CN102250843B

CN102250843B - Genetic engineering marked attenuated vaccine strain of porcine reproductive and respiratory syndrome virus and application thereof

Info

Publication number: CN102250843B
Application number: CN 201110146821
Authority: CN
Inventors: 童光志; 周艳君; 徐彦召; 张善瑞
Original assignee: Shanghai Veterinary Research Institute CAAS
Current assignee: Shanghai Veterinary Research Institute CAAS
Priority date: 2011-06-01
Filing date: 2011-06-01
Publication date: 2013-02-27
Anticipated expiration: 2031-06-01
Also published as: CN102250843A

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

Porcine reproductive and respiratory syndrome virus genetically engineered mark attenuated vaccine strain and application

Technical field

The present invention relates to technical field of bioengineering, relate in particular to a kind of highly pathogenic PRRSV 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, the 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 the symptoms such as obvious dyspnoea.Up to now, PRRS is still popular at global most areas, 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 highly pathogenic PRRSV obviously strengthens, and 482 and 534～562 in its Nonstructural Protein Nsp2 exist respectively a characteristic 1+29 aminoacid deletion.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 although have, the reason that at present this virus virulence is strengthened still 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 for the prevention of high-pathogenicity porcine reproductive and respiration syndrome and control, the existing several attenuated vaccines for high-pathogenicity porcine reproductive and respiration syndrome of China, all virulent strain is passed through external continuous passage to weak acquisition at cell, such 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 in the future and purify the high PRRSV of causing a disease, and 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 restructuring 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 gene region of coding porcine reproductive and respiratory syndrome virus Nsp2 albumen

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) at the regional nucleotide sequence that inserts coding Newcastle disease virus NP albumen of (1) described disappearance;

(b) at the regional nucleotide sequence that inserts coding Newcastle disease virus NP albumen of (2) described disappearance.

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 gene region of coding porcine reproductive and respiratory syndrome virus Nsp2 albumen

Described disappearance is selected from:

Described sudden change is selected from:

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 gene region of coding porcine reproductive and respiratory syndrome virus Nsp2 albumen, this sudden change is: in the disappearance zone of the nucleotide sequence of coding Nsp2 albumen 480-532 amino acids, insert the nucleotide sequence of coding Newcastle disease virus NP albumen; Or in the disappearance zone of the nucleotide sequence of coding Nsp2 albumen 508-532 amino acids, insert the nucleotide sequence of coding Newcastle disease virus NP albumen.

In another aspect of this invention, also provide the application of a kind of above-mentioned restructuring 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 detectable antigens, set up the ELISA antibody detection method, detect the antibody that immune animal produces for NP49 albumen with the method, to distinguish attenuated vaccine strain and the 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 highly pathogenic PRRSV 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 of the present invention's infections clone strain of saving different genes deletion 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 the infections clone strain genetic stability sequential analysis figure that the present invention rescues the different genes deletion and insertion mark that obtains;

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 immune swine and the natural histogram of strong malicious infected pigs;

Fig. 8 is that the embodiment of the invention 3 is utilized RT-PCR to cut in conjunction with the MluI enzyme to differentiate immune swine and the natural electrophorogram of strong malicious infected pigs;

Fig. 9 is that the embodiment of the invention 3 genetically engineered mark attenuated vaccine strains are attacked the rear dead protection ratio statistics figure of poison.

Embodiment

In the following example, the experimental technique of unreceipted actual conditions, usually condition routinely, such as " fine works molecular biology experiment guide " (F.M. Ao Sibai, R.E. James Kingston, the 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 obtains 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 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 lack the epi-position (49 amino acid of C-terminal) of inserting respectively the more intense Newcastle disease virus NP albumen of antigenicity in the zones at above-mentioned two simultaneously, 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 obtain can be used as genetically engineered mark attenuated vaccine and is applied to control to high-pathogenicity porcine reproductive and respiration syndrome.

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 is by this laboratory Attenuation 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 domestic analytical pure.

1.2 design of primers

According to PRRSV HuN4 strain (GenBank accession number: EF635006) Nsp2 gene order, with Olio 6.0 software designs the 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

Utilize respectively F Δ 52/

R Δ

52 and 25 two pairs of primers of R Δ 25/F Δ, the PCR that suddenlys change take the pSK-HuN4-F112-ABC plasmid as template operation, 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 the structure of virogene deletion mutantion full-length cDNA 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.Then respectively with two linearizing PCR product transformed competence colibacillus bacillus coli DH 5 alphas of above-mentioned acquisition, obtain positive recombinant plasmid 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 the enzyme of ABC-Δ 52, ABC-Δ 25 and pSK-HuN4-F112-DEF fragment 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 respectively linearizing, synthesize respectively viral RNA according to in-vitro transcription test kit mMessageHigh Yield Capped RNA Transcription kit operation instructions, cultivate simultaneously the BHK21 cell 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

The cell of gathering in the crops above-mentioned transfection is stored in-80 ℃, behind multigelation, is inoculated in advance on the cultured 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 respectively individual layer Macr-145 cell, and 48h discards nutrient solution after infecting.With icing fixedly 10min of methyl alcohol, add the monoclonal antibody for PRRSV nucleocapsid protein and GP5 albumen (Zhou YJ et al., 2005 after diluting; 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 titer

Reference literature carries out the mensuration (Yin Zhen etc., 1997) of infection titer with 96 hole tissue culturing plate methods.After sample to be checked made 10 times of serial dilutions with maintenance medium, 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 virus liquid with maintenance medium), 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 when stopping CPE to occur observing.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 every 12h and carry out virus titer mensuration, each time point virus median infective dose (TCID that collects 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 the 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 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 obvious cytopathy (Fig. 2), the cell main manifestations contracts, comes off for gathering, circle, become the hauling type cytopathy, basically identical with the caused cytopathy 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 respectively Δ 52 and Δ 25 aminoacid deletion, 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 obtains is that PRRS is viral.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 rear 48 hours levels of replication of infection, after this substantially remain on a plateau, whole replicative cycle and parent's poison substantially consistent (Fig. 5).

Above result shows, the recombinant virus of above-mentioned 2 Nsp2 genetically deficients that the present invention obtains can genetic stability, and the biological characteristics of its genetically deficient virus strain and parent's poison are basically identical.

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 domestic 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 namely: F-NDVNP/R-NDVNP, F Δ 52+NP49/R Δ 52+NP49 and F Δ 25+NP49/R Δ 25+NP49 (seeing Table 1), wherein carried the NP49 gene region that carries out insertion mutation in rear two pairs of primers.

1.3 insert the pcr amplification of gene specific fragment

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.Take cDNA as template, utilize F-NDV NP/R-NDV NP primer, reaction system is 50 μ L, 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.Then take the NP gene that obtains as template, utilize respectively F Δ 52+NP49/R Δ 52+NP49 and F Δ 25+NP49/R Δ 25+NP49 to be primer, amplify respectively the gene order of Δ 52+NP49 and Δ 25+NP49, and reclaim these two gene fragments as mutant primer.

Utilize respectively above-mentioned two pairs of mutant primers, the PCR that suddenlys change take the pSK-HuN4-F112-ABC plasmid as template operation, reaction system is 50 μ L, reaction conditions is: 92 ℃/2min, 92 ℃/10s, 55 ℃/20s, 68 ℃/6min, circulate 18 times; Extend 5min in 68 ℃ at last.

1.4 insert the structure of NP49 gene viruses full-length cDNA recombinant plasmid

Respectively above-mentioned reaction product is carried out Dpn I digestion with restriction enzyme, then respectively with two linearizing PCR product transformed competence colibacillus bacillus coli DH 5 alphas of above-mentioned acquisition, identify positive recombinant plasmid 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 the enzyme of ABC-Δ 52+NP49, ABC-Δ 25+NP49 and pSK-HuN4-F112-DEF fragment 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 respectively linearizing, synthesize respectively viral RNA according to in-vitro transcription test kit mMessage High Yield Capped RNA Transcription kit operation instructions, cultivate simultaneously the BHK21 cell 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

The cell of gathering in the crops above-mentioned transfection is stored in-80 ℃, behind multigelation, is inoculated in advance on the cultured 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 respectively 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, 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 respectively the Macr-145 cell monolayer, and 48h discards nutrient solution after infecting.With icing fixedly 10min of methyl alcohol, add (the Zhou YJ et al. of the monoclonal antibody for the PRRSV nucleocapsid protein after diluting, 2005) and for 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 organism Epidemiological Analysis

1.8.1 the mensuration of viral titer

Reference literature carries out the mensuration (Yin Zhen etc., 1997) of infection titer with 96 hole tissue culturing plate methods.After sample to be checked made 10 times of serial dilutions with maintenance medium, 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 virus liquid with maintenance medium), 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 when stopping CPE to occur observing.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

(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 every 12h and carry out virus titer mensuration, each time point virus median infective dose (TCID that collects 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 the 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 of getting different generations uses the RT-PCR amplification to contain 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 zone of the Nsp2 gene of PRRSV attenuated vaccine strain HuN4-F112 strain, respectively successful 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 obvious cytopathy (Fig. 2), the cell main manifestations contracts, comes off for gathering, circle, be the hauling type cytopathy, basically identical with the caused cytopathy 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 the equal stable existence of this insertion mutation of 20 generations, do not undergo mutation and the 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 specificity fluorescent of N albumen and express the specificity fluorescent (Fig. 4) that inserts newcastle disease NP49 albumen in PRRSV, confirm that further the virus of saving 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 rear 48 hours levels of replication of infection, maintain afterwards a higher levels of replication always, substantially remain on a plateau, whole replicative cycle and parent's poison HuN4-F112 substantially consistent (Fig. 5) show with parent's poison HuN4-F112 to have similar biologic activity at the virus titer that copies, 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 all negative sodium selenites of the cause of diseases such as 30 35 age in days PRRSV, CSFV, PCV2 and antibody, be divided at random 4 groups, 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 challenge test after the immunity

In inoculation rear 28 days is 10 with the strong poison dilution of HuN4-F5 strain ^4.0TCID ₅₀/ ml attacks by force poison of HuN4-F5 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 measurement

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 the rear clinical manifestation of poison, every day all test pig was carried out body temperature measurement simultaneously every day, continued thermometric to attacking poison rear 14 days.

1.5 test pig PRRSV detection of specific antibody

Before the immunity of genetically engineered mark attenuated vaccine test group and the immunity after 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 also gathered serum and the antibody test same period.

1.6 the discriminating of test pig traget antibody detects

Before the immunity of genetically engineered mark attenuated vaccine test group and after the immunity, gathered the serum of all test group pigs in the 7th, 14,21,28 days, utilize the NP49 albumen of amalgamation and expression mark as detectable antigens, the antiserum(antisera) antibody of all collections is carried out differential diagnosis.

2. result

Utilize respectively 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; within rear 28 days of inoculation (before namely attacking poison); the pig of test group is not all found body temperature and the unusual phenomenon of Symptoms; 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 wherein the DMEM control group then after attacking poison all pigs of second day the fervescence phenomenon all appears, continue nearly about 10 days always, the pig main manifestations of heating be appetite stimulator, thick disorderly by hair, present the 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 rHN4-Δ 25+NP49 immune group and parent's poison HuN4-F112 vaccine group are basically identical, the reactions such as obvious fervescence do not occur, and the test pig is also there are no unusual clinical manifestation.

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 the DMEM control group each experimental group the 7th day antibody after immunity and begins to occur, after this antibody horizontal continues to raise, until maintain higher level (seeing Fig. 6) before attacking poison always, show that above-mentioned two kinds of genetically engineered mark attenuated vaccines are consistent with parent's vaccine virus, pig is had preferably immunogenicity, can stimulate body to produce stronger humoral immune reaction.

Utilize the NP49 albumen of expression as the ELISA antibody detection method of detectable antigens foundation, detect the serum antibody of each immune group test pig in the different rear times of immunity, the result shows, two genetically engineered mark attenuated vaccine strain immune group can detect the serum antibody of anti-NP49 albumen on the 14th day after immunity, obviously raise to rear the 28th day antibody horizontal of immunity, 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 the wild poison of difference natural infection and vaccine immunity poison.

After immunity, gathered respectively the blood sample of each test group pig on the 14th day, extract RNA and carry out the RT-PCR detection of virogene, and utilize the MluI restriction enzyme to carry out the characteristic mark that enzyme cuts to detect immune strain to its amplified production, the result shows, except 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 only has 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 immune protective effect evaluation behind the poison, according to the mortality statistics result, can see that the DMEM control group attacking poison rear 11 days, all pigs are all dead, and mortality ratio reaches 100%; RHN4-Δ 52+NP49 immune group is each dead one of the 7th day and the 11st day after attacking poison respectively, and all the other are healthy the survival all, and 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 pigs are all healthy survivals all, 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 preferably immune protective effect to piglet.

In sum; genetically engineered mark attenuated vaccine strain rHN4-Δ 25+NP4 strain based on construction of recombinant plasmid has preferably security to pig; can effectively induce body to produce protective immunological reaction after the immunity, and provide 100% immunoprotection to its opposing highly pathogenic PRRSV virulent strain.

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

One kind the restructuring 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 gene region of coding porcine reproductive and respiratory syndrome virus Nsp2 albumen

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 terminal 49 the amino acid whose nucleotide sequences of coding Newcastle disease virus NP PROTEIN C in (1) described disappearance zone;

(b) insert terminal 49 the amino acid whose nucleotide sequences of coding Newcastle disease virus NP PROTEIN C in (2) described disappearance zone.
2. restructuring 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.
3. 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 comprises a disappearance or sudden change in the gene region of coding porcine reproductive and respiratory syndrome virus Nsp2 albumen

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 terminal 49 the amino acid whose nucleotide sequences of coding Newcastle disease virus NP PROTEIN C in (1) described disappearance zone;

(b) insert terminal 49 the amino acid whose nucleotide sequences of coding Newcastle disease virus NP PROTEIN C in (2) described disappearance zone.
4. recombinant vectors that comprises the described nucleic acid molecule of claim 3.
5. 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 gene region of coding porcine reproductive and respiratory syndrome virus Nsp2 albumen, this sudden change is: in the disappearance zone of the nucleotide sequence of coding Nsp2 albumen 480-532 amino acids, insert terminal 49 the amino acid whose nucleotide sequences of coding Newcastle disease virus NP PROTEIN C; Or in the disappearance zone of the nucleotide sequence of coding Nsp2 albumen 508-532 amino acids, insert terminal 49 the amino acid whose nucleotide sequences of coding Newcastle disease virus NP PROTEIN C.
6. the application of restructuring porcine reproductive and respiratory syndrome virus claimed in claim 1 in the vaccine of preparation prevention or treatment high-pathogenicity porcine reproductive and respiration syndrome.
7. the application of porcine reproductive and respiratory syndrome virus attenuated vaccine strain claimed in claim 5 in the vaccine of preparation prevention or treatment high-pathogenicity porcine reproductive and respiration syndrome.