1) described disappearance zone;
(b) insert the proteic nucleotide sequence of coding Newcastle disease virus NP in (2) described disappearance zone.
Preferably, the nucleotide sequence of described disappearance zone insertion is terminal 49 the amino acid whose nucleotide sequences (SEQ ID NO.19) of coding Newcastle disease virus NP PROTEIN C.
Preferably, described HuN4-F112 genome also comprises the MluI restriction endonuclease sites of mark.
Preferred, the Nsp2 gene is after the nucleotide sequence of disappearance coding 480-532 amino acids (Δ 52 amino acid), and its gene order is shown in SEQ ID NO.1; The Nsp2 gene is after the nucleotide sequence of disappearance coding 508-532 amino acids (Δ 25 amino acid), and its gene order is shown in SEQ ID NO.2.
Preferred, the Nsp2 gene is after terminal 49 the amino acid whose nucleotide sequences of coding Newcastle disease virus NP PROTEIN C are inserted in its Δ 52 aminoacid deletion zone, and its gene order is shown in SEQ ID NO.3; The Nsp2 gene is after terminal 49 the amino acid whose nucleotide sequences of coding Newcastle disease virus NP PROTEIN C are inserted in its Δ 25 aminoacid deletion zone, and its gene order is shown in SEQ ID NO.4.
In another aspect of this invention, a kind of nucleic acid molecule is provided, the genome polynucleotide sequence that comprises porcine reproductive and respiratory syndrome virus attenuated vaccine strain HuN4-F112, this polynucleotide sequence comprises a disappearance or sudden change in the proteic gene region of coding porcine reproductive and respiratory syndrome virus Nsp2
Described disappearance is selected from:
(1) disappearance of the nucleotide sequence of coding Nsp2 albumen 480-532 amino acids;
(2) disappearance of the nucleotide sequence of coding Nsp2 albumen 508-532 amino acids;
Described sudden change is selected from:
(a) insert the proteic nucleotide sequence of coding Newcastle disease virus NP in (1) described disappearance zone;
(b) insert the proteic nucleotide sequence of coding Newcastle disease virus NP in (2) described disappearance zone.
In another aspect of this invention, also provide a kind of recombinant vectors that comprises above-mentioned nucleic acid molecule.
In another aspect of this invention, a kind of porcine reproductive and respiratory syndrome virus attenuated vaccine strain also is provided, the genomic nucleic acids that comprises porcine reproductive and respiratory syndrome virus attenuated vaccine strain HuN4-F112, described HuN4-F112 genome comprises a sudden change in the proteic gene region of coding porcine reproductive and respiratory syndrome virus Nsp2, this sudden change is: in the disappearance zone of the nucleotide sequence of coding Nsp2 albumen 480-532 amino acids, insert the proteic nucleotide sequence of coding Newcastle disease virus NP; Or, insert the proteic nucleotide sequence of coding Newcastle disease virus NP in the disappearance zone of the nucleotide sequence of coding Nsp2 albumen 508-532 amino acids.
Preferably, the nucleotide sequence of described disappearance zone insertion is terminal 49 the amino acid whose nucleotide sequences (SEQ ID NO.19) of coding Newcastle disease virus NP PROTEIN C.
In another aspect of this invention, also provide the application of a kind of above-mentioned reorganization porcine reproductive and respiratory syndrome virus in the vaccine of preparation prevention or treatment high-pathogenicity porcine reproductive and respiration syndrome.
In another aspect of this invention, also provide the application of a kind of above-mentioned porcine reproductive and respiratory syndrome virus attenuated vaccine strain in the vaccine of preparation prevention or treatment high-pathogenicity porcine reproductive and respiration syndrome.
In another aspect of this invention, also provide a kind of detection method of distinguishing above-mentioned porcine reproductive and respiratory syndrome virus attenuated vaccine strain and natural infection strain, may further comprise the steps:
Utilize the Newcastle disease virus NP albumen of expressing as detecting antigen, set up the ELISA antibody detection method, detect the antibody that immune animal produces at NP49 albumen with this method, to distinguish attenuated vaccine strain and natural infection strain with genetically engineered mark.
Porcine reproductive and respiratory syndrome virus genetically engineered mark attenuated vaccine strain of the present invention; behind the immune swine body, can effectively induce body to produce immunne response; lethal hit to immune swine opposing high-pathogenicity porcine reproductive and breath syndrome virus provides 100% immune protective rate; safe and reliable; and genetically engineered mark attenuated vaccine strain of the present invention; can effectively distinguish porcine reproductive and respiratory syndrome natural infection pig and porcine reproductive and respiratory syndrome vaccine immunity pig; satisfy clinically to the differential diagnosis of highly pathogenic PRRSV vaccine immunity strain and natural infection strain, be very beneficial for prevention and the control of highly pathogenic PRRSV.
Description of drawings
The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.
Fig. 1 is the RT-PCR detected result figure that the present invention saves the infections clone strain of different genes disappearance and insertion mark;
Fig. 2 is the cytopathy figure after genetically deficient of the present invention and genetic marker infections clone poison infect the Macr-145 cell;
Fig. 3 is that the present invention rescues different genes disappearance that obtains and the infections clone strain genetic stability sequential analysis figure that inserts mark;
Fig. 4 is that the present invention utilizes anti-PRRSV N albumen monoclonal antibody that genetically deficient and the infections clone strain that inserts mark are carried out indirect immunofluorescence qualification result figure;
Fig. 5 is the different infections clone strains of the present invention and parent's poison HuN4-F112 strain growth curve comparison diagram;
Fig. 6 is that the embodiment of the invention 3 genetically engineered mark attenuated vaccine strain immune swine ELISA detect PRRSV antibody horizontal variation diagram;
Fig. 7 is that the embodiment of the invention 3 is utilized indirect ELISA method to detect the anti-NP49 protein antibodies of immune swine to differentiate the immune swine and the histogram of strong malicious infected pigs naturally;
Fig. 8 is that the embodiment of the invention 3 is utilized RT-PCR to cut in conjunction with the MluI enzyme to differentiate the immune swine and the electrophorogram of strong malicious infected pigs naturally;
Fig. 9 is that the embodiment of the invention 3 genetically engineered mark attenuated vaccine strains are attacked the dead protection ratio statistics figure in poison back.
Embodiment
In the following example, the experimental technique of unreceipted actual conditions, usually condition routinely, as " fine works molecular biology experiment guide " (F.M. Ao Sibai, R.E. James Kingston, chief editors such as J.G. Sai Deman, Ma Xuejun, the Su Yuelong translates. Beijing: Science Press, 2004) described in method carry out.
Because there is the problem that can not effectively distinguish natural infection and vaccine immunity animal in the attenuated vaccine of present porcine reproductive and respiratory syndrome virus, be necessary to utilize new technology development PRRS new generation vaccine.This laboratory is separated to the pathogenic PRRSV strain of plant height HuN4 several years ago, compares with the american type strain VR-2332 sequence of classics the 483rd of the Nsp2 gene and 535～563 amino acids to lack, and be the strong malicious variant of the typical PRRSV of a strain.By the strong malicious HuN4 strain subculture in vitro separately of highly pathogenic PRRSV being caused the weak attenuated live vaccine HuN4-F112 that obtains; confirm that by a large amount of clinical experiments the HuN4-F112 attenuated vaccine strain that is obtained has good immune protective efficiency, the experimental group animal can be resisted the lethality strong virus attack and the clinical symptom (patent No. is 200810097546.8 Chinese invention patent) of the high PRRS of causing a disease do not occurred.Just on this basis, the present invention utilizes porcine reproductive and respiratory syndrome virus HuN4-F112 vaccine strain genome full length cDNA clone pSK-HuN4-F112, by technique construction such as sudden change PCR etc. the mutant infections clone in stable disappearance zone of two Nsp2 genes, that is: dHN4-Δ 52 and dHN4-Δ 25, gene serves as a mark to insert the proteic epi-position of the more intense Newcastle disease virus NP of antigenicity (49 amino acid of C-terminal) respectively simultaneously in above-mentioned two disappearance zones, and successfully obtained can genetic stability genetically engineered mark attenuated vaccine strain, be rHN4-Δ 52+NP49 and rHN4-Δ 25+NP49, confirm that by experiment the above-mentioned infections clone strain with genetic marker obtained can be used as genetically engineered mark attenuated vaccine and is applied to control to high-pathogenicity porcine reproductive and respiration syndrome.
The structure and the evaluation of embodiment 1 porcine reproductive and respiratory syndrome virus genetically deficient strain
1. materials and methods
1.1 virus, carrier and reagent
PRRSV vaccine strain HuN4-F112 by this laboratory go down to posterity cause weak and preserve, infections clone carrier pSK-Hun4-F112 makes up and preserved that (Tong Guangzhi etc. 2007 by this laboratory; Tong GZ et al., 2007; Zhou YJ et al., 2008; TianZJ et al., 2009), RNeasy Plus Mini Kit test kit is available from QIAGEN; Glue reclaims test kit available from Shanghai China Shun bio tech ltd; SupersciptIII reverse transcriptase and
Pfx DNA polymerase is available from Invitrogen; RNase H, T4DNA ligase enzyme, restriction enzyme are available from NEB company; DL-15000, DL-2000DNA Marker is available from TakaRa; Other chemical reagent all is import or homemade analytical pure.
1.2 design of primers
According to PRRSV HuN4 strain (the GenBank accession number: EF635006) Nsp2 gene order, with Olio 6.0 software designs 2 pairs of primers F Δ 52/R Δs 52 and R Δ 25/F Δ 25 (seeing Table 1), wherein primer has been contained the gene region that carries out deletion mutantion.
Table 1Nsp2 transgenation primer title and sequence
1.3 the pcr amplification of missing gene specific fragment
Utilizing F Δ 52/ R Δ 52 and 25 two pairs of primers of R Δ 25/F Δ respectively, is the template PCR operation that suddenlys change with the pSK-HuN4-F112-ABC plasmid, and reaction system is 50 μ L, reaction conditions is: 92 ℃/2min, and 92 ℃/10s, 55 ℃/20s, 68 ℃/6min, circulate 18 times; Extend 5min in 68 ℃ at last.
1.4 virogene deletion mutantion full-length cDNA construction of recombinant plasmid
Respectively above-mentioned reaction product is carried out Dpn I digestion with restriction enzyme, reaction system is: 50 μ L PCR products, 5.8 μ L, 10 * reaction buffer, 2.2 μ L Dpn I, 37 ℃ of effect 4.5h.Respectively with two linearizing PCR product transformed competence colibacillus bacillus coli DH 5 alphas of above-mentioned acquisition, obtain positive recombinant plasmid then through plasmid extraction, PCR evaluation and sequencing analysis etc.
Utilizing Pac I and NheI to carry out enzyme the positive recombinant plasmid of above-mentioned acquisition and pSK-HuN4-F112 plasmid respectively cuts digestion, reclaims ABC-Δ 52, ABC-Δ 25 and the segmental enzyme of pSK-HuN4-F112-DEF and cut product.The gene fragment that is recovered to is connected with the pSK-HuN4-F112-DEF fragment respectively and the transformed competence colibacillus bacillus coli DH 5 alpha, filter out positive recombinant plasmid through plasmid extraction, PCR evaluation and sequencing analysis etc., and it is distinguished called after pSK-HN4vac-Δ 52 and pSK-HN4vac-Δ 25.
1.5RNA external synthetic and transfection
Plasmid pSK-HN4vac-Δ 52 and pSK-HN4vac-Δ 25 that the SwaI restriction enzyme site that utilizes 3 ' latter end Poly (A) tail to introduce will comprise the viral genome full-length cDNA carry out linearizing respectively, according to the synthetic respectively viral RNA of in-vitro transcription test kit mMessageHigh Yield Capped RNA Transcription kit operation instructions, cultivate the BHK21 cell simultaneously and make its density reach 70%～90%, with above-mentioned synthetic viral RNA respectively according to DMRI-C transfection reagent specification sheets transfection BHK21 cell.Cell after the transfection places 37 ℃ of CO
2Cultivated 48 hours in the incubator.
1.6 the rescue of genetically deficient virus strain
Gather in the crops above-mentioned cells transfected and be stored in-80 ℃, behind multigelation, be inoculated on the cultured in advance Macr-145 cell monolayer in 37 ℃ of CO
2Sense is done 1 hour in the incubator, adds cell maintenance medium in 37 ℃ of CO
2Continue in the incubator to cultivate, carry out the rescue of virus, simultaneously observation of cell pathology situation.
1.7 the evaluation of virus
1.7.1 rescuing the RT-PCR that obtains genetically deficient virus identifies
The RNA that the genetically deficient that obtains is cloned malicious dHN4-Δ 52, dHN4-Δ 25 and parent's poison HuN4-F112 cells infected is rescued in extraction, reverse transcription, utilize primer JD1/JD2 (seeing Table 1) amplification to comprise the fragment of MIuI, utilize MIuI to carry out enzyme and cut evaluation, simultaneously, utilize primers F 2701/R3192 (seeing Table 1) to carry out RT-PCR amplification, and product is connected the pMD18-T carrier check order.
1.7.2 rescuing the indirect immunofluorescence that obtains genetically deficient virus identifies
The genetically deficient of rescue is cloned malicious dHN4-Δ 52 and dHN4-Δ 25 is inoculated individual layer Macr-145 cell respectively, infect back 48h and discard nutrient solution.With the fixing 10min of ice methyl alcohol, add after diluting at PRRSV nucleocapsid protein and the proteic monoclonal antibody of GP5 (Zhou YJ et al., 2005; Zhou YJ et al., 2006), incubated at room 1h, PBS washing 5 times adds two of FITC mark sheep anti mouse again and resists, incubated at room 1h, PBS washing 5 times, observations under fluorescent microscope.
1.8 rescue viral biology specificity analysis
1.8.1 the mensuration of viral malicious valency
Reference literature carries out the mensuration (Yin Zhen etc., 1997) of infection titer with 96 hole tissue culturing plate methods.With sample to be checked with after keeping liquid and making 10 times of serial dilutions, with the Marc-145 monolayer cell on the virus inoculation 96 porocyte culture plates of serial dilution.Every extent of dilution is inoculated 8 holes, establishes 8 holes contrast (replacing viral liquid with keeping liquid), puts in 37 ℃ 5% the CO2gas incubator and cultivates, and observes cells infected every day, and the hole count of CPE (cytopathy) appears in record, stops observation when stopping CPE to occur.Press the Reed-Muench method according to the result and calculate TCID
50(Yin Zhen etc., 1997).
1.8.2 the drafting of viral multistep growth curve
Infect the Macr-145 cell with low dosage (0.1MOI) virus (genetically deficient of parent poison HuN4-F112 and rescue is cloned malicious dHN4-Δ 52 and dHN4-Δ 25), after infection, collect 200 μ L cell conditioned medium liquid and carry out virus titer mensuration, each time point virus median infective dose (TCID that collects every 12h
50/ mL) calculate titre, draw viral multistep growth curve according to the titre of different time points virus.
1.8.3 genetically deficient strain genetic stability is analyzed
In order further to study the stability of missing gene in viral dHN4-Δ 52 of rescue and the dHN4-Δ 25, respectively this two strains rescue virus is passed 20 generations continuously at Macr-145, utilization RT-PCR amplifies the zone of missing gene, and its amplified production is carried out sequencing analysis.
2. result
According to embodiment 1 above-mentioned test method, by sudden change PCR operation equimolecular biological experiment technique means, two full length cDNA clone plasmids of the Nsp2 gene Δ 480-532 amino acids and the Δ 508-532 amino acids disappearance of PRRSV attenuated vaccine strain HuN4-F112 strain have successfully been made up respectively, that is: pSK-HN4vac-Δ 52 and pSK-HN4vac-Δ 25, wherein, the Nsp2 gene order of the Δ 52 of disappearance 480-532 amino acids is shown in SEQ ID NO.1, and the Nsp2 gene order of the Δ 25 of disappearance 508-532 amino acids is shown in SEQ ID NO.2.Utilize pSK-HN4vac-Δ 52 and pSK-HN4vac-Δ 25 these two recombinant plasmids, through in-vitro transcription, transfection and virus rescue, all can rescue capacitation and enough infect the genetically deficient virus strain dHN4-Δ 52 of Macr-145 cell and the live virus of dHN4-Δ 25, detect through RT-PCR and all can amplify specific gene fragment (Fig. 1).In Fig. 1, M.DNA molecular weight standard DL 2000; 1.HuN4-F112 the RT-PCR detected result; 2.dHN4-the RT-PCR detected result of Δ 52; 3.dHN4-the RT-PCR detected result of Δ 25; 4.rHN4-the RT-PCR detected result of Δ 52+NP49; 5.rHN4-the RT-PCR detected result of Δ 25+NP49; 6. the RT-PCR detected result of negative control.
Rescue behind the genetically deficient virus infection Macr-145 cell that obtains 48 hours and just can observe tangible cytopathy (Fig. 2), cell mainly shows as gathering, circle contracts, comes off, and becomes the hauling type cytopathy, with the caused cytopathy basically identical of parent's poison.The live virus of rescuing the different generations that obtain is carried out RT-PCR and order-checking evaluation, confirm to rescue its Nsp2 gene of two strain live viruses that obtains and have Δ 52 and Δ 25 aminoacid deletion respectively, and reach the equal stable existence of this deletion mutantion of 20 generations (Fig. 3).
Utilize anti-PRRSV N protein-specific monoclonal antibody to carry out indirect immunofluorescence assay, result's confirmation can detect the specificity fluorescent (Fig. 4) of the N albumen generation of genetically deficient expressing viral, confirms that further the genetically deficient virus that is obtained is PRRS virus.TCID to two pnca genes disappearance virus strain
50Measurement result is basic consistent with the malicious valency of parent's poison, wherein the TCID of dHN4-Δ 52
50Be 10
6The TCID of/0.1ml and dHN4-Δ 25
50Be 10
6.22/ 0.1ml.Further the duplicating dynamics of two pnca genes disappearance virus different time points behind cells infected is drawn the multistep growth curve, the result shows that this two pnca genes disappearance virus has just reached the peak in back 48 hours levels of replication of infection, after this remain on a plateau substantially, whole replicative cycle and parent's poison consistent substantially (Fig. 5).
Above result shows that the recombinant virus of above-mentioned 2 Nsp2 genetically deficients that the present invention obtains can genetic stability, the biological characteristics of its genetically deficient virus strain and parent's poison basically identical.
The structure and the evaluation of embodiment 2 porcine reproductive and respiratory syndrome virus genetically engineered mark attenuated vaccine strains
1. materials and methods
1.1 virus, carrier and reagent
Avian pneumo-encephalitis virus LaSota strain by China Agriculture Academe Shanghai Veterinary Institute's fowl Infectious Diseases Lab provide, PRRSV vaccine strain and infections clone carrier pSK-Hun4-F112 thereof make up and preserved (Tong GZ et al., 2007 by this laboratory; ZhouYJ et al., 2008), Newcastle disease virus NP albumen monoclonal antibody is provided by poultry diease chamber avian paramyxoviruses study group of Harbin Veterinary Medicine Inst., China Academy of Agriculture; RNeasy Plus Mini Kit test kit is available from QIAGEN; Glue reclaims test kit available from Shanghai China Shun bio tech ltd; SupersciptIII reverse transcriptase and
Pfx DNApolymerase is available from Invitrogen; RNase H, T4DNA ligase enzyme, restriction enzyme are available from NEB company; DL-15000, DL-2000DNA Marker is available from TakaRa; Other chemical reagent all is import or homemade analytical pure.
1.2 design of primers
According to Avian pneumo-encephalitis virus LaSota strain (GenBank accession number: AY845400) NP gene order and PRRSV HuN4-F112 strain Nsp2 gene order, design 3 pairs of primers promptly: F-NDVNP/R-NDVNP, F Δ 52+NP49/R Δ 52+NP49 and F Δ 25+NP49/R Δ 25+NP49 (seeing Table 1), carried the NP49 gene region that inserts sudden change in wherein back two pairs of primers.
1.3 insert the segmental pcr amplification of gene specific
1.3.1 the acquisition of mutant primer
Adopt Rneasy kit (Qiagen) test kit to extract total RNA of Avian pneumo-encephalitis virus LaSota strain, through the synthetic cDNA of reverse transcription.With cDNA is template, utilizes F-NDV NP/R-NDV NP primer, and reaction system is 50 μ L, and reaction conditions is: 92 ℃ of 2min; 92 ℃ of 10s, 60 ℃ of 20s, 72 ℃ of 1min circulate 30 times; Extend 5min in 72 ℃ at last.Amplify Newcastle disease virus NP gene.Be template with the NP gene that obtains then, utilize F Δ 52+NP49/R Δ 52+NP49 and F Δ 25+NP49/R Δ 25+NP49 to be primer respectively, amplify the gene order of Δ 52+NP49 and Δ 25+NP49 respectively, and reclaim these two gene fragments as mutant primer.
Utilizing above-mentioned two pairs of mutant primers respectively, is suddenly change PCR operation of template with the pSK-HuN4-F112-ABC plasmid, and reaction system is 50 μ L, and reaction conditions is: 92 ℃/2min, 92 ℃/10s, 55 ℃/20s, 68 ℃/6min, circulate 18 times; Extend 5min in 68 ℃ at last.
1.4 insert NP49 gene viruses full-length cDNA construction of recombinant plasmid
Respectively above-mentioned reaction product is carried out Dpn I digestion with restriction enzyme, respectively with two linearizing PCR product transformed competence colibacillus bacillus coli DH 5 alphas of above-mentioned acquisition, identify positive recombinant plasmid then through plasmid extraction, PCR evaluation and sequencing analysis etc.
Utilizing Pac I and NheI to carry out enzyme the positive recombinant plasmid of above-mentioned acquisition and pSK-HuN4-F112 plasmid respectively cuts digestion, reclaims ABC-Δ 52+NP49, ABC-Δ 25+NP49 and the segmental enzyme of pSK-HuN4-F112-DEF and cut product.The gene fragment that is recovered to is connected with the pSK-HuN4-F112-DEF fragment respectively and the transformed competence colibacillus bacillus coli DH 5 alpha, through plasmid extraction, PCR evaluation and sequencing analysis etc., filter out positive recombinant plasmid, respectively called after pSK-HN4vac-Δ 52+NP49 and pSK-HN4vac-Δ 25+NP49.
1.5RNA external synthetic and transfection
Plasmid pSK-HN4vac-Δ 52+NP49 and pSK-HN4vac-Δ 25+NP49 that the SwaI restriction enzyme site that utilizes 3 ' latter end Poly (A) tail to introduce will comprise the viral genome full-length cDNA carry out linearizing respectively, according to the synthetic respectively viral RNA of in-vitro transcription test kit mMessage High Yield Capped RNA Transcription kit operation instructions, cultivate the BHK21 cell simultaneously and make its density reach 70%～90%, with above-mentioned synthetic viral RNA respectively according to DMRI-C transfection reagent specification sheets transfection BHK21 cell.Cell after the transfection places 37 ℃ of CO
2Cultivated 48 hours in the incubator.
1.6 insert the rescue of NP49 genetic marker virus strain
Gather in the crops above-mentioned cells transfected and be stored in-80 ℃, behind multigelation, be inoculated on the cultured in advance Macr-145 cell monolayer in 37 ℃ of CO
2Sense is done 1 hour in the incubator, adds cell maintenance medium in 37 ℃ of CO
2Continue in the incubator to cultivate, carry out the rescue of virus.
1.7 the evaluation of virus
Obtain the RT-PCR evaluation of inserting genetic marker virus 1.7.1 rescue
Extract the RNA that two strains have infective genetically engineered mark attenuated vaccine strain rHN4-Δ 52+NP49, rHN4-Δ 25+NP49 and parent's poison HuN4-F112 cells infected respectively, through reverse transcription, utilize primers F 2701/R3192 to carry out RT-PCR amplification, and product is connected the pMD18-T carrier check order.
Obtain the indirect immunofluorescence evaluation of inserting genetic marker virus 1.7.2 rescue
Two strains of rescue are had infective genetically engineered mark attenuated vaccine strain rHN4-Δ 52+NP49 and rHN4-Δ 25+NP49 inoculates the Macr-145 cell monolayer respectively, infect back 48h and discard nutrient solution.With icing fixedly 10min of methyl alcohol, add (the Zhou YJ et al. of the monoclonal antibody at the PRRSV nucleocapsid protein after diluting, 2005) and at the specific monoclonal antibody of Newcastle disease virus NP gene, incubated at room 1h, PBS washing 5 times adds two of FITC mark sheep anti mouse again and resists incubated at room 1h, PBS washing 5 times, observations under fluorescent microscope.
1.8 rescue viral biology analysis
1.8.1 the mensuration of viral malicious valency
Reference literature carries out the mensuration (Yin Zhen etc., 1997) of infection titer with 96 hole tissue culturing plate methods.With sample to be checked with after keeping liquid and making 10 times of serial dilutions, with the Marc-145 monolayer cell on the virus inoculation 96 porocyte culture plates of serial dilution.Every extent of dilution is inoculated 8 holes, establishes 8 holes contrast (replacing viral liquid with keeping liquid), puts in 37 ℃ 5% the CO2gas incubator and cultivates, and observes cells infected every day, and the hole count of CPE appears in record, stops observation when stopping CPE to occur.Press the Reed-Muench method according to the result and calculate TCID
50(Yin Zhen etc., 1997).
1.8.2 the drafting of viral multistep growth curve
(the genetically engineered mark attenuated vaccine strain rHN4-Δ 52+NP49 and the rHN4-Δ 25+NP49 of parent poison HuN4-F112 and insertion NP49 gene) infects the Macr-145 cell with low dosage (0.1MOI) virus, after infection, collect 200 μ L cell conditioned medium liquid and carry out virus titer mensuration, each time point virus median infective dose (TCID that collects every 12h
50/ mL) calculate titre, draw viral multistep growth curve according to the titre of different time points virus.
1.8.3 the marker gene genetic stability detects
In order further to study the stability of the marker gene NP49 that inserts among viral rHN4-Δ 52+NP49 of rescue and the rHN4-Δ 25+NP49, respectively this two strains rescue virus was passed for 20 generations continuously on the Macr-145 cell, and the virus utilization RT-PCR amplification of getting different generations contains the fragment of NP49 gene, and the gene fragment of amplification has been carried out sequencing analysis.
2. result
According to embodiment 2 described test methods, by sudden change PCR operation equimolecular biological experiment technique means, respectively at the Δ 52 and Δ 25 aminoacid deletion zones of the Nsp2 gene of PRRSV attenuated vaccine strain HuN4-F112 strain, successful respectively insertion Newcastle disease virus NP 49 gene fragments that serve as a mark, two full length cDNA clone plasmids that contain the NP49 gene have been made up, that is: pSK-HN4vac-Δ 52+NP49 and pSK-HN4vac-Δ 25+NP49.Nsp2 gene order after the Δ 52 aminoacid deletion zone insertion NP49 marker gene fragment of Nsp2 gene is shown in SEQ ID NO.3, and the Nsp2 gene order after Δ 25 aminoacid deletion of the Nsp2 gene zone insertion NP49 marker gene fragment is shown in SEQ ID NO.4.Utilize these two recombinant plasmids of pSK-HN4vac-Δ 52+NP49 and pSK-HN4vac-Δ 25+NP49, through in-vitro transcription, transfection and virus rescue, all can rescue capacitation and enough infect the live virus of the insertion NP49 genetic marker virus of Macr-145 cell, RT-PCR detects can detect specific gene fragment (Fig. 1).
Rescue behind the insertion genetic marker virus infection Macr-145 cell that obtains 48 hours and just can observe tangible cytopathy (Fig. 2), cell mainly shows as gathering, circle contracts, comes off, be the hauling type cytopathy, with the caused cytopathy basically identical of parent's poison.The live virus of rescuing the different generations that obtain is carried out RT-PCR and order-checking evaluation, confirmation is rescued the two strain live viruses that obtain and is had the NP49 gene that inserts in Nsp2 gene Δ 52 aminoacid deletion zone and Δ 25 aminoacid deletion zone, and reach 20 generations this insert the equal stable existence of sudden change, do not undergo mutation and phenomenon (Fig. 3) such as disappearance.
Utilizing anti-PRRSV N protein-specific monoclonal antibody and Newcastle disease virus NP protein-specific monoclonal antibody to carry out indirect immunofluorescence assay detects, the result confirms, can detect PRRSV expresses the proteic specificity fluorescent of N and express the insertion proteic specificity fluorescent of newcastle disease NP49 (Fig. 4) in PRRSV, confirm that further the virus of being saved is PRRS virus, and rescue the PRRS virus that obtains and to express Newcastle disease virus NP 49 genes.Two strains had the TCID that inserts the genetic marker virus strain
50Measurement result is basic consistent with the malicious valency of parent's poison, wherein the TCID of rHN4-Δ 52+NP49
50Be 10
5.67The TCID of/0.1ml and rHN4-Δ 25+NP49
50Be 10
5.81/ 0.1ml.Two strain virus growth kinetics curve plotting results as shown in Figure 5, the result shows that this two strains insertion genetic marker virus has just reached the peak in back 48 hours levels of replication of infection, maintain a higher levels of replication afterwards always, substantially remain on a plateau, whole replicative cycle and parent's poison HuN4-F112 consistent substantially (Fig. 5) show with parent's poison HuN4-F112 to have similar biologic activity at the virus titer that duplicates, breeds each time of virus.
Above result shows that the rHN4-Δ 52+NP49 of NP49 gene and candidate's strain that rHN4-Δ 25+NP49 recombinant virus can be used as PRRSV genetically engineered mark attenuated vaccine are inserted in two strains that the present invention obtains in the Nsp2 gene.
The test of embodiment 3 porcine reproductive and respiratory syndrome virus genetically engineered mark attenuated vaccine strain immunoprotections
1. materials and methods
1.1 the selection of test pig and grouping
Choose cause of disease and all negative healthy piglets of antibody such as 30 35 age in days PRRSV, CSFV, PCV2, be divided into 4 groups at random, two clone's poison that insert NP49 that embodiment 2 obtains are used genetically engineered marker vaccine poison as testing, laboratory animal is divided into: rHN4-Δ 52+NP49 (A group) test group, rHN4-Δ 25+NP49 (B group) test group, HuN4-F112 (F group) vaccine control group, DMEM (K group) control group.Every group of 5 pigs carry out isolated rearing.
1.2 the immunization of genetically engineered mark attenuated vaccine strain
The virus titer of genetically engineered mark attenuated vaccine strain and HuN4-F112 vaccine control group is adjusted into TCID
5010
5/ ml inoculates by the mode of musculi colli injection respectively, and dosage of inoculation is the 1ml/ head, blank group musculi colli injection DMEM nutrient solution 1ml.
1.3 immunity back challenge test
In inoculation back 28 days is 10 with the strong poison dilution of HuN4-F5 strain
4.0TCID
50/ ml attacks HuN4-F5 poison by force to each test pig according to the dosage of every incidence intramuscular injection 3ml, and the pig of blank group also inoculates with same vaccination ways and dosage of inoculation.
1.4 test pig clinicing symptom observation and body temperature are measured
Behind the test pig inoculation genetically engineered marker vaccine poison, day by day viewing test pig clinical manifestation, after 28 days all test pig of inoculation were attacked poison with the HuN4-F5 strain, the viewing test pig was attacked poison back clinical manifestation, every day all test pig was carried out body temperature mensuration simultaneously every day, continued thermometric to attacking poison back 14 days.
1.5 test pig PRRSV specific antibody detects
Before the immunity of genetically engineered mark attenuated vaccine test group and immunity back the 7th, gathered the serum of all test group pigs in 14,21,28 days, utilize IDEXX PRRSV antibody assay kit to carry out antibody test, simultaneously the pig of inoculation DMEM control group is also gathered serum and antibody test the same period.
1.6 the discriminating of test pig traget antibody detects
Before the immunity of genetically engineered mark attenuated vaccine test group and immunity back gathered the serum of all test group pigs in the 7th, 14,21,28 days, the NP49 albumen that utilizes the amalgamation and expression mark is as detecting antigen, and the antiserum(antisera) antibody of all collections is carried out differential diagnosis.
2. result
Utilize two strain PRRSV genetically engineered mark attenuated vaccine strains (rHN4-Δ 52+NP49 and rHN4-Δ 25+NP49) that the piglet of 35 ages in days has been carried out the immune protective test respectively; within back 28 days of inoculation (before promptly attacking poison); the pig of test group is not all found body temperature and the unusual phenomenon of symptom performance; illustrate that above-mentioned two kinds of genetically engineered mark attenuated vaccine strains are consistent with parent's poison; pig not being had pathogenic, is safe and reliable.After the test pig immunity was attacked poison in 28 days, find that wherein the DMEM control group then the fervescence phenomenon only all occurs attacking back second day all pigs of poison, continue nearly about 10 days always, the pig of heating mainly show as appetite stimulator, thick disorderly by hair, present clinical manifestations such as typical ventral breathing, skin cyanosis; RHN4-Δ 52+NP49 immune group is after attacking poison with highly pathogenic PRRSV HuN4-F5, and second day begins to have 2 pigs the fervescence phenomenon to occur, has reached 41 ℃, continues nearly five days; And reactions such as tangible fervescence do not appear in rHN4-Δ 25+NP49 immune group and parent's poison HuN4-F112 vaccine group basically identical, and test pig does not only see that unusual clinical manifestation is arranged yet.
All test group are before immunity and immunity afterwards the 7th, 14,21, gathering serum in 28 days utilizes IDEXX PRRSV antibody assay kit to detect PRRSV antibody, the result is presented at except that the DMEM control group each experimental group the 7th day antibody after immunity and begins to occur, after this antibody horizontal continues to raise, before attacking poison, maintain the higher level (see figure 6) always, show that above-mentioned two kinds of genetically engineered mark attenuated vaccines are consistent with parent's vaccine virus, pig is had better immunogenicity, can stimulate body to produce stronger humoral immune reaction.
Utilize the NP49 albumen of expressing as detecting the ELISA antibody detection method that antigen is set up, detect the serum antibody of each immune group test pig in the different immunity back times, the result shows, two genetically engineered mark attenuated vaccine strain immune group can detect the proteic serum antibody of anti-NP49 on the 14th day after immunity, obviously raise to the 28th day antibody horizontal in immunity back, parent's vaccine virus HuN4-F112 immune group and DMEM control group then present feminine gender (Fig. 7) all the time, show that NP49 gene that the present invention inserts can induce body to produce specific antibody in parent's vaccine virus, and can be with its mark as wild poison of difference natural infection and vaccine immunity poison.
After immunity, gathered the blood sample of each test group pig on the 14th day respectively, extract RNA and carry out the RT-PCR detection of virogene, and utilize the MluI restriction enzyme to carry out enzyme to its amplified production and cut to detect the characteristic mark of immune strain, the result shows, except that the DMEM control group does not have the RT-PCR amplified production, remaining vaccine immunity group all can amplify DNA fragment specific, after the PCR product is cut through the MluI enzyme, discovery has only the PCR product of parent's vaccine virus HuN4-F112 immune group not to be cut open, the PCR product of two other genetically engineered mark attenuated vaccine immunity group all can be cut into the band (Fig. 8) of two clauses and subclauses, this conforms to experimental design, and there is not wild virus infection in genetically engineered mark attenuated vaccine immunity pig.In Fig. 8 (1), M.DNA molecular weight standard DL 2000; RT-PCR detected result after F1～F5.HuN4-F112 immunity; RT-PCR detected result after A1～A5.rHH4-Δ 52+NP49 immunity; RT-PCR detected result after B1～B5.rHN4-Δ 25+NP49 immunity; RT-PCR detected result after K1～K5.DMEM immunity.In Fig. 8 (2), M.DNA molecular weight standard DL 2000; RT-PCR product Mlu I enzyme after F1～F5.rHuN4-F112 immunity is cut detected result; RT-PCR detected result product Mlu I enzyme after A1～A5.rHN4-Δ 52+NP49 immunity is cut detected result; RT-PCR product Mlu I enzyme after B1～B5.rHN4-Δ-25+NP49 immunity is cut the detected result detected result.
The pig of each test group is attacked the immune protective effect evaluation of poison back, according to the mortality statistics result, can see that the DMEM control group attacking poison back 11 days, all pigs are all dead, and mortality ratio reaches 100%; Each dead one respectively of rHN4-Δ 52+NP49 immune group in attack poison back the 7th day and the 11st day, all the other all healthy survivals, dead protection ratio is 60%; RHN4-Δ 25+NP49 immune group and parent's vaccine virus HuN4-F112 strain immune group are then until off-test, and all immune swines are only all healthy survives, and dead protection ratio reaches 100% (Fig. 9).The result shows that genetically engineered mark attenuated vaccine rHN4-of the present invention Δ 25+NP4 strain and commercial HuN4-F112 vaccine strain have good immunogenicity equally; can stimulate body to produce very strong immune response; can resist highly pathogenic PRRSV virulent strain HuN4 and attack, provide immune protective effect preferably piglet.
In sum; genetically engineered mark attenuated vaccine strain rHN4-Δ 25+NP4 strain based on construction of recombinant plasmid has security preferably to pig; can induce body to produce protective immunological reaction after the immunity effectively, and it be resisted highly pathogenic PRRSV virulent strain 100% immunoprotection is provided.
The above embodiment has only expressed embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Sequence table
＜110〉China Agriculture Academe Shanghai Veterinary Institute
＜120〉porcine reproductive and respiratory syndrome virus genetically engineered mark attenuated vaccine strain and application
<160>14
<170>PatentIn?version?3.3
<210>1
<211>3342
<212>DNA
＜213〉porcine reproductive and respiratory syndrome virus genetically deficient strain dHN4-Δ 52
<400>1
gccggaaaga?gagcaaggaa?aacacgctct?ggtgcgacta?ctatggtcgc?tcgtcacgct 60
tcgtccgctc?atgaaacccg?gcaggccacg?aagcacgagg?gtgccggcgc?taacaaggct 120
gagcatctca?agcgctactc?tccgcctgcc?gaagggaact?gtggttggca?ctgcatttcc 180
gccatcgcca?accggatggt?gaattccaac?tttgagacca?cccttcctga?aagagtaagg 240
ccttcagatg?actgggccac?tgacgaggat?cttgtgaaca?tcatccaaat?cctcaggctc 300
cctgcggcct?tggacaggaa?cggcgcttgc?ggtagcgcca?agtacgtgct?taaactggag 360
ggtgagcatt?ggactgtctc?tgtgatccct?gggatgtccc?ctactttgct?cccccttgaa 420
tgtgttcagg?gttgttgtga?gcataagggc?ggtcttgttt?ccccggatgc?ggtcgaaatt 480
tccggatttg?atcctgcctg?ccttgaccga?ctggctaagg?taatgcactt?gcctagcagt 540
accatcccag?ccgctctggc?cgaattgtcc?gacgactcct?accgtccggt?ttccccggcc 600
gctactacgt?ggactgtttc?gcaattctat?gctcgttata?gaggaggaga?tcatcatgac 660
caggtgtgct?tggggaaaat?catcagcctt?tgtcaagtta?ttgaggattg?ctgctgccat 720
cagaataaaa?ccaaccgggc?tactccggaa?gaggtcgcgg?caaagattga?tcagtacctc 780
cgtggcgcaa?caagtcttga?ggaatgcttg?gccaaacttg?agagagtttc?cccgccgagc 840
gctgcggaca?cctcctttga?ttggaatgtt?gtgcttcctg?gggttgaggc?ggcgaatcag 900
acaaccgaac?aacctcacgt?caactcatgc?tgcaccctgg?tccctcccgt?gactcaagag 960
cctttgggca?aggactcggt?ccctctgacc?gccttctcac?tgtccaattg?ctattaccct 1020
gcacaaggtg?acgaggttca?tcaccgtgag?aggttaaatt?ccgtactctc?taagttggaa 1080
gaggttgtcc?tggaagaata?tgggctcatg?tccactggac?ttggcccgcg?acccgtgctg 1140
ccgagcgggc?tcgacgagct?taaagaccag?atggaggagg?atctgctaaa?actagccaac 1200
acccaggcga?cttcagaaat?gatggcctgg?gcggctgagc?aggtcaattt?aaaagcttgg 1260
gtcaaaagct?acccgcggtg?gacaccacca?ccccctccac?caagagttca?acctcgaaga 1320
acaaagtctg?tcaaaagctt?gccagagggc?aagcctgtcc?ctgctccgcg?caggaaggtc 1380
agatccgatt?gcggcagccc?ggttttgatg?ggcgacaatg?tccctaacgg?ttcggaagaa 1440
acaacgctga?cgcaccagga?tgagcctctg?gatttgcctg?cgtcctcaca?gacggaatat 1500
gaggctttcc?ccctagcacc?atcgcagaac?atgggcatcc?tggaggcggg?ggggcaagaa 1560
gttgaggaag?tcctgagtga?aatctcggat?atactaaatg?acaccaaccc?tgcacctgtg 1620
tcatcaagca?gccccctgtc?aagtgttaag?atcacacgcc?caaaatactc?agctcaagcc 1680
atcatcgact?ctggcgggcc?ttgcagtggg?catctccaaa?aggaaaaaga?agcatgcctc 1740
agcatcatgc?gtgaggcttg?tgatgcgtcc?aagcttggtg?atcctgctac?gcaggagtgg 1800
ctctctcgca?tgtgggatag?ggttgacatg?ctgacttggc?gcaacacgtc?tgcttaccag 1860
gcgtttcgca?tcttaagtgg?caggtttgag?tttctcccaa?agatgattct?cgagacaccg 1920
ccgccccacc?cgtgcgggtt?tgtgatgtta?cctcgcacgc?ctgcaccttc?cgtgagtgca 1980
gagagtgacc?tcaccattgg?ttcagtggcc?accgaggatg?ttccacgcat?cctcgggaaa 2040
ataggagaca?ctgacgagct?gcttgaccgg?ggtccctcgg?caccctccaa?gggagaaccg 2100
gtcagtgacc?aacctgccaa?agatccccgg?atgtcgccgc?gggagtctga?cgagagcatg 2160
atagctccgc?ccgcagatac?aggtggtgtc?ggctcattca?ctgatttgcc?gtcttcagat 2220
ggtgtggatg?tggacggggg?ggggccgtta?agaacggtaa?aaacaaaagc?ggggaggctc 2280
ttagaccaac?tgagctgcca?ggtttttagc?ctcgtttccc?atctccctat?tttcttctca 2340
cacctcttca?aatctgacag?tggttattct?ccgggtgatt?ggggttttgc?agcttttact 2400
ctattttgcc?tctttctatg?ttacagttac?ccattcttcg?gttttgctcc?cctcttgggt 2460
gtattttctg?ggtcttctcg?gcgtgtgcga?atgggggttt?ttggctgctg?gttggctttt 2520
gctgttggtc?tgttcaagcc?tgtgtccgac?ccagtcggca?ctgcttgtga?gtttgactcg 2580
ccagagtgta?ggaacgtact?tcattctttt?gagcttctca?aaccttggga?ccctgtccgc 2640
agccttgttg?tgggccccgt?cggtctcggc?cttgccattc?ttggcaggtt?actgggcggg 2700
gcacgctata?tctggcactt?tttgcttagg?cttggcattg?ttacagactg?tatcttggct 2760
ggagcttatg?tgctttctca?aggtaggtgt?aaaaagtgct?ggggatcttg?tgtaagaact 2820
gctcctaatg?agatcgcctt?caacgtgttc?ccttttacac?gtgcgaccag?gtcgtcactc 2880
atcgacctgt?gcgatcggtt?ttgcgcacca?aaaggcatgg?accccatttt?tctcgccact 2940
gggtggcgtg?ggtgctggac?cggccggagt?cccattgagc?aaccttctga?aaaacccatc 3000
gcgttcgccc?agctggatga?gaagaggatt?acggctagaa?ctgtggtcgc?tcagccttat 3060
gatcccaacc?aggccgtaaa?gtgcttgcgg?gtattacagg?cgggtggggc?gatggtggcc 3120
gaggcagtcc?caaaagtggt?caaagtttcc?gctattccat?tccgagctcc?tttctttccc 3180
gctggagtga?aagttgatcc?tgagtgcaga?atcgtggttg?atcccgatac?ttttactaca 3240
gccctccggt?ctggctattc?caccgcgaac?ctcgtccttg?gtacggggga?ctttgcccag 3300
ctgaatggac?taaagatcag?gcaaatttcc?aagccttcag?gg 3342
<210>2
<211>3420
<212>DNA
＜213〉porcine reproductive and respiratory syndrome virus genetically deficient strain dHN4-Δ 25
<400>2
gccggaaaga?gagcaaggaa?aacacgctct?ggtgcgacta?ctatggtcgc?tcgtcacgct 60
tcgtccgctc?atgaaacccg?gcaggccacg?aagcacgagg?gtgccggcgc?taacaaggct 120
gagcatctca?agcgctactc?tccgcctgcc?gaagggaact?gtggttggca?ctgcatttcc 180
gccatcgcca?accggatggt?gaattccaac?tttgagacca?cccttcctga?aagagtaagg 240
ccttcagatg?actgggccac?tgacgaggat?cttgtgaaca?tcatccaaat?cctcaggctc 300
cctgcggcct?tggacaggaa?cggcgcttgc?ggtagcgcca?agtacgtgct?taaactggag 360
ggtgagcatt?ggactgtctc?tgtgatccct?gggatgtccc?ctactttgct?cccccttgaa 420
tgtgttcagg?gttgttgtga?gcataagggc?ggtcttgttt?ccccggatgc?ggtcgaaatt 480
tccggatttg?atcctgcctg?ccttgaccga?ctggctaagg?taatgcactt?gcctagcagt 540
accatcccag?ccgctctggc?cgaattgtcc?gacgactcct?accgtccggt?ttccccggcc 600
gctactacgt?ggactgtttc?gcaattctat?gctcgttata?gaggaggaga?tcatcatgac 660
caggtgtgct?tggggaaaat?catcagcctt?tgtcaagtta?ttgaggattg?ctgctgccat 720
cagaataaaa?ccaaccgggc?tactccggaa?gaggtcgcgg?caaagattga?tcagtacctc 780
cgtggcgcaa?caagtcttga?ggaatgcttg?gccaaacttg?agagagtttc?cccgccgagc 840
gctgcggaca?cctcctttga?ttggaatgtt?gtgcttcctg?gggttgaggc?ggcgaatcag 900
acaaccgaac?aacctcacgt?caactcatgc?tgcaccctgg?tccctcccgt?gactcaagag 960
cctttgggca?aggactcggt?ccctctgacc?gccttctcac?tgtccaattg?ctattaccct 1020
gcacaaggtg?acgaggttca?tcaccgtgag?aggttaaatt?ccgtactctc?taagttggaa 1080
gaggttgtcc?tggaagaata?tgggctcatg?tccactggac?ttggcccgcg?acccgtgctg 1140
ccgagcgggc?tcgacgagct?taaagaccag?atggaggagg?atctgctaaa?actagccaac 1200
acccaggcga?cttcagaaat?gatggcctgg?gcggctgagc?aggtcaattt?aaaagcttgg 1260
gtcaaaagct?acccgcggtg?gacaccacca?ccccctccac?caagagttca?acctcgaaga 1320
acaaagtctg?tcaaaagctt?gccagagggc?aagcctgtcc?ctgctccgcg?caggaaggtc 1380
agatccgatt?gcggcagccc?ggttttgatg?ggcgacaatg?tccctaacgg?ttcggaagaa 1440
actgtcggtg?gtcccctcaa?ttttccgaca?ccatccgagc?cgatgacacc?tatgagtgag 1500
cccgtactta?tgcccgcgac?aacgctgacg?caccaggatg?agcctctgga?tttgcctgcg 1560
tcctcacaga?cggaatatga?ggctttcccc?ctagcaccat?cgcagaacat?gggcatcctg 1620
gaggcggggg?ggcaagaagt?tgaggaagtc?ctgagtgaaa?tctcggatat?actaaatgac 1680
accaaccctg?cacctgtgtc?atcaagcagc?cccctgtcaa?gtgttaagat?cacacgccca 1740
aaatactcag?ctcaagccat?catcgactct?ggcgggcctt?gcagtgggca?tctccaaaag 1800
gaaaaagaag?catgcctcag?catcatgcgt?gaggcttgtg?atgcgtccaa?gcttggtgat 1860
cctgctacgc?aggagtggct?ctctcgcatg?tgggataggg?ttgacatgct?gacttggcgc 1920
aacacgtctg?cttaccaggc?gtttcgcatc?ttaagtggca?ggtttgagtt?tctcccaaag 1980
atgattctcg?agacaccgcc?gccccacccg?tgcgggtttg?tgatgttacc?tcgcacgcct 2040
gcaccttccg?tgagtgcaga?gagtgacctc?accattggtt?cagtggccac?cgaggatgtt 2100
ccacgcatcc?tcgggaaaat?aggagacact?gacgagctgc?ttgaccgggg?tccctcggca 2160
ccctccaagg?gagaaccggt?cagtgaccaa?cctgccaaag?atccccggat?gtcgccgcgg 2220
gagtctgacg?agagcatgat?agctccgccc?gcagatacag?gtggtgtcgg?ctcattcact 2280
gatttgccgt?cttcagatgg?tgtggatgtg?gacggggggg?ggccgttaag?aacggtaaaa 2340
acaaaagcgg?ggaggctctt?agaccaactg?agctgccagg?tttttagcct?cgtttcccat 2400
ctccctattt?tcttctcaca?cctcttcaaa?tctgacagtg?gttattctcc?gggtgattgg 2460
ggttttgcag?cttttactct?attttgcctc?tttctatgtt?acagttaccc?attcttcggt 2520
tttgctcccc?tcttgggtgt?attttctggg?tcttctcggc?gtgtgcgaat?gggggttttt 2580
ggctgctggt?tggcttttgc?tgttggtctg?ttcaagcctg?tgtccgaccc?agtcggcact 2640
gcttgtgagt?ttgactcgcc?agagtgtagg?aacgtacttc?attcttttga?gcttctcaaa 2700
ccttgggacc?ctgtccgcag?ccttgttgtg?ggccccgtcg?gtctcggcct?tgccattctt 2760
ggcaggttac?tgggcggggc?acgctatatc?tggcactttt?tgcttaggct?tggcattgtt 2820
acagactgta?tcttggctgg?agcttatgtg?ctttctcaag?gtaggtgtaa?aaagtgctgg 2880
ggatcttgtg?taagaactgc?tcctaatgag?atcgccttca?acgtgttccc?ttttacacgt 2940
gcgaccaggt?cgtcactcat?cgacctgtgc?gatcggtttt?gcgcaccaaa?aggcatggac 3000
cccatttttc?tcgccactgg?gtggcgtggg?tgctggaccg?gccggagtcc?cattgagcaa 3060
ccttctgaaa?aacccatcgc?gttcgcccag?ctggatgaga?agaggattac?ggctagaact 3120
gtggtcgctc?agccttatga?tcccaaccag?gccgtaaagt?gcttgcgggt?attacaggcg 3180
ggtggggcga?tggtggccga?ggcagtccca?aaagtggtca?aagtttccgc?tattccattc 3240
cgagctcctt?tctttcccgc?tggagtgaaa?gttgatcctg?agtgcagaat?cgtggttgat 3300
cccgatactt?ttactacagc?cctccggtct?ggctattcca?ccgcgaacct?cgtccttggt 3360
acgggggact?ttgcccagct?gaatggacta?aagatcaggc?aaatttccaa?gccttcaggg 3420
<210>3
<211>3489
<212>DNA
＜213〉porcine reproductive and respiratory syndrome virus genetically engineered mark infections clone strain rHN4-Δ 52+NP49
<400>3
gccggaaaga?gagcaaggaa?aacacgctct?ggtgcgacta?ctatggtcgc?tcgtcacgct 60
tcgtccgctc?atgaaacccg?gcaggccacg?aagcacgagg?gtgccggcgc?taacaaggct 120
gagcatctca?agcgctactc?tccgcctgcc?gaagggaact?gtggttggca?ctgcatttcc 180
gccatcgcca?accggatggt?gaattccaac?tttgagacca?cccttcctga?aagagtaagg 240
ccttcagatg?actgggccac?tgacgaggat?cttgtgaaca?tcatccaaat?cctcaggctc 300
cctgcggcct?tggacaggaa?cggcgcttgc?ggtagcgcca?agtacgtgct?taaactggag 360
ggtgagcatt?ggactgtctc?tgtgatccct?gggatgtccc?ctactttgct?cccccttgaa 420
tgtgttcagg?gttgttgtga?gcataagggc?ggtcttgttt?ccccggatgc?ggtcgaaatt 480
tccggatttg?atcctgcctg?ccttgaccga?ctggctaagg?taatgcactt?gcctagcagt 540
accatcccag?ccgctctggc?cgaattgtcc?gacgactcct?accgtccggt?ttccccggcc 600
gctactacgt?ggactgtttc?gcaattctat?gctcgttata?gaggaggaga?tcatcatgac 660
caggtgtgct?tggggaaaat?catcagcctt?tgtcaagtta?ttgaggattg?ctgctgccat 720
cagaataaaa?ccaaccgggc?tactccggaa?gaggtcgcgg?caaagattga?tcagtacctc 780
cgtggcgcaa?caagtcttga?ggaatgcttg?gccaaacttg?agagagtttc?cccgccgagc 840
gctgcggaca?cctcctttga?ttggaatgtt?gtgcttcctg?gggttgaggc?ggcgaatcag 900
acaaccgaac?aacctcacgt?caactcatgc?tgcaccctgg?tccctcccgt?gactcaagag 960
cctttgggca?aggactcggt?ccctctgacc?gccttctcac?tgtccaattg?ctattaccct 1020
gcacaaggtg?acgaggttca?tcaccgtgag?aggttaaatt?ccgtactctc?taagttggaa 1080
gaggttgtcc?tggaagaata?tgggctcatg?tccactggac?ttggcccgcg?acccgtgctg 1140
ccgagcgggc?tcgacgagct?taaagaccag?atggaggagg?atctgctaaa?actagccaac 1200
acccaggcga?cttcagaaat?gatggcctgg?gcggctgagc?aggtcaattt?aaaagcttgg 1260
gtcaaaagct?acccgcggtg?gacaccacca?ccccctccac?caagagttca?acctcgaaga 1320
acaaagtctg?tcaaaagctt?gccagagggc?aagcctgtcc?ctgctccgcg?caggaaggtc 1380
agatccgatt?gcggcagccc?ggttttgatg?ggcgacaatg?tccctaacgg?ttcggaagaa 1440
ggggatgggg?agacccaatt?cctggatctg?atgagagcgg?tagcaaatag?catgagggag 1500
gcgccaaact?ctgcacaggg?cactccccaa?tcggggcctc?ccccaactcc?tgggccatcc 1560
caagataacg?acaccgactg?ggggtataca?acgctgacgc?accaggatga?gcctctggat 1620
ttgcctgcgt?cctcacagac?ggaatatgag?gctttccccc?tagcaccatc?gcagaacatg 1680
ggcatcctgg?aggcgggggg?gcaagaagtt?gaggaagtcc?tgagtgaaat?ctcggatata 1740
ctaaatgaca?ccaaccctgc?acctgtgtca?tcaagcagcc?ccctgtcaag?tgttaagatc 1800
acacgcccaa?aatactcagc?tcaagccatc?atcgactctg?gcgggccttg?cagtgggcat 1860
ctccaaaagg?aaaaagaagc?atgcctcagc?atcatgcgtg?aggcttgtga?tgcgtccaag 1920
cttggtgatc?ctgctacgca?ggagtggctc?tctcgcatgt?gggatagggt?tgacatgctg 1980
acttggcgca?acacgtctgc?ttaccaggcg?tttcgcatct?taagtggcag?gtttgagttt 2040
ctcccaaaga?tgattctcga?gacaccgccg?ccccacccgt?gcgggtttgt?gatgttacct 2100
cgcacgcctg?caccttccgt?gagtgcagag?agtgacctca?ccattggttc?agtggccacc 2160
gaggatgttc?cacgcatcct?cgggaaaata?ggagacactg?acgagctgct?tgaccggggt 2220
ccctcggcac?cctccaaggg?agaaccggtc?agtgaccaac?ctgccaaaga?tccccggatg 2280
tcgccgcggg?agtctgacga?gagcatgata?gctccgcccg?cagatacagg?tggtgtcggc 2340
tcattcactg?atttgccgtc?ttcagatggt?gtggatgtgg?acgggggggg?gccgttaaga 2400
acggtaaaaa?caaaagcggg?gaggctctta?gaccaactga?gctgccaggt?ttttagcctc 2460
gtttcccatc?tccctatttt?cttctcacac?ctcttcaaat?ctgacagtgg?ttattctccg 2520
ggtgattggg?gttttgcagc?ttttactcta?ttttgcctct?ttctatgtta?cagttaccca 2580
ttcttcggtt?ttgctcccct?cttgggtgta?ttttctgggt?cttctcggcg?tgtgcgaatg 2640
ggggtttttg?gctgctggtt?ggcttttgct?gttggtctgt?tcaagcctgt?gtccgaccca 2700
gtcggcactg?cttgtgagtt?tgactcgcca?gagtgtagga?acgtacttca?ttcttttgag 2760
cttctcaaac?cttgggaccc?tgtccgcagc?cttgttgtgg?gccccgtcgg?tctcggcctt 2820
gccattcttg?gcaggttact?gggcggggca?cgctatatct?ggcacttttt?gcttaggctt 2880
ggcattgtta?cagactgtat?cttggctgga?gcttatgtgc?tttctcaagg?taggtgtaaa 2940
aagtgctggg?gatcttgtgt?aagaactgct?cctaatgaga?tcgccttcaa?cgtgttccct 3000
tttacacgtg?cgaccaggtc?gtcactcatc?gacctgtgcg?atcggttttg?cgcaccaaaa 3060
ggcatggacc?ccatttttct?cgccactggg?tggcgtgggt?gctggaccgg?ccggagtccc 3120
attgagcaac?cttctgaaaa?acccatcgcg?ttcgcccagc?tggatgagaa?gaggattacg 3180
gctagaactg?tggtcgctca?gccttatgat?cccaaccagg?ccgtaaagtg?cttgcgggta 3240
ttacaggcgg?gtggggcgat?ggtggccgag?gcagtcccaa?aagtggtcaa?agtttccgct 3300
attccattcc?gagctccttt?ctttcccgct?ggagtgaaag?ttgatcctga?gtgcagaatc 3360
gtggttgatc?ccgatacttt?tactacagcc?ctccggtctg?gctattccac?cgcgaacctc 3420
gtccttggta?cgggggactt?tgcccagctg?aatggactaa?agatcaggca?aatttccaag 3480
ccttcaggg 3489
<210>4
<211>3567
<212>DNA
＜213〉porcine reproductive and respiratory syndrome virus genetically engineered mark infections clone strain rHN4-Δ 25+NP49
<400>4
gccggaaaga?gagcaaggaa?aacacgctct?ggtgcgacta?ctatggtcgc?tcgtcacgct 60
tcgtccgctc?atgaaacccg?gcaggccacg?aagcacgagg?gtgccggcgc?taacaaggct 120
gagcatctca?agcgctactc?tccgcctgcc?gaagggaact?gtggttggca?ctgcatttcc 180
gccatcgcca?accggatggt?gaattccaac?tttgagacca?cccttcctga?aagagtaagg 240
ccttcagatg?actgggccac?tgacgaggat?cttgtgaaca?tcatccaaat?cctcaggctc 300
cctgcggcct?tggacaggaa?cggcgcttgc?ggtagcgcca?agtacgtgct?taaactggag 360
ggtgagcatt?ggactgtctc?tgtgatccct?gggatgtccc?ctactttgct?cccccttgaa 420
tgtgttcagg?gttgttgtga?gcataagggc?ggtcttgttt?ccccggatgc?ggtcgaaatt 480
tccggatttg?atcctgcctg?ccttgaccga?ctggctaagg?taatgcactt?gcctagcagt 540
accatcccag?ccgctctggc?cgaattgtcc?gacgactcct?accgtccggt?ttccccggcc 600
gctactacgt?ggactgtttc?gcaattctat?gctcgttata?gaggaggaga?tcatcatgac 660
caggtgtgct?tggggaaaat?catcagcctt?tgtcaagtta?ttgaggattg?ctgctgccat 720
cagaataaaa?ccaaccgggc?tactccggaa?gaggtcgcgg?caaagattga?tcagtacctc 780
cgtggcgcaa?caagtcttga?ggaatgcttg?gccaaacttg?agagagtttc?cccgccgagc 840
gctgcggaca?cctcctttga?ttggaatgtt?gtgcttcctg?gggttgaggc?ggcgaatcag 900
acaaccgaac?aacctcacgt?caactcatgc?tgcaccctgg?tccctcccgt?gactcaagag 960
cctttgggca?aggactcggt?ccctctgacc?gccttctcac?tgtccaattg?ctattaccct 1020
gcacaaggtg?acgaggttca?tcaccgtgag?aggttaaatt?ccgtactctc?taagttggaa 1080
gaggttgtcc?tggaagaata?tgggctcatg?tccactggac?ttggcccgcg?acccgtgctg 1140
ccgagcgggc?tcgacgagct?taaagaccag?atggaggagg?atctgctaaa?actagccaac 1200
acccaggcga?cttcagaaat?gatggcctgg?gcggctgagc?aggtcaattt?aaaagcttgg 1260
gtcaaaagct?acccgcggtg?gacaccacca?ccccctccac?caagagttca?acctcgaaga 1320
acaaagtctg?tcaaaagctt?gccagagggc?aagcctgtcc?ctgctccgcg?caggaaggtc 1380
agatccgatt?gcggcagccc?ggttttgatg?ggcgacaatg?tccctaacgg?ttcggaagaa 1440
actgtcggtg?gtcccctcaa?ttttccgaca?ccatccgagc?cgatgacacc?tatgagtgag 1500
cccgtactta?tgcccgcggg?ggatggggag?acccaattcc?tggatctgat?gagagcggta 1560
gcaaatagca?tgagggaggc?gccaaactct?gcacagggca?ctccccaatc?ggggcctccc 1620
ccaactcctg?ggccatccca?agataacgac?accgactggg?ggtatacaac?gctgacgcac 1680
caggatgagc?ctctggattt?gcctgcgtcc?tcacagacgg?aatatgaggc?tttcccccta 1740
gcaccatcgc?agaacatggg?catcctggag?gcgggggggc?aagaagttga?ggaagtcctg 1800
agtgaaatct?cggatatact?aaatgacacc?aaccctgcac?ctgtgtcatc?aagcagcccc 1860
ctgtcaagtg?ttaagatcac?acgcccaaaa?tactcagctc?aagccatcat?cgactctggc 1920
gggccttgca?gtgggcatct?ccaaaaggaa?aaagaagcat?gcctcagcat?catgcgtgag 1980
gcttgtgatg?cgtccaagct?tggtgatcct?gctacgcagg?agtggctctc?tcgcatgtgg 2040
gatagggttg?acatgctgac?ttggcgcaac?acgtctgctt?accaggcgtt?tcgcatctta 2100
agtggcaggt?ttgagtttct?cccaaagatg?attctcgaga?caccgccgcc?ccacccgtgc 2160
gggtttgtga?tgttacctcg?cacgcctgca?ccttccgtga?gtgcagagag?tgacctcacc 2220
attggttcag?tggccaccga?ggatgttcca?cgcatcctcg?ggaaaatagg?agacactgac 2280
gagctgcttg?accggggtcc?ctcggcaccc?tccaagggag?aaccggtcag?tgaccaacct 2340
gccaaagatc?cccggatgtc?gccgcgggag?tctgacgaga?gcatgatagc?tccgcccgca 2400
gatacaggtg?gtgtcggctc?attcactgat?ttgccgtctt?cagatggtgt?ggatgtggac 2460
gggggggggc?cgttaagaac?ggtaaaaaca?aaagcgggga?ggctcttaga?ccaactgagc 2520
tgccaggttt?ttagcctcgt?ttcccatctc?cctattttct?tctcacacct?cttcaaatct 2580
gacagtggtt?attctccggg?tgattggggt?tttgcagctt?ttactctatt?ttgcctcttt 2640
ctatgttaca?gttacccatt?cttcggtttt?gctcccctct?tgggtgtatt?ttctgggtct 2700
tctcggcgtg?tgcgaatggg?ggtttttggc?tgctggttgg?cttttgctgt?tggtctgttc 2760
aagcctgtgt?ccgacccagt?cggcactgct?tgtgagtttg?actcgccaga?gtgtaggaac 2820
gtacttcatt?cttttgagct?tctcaaacct?tgggaccctg?tccgcagcct?tgttgtgggc 2880
cccgtcggtc?tcggccttgc?cattcttggc?aggttactgg?gcggggcacg?ctatatctgg 2940
cactttttgc?ttaggcttgg?cattgttaca?gactgtatct?tggctggagc?ttatgtgctt 3000
tctcaaggta?ggtgtaaaaa?gtgctgggga?tcttgtgtaa?gaactgctcc?taatgagatc 3060
gccttcaacg?tgttcccttt?tacacgtgcg?accaggtcgt?cactcatcga?cctgtgcgat 3120
cggttttgcg?caccaaaagg?catggacccc?atttttctcg?ccactgggtg?gcgtgggtgc 3180
tggaccggcc?ggagtcccat?tgagcaacct?tctgaaaaac?ccatcgcgtt?cgcccagctg 3240
gatgagaaga?ggattacggc?tagaactgtg?gtcgctcagc?cttatgatcc?caaccaggcc 3300
gtaaagtgct?tgcgggtatt?acaggcgggt?ggggcgatgg?tggccgaggc?agtcccaaaa 3360
gtggtcaaag?tttccgctat?tccattccga?gctcctttct?ttcccgctgg?agtgaaagtt 3420
gatcctgagt?gcagaatcgt?ggttgatccc?gatactttta?ctacagccct?ccggtctggc 3480
tattccaccg?cgaacctcgt?ccttggtacg?ggggactttg?cccagctgaa?tggactaaag 3540
atcaggcaaa?tttccaagcc?ttcaggg 3567
<210>5
<211>40
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(40)
＜223〉primer
<400>5
tccctaacgg?ttcggaagaa?acaacgctga?cgcaccagga 40
<210>6
<211>40
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(40)
＜223〉primer
<400>6
tcctggtgcg?tcagcgttgt?ttcttccgaa?ccgttaggga 40
<210>7
<211>40
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(40)
＜223〉primer
<400>7
agcccgtact?tatgcccgcg?acaacgctga?cgcaccagga 40
<210>8
<211>40
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(40)
＜223〉primer
<400>8
tcctggtgcg?tcagcgttgt?cgcgggcata?agtacgggct 40
<210>9
<211>31
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(31)
＜223〉primer
<400>9
cgcgaattca?tgtcttccgt?atttgatgag?t 31
<210>10
<211>29
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(29)
＜223〉primer
<400>10
atactcgagt?caataccccc?agtcggtgt 29
<210>11
<211>39
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(39)
＜223〉primer
<400>11
tccctaacgg?ttcggaagaa?ggggatgggg?agacccaat 39
<210>12
<211>40
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(40)
＜223〉primer
<400>12
tcctggtgcg?tcagcgttgt?atacccccag?tcggtgtcgt 40
<210>13
<211>39
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(39)
＜223〉primer
<400>13
agcccgtact?tatgcccgcg?ggggatgggg?agacccaat 39
<210>14
<211>40
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(40)
＜223〉primer
<400>14
tcctggtgcg?tcagcgttgt?atacccccag?tcggtgtcgt 40
<210>15
<211>17
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(17)
＜223〉primer
<400>15
gctccgcgca?ggaaggt 17
<210>16
<211>17
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(17)
＜223〉primer
<400>16
ggagatgccc?actgcaa 17
<210>17
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(22)
＜223〉primer
<400>17
agagagttgt?gcttgatggt?tc 22
<210>18
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(22)
＜223〉primer
<400>18
gatgatctta?cccagcattt?gg 22
<210>19
<211>147
<212>DNA
＜213〉Newcastle disease virus NP 49
<400>19
ggggatgggg?agacccaatt?cctggatctg?atgagagcgg?tagcaaatag?catgagggag 60
gcgccaaact?ctgcacaggg?cactccccaa?tcggggcctc?ccccaactcc?tgggccatcc 120
caagataacg?acaccgactg?ggggtat 147
Claims (10)
- One kind the reorganization porcine reproductive and respiratory syndrome virus, it is characterized in that, the genomic nucleic acids that comprises porcine reproductive and respiratory syndrome virus attenuated vaccine strain HuN4-F112, described HuN4-F112 genome comprises a disappearance or sudden change in the proteic gene region of coding porcine reproductive and respiratory syndrome virus Nsp2Described disappearance is selected from:(1) disappearance of the nucleotide sequence of coding Nsp2 albumen 480-532 amino acids;(2) disappearance of the nucleotide sequence of coding Nsp2 albumen 508-532 amino acids;Described sudden change is selected from:(a) insert the proteic nucleotide sequence of coding Newcastle disease virus NP in (1) described disappearance zone;(b) insert the proteic nucleotide sequence of coding Newcastle disease virus NP in (2) described disappearance zone.
- 2. reorganization porcine reproductive and respiratory syndrome virus according to claim 1 is characterized in that, the nucleotide sequence that insert in described disappearance zone is terminal 49 the amino acid whose nucleotide sequences of coding Newcastle disease virus NP PROTEIN C.
- 3. reorganization porcine reproductive and respiratory syndrome virus according to claim 1 is characterized in that, described HuN4-F112 genome also comprises the MluI restriction endonuclease sites of mark.
- 4. nucleic acid molecule, it is characterized in that, the genome polynucleotide sequence that comprises porcine reproductive and respiratory syndrome virus attenuated vaccine strain HuN4-F112, this polynucleotide sequence comprise a disappearance or sudden change in the proteic gene region of coding porcine reproductive and respiratory syndrome virus Nsp2Described disappearance is selected from:(1) disappearance of the nucleotide sequence of coding Nsp2 albumen 480-532 amino acids;(2) disappearance of the nucleotide sequence of coding Nsp2 albumen 508-532 amino acids;Described sudden change is selected from:(a) insert the proteic nucleotide sequence of coding Newcastle disease virus NP in (1) described disappearance zone;(b) insert the proteic nucleotide sequence of coding Newcastle disease virus NP in (2) described disappearance zone.
- 5. recombinant vectors that comprises the described nucleic acid molecule of claim 4.
- 6. porcine reproductive and respiratory syndrome virus attenuated vaccine strain, it is characterized in that, the genomic nucleic acids that comprises porcine reproductive and respiratory syndrome virus attenuated vaccine strain HuN4-F112, described HuN4-F112 genome comprises a sudden change in the proteic gene region of coding porcine reproductive and respiratory syndrome virus Nsp2, this sudden change is: in the disappearance zone of the nucleotide sequence of coding Nsp2 albumen 480-532 amino acids, insert the proteic nucleotide sequence of coding Newcastle disease virus NP; Or, insert the proteic nucleotide sequence of coding Newcastle disease virus NP in the disappearance zone of the nucleotide sequence of coding Nsp2 albumen 508-532 amino acids.
- 7. porcine reproductive and respiratory syndrome virus attenuated vaccine strain according to claim 6 is characterized in that, the nucleotide sequence that insert in described disappearance zone is terminal 49 the amino acid whose nucleotide sequences of coding Newcastle disease virus NP PROTEIN C.
- 8. the application of the described reorganization porcine reproductive and respiratory syndrome virus of claim 1 in the vaccine of preparation prevention or treatment high-pathogenicity porcine reproductive and respiration syndrome.
- 9. the application of the described porcine reproductive and respiratory syndrome virus attenuated vaccine strain of claim 6 in the vaccine of preparation prevention or treatment high-pathogenicity porcine reproductive and respiration syndrome.
- 10. a detection method of distinguishing described porcine reproductive and respiratory syndrome virus attenuated vaccine strain of claim 6 and natural infection strain is characterized in that, may further comprise the steps:Utilize the Newcastle disease virus NP albumen of expressing as detecting antigen, set up the ELISA antibody detection method, detect the antibody that immune animal produces at NP49 albumen with this method, to distinguish attenuated vaccine strain and natural infection strain with genetically engineered mark.
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| CN 201110146821 CN102250843B (en) | 2011-06-01 | 2011-06-01 | Genetic engineering marked attenuated vaccine strain of porcine reproductive and respiratory syndrome virus and application thereof |
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| CN103087996A (en) * | 2013-01-18 | 2013-05-08 | 中国农业大学 | Recombinant porcine reproductive and respiratory syndrome virus as well as preparation method and application thereof |
| CN103773740A (en) * | 2013-12-19 | 2014-05-07 | 广东省农业科学院动物卫生研究所 | Construction and application of porcine reproductive and respiratory syndrome virus replication defective virus vaccine strain |
| CN104152417A (en) * | 2014-02-19 | 2014-11-19 | 中国农业科学院上海兽医研究所 | Attenuated vaccine strain expressing GM-CSF (granulocyte-macrophage colony-stimulating factor) recombinant PRRSV (porcine reproductive and respiratory syndrome virus) as well as preparation method and application thereof |
| CN104165997A (en) * | 2013-06-03 | 2014-11-26 | 中国农业科学院上海兽医研究所 | Kit and method for porcine reproductive and respiratory syndrome virus (PRRSV) gene-labeled vaccine strain ELISA differential diagnosis and use of kit |
| CN104165998A (en) * | 2013-06-17 | 2014-11-26 | 中国农业科学院上海兽医研究所 | Kit and method for porcine reproductive and respiratory syndrome virus (PRRSV) gene-labeled vaccine strain ELISA differential diagnosis and use of kit |
| CN104717978A (en) * | 2012-07-18 | 2015-06-17 | 南达科他州评议委员会 | Arterivirus protein and expression mechanisms |
| CN106929519A (en) * | 2015-12-29 | 2017-07-07 | 普莱柯生物工程股份有限公司 | A kind of nucleotide sequence, the albumen of expression, the vaccine combination of strain and its preparation and application |
| CN110628817A (en) * | 2019-09-19 | 2019-12-31 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Construction method and application of recombinant porcine reproductive and respiratory syndrome virus for expressing African swine fever virus p30 protein |
| CN110904152A (en) * | 2019-11-23 | 2020-03-24 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Construction method and application of recombinant porcine reproductive and respiratory syndrome virus for expressing African swine fever virus p54 protein |
| CN110904153A (en) * | 2019-11-23 | 2020-03-24 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Construction method and application of recombinant porcine reproductive and respiratory syndrome virus for expressing African swine fever virus p12 or p17 protein |
| CN110904055A (en) * | 2019-11-15 | 2020-03-24 | 华南农业大学 | Porcine reproductive and respiratory syndrome virus recombinant vaccine strain PRRSV-SP and preparation method and application thereof |
| CN113215192A (en) * | 2020-02-06 | 2021-08-06 | 广西大学 | Construction method of porcine reproductive and respiratory syndrome virus double-fluorescence labeling gene recombinant strain |
| CN113321712A (en) * | 2021-07-20 | 2021-08-31 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Antigen polypeptides of porcine reproductive and respiratory syndrome virus and application thereof |
| CN116036253A (en) * | 2022-11-24 | 2023-05-02 | 畜科生物工程有限公司 | Live vaccine for porcine reproductive and respiratory syndrome and preparation method thereof |
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