CN111218427B

CN111218427B - Recombinant PRRS virus HV-NSP2(500-596) and application thereof

Info

Publication number: CN111218427B
Application number: CN201811416015.0A
Authority: CN
Inventors: 封文海; 杜丽; 唐军; 王红蕾; 刘芳; 魏泽宇
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2018-11-26
Filing date: 2018-11-26
Publication date: 2022-05-24
Anticipated expiration: 2038-11-26
Also published as: CN111218427A

Abstract

The invention discloses a recombinant PRRS virus HV-NSP2(500-596) and application thereof. The cDNA sequence corresponding to the genome of the recombinant PRRS virus is obtained by deleting the cDNA sequence corresponding to the genome of the wild type PRRS virus from the DNA molecule shown in the 2833-3036 th site of the 5' end. The invention discloses a molecular mechanism of high fever caused by highly pathogenic PRRSV, and simultaneously, compared with the highly pathogenic PRRSV, the recombinant PRRS virus has reduced virulence and greatly slowed replication capacity, thereby providing a foundation for developing novel attenuated vaccines.

Description

Recombinant PRRS virus HV-NSP2(500-596) and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to recombinant PRRS virus HV-NSP2(500-596) and application thereof.

Background

Porcine Reproductive and Respiratory Syndrome (PRRS), commonly known as porcine reproductive and respiratory syndrome, is one of the important viral diseases which harm the swine industry all over the world at present. The cause of porcine reproductive and respiratory syndrome is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). Porcine reproductive and respiratory syndrome virus is a single-stranded plus-sense RNA virus belonging to the order Nidovirales, the family of arteriviruses, the genus arterivirus. The virus comprises two subtypes, namely European type (type I) and American type (type II), wherein the European type is mainly distributed in Europe at present, and the American type is distributed in North America, south America and Asia. In 1995, outbreaks of porcine reproductive and respiratory syndrome were first reported in northern China and subsequently spread across the country. Typically, classical PRRSV strains cause clinically significant reproductive disorders in pregnant sows (e.g. miscarriage, stillbirth, mummy and boredom) and respiratory disorders in piglets. Due to the rapid mutation speed of the virus, many strains with different virulence are derived besides the European and American prototypes. In 2006, highly pathogenic porcine reproductive and respiratory syndrome (also called innominate hyperpyrexia) caused by PRRS virus variant strains (highly pathogenic PRRSV viruses) was developed in China. Compared with the classical porcine reproductive and respiratory syndrome, the sick pigs infected with the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) are mainly characterized by persistent high fever, the body temperature reaches 40-42 ℃, the sick pigs have high mortality rate and acute death rate in all ages, and the morbidity of the infected piglets can reach 100%. The mortality rate is up to 50-100%, the abortion rate of sows can be up to 100%, and the mortality rate of sows and fattening pigs is up to 30% or more.

Studies have shown that hyperpyrexia caused by porcine reproductive and respiratory syndrome virus is directly proportional to mortality, i.e., the higher the temperature, the faster the death rate. The severity of porcine reproductive and respiratory syndrome disease is related to the virulence of PRRSV. The hyperpyrexia phenomenon of sick pigs infected with the highly pathogenic porcine reproductive and respiratory syndrome virus occurs the next day after infection and persists. The body temperature of sick pigs infected with the traditional porcine reproductive and respiratory syndrome virus is not obviously changed and does not reach the phenomenon of high fever. Therefore, from the mechanism of causing high fever, the search for a method of weakening highly pathogenic porcine reproductive and respiratory syndrome virus may be a starting point.

Disclosure of Invention

The invention aims to solve the technical problem of how to weaken the highly pathogenic porcine reproductive and respiratory syndrome virus.

In order to solve the technical problems, the invention firstly provides a recombinant PRRS virus.

The cDNA sequence corresponding to the genome of the recombinant PRRS virus is obtained by deleting the cDNA sequence corresponding to the genome of the wild type PRRS virus from the DNA molecule shown in the 2833-3036 th site at the 5' end.

In the recombinant PRRS virus, a cDNA sequence corresponding to the genome of the wild type PRRS virus is shown as a sequence 1 in a sequence table.

In order to solve the technical problems, the invention also provides a new application of the recombinant PRRS virus.

The invention provides an application of the recombinant PRRS virus in preparation of PRRS vaccines.

The invention also provides application of the recombinant PRRS virus in preparation of PRRS attenuated vaccines.

The invention also provides application of the recombinant PRRS virus in preparation of a medicament for preventing porcine reproductive and respiratory syndrome.

In order to solve the technical problems, the invention also provides a DNA fragment.

The DNA fragment provided by the invention is obtained by deleting the DNA molecule shown in the 2833-3036 th site from the 5' end of the sequence 1 in the sequence table.

The application of the DNA fragment in the preparation of the recombinant PRRS virus and/or the preparation of PRRS vaccine and/or the preparation of PRRS attenuated vaccine also belongs to the protection scope of the invention.

In order to solve the technical problems, the invention also provides a recombinant plasmid, a recombinant bacterium or a recombinant cell containing the DNA segment.

The recombinant plasmid is a sequence obtained by deleting DNA molecules which correspond to the 2833-3036 th site at the 5' tail end in the sequence 1 in the plasmid pcDNA3.1-HV.

Furthermore, the pcDNA3.1-HV vector is obtained by replacing a small fragment between the Smi I and Not I enzyme cutting sites of the pcDNA3.1 vector with a genome full-length cDNA sequence of an HV strain shown in a sequence 1 in a sequence table. The recombinant cell is an HEK-293T cell containing the recombinant plasmid.

The application of the recombinant plasmid, the recombinant bacterium or the recombinant cell in the preparation of the recombinant PRRS virus and/or the preparation of PRRS vaccine and/or the preparation of PRRS attenuated vaccine also belongs to the protection scope of the invention.

In order to weaken the highly pathogenic porcine reproductive and respiratory syndrome virus, the invention uses the amino acid regions shown in the 500-596 th site and/or the 658-777 th site in the non-structural protein 2(NSP2) of the highly pathogenic porcine reproductive and respiratory syndrome virus HV as target segments to reconstruct and obtain the recombinant PRRS virus. Compared with highly pathogenic PRRSV, the recombinant PRRS virus disclosed by the invention has the advantages that the toxicity is reduced, the replication capacity is greatly reduced, and a foundation is provided for the research and development of a novel attenuated vaccine.

Drawings

FIG. 1 shows the successful rescue of viruses by indirect immunofluorescence detection.

FIG. 2 shows that supernatants were collected at the indicated times after infection and virus titers were measured by the method of TCID 50.

FIG. 3 is virus-induced cytopathic effect 96 hours post-infection.

FIG. 4 is the rectal temperature change after infection.

FIG. 5 is a comparison of survival rates after infection.

FIG. 6 is the change in body weight after infection.

FIG. 7 shows the change in viral titer in serum after infection.

FIG. 8 shows the viral RNA content in the infected tissue.

FIG. 9 shows the results of H & E staining of tissues after infection.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are all conventional ones unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The wild type PRRSV highly pathogenic strain HV used in the following examples: genbank number JX317648.1, publicly available from the chinese university of agriculture.

Data analysis methods for the following examples: all data are averages from three independent replicates and the significance of the difference was analyzed by two-tailed T-test (pairwise), with P.ltoreq.0.05 considered significant, 0.01< P.ltoreq.0.05 (x), 0.005< P.ltoreq.0.01 (x), and P.ltoreq.0.005 (x).

Example 1 obtaining of recombinant PRRS viruses HV-NSP2(500-

First, selection of target section

Through the research on the molecular mechanism of PRRS virus causing high fever, the two sections of amino acid regions, namely the 500 th 596 th site and the 658 th 777 th site in NSP2 of a typical PRRSV strain VR2332, in the non-structural protein 2(NSP2) of the highly pathogenic PRRSV (HP-PRRSV, HV for short) are found to be the functional regions of the highly pathogenic PRRSV for inducing high fever. In order to weaken the highly pathogenic porcine reproductive and respiratory syndrome virus, the amino acid regions shown at the 500-596 th site and the 658-777 th site in the non-structural protein 2(NSP2) of the highly pathogenic porcine reproductive and respiratory syndrome virus HV are used as target segments for modification to construct the recombinant PRRS virus.

Second, construction of infectious cDNA clone

1. Construction of pcDNA3.1-HV

(1) Selection of cleavage sites

Analyzing the genome full-length cDNA sequence (shown as a sequence 1 in a sequence table) of the HV strain, and selecting the following 6 enzyme cutting sites for segmenting and splicing the genome full-length sequence: smi I, Xho I, Afl II, Cla I, EcoR V and Not I. Wherein the restriction sites Xho I, Afl II, Cla I and EcoR V are original in the genome and are contained in the amplified sequence; the cleavage sites Smi I and Not I are designed and added to both ends of the viral genome. The 6 enzyme sites divided the entire HV genome into five fragments.

(2) Construction of pcDNA3.1-HV

The full-length cDNA of the HV virus strain was used as a template, and PCR was performed using the following primers to obtain five fragments of the full-length HV genome. Primer pair HP1F/HP1R was used to amplify fragment 1, primer pair HP2F/HP2R was used to amplify fragment 2, primer pair HP3F/HP3R was used to amplify fragment 3, primer pair HP4F/HP4R was used to amplify fragment 4, and primer pair HP5F/HP5R was used to amplify fragment 5. The primer sequences are as follows:

HP1F：TAAATTTAA ATGACGTATAGGTGTTGGCTCT；

HP1R：GCGTGCGAGGTAACATCA；

HP2F：TTCTCCCAAAGATGATTCTCG；

HP2R：GGCAGCGGTACTTAAACAAG；

HP3F：GCCCTTAACAGAAACAGATGG；

HP3R：TACGACGGTAGATGCTCCTC；

HP4F：GGGAAGAAGAAGACTAGGACAAT；

HP4R：TCAGGGTGAACGGTAGAGC；

HP5F：GCCCTGTCATTGAACCAACT；

HP5R：TTATTAGCGGCCGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAATTACGGCCGCATGGTT。

the five amplified fragments were cloned into the backbone of eukaryotic expression vector pcDNA3.1 (purchased from Invitrogen) to obtain pcDNA3.1-HV vector. The pcDNA3.1-HV vector is obtained by replacing a small fragment between the Smi I and Not I enzyme cutting sites of the pcDNA3.1 vector with a genome full-length cDNA sequence of an HV strain shown in a sequence 1 in a sequence table.

2. Obtaining recombinant fragments

(1) Construction of DNA fragment 1-pEASY

And (2) taking a pcDNA3.1-HV vector as a template, and respectively amplifying by using F1F/F1R, F2F/F2R and F3F/F3R primer pairs (for convenience of subsequent experiments, the designed primers all contain homologous arm sequences) to respectively obtain a DNA fragment 1 shown in a sequence 2 of a sequence table, a DNA fragment 2 shown in the sequence 3 of the sequence table and a DNA fragment 3 shown in the sequence 4 of the sequence table. The primer sequences are as follows:

F1F：GCCGGAAAGAGAGCAAGGAAAACACGC；

F1R：CCGCGGCGACATCCGGGGATCTTTGGCAGGTTG；

F2F：CGATCTTTCCGATTGCACGAATGA；

F2R：GCGTGTTTTCCTTGCTCTCTTTCCGGC；

F3F：CCAAAGATCCCCGGATGTCGCCGCGGGAGTC；

F3R：GGGGCAGGAAGGCATAGGTGCTTAA。

the resulting DNA fragment 1 was ligated into pEASY-Blunt Zero Cloning Vector in the following reaction system: PCR product (DNA fragment 1): 4 microliter; pEASY-Blunt Zero Cloning Vector Mix: 1 microliter, ligation conditions were as follows: the reaction was carried out at room temperature for 5 minutes, and after completion of ligation, the ligation product was transformed into DH-5. alpha. to obtain DNA fragment 1-pEASY.

(2) Construction of vector clones for DNA fragment 1 deletion (500-

The DNA fragment 1-pEASY was subjected to deletion mutation using Q5Hot Start High-Fidelity 2X Master Mix of NEB. The method comprises the following specific steps: the DNA fragment 1-pEASY is taken as a template, and primers of delete500-596F/delete500-596R and delete658-777F/delete658-777R are respectively adopted for amplification, so as to respectively obtain the DNA fragment 1 deletion (500-. The primer sequences are as follows:

delete500-596F：GCGGGGGGGCAAGAAGTTGAGGAAGTCCTGAGTGAAAT；

delete500-596R：CATAGGTGTCATCGGCTCGGATGGT；

delete658-777F：AAGGGAGAACCGGTCTGCGACCAACCT；

delete658-777R：TTTTTCCTTTTGGAGATGCCCACTGC。

the PCR reaction system is as follows: and (3) primer F: 1.25 microliters; and (3) primer R: 1.25 microliters; template (DNA fragment 1-pEASY less than 20 ng): 1 microliter; water: 9 microliter; q5Hot Start High-Fidelity 2X Master Mix: 12.5 microliters;

the PCR reaction conditions were as follows: 98 degrees 30s, 98 degrees 10s, 60 degrees 20s, 72 degrees 4min, 25 cycles, 72 degrees 2 min, 4 degrees of storage.

Taking out the PCR product and then connecting, wherein a connecting system is as follows: PCR products: 1 microliter, 2X KLD buffer: 5 microliter, 10 XKLD Mix: 1 microliter, water: 3 microliter; after 5 minutes reaction at room temperature, the DNA fragment was transformed into DH-5. alpha. to finally obtain vector clones with DNA fragment 1 deletion (500-.

(3) Construction of vector clone with deletion of DNA fragment 1(56)

The vector clone with the DNA fragment 1 deletion (500-596) was subjected to the deletion of 658-777 as described in (2) to obtain the vector clone with the DNA fragment 1 deletion (56).

(4) DNA fragment 1 deletion (500-

The vector clone with the DNA fragment 1 deletion (500-. The primer sequences are as follows:

F1F：GCCGGAAAGAGAGCAAGGAAAACACGC；

F1R：CCGCGGCGACATCCGGGGATCTTTGGCAGGTTG。

3. construction of pcDNA3.1-HV-NSP2(500-

(1) Linearization of vectors

The full-length genome sequence (shown as sequence 1 in the sequence table) of the HV strain is analyzed, and 2 single enzyme cutting sites are selected and are respectively Spe I and Afl II. And (3) linearizing the pcDNA3.1-HV by using a double enzyme digestion mode to obtain the linearized pcDNA3.1-HV.

(2) Construction of pcDNA3.1-HV-NSP2(500-596)

The DNA fragment 1 was deleted (500-596), the DNA fragment 2 and the DNA fragment 3 were subjected to homologous recombination with the linearized pcDNA3.1-HV using the NEB HiFi DNA Assembly Master Mix to obtain the recombinant vector pcDNA3.1-HV-NSP2 (500-596). The recombinant vector pcDNA3.1-HV-NSP2(500-596) is obtained by deleting the DNA molecule shown in 2833-3036 site in the full-length cDNA sequence of the genome of the HV strain shown in the sequence 1 in the vector pcDNA3.1-HV.

The DNA fragment 1 was deleted (658; 777), the DNA fragment 2 and the DNA fragment 3 were subjected to homologous recombination with linearized pcDNA3.1-HV using the NEB HiFi DNA Assembly Master Mix to obtain the recombinant vector pcDNA3.1-HV-NSP2 (658; 777). The recombinant vector pcDNA3.1-HV-NSP2(658- 777) is obtained by deleting the DNA molecule shown in the 3220- 3579 position in the full-length cDNA sequence of the genome of the HV strain shown in the sequence 1 in the vector pcDNA3.1-HV.

The DNA fragment 1 was deleted (56), the DNA fragment 2 and the DNA fragment 3 were subjected to homologous recombination with linearized pcDNA3.1-HV using NEB HiFi DNA Assembly Master Mix to obtain a recombinant vector pcDNA3.1-HV-NSP2 (56). The recombinant vector pcDNA3.1-HV-NSP2(56) is obtained by deleting the DNA molecule shown in 2833-3036 th site in the full-length genome sequence of the HV strain shown in the sequence 1 in the vector pcDNA3.1-HV and deleting the DNA molecule shown in 3220-3579 th site.

Third, virus rescue

The recombinant plasmids pcDNA3.1-HV-NSP2 (500-. Cell lysates were added to Marc-145 cells for visualization, and indirect immunofluorescence detection of recombinant PRRS virus was performed after the cells developed lesions using a murine anti-PRRSV N protein specific antibody SDOW-17 (available from Rural Technologies).

The results are shown in FIG. 1. The results show that: the recombinant plasmids pcDNA3.1-HV-NSP2 (500-. The PRRSV successfully rescued by the recombinant plasmids pcDNA3.1-HV-NSP2 (500-.

Fourth, determination of growth curve of recombinant virus

Inoculating the cell lysate obtained in the third step into PAMs for continuous virus propagation for 4-5 times to obtain culture supernatant. Culture supernatants containing wild-type PRRSV highly pathogenic strain HV (hereinafter abbreviated as HV-wt), recombinant PRRS virus HV-NSP2 (500-.

The method for determining the virus titer is as follows: inoculating PAM cells in 96-well cell culture plate (containing RPMI1640 culture solution), culturing at 37 deg.C with 5% CO₂Culturing for 24h in an incubator; the virus solution was serially diluted 10-fold in RPMI1640 medium in 1.5mL EP tubes from 10^-1To 10^-9(ii) a Inoculating the diluted virus solution into a cell culture plate, wherein each dilution is inoculated into 8 holes, and each hole is inoculated with 100 mu L; setting 8 control wells of normal cells without virus inoculation; taking a small amount of cell culture supernatant at 12h, 24h, 36h, 48h, 72h and 96h after virus inoculation, carrying out indirect immunofluorescence detection on the PRRS virus by using a mouse anti-PRRSV N protein specific antibody SDOW-17 (purchased from Rural Technologies), counting the number of virus positive holes, and calculating the infection amount of virus half Tissue (TCID) in each milliliter of virus liquid₅₀/mL)。

The method for propagating the virus comprises the following steps: inoculating PAM into a cell culture bottle (containing RPMI1640 culture solution) for adherent culture for 24h, sucking supernatant, and inoculating virus solution; adsorbing at 37 ℃ for 120 minutes, and slightly shaking the culture bottle every 30 minutes to ensure that the virus liquid is fully contacted with the PAM cells; then supplementing RPMI1640 culture solution, culturing at 37 ℃ for at least 48h, and freezing and thawing the cells for three times between-80 ℃ and room temperature to lyse the cells; transferring the cell lysate to a 50mL centrifuge tube, centrifuging at 4 ℃ and 5000g for 10 minutes to obtain supernatant, namely virus solution, subpackaging the supernatant into 1.5mL EP tubes, and storing at-80 ℃ for later use.

The results of the virus titer test are shown in fig. 2 and table 1, while the cytopathic effect was observed under white light and photographed as shown in fig. 3. The results show that: the wild PRRSV highly pathogenic strain HV grows rapidly on PAM, and the virus titer reaches the peak value of 10 within 48 hours after infection^8.5TCID₅₀mL, the replication speed of the recombinant PRRS virus is obviously lower than that of a wild type PRRSV highly pathogenic strain HV, and the virus titers of the recombinant PRRS virus HV-NSP2 (500-. Cell lesions are observed at different times after PAM infection, and PAM cleavage can be quickly induced after wild type PRRSV highly pathogenic strain HV infection, but the cell lesion effect is not better than that of the wild type PRRSV highly pathogenic strain HV infection until 96 hours after infection of the same amount of recombinant PRRS virus HV-NSP2 (500-. The above results show that: compared with the wild type PRRSV highly pathogenic strain HV, the recombinant PRRS viruses HV-NSP2 (500-.

Table 1, results of viral titers (mean values) at different times after infection of PAM with each recombinant PRRS virus (log)₁₀(TCID₅₀/mL))

PRRSV

12h

24h

36h

48h

72h

96h

HV-wt

3.0697

6.673

7.0117

8.5

HV-nsp2(500-596)

2.222

3.553*

4.1933*

4.3323*

4.6667

5.182

HV-nsp2(658-777)

2.445

4.22*

4.262*

4.445*

4.555

5.1667

HV-nsp2(56)

2

3.663*

4.2767*

4.999*

4.743667

5

Example 2 detection of virulence of recombinant PRRS virus in pigs

30 healthy 4-week-old plain white pigs were purchased from an SPF pig farm and all pigs were confirmed to be PRRSV negative by PCR. All pigs were randomly divided into 5 groups (6 pigs per group) and inoculated nasally with a virus dose of 10⁵TCID₅₀2mL of virus solution per nostril, 1 mL. The virus liquid inoculated in each group is divided into the following groups according to the difference:

blank control (MOCK): inoculating RPMI1640 culture solution;

wild-type virus control group: inoculating a wild type PRRSV highly pathogenic strain HV;

recombinant virus panel (500-596): inoculating recombinant PRRS virus HV-NSP2 (500-596);

recombinant virus experimental group (658-777): inoculating recombinant PRRS virus HV-NSP2(658 and 777);

recombinant virus experimental group (56): inoculation with recombinant PRRS virus HV-NSP2 (56).

Observing whether clinical symptoms such as cough, dyspnea, anorexia, diarrhea, unstable standing, shivering and the like appear every day after toxin attack; rectal temperature, body weight were monitored and survival was counted. The forechamber venous blood was collected every two days after challenge (2, 4, 6, 10, 13, 16, 21, 24, 28 days after challenge) for serum isolation and virus titer detection therein. 3 piglets were each necropsied 6 days after challenge and were necropsied when the HV-vaccinated piglets died. And (3) observing pathological changes of each organ during the autopsy and sampling, fixing 1 part of sample with 4% formaldehyde solution, slicing the tissue and performing HE staining, quickly freezing the other 1 part of sample with liquid nitrogen, and storing the sample at-80 ℃ for extracting tissue RNA and real-time PCR (polymerase chain reaction) for determining the content of viral RNA in the tissue.

The method for detecting the virus titer in the serum comprises the following steps:

adding 750 microliter Trizol (invitrogen) into every 250 microliter, repeatedly blowing by using a gun, and standing for 5 minutes at room temperature; adding 0.2mL of chloroform into the EP tube, covering the lid of the EP tube, shaking vigorously in hands for 15 seconds, standing at room temperature for 10 minutes, and centrifuging at 12000g and 4 ℃ for 15 minutes; placing the upper water phase in a new EP tube, adding 0.5mL of isopropanol, standing at room temperature for 10 minutes, and centrifuging at 12000g and 4 ℃ for 10 minutes; carefully abandoning the supernatant, adding 1mL of 75% ethanol for washing, centrifuging at 7500g and 4 ℃ for 5 minutes, and abandoning the supernatant; allowing the precipitated RNA to dry naturally at room temperature; finally 20. mu.l of DEPC water was added to dissolve the RNA and RNA was quantified using Nanodrop 1000(Thermo scientific).

5 microliters of RNA was taken for RT-PCR. Add 5. mu.l of RNA and 1. mu.l of Random Primer (10. mu.M, TaKaRa) into a 250. mu.l PCR tube and mix well; reacting at 70 ℃ for 5 minutes, and quickly cooling on ice for 2 minutes; to the above mixture was added the following components: 5. mu.l M-MLV 5 × Reaction Buffer, 5. mu.l dNTP (2.5mM), 25units of Recombinant RNase Ribonuclense Inhibitor, 200units of M-MLV RT (Promega), and finally adding DEPC water to make up the volume to 25. mu.l; mixing the mixture lightly, and reacting the mixture for 60 minutes at 37 ℃; transferring the PCR tube to 70 ℃ for reaction for 15 minutes; the cDNA product was cooled on ice and stored at-20 ℃.

The Real-Time PCR was performed using the SYBR Premix Ex TaqTM II (Perfect Real Time) reaction system from TaKaRa.

Premix Ex TaqTM (2X) 10. mu.l, PCR primers ORF7F (AATAACAACGGCAAGCAGCA), ORF7R (GCACAGTATGATGCGTCGGC) (10. mu.M) 0.8. mu.l each, ROX Reference Dye II (50X) 0.4. mu.l, cDNA template 2. mu.l, sterile double distilled water 6. mu.l, total volume 20. mu.l. Reacting on an ABI 7900Real-Time PCR System by adopting a two-step PCR amplification standard program: 30s at 95 ℃; 95 ℃ for 5s, 60 ℃ for 30s (40 cycles). Each experiment contained a single 10 diluted with a gradient⁰–10⁷Standard curves of TCID50/mL PRRSV particles were analyzed with SDS2.2.2for 7900 software.

The method for measuring the content of the viral RNA in the tissues comprises the following steps:

adding 1mL of Trizol (invitrogen) into each 5mg of tissue, grinding, blowing by using a gun in a reversed mode, and standing for 5 minutes at room temperature; adding 0.2mL of chloroform into the EP tube, covering the lid of the EP tube, shaking vigorously in hands for 15 seconds, standing at room temperature for 10 minutes, and centrifuging at 12000g and 4 ℃ for 15 minutes; placing the upper water phase in a new EP tube, adding 0.5mL of isopropanol, standing at room temperature for 10 minutes, and centrifuging at 12000g and 4 ℃ for 10 minutes; carefully abandoning the supernatant, adding 1mL of 75% ethanol for washing, centrifuging at 7500g and 4 ℃ for 5 minutes, and abandoning the supernatant; allowing the precipitated RNA to dry naturally at room temperature; finally 20. mu.l of DEPC water was added to dissolve the RNA and RNA was quantified using Nanodrop 1000(Thermo scientific).

500ng of RNA was used for RT-PCR. Adding 500ng of RNA and 1 microliter of Random Primer (10. mu.M, TaKaRa) into a 250 microliter PCR tube, and uniformly mixing; reacting for 5 minutes at 70 ℃, and rapidly cooling for 2 minutes on ice; to the above mixture was added the following components: 5. mu.l M-MLV 5 × Reaction Buffer, 5. mu.l dNTP (2.5mM), 25units of recombined RNase Ribonucleae Inhibitor, 200units M-MLV RT (Promega), and finally adding DEPC water to make up the volume to 25. mu.l; mixing the mixture lightly, and reacting the mixture for 60 minutes at 37 ℃; transferring the PCR tube to 70 ℃ for reaction for 15 minutes; the cDNA product was cooled on ice and stored at-20 ℃.

The detection results of onset symptoms and rectal temperature after challenge are shown in fig. 4-6. The results show that: piglets infected with wild type HV strain quickly develop symptoms such as high fever, dyspnea, cough, anorexia, diarrhea, unstable standing and the like, and the rectal temperature of the piglets rises to about 40 ℃ 2 days after challenge (as shown in figure 4); all died 15-18 days after infection (as shown in figure 5). The rectal temperature of the piglets infected with HV-NSP2(500-596) is still in the normal temperature range (38-39.5 ℃) within 5-6 days after challenge, but the rectal temperature of the piglets rises up to about 40 ℃ after 6 days of challenge, but the rectal temperature of the piglets falls back to the normal temperature range (as shown in figure 4) after 19 days of challenge, wherein one piglet dies after 21 days of challenge (as shown in figure 5), and the appetite of the piglets is normal in the whole experimental process (as shown in figure 5). The piglets infected with HV-NSP2(658-777) and HV-NSP2(56) virus had large rectal temperature changes after 12-16 days of infection, and all were within normal temperature range (as shown in FIG. 4), and all piglets survived healthily until the end of the experiment (as shown in FIG. 5), had good mental status, normal coat and good appetite throughout the experiment (FIG. 6).

The results of the virus titer after challenge are shown in fig. 7. The results show that: after 2 days of virus challenge, the titer of the piglet virus infected with the wild type HV strain reaches 10⁴TCID50/mL, but with the increase of the challenge time, the virus titer in the serum of the piglet infected with the wild type HV strain is always increased, and after 10 days of challenge, the virus titer reaches 10⁷TCID50/mL, and maintain high titers until death. In piglets infected with recombinant virus, the virus titer in serum reaches 10 days after challenge³About TCID50/mL, with the increase of the challenge time, the virus titer in the serum of the piglet infected with the recombinant virus is increased, but the increase rate is very slow compared with the wild type HV strain, although the peak value is reached 10-16 days after the challenge, the titer is only 10⁵ TCID 50/mL. And the virus titer in the serum of the piglet infected with the recombinant virus gradually decreases after 16 days of challenge, and the virus titer in the serum already decreases to 10 days after 28 days of challenge²TCID50/mL。

The results of the detection of viral RNA content and pathological changes in organ tissues after challenge are shown in fig. 8 and 9. The results show that: in lung and brain, the amount of viral RNA in the recombinant PRRS viruses HV-nsp2 (500-.

Sequence listing

<110> university of agriculture in China

<120> recombinant PRRS virus HV-NSP2(500-596) and application thereof

<160>4

<170>PatentIn version 3.5

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<211>15320

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<213> Artificial Sequence (Artificial Sequence)

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cctttaacca tgtctgggat acttgatcgg tgcacgtgta cccccaatgc cagggtgttt 240

gtggcggagg gccaggtcta ctgcacacga tgtctcagtg cacggtctct ccttcctctg 300

aatctccaag ttcctgagct tggggtgctg ggtctatttt ataggcccga agagccactc 360

cggtggacgt tgccacgtgc attccccact gtcgagtgct cccccgccgg ggcctgctgg 420

ctttctgcga tctttccgat tgcacgaatg actagtggaa acctgaactt tcaacaaaga 480

atggtgcggg tcgcagctga aatctacaga gccggccaac tcacccctac agttctaaag 540

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gtgggcgttt acgccaactc cctacatgtg agtgacaaac ctttcccggg agcaactcat 660

gtgttaacca acttgccgct cccgcagagg cccaaacctg aggacttttg cccttttgag 720

tgtgctatgg ctgacgtcta tgacattggt cgtggcgccg tcatgtatgt ggccggagga 780

aaggtctctt gggcccctcg tggtgggaat gaagtgaaat ttgaacctgt ccccaaggag 840

ttgaagttgg ttgcgaaccg actccacacc tccttcccgc cccatcacgt agtggacatg 900

tccaggttta ccttcatgac ccctgggagt ggtgtctcca tgcgggttga gtaccaacac 960

ggctgcctcc ccgctgacac tgtccctgaa ggaaactgct ggtggtgctt gtttgactcg 1020

ctcccaccgg aagttcagta caaagaaatt cgccatgcta accaatttgg ctatcaaacc 1080

aagcatggtg tccctggcaa atacctacag cggaggctgc aagttaatgg tcttcgagca 1140

gtgaccgaca cacatggacc tatcgtcata cagtacttct ctgttaagga gagttggatc 1200

cgccacctga agttggtgga agagcccagt ctccccgggt ttgaggatct cctcagaatc 1260

agggttgagc ccaatacgtc accactggct ggaaaggatg agaagatttt ccggtttggc 1320

agtcataagt ggtacggtgc cggaaagaga gcaaggaaaa cacgctctgg tgcgactact 1380

gtggtcgctc atcacgcttc gtccgctcat gaaacccggc aggccacgaa gcacgagggt 1440

gccggcgcta acaaggctga gcatctcaag cgctactctc cgcctgccga agggaactgt 1500

ggttggcact gcatttccgc catcgccaac cggatggtga attccaactt tgagaccacc 1560

cttcctgaaa gagtaaggcc ttcagatgac tgggccactg acgaggatct tgtgaacacc 1620

atccaaatcc tcaggctccc tgcggccttg gacaggaacg gcgcttgcgg tagcgccaag 1680

tacgtgctta aactggaggg tgagcattgg actgtctctg tgatccctgg gatgtcccct 1740

actttgctcc cccttgaatg tgttcagggt tgttgtgagc ataagggcag tcttgtttcc 1800

ccggatgcgg tcgaaatttc cggatttgat cctgcctgcc ttgaccgact ggctaaggta 1860

atgcacttgc ctagcagtac catcccagcc gctctggccg aattgtccga cgactccaac 1920

cgtccggttt ccccggccgc tactacgtgg actgtttcgc aattctatgc tcgtcataga 1980

ggaggagatc atcatgacca agtgtgcttg gggaaaatca tcagcctttg tcaagttatt 2040

gaggattgct gctgccatca gaataaaacc aaccgggcta ctccggaaga ggtcgcggca 2100

aagattgatc agtacctccg tggcgcaaca agtcttgagg aatgcttggc caaacttgag 2160

agagtttccc cgccgagcgc tgcggacacc tcctttgatt ggaatgttgt gcttcctggg 2220

gttgaggcgg cgaatcagac aaccgaacaa cctcacgtca actcatgctg caccctggtc 2280

cctcccgtga ctcaagagcc tttgggcaag gactcggtcc ctctgaccgc cttctcactg 2340

tccaattgct attaccctgc acaaggtgac gaggttcatc accgtgagag gttaaattcc 2400

gtactctcta agttggaaga ggttgtcctg gaagaatatg ggctcatgtc cactggactt 2460

ggcccgcgac ccgtgctgcc gagcgggctc gacgagctta aagaccagat ggaggaggat 2520

ctgctaaaac tagccaacac ccaggcgact tcagaaatga tggcctgggc ggctgagcag 2580

gtcgatttaa aagcttgggt caaaagctac ccgcggtgga caccaccacc ccctccacca 2640

agagttcaac ctcgaagaac aaagtctgtc aaaagcttgt cagagggcaa gcctgtccct 2700

gctccgcgca ggaaggtcag atccgattgc ggcagcccgg ttttgatggg cgacaatgtc 2760

cctaacggtt cggaagaaac tgtcggtggt cccctcaatt ttcctacacc atccgagccg 2820

atgacaccta tgagtgagcc cgtacttgtg cccgcgtcgc gacgtgtccc caagctgatg 2880

acacctttga gtgggtcggc accagttcct gcaccgcgta gaactatgac aacaacgccg 2940

acgcaccagg atgaacctct ggatttgtct gcgtcctcac agacggaata tgaggctttc 3000

cccctggcac catcgcagaa catgggcatc ctggaggcgg gggggcaaga agttgaggaa 3060

gtcctgagtg aaatctcgga tatactaaat gacaccaacc ctgcacctgt gtcatcaagc 3120

agctccctgt caagtattaa gatcacacgc ccaaaatact cagctcaagc catcatcgac 3180

tctggcgggc cttgcagtgg gcatctccaa aaggaaaaag aagcatgcct cagcatcatg 3240

cgtgaggctt gtgatgcgtc caagcttggt gatcctgcta cgcaggaatg gctctctcgc 3300

atgtgggata gggttgacat gctgacttgg cgcaacacgt ctgcttacca ggcgtttcgc 3360

atcttaaatg gcaggtttga gtttctccca aagatgattc tcgagacacc gccgccccac 3420

ccgtgcgggt ttgtgatgtt acctcgcacg cctgcacctt ccgtgagtgc agagagtgac 3480

ctcaccattg gttcagtggc caccgaggat gttccacgca tcctcgggaa agtaggagac 3540

actgacgagc tgcttgaccg gggtccttcg gcaccctcca agggagaacc ggtctgcgac 3600

caacctgcca aagatccccg gatgtcgccg cgggagtctg acgagagcat gatagctccg 3660

cccgcagata caggtggtat cggctcattc actgatttgc cgtcttcaga tggtgtggat 3720

gtggacgggg gggggccgtt aagaacggta aaaacaaaag caggaaggct cttagaccaa 3780

ctgagctgcc aggtttttag cctcgtttcc catctcccta ttttcttctc acacctcttc 3840

aaatctgaca gtggttattc tccgggtgat tggggttttg cagcttttac tctattttgc 3900

ctctttctat gttacagtta cccattcttc ggttttgctc ccctcttggg tgtattttct 3960

gggtcttctc ggcgtgtgcg aatgggggtt tttggctgct ggttggcttt tgctgttggt 4020

ctgttcaagc ctgtgtccga cccagtcggc actgcttgtg agtttgactc gccagagtgt 4080

aggaacgtcc ttcattcttt tgagcttctc aaaccttggg accctgtccg cagccttgtt 4140

gtgggccccg tcggtctcgg ccttgccatt cttggcaggt tactgggcgg ggcacgctac 4200

atctggcact ttttgcttag gcttggcatt gttgcagact gtatcttggc tggagcttat 4260

gtgctttctc aaggtaggtg taaaaggtgc tggggatctt gtgtaagaac tgctcctaat 4320

gagatcgcct tcaacgtgtt cccttttaca cgtgcgacca ggtcgtcact catcgacctg 4380

tgcgatcggt tttgcgcacc aaaaggcatg gaccccattt ttctcgccac tgggtggcgt 4440

gggtgctgga ccggccggag tcccattgag caaccttctg aaaaacccat cgcgttcgcc 4500

cagctggatg agaagagaat tacggctaga actgtggtcg ctcagcctta tgatcccaac 4560

caggccgtaa agtgcttgcg agtattacag gcgggtgggg cgatggtggc cgaggcagtc 4620

ccaaaagtgg tcaaagtttc cgctattcca ttccgagctc ctttctttcc cgctggagtg 4680

aaagttgatc ctgaatgcag aatcgtggtt gatcccgata cttttactac agccctccgg 4740

tctggctatt ccaccgcgaa cctcgtcctt ggtacggggg actttgccca gctgaatgga 4800

ctaaagatca ggcaaatttc caagccttca gggggaggcc cacacctcat tgctgccttg 4860

catgttgcct gctcgatggc gttacacatg cttgctggtg tttatgtaac tgcagtgggg 4920

tcctgcggta ccggtaccaa cgatccgtgg tgcactaacc cgtttgccgt ccctggctac 4980

ggacctggct ctctttgcac gtctagattg tgcatctccc aacacggcct caccttgccc 5040

ttgacagcac ttgtggcggg attcggcctt caagagattg ccttggtcgt tttgattttt 5100

gtctccatcg gaggcatggc tcataggttg agttgtaagg ctgacatgtt gtgcatctta 5160

ctcgcaatcg ctagttatgt ttgggtacct cttacctggt tgctttgtgt gtttccttgt 5220

tggttgcgct ggttctcttt gcaccccctc accatcctgt ggttggtgtt tttcttgatt 5280

tctgtaaata taccctcggg aatcttggcc gtggtgttat tggtttctct ctggctttta 5340

ggtcgttata ctaacattgc tggtctcgtc accccctatg acattcatca ttacaccagc 5400

ggcccccgcg gtgtcgccgc cttggccacc gcaccagatg gaacctactt ggctgccgtc 5460

cgccgtgctg cgctgactgg tcgtaccatg cttttcaccc cgtctcagct cgggtccctc 5520

cttgagggcg ctttcagaac tcaaaagccc tcactgaaca ccgtcaatgt ggtcgggtcc 5580

tccatgggct ctggcggagt gttcactatt gacgggaaaa tcaagtgcgt gactgccgca 5640

catgtcctta cgggtaactc agctagggtt tccggggtcg gcttcaatca aatgcttgac 5700

tttgatgtaa aaggggactt cgccatagct gattgcccga attggcaagg ggttgctccc 5760

aaggcccagt tctgcgagga tgggtggact ggtcgcgcct attggctgac atcctctggc 5820

gttgaacccg gtgttattgg gaatgggttc gccttctgct tcaccgcgtg tggcgattct 5880

ggatccccag tgattaccga agccggtgag cttgtcggcg ttcacacagg atcaaacaaa 5940

caaggaggag gcattgtcac gcgcccctca ggccagtttt gtaatgtgaa gcccatcaag 6000

ctgagcgagt tgagtgaatt cttcgctgga cctaaggtcc cgctcggtga tgtgaaaatt 6060

ggcagtcaca taattaaaga cacatgcgag gtgccttcag atctttgtgc cctacttgct 6120

gccaaacccg aactggaagg aggcctttcc acagttcaac ttctgtgtgt gtttttcctc 6180

ctgtggagaa tgatggggca tgcttggacg cccttggttg ctgtggggtt tttcatcctg 6240

aatgagattc tcccagctgt cctggtccgg agtgttttct cctttgggat gtttgtgcta 6300

tcttggctca caccatggtc tgcgcaagtc ctgatgatca ggcttctgac agcagccctt 6360

aacagaaaca gatggtctct tggtttttat agccttggtg cagtaaccag ttttgtcgca 6420

gatcttgcgg taactcaagg gcatccgtta caggtggtaa tgaacttaag cacctatgcc 6480

ttcctgcccc ggatgatggt tgtgacctcg ccagtcccag tgatcgcgtg tggtgttgtg 6540

cacctccttg ccataatttt gtacttgttt aagtaccgct gccttcacaa tgtccttgtt 6600

ggcgatgggg tgttctcttc ggctttcttc ttgcgatact ttgccgaggg aaagttgagg 6660

gaaggggtgt cgcaatcctg cgggatgagt catgagtcgc tgactggtgc cctcgccatg 6720

agactcactg acgaggactt ggatttcctt acgaaatgga ctgattttaa gtgctttgtt 6780

tctgcgtcca acatgaggaa tgcagcgggc caatttatcg aggctgctta tgcaaaagca 6840

ctaagaattg aacttgctca gttggtacag gttgataagg tccgaggcac catggccaaa 6900

ctcgaggctt ttgccgatac cgtggcaccc caactctcgc ccggtgacat tgttgttgcc 6960

cttggccaca cgcctgttgg cagcatcttc gacctaaagg ttggtagcac taagcatact 7020

ctccaagcca ttgagactag agtccttgcc gggtccaaaa tgactgtggc gcgtgtcgtt 7080

gacccaaccc ccgcaccccc acccgtacct gtgcccatcc ctctcccacc gaaagttctg 7140

gagaacggtc ccaatgcctg gggggatgag gaccgtttga acaagaagaa gaggcgcagg 7200

atggaagccg tcggcatttt tgtcatggac gggaaaaagt accagaaatt ttgggacaag 7260

aattccggtg atgtgtttta tgaggaggtc catattagca cagacgagtg ggagtgcctt 7320

aggactggcg accctgtcga ctttgatcct gagacaggga ttcagtgtgg gcatatcacc 7380

attgaagata aggtttacaa tgtcttcacc tccccatctg gtaggagatt cttggtcccc 7440

gccaaccccg agaatagaag agctcagtgg gaagccgcca agctttccgt ggagcaagcc 7500

cttggtatga tgaacgtcga cggcgaactg actgccaaag aactggagaa actgaaaaga 7560

ataattgaca aactccaggg cctgactaag gagcagtgtt taaactgcta gccgccagcg 7620

gcttgacccg ctgtggtcgc ggcggcttag ttgttactga gacagcggta aaaatagtca 7680

aatttcacaa ccggaccttc accctaggac ctgtgaactt aaaagtggcc agtgaggttg 7740

agctaaaaga cgcggttgag cacaaccaac atccggttgc caaaccggtt gatggtggtg 7800

ttgtgctcct gcgctctgca gttccttcgc ttatagatgt cttgatctcc ggcgctgatg 7860

catctcctaa gttactcgcc cgccacgggc cgggaaacac tgggattgat ggcacgcttt 7920

gggattttga ggccgaggct actaaagagg aagtcgcact cagtgcgcaa ataatacagg 7980

cttgtgatat taggcgcggc gacgcgcctg aaattggtct cccttataag ttgtaccctg 8040

ttaggggcaa ccctgagcgg gtaaaaggag ttttacagaa tacaaggttt ggagacatac 8100

cttacaaaac ccctagtgac actggaagcc cggtgcatgc ggctgcctgc ctcacgccta 8160

atgctactcc ggtgactgat gggcgctccg tcttggctac aaccatgccc tctggctttg 8220

agttgtatgt gccgaccatt ccagcgtccg tccttgatta tcttgattct aggcctgact 8280

gccctaaaca gttaacagag cacggttgtg aggatgctgc attaagagac ctctccaagt 8340

atgatttgtc cacccaaggc tttgttttgc ctggagttct tcgcctcgtg cggaagtacc 8400

tgttcgccca cgtgggtaag tgcccgcccg ttcatcggcc ttccacttac cctgctaaga 8460

attctatggc tggaataaat gggaacaggt ttccaaccaa ggacattcag agcgtccctg 8520

aaatcgacgt tctgtgcgca caggctgtgc gagaaaactg gcaaactgtt accccttgta 8580

ccctcaagaa acagtactgt gggaagaaga agactaggac aatacttggc accaataact 8640

tcattgcgtt ggcccatcgg gcagcgttga gtggtgttac ccagggcttc atgaaaaagg 8700

cgttcaactc gcccatcgcc ctcgggaaaa acaaatttaa ggagctacaa gccccggtcc 8760

taggcaggtg ccttgaagcc gatcttgcgt cctgcgatcg atccacacct gcaattgtcc 8820

gctggtttgc cgccaatctt ctttatgaac tcgcctgtgc tgaggagcat ctaccgtcgt 8880

acgtgcttaa ctgctgccac gacttactgg tcacgcagtc cggcgcggtg actaagagag 8940

gtggcctgtc gtctggcgac ccgattacct ctgtgtcaaa caccatttac agcttagtga 9000

tatatgcaca gcacatggtg ctcagttact tcaaaagtgg tcaccctcat ggccttctgt 9060

ttctgcaaga ccagctaaag tttgaggaca tgctcaaggt tcaacccctg atcgtctatt 9120

ccgacgacct tgtgctgtat gccgagtctc cctccatgcc aaactaccac tggtgggttg 9180

aacatctgaa tcttatgctg ggtttccaga cggacccaaa gaagacaacc atcacagact 9240

caccatcatt cctaggttgc aggataataa atgggcgcca gctagtccct aaccgtgaca 9300

ggatcctcgc ggccctcgcc tatcacatga aggcaagtaa tgtttctgaa tactacgcct 9360

cggcggctgc aatactcatg gacagctgtg cttgtttaga gtatgatcct gaatggtttg 9420

aagagctcgt ggttgggata gcgcagtgcg cccgcaagga cggctacagc tttcctggcc 9480

caccgttctt cttgtccatg tgggaaaaac tcaggtccaa tcatgagggg aagaagtcca 9540

gaatgtgcgg gtactgcggg gccccggctc cgtacgccac tgcctgtggt ctcgatgtct 9600

gtgtttacca cacccacttc caccagcatt gtcctgttat aatctggtgt ggccacccgg 9660

cgggttctgg ttcttgtagt gagtgcgaac cccccctagg aaaaggcaca agccctctag 9720

atgaggtgtt agaacaagtt ccgtacaagc ctccgcggac tgtgatcatg catgtggagc 9780

agggtctcac ccctcttgac ccaggtagat accagactcg ccgcggatta gtctccgtta 9840

ggcgtggcat caggggaaat gaagtcgacc taccagacgg tgattacgct agtaccgcct 9900

tgctccctac ttgtaaagag atcaacatgg tcgctgtcgc ctctaacgtg ttgcgcagca 9960

ggtttatcat cggcccaccc ggtgctggga aaacacactg gcttcttcaa caagtccagg 10020

atggtgatgt catttacacg ccaactcacc agaccatgct cgacatgatt agggctttgg 10080

ggacgtgccg gttcaacgtt ccagcaggta caacgctgca attccctgcc ccctcccgta 10140

ccggcccatg ggttcgcatc ttggccggcg gttggtgtcc tggcaagaac tccttcctgg 10200

atgaagcggc gtattgcaat caccttgatg tcttgaggct tctcagtaaa acaactctca 10260

cttgcctagg ggacttcaaa caactccacc ctgtgggttt tgactcccat tgctatgtat 10320

ttgacatcat gcctcagacc caattaaaga ccatttggag gttcgggcag aatatctgtg 10380

atgccattca accagattac agggacaaac ttatgtccat ggtcaacacg acccgtgtga 10440

cctatgtgga aaaacctgtc aggtatgggc aagtcctcac cccctaccac agggaccgag 10500

aggacagcgc cattactatc gactccagtc aaggcgccac atttgatgtg gttacactgc 10560

atttgcccac taaagattca ctcaacaggc aaagagctct tgttgctatc accagggcaa 10620

gacatgctat cttcgtgtat gacccacaca ggcaattgca gagtatgttt gatctccccg 10680

cgaaaggcac acccgtcaac ctcgcagtgc accgtgacga acagctgatc gtattagaca 10740

gaaacaacag agaaatcacg gttgctcagg ctctaggcaa tggagataaa ttcagggcca 10800

cagataagcg cgttgtagat tctctccgcg ctatttgcgc agacctggaa gggtcgagct 10860

ccccgctccc caaggtcgcg cataacttgg gattctattt ctcacctgat ttgactcagt 10920

ttgctaaact cccggcagaa cttgcacccc actggcccgt ggtgacaacc cagaacaatg 10980

aaaggtggcc agatcggctg gtagccagcc ttcgccctat ccataaatat agccgcgcgt 11040

gcattggtgc cggctatatg gtgggcccct cggtgttttt aggcacccct ggggttgtgt 11100

catactatct cacaaaattt gttagaggcg aggctcaagt gcttccggag acagtcttca 11160

gcaccggccg aattgaggta gattgtcgag agtatcttga tgatcgggag cgagaagttg 11220

ctgagtccct cccacatgcc ttcatcggcg atgtcaaagg taccaccgtt gggggatgtc 11280

atcacgttac ctccaaatac cttccgcgct tccttcccaa ggaatcagtt gcggtggtcg 11340

gggtttcgag ccccgggaaa gccgcgaaag cagtttgcac attgacggat gtgtacctcc 11400

cagaccttga agcgtacctc cacccagaga cccaatccag gtgctggaaa gtgatgttgg 11460

actttaagga ggttcgactg atggtatgga aagacaagac ggcctatttt caacttgaag 11520

gccgccattt cacctggtat caacttgcaa gctacgcctc atacatccga gttcctgtta 11580

attctactgt gtacttggac ccctgcatgg gccctgctct ttgcaacaga agggttgtcg 11640

ggtccaccca ttggggagct gacctcgcag tcacccctta tgattacggt gccaaaatta 11700

ttctgtctag tgcataccat ggtgaaatgc ctccaggtta caaaattctg gcgtgcgcgg 11760

agttctcgct tgatgatcca gtaaggtaca aacacacctg gggatttgaa tcggatacag 11820

cgtatctgta cgagtttact ggaaatggtg aggactggga ggattacaat gatgcgtttc 11880

gggcgcgcca gaaagggaaa atttataaag ctaatgccac cagcatgagg tttcattttc 11940

ccccgggccc tgtcattgaa ccaactttag gcctgaattg aaatgaaatg gggtctatgc 12000

aaagcctctt tgacaaaatt ggccaacttt ttgtggatgc tttcacggaa tttctggtgt 12060

ccattgttga tatcatcata tttttggcca ttttgtttgg cttcacaatc gccggttggc 12120

tggtggtctt atgcatcaga ctggtttgct ccgcggtact ccgtgcgcgc tctaccgttc 12180

accctgagca attacagaag atcttatgag gtctttcttt ctcagtgtca ggtggacatt 12240

cccacctggg gcgtcaaaca ccctttgggg gtgctttggc accataaggt gtcaaccctg 12300

attgatgaaa tggtgtcgcg tcgaatgtac cgcatcatgg aaaaagcagg gcaggctgcc 12360

tggaaacagg tggtgagcga ggctacattg tctcgcatta gtggtttgga tgtggtggct 12420

cactttcaac atcttgccgc tattgaagcc gagacttgta aatatttggc ttcccggcta 12480

cccatgctgc ataacctgcg cttgacaggg tcaaatgtaa ccatagtgta taatagtact 12540

ttggatcagg tgtttgccat tttcccaacc cctggttccc ggccaaagct tcatgacttt 12600

cagcaatggc taatagctgt acattcctcc atattttcct ccgttgcagc ttcttgtact 12660

ctttttgttg tgctgtggtt gcgaattcca atgctacgtt ctgtttttgg tttccgctgg 12720

ttaggggcaa cttttctttt gaactcatgg tgaattacac ggtatgcccg ctttgcccaa 12780

cccggcaggc agccgctgag atccttgaac ccggcaagtc tttttggtgc aggatagggc 12840

atgaccgatg tagtgagaac gatcatgacg aactagggtt catggttccg cccggcctct 12900

ccagcgaagg ccacttgacc agtgtttacg cctggttggc gttcctgtcc ttcagctaca 12960

cggcccagtt ccatcccgag atatttggga tagggaatgt gagtcaagtt tatgttgaca 13020

tcaagcacca attcatctgc gctgttcacg acggggataa cgccaccttg cctcgccatg 13080

acaatatttc agccgtattt cagacctact accaacacca ggtcgacggc ggcaattggt 13140

ttcacctgga atggctgcgt cctttctttt cctcttggtt ggttttaaat gtttcgtggt 13200

ttctcaggcg ttcgcctgca agccatgttt cagttcgagt ctttcggaca tcaaaaccaa 13260

caccaccgca gcatcagact tcgttgtcct ccaggacatc agctgcctta ggcatggcga 13320

ctcgtcctct ccgacgattc gcaaaagttc tcagtgccgc acggcgatag ggacgcccgt 13380

gtatatcacc atcaccgcca atgtcacaga tgaaaattat ctacattctt ctgatctcct 13440

catgctttct tcttgccttt tctacgcttc cgagatgagt gaaaagggat tcaaagtggt 13500

gtttggcaat gtgtcaggca tcgtggctgt gtgcgtcaac tttaccagct acgtccaaca 13560

cgtcaaggag tttactcaac gctccttagt ggtcgatcat gtgcgactgc ttcatttcat 13620

gacacctgag accatgaggt gggcaaccgt tttagcctgt ctttttgcca tcctactggc 13680

aatttgaatg ttcaagtatg ttggggaagt gcttgaccgc gtgctgttgc tcgcgattgc 13740

tttttttgtg gtgtatcgtg ccgttctatc ttgctgtgct cgtcaacgcc agcaacaaca 13800

acagctctca tattcagttg atttataact taacgctatg tgagctgaat ggcacagatt 13860

ggctggcgca aaaatttgac tgggcagtgg agacttttgt catcttcccc gtgttgactc 13920

acattgtttc ctatggggca ctcaccacca gccatttcct tgacacagtt ggtctggcca 13980

ctgtgtccac cgccggatat tatcacgggc ggtatgtctt gagtagcatt tacgcagtct 14040

gtgctctggc tgcgctgatt tgctttgtca ttaggcttgc gaagaactgc atgtcctggc 14100

gctactcttg taccagatat accaacttcc ttctggacac taagggcaga ctctatcgtt 14160

ggcggtcgcc cgtcattgtg gagaaagggg gtaaggttga ggtcgaaggt cacctgatcg 14220

acctcaagag agttgtgctt gatggttccg cggcaacccc tttaaccaga gtttcagcgg 14280

aacaatgggg tcgtctctag acgacttctg caatgatagc acagctccac agaaggtgct 14340

tttggcgttt tccattacct acacgccagt gatgatatat gctctaaagg taagtcgcgg 14400

ccgactgcta gggcttctgc accttttgat ctttctgaat tgtgctttta ccttcgggta 14460

catgacattc gtgcactttg agagcacaaa tagggtcgcg ctcactatgg gagcagtagt 14520

tgcacttctt tggggagtgt actcagccat agaaacctgg aaattcatca cctccagatg 14580

ccgtttgtgc ttgctaggcc gcaagtacat tctggcccct gcccaccacg tcgaaagtgc 14640

cgcgggcttt catccgattg cggcaaatga taaccacgca tttgtcgtcc ggcgtcccgg 14700

ctccactacg gtcaacggca cattggtgcc cgggttgaaa agcctcgtgt tgggtggcag 14760

aaaagctgtt aagcagggag tggtaaacct tgttaagtat gccaaataac aacggcaagc 14820

agcaaaagaa aaagaagggg aatggccagc cagtcaatca gctgtgccaa atgctgggta 14880

agatcatcgc ccaacaaaac cagtccagag gcaagggacc ggggaagaaa aataggaaga 14940

aaaacccgga gaagccccat ttccctctag cgactgaaga tgacgtcagg catcacttta 15000

cccctagtga gcggcaattg tgtctgtcgt cgatccagac tgcctttaat cagggcgctg 15060

gaacttgtgc cctgtcagat tcagggagga taagttacac tgtggagttt agtttgccga 15120

cgcaacatac tgtgcgtctg atccgcgcca cagcatcacc ctcagcgtga tgggctggca 15180

ttctttggca cctcagtgtt agaattggga gaatgtgtgg tgaatggcac tgattgacac 15240

tgtgcctcta agtcacctat tcaattaggg cgaccgtgtg ggggtaaagt ttaattggcg 15300

agaaccatgc ggccgtaatt 15320

<210>2

<211>3206

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

cgatctttcc gattgcacga atgactagtg gaaacctgaa ctttcaacaa agaatggtgc 60

gggtcgcagc tgaaatctac agagccggcc aactcacccc tacagttcta aagactctgc 120

aagtttatga acggggttgt cgttggtacc ccattgtcgg gcccgtccct ggggtgggcg 180

tttacgccaa ctccctacat gtgagtgaca aacctttccc gggagcaact catgtgttaa 240

ccaacttgcc gctcccgcag aggcccaaac ctgaggactt ttgccctttt gagtgtgcta 300

tggctgacgt ctatgacatt ggtcgtggcg ccgtcatgta tgtggccgga ggaaaggtct 360

cttgggcccc tcgtggtggg aatgaagtga aatttgaacc tgtccccaag gagttgaagt 420

tggttgcgaa ccgactccac acctccttcc cgccccatca cgtagtggac atgtccaggt 480

ttaccttcat gacccctggg agtggtgtct ccatgcgggt tgagtaccaa cacggctgcc 540

tccccgctga cactgtccct gaaggaaact gctggtggtg cttgtttgac tcgctcccac 600

cggaagttca gtacaaagaa attcgccatg ctaaccaatt tggctatcaa accaagcatg 660

gtgtccctgg caaataccta cagcggaggc tgcaagttaa tggtcttcga gcagtgaccg 720

acacacatgg acctatcgtc atacagtact tctctgttaa ggagagttgg atccgccacc 780

tgaagttggt ggaagagccc agtctccccg ggtttgagga tctcctcaga atcagggttg 840

agcccaatac gtcaccactg gctggaaagg atgagaagat tttccggttt ggcagtcata 900

agtggtacgg tgccggaaag agagcaagga aaacacgctc tggtgcgact actgtggtcg 960

ctcatcacgc ttcgtccgct catgaaaccc ggcaggccac gaagcacgag ggtgccggcg 1020

ctaacaaggc tgagcatctc aagcgctact ctccgcctgc cgaagggaac tgtggttggc 1080

actgcatttc cgccatcgcc aaccggatgg tgaattccaa ctttgagacc acccttcctg 1140

aaagagtaag gccttcagat gactgggcca ctgacgagga tcttgtgaac accatccaaa 1200

tcctcaggct ccctgcggcc ttggacagga acggcgcttg cggtagcgcc aagtacgtgc 1260

ttaaactgga gggtgagcat tggactgtct ctgtgatccc tgggatgtcc cctactttgc 1320

tcccccttga atgtgttcag ggttgttgtg agcataaggg cagtcttgtt tccccggatg 1380

cggtcgaaat ttccggattt gatcctgcct gccttgaccg actggctaag gtaatgcact 1440

tgcctagcag taccatccca gccgctctgg ccgaattgtc cgacgactcc aaccgtccgg 1500

tttccccggc cgctactacg tggactgttt cgcaattcta tgctcgtcat agaggaggag 1560

atcatcatga ccaagtgtgc ttggggaaaa tcatcagcct ttgtcaagtt attgaggatt 1620

gctgctgcca tcagaataaa accaaccggg ctactccgga agaggtcgcg gcaaagattg 1680

atcagtacct ccgtggcgca acaagtcttg aggaatgctt ggccaaactt gagagagttt 1740

ccccgccgag cgctgcggac acctcctttg attggaatgt tgtgcttcct ggggttgagg 1800

cggcgaatca gacaaccgaa caacctcacg tcaactcatg ctgcaccctg gtccctcccg 1860

tgactcaaga gcctttgggc aaggactcgg tccctctgac cgccttctca ctgtccaatt 1920

gctattaccc tgcacaaggt gacgaggttc atcaccgtga gaggttaaat tccgtactct 1980

ctaagttgga agaggttgtc ctggaagaat atgggctcat gtccactgga cttggcccgc 2040

gacccgtgct gccgagcggg ctcgacgagc ttaaagacca gatggaggag gatctgctaa 2100

aactagccaa cacccaggcg acttcagaaa tgatggcctg ggcggctgag caggtcgatt 2160

taaaagcttg ggtcaaaagc tacccgcggt ggacaccacc accccctcca ccaagagttc 2220

aacctcgaag aacaaagtct gtcaaaagct tgtcagaggg caagcctgtc cctgctccgc 2280

gcaggaaggt cagatccgat tgcggcagcc cggttttgat gggcgacaat gtccctaacg 2340

gttcggaaga aactgtcggt ggtcccctca attttcctac accatccgag ccgatgacac 2400

ctatgagtga gcccgtactt gtgcccgcgt cgcgacgtgt ccccaagctg atgacacctt 2460

tgagtgggtc ggcaccagtt cctgcaccgc gtagaactat gacaacaacg ccgacgcacc 2520

aggatgaacc tctggatttg tctgcgtcct cacagacgga atatgaggct ttccccctgg 2580

caccatcgca gaacatgggc atcctggagg cgggggggca agaagttgag gaagtcctga 2640

gtgaaatctc ggatatacta aatgacacca accctgcacc tgtgtcatca agcagctccc 2700

tgtcaagtat taagatcaca cgcccaaaat actcagctca agccatcatc gactctggcg 2760

ggccttgcag tgggcatctc caaaaggaaa aagaagcatg cctcagcatc atgcgtgagg 2820

cttgtgatgc gtccaagctt ggtgatcctg ctacgcagga atggctctct cgcatgtggg 2880

atagggttga catgctgact tggcgcaaca cgtctgctta ccaggcgttt cgcatcttaa 2940

atggcaggtt tgagtttctc ccaaagatga ttctcgagac accgccgccc cacccgtgcg 3000

ggtttgtgat gttacctcgc acgcctgcac cttccgtgag tgcagagagt gacctcacca 3060

ttggttcagt ggccaccgag gatgttccac gcatcctcgg gaaagtagga gacactgacg 3120

agctgcttga ccggggtcct tcggcaccct ccaagggaga accggtctgc gaccaacctg 3180

ccaaagatcc ccggatgtcg ccgcgg 3206

<210>3

<211>938

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

cgatctttcc gattgcacga atgactagtg gaaacctgaa ctttcaacaa agaatggtgc 60

gggtcgcagc tgaaatctac agagccggcc aactcacccc tacagttcta aagactctgc 120

aagtttatga acggggttgt cgttggtacc ccattgtcgg gcccgtccct ggggtgggcg 180

tttacgccaa ctccctacat gtgagtgaca aacctttccc gggagcaact catgtgttaa 240

ccaacttgcc gctcccgcag aggcccaaac ctgaggactt ttgccctttt gagtgtgcta 300

tggctgacgt ctatgacatt ggtcgtggcg ccgtcatgta tgtggccgga ggaaaggtct 360

cttgggcccc tcgtggtggg aatgaagtga aatttgaacc tgtccccaag gagttgaagt 420

tggttgcgaa ccgactccac acctccttcc cgccccatca cgtagtggac atgtccaggt 480

ttaccttcat gacccctggg agtggtgtct ccatgcgggt tgagtaccaa cacggctgcc 540

tccccgctga cactgtccct gaaggaaact gctggtggtg cttgtttgac tcgctcccac 600

cggaagttca gtacaaagaa attcgccatg ctaaccaatt tggctatcaa accaagcatg 660

gtgtccctgg caaataccta cagcggaggc tgcaagttaa tggtcttcga gcagtgaccg 720

acacacatgg acctatcgtc atacagtact tctctgttaa ggagagttgg atccgccacc 780

tgaagttggt ggaagagccc agtctccccg ggtttgagga tctcctcaga atcagggttg 840

agcccaatac gtcaccactg gctggaaagg atgagaagat tttccggttt ggcagtcata 900

agtggtacgg tgccggaaag agagcaagga aaacacgc 938

<210>4

<211>2883

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

ccaaagatcc ccggatgtcg ccgcgggagt ctgacgagag catgatagct ccgcccgcag 60

atacaggtgg tatcggctca ttcactgatt tgccgtcttc agatggtgtg gatgtggacg 120

ggggggggcc gttaagaacg gtaaaaacaa aagcaggaag gctcttagac caactgagct 180

gccaggtttt tagcctcgtt tcccatctcc ctattttctt ctcacacctc ttcaaatctg 240

acagtggtta ttctccgggt gattggggtt ttgcagcttt tactctattt tgcctctttc 300

tatgttacag ttacccattc ttcggttttg ctcccctctt gggtgtattt tctgggtctt 360

ctcggcgtgt gcgaatgggg gtttttggct gctggttggc ttttgctgtt ggtctgttca 420

agcctgtgtc cgacccagtc ggcactgctt gtgagtttga ctcgccagag tgtaggaacg 480

tccttcattc ttttgagctt ctcaaacctt gggaccctgt ccgcagcctt gttgtgggcc 540

ccgtcggtct cggccttgcc attcttggca ggttactggg cggggcacgc tacatctggc 600

actttttgct taggcttggc attgttgcag actgtatctt ggctggagct tatgtgcttt 660

ctcaaggtag gtgtaaaagg tgctggggat cttgtgtaag aactgctcct aatgagatcg 720

ccttcaacgt gttccctttt acacgtgcga ccaggtcgtc actcatcgac ctgtgcgatc 780

ggttttgcgc accaaaaggc atggacccca tttttctcgc cactgggtgg cgtgggtgct 840

ggaccggccg gagtcccatt gagcaacctt ctgaaaaacc catcgcgttc gcccagctgg 900

atgagaagag aattacggct agaactgtgg tcgctcagcc ttatgatccc aaccaggccg 960

taaagtgctt gcgagtatta caggcgggtg gggcgatggt ggccgaggca gtcccaaaag 1020

tggtcaaagt ttccgctatt ccattccgag ctcctttctt tcccgctgga gtgaaagttg 1080

atcctgaatg cagaatcgtg gttgatcccg atacttttac tacagccctc cggtctggct 1140

attccaccgc gaacctcgtc cttggtacgg gggactttgc ccagctgaat ggactaaaga 1200

tcaggcaaat ttccaagcct tcagggggag gcccacacct cattgctgcc ttgcatgttg 1260

cctgctcgat ggcgttacac atgcttgctg gtgtttatgt aactgcagtg gggtcctgcg 1320

gtaccggtac caacgatccg tggtgcacta acccgtttgc cgtccctggc tacggacctg 1380

gctctctttg cacgtctaga ttgtgcatct cccaacacgg cctcaccttg cccttgacag 1440

cacttgtggc gggattcggc cttcaagaga ttgccttggt cgttttgatt tttgtctcca 1500

tcggaggcat ggctcatagg ttgagttgta aggctgacat gttgtgcatc ttactcgcaa 1560

tcgctagtta tgtttgggta cctcttacct ggttgctttg tgtgtttcct tgttggttgc 1620

gctggttctc tttgcacccc ctcaccatcc tgtggttggt gtttttcttg atttctgtaa 1680

atataccctc gggaatcttg gccgtggtgt tattggtttc tctctggctt ttaggtcgtt 1740

atactaacat tgctggtctc gtcaccccct atgacattca tcattacacc agcggccccc 1800

gcggtgtcgc cgccttggcc accgcaccag atggaaccta cttggctgcc gtccgccgtg 1860

ctgcgctgac tggtcgtacc atgcttttca ccccgtctca gctcgggtcc ctccttgagg 1920

gcgctttcag aactcaaaag ccctcactga acaccgtcaa tgtggtcggg tcctccatgg 1980

gctctggcgg agtgttcact attgacggga aaatcaagtg cgtgactgcc gcacatgtcc 2040

ttacgggtaa ctcagctagg gtttccgggg tcggcttcaa tcaaatgctt gactttgatg 2100

taaaagggga cttcgccata gctgattgcc cgaattggca aggggttgct cccaaggccc 2160

agttctgcga ggatgggtgg actggtcgcg cctattggct gacatcctct ggcgttgaac 2220

ccggtgttat tgggaatggg ttcgccttct gcttcaccgc gtgtggcgat tctggatccc 2280

cagtgattac cgaagccggt gagcttgtcg gcgttcacac aggatcaaac aaacaaggag 2340

gaggcattgt cacgcgcccc tcaggccagt tttgtaatgt gaagcccatc aagctgagcg 2400

agttgagtga attcttcgct ggacctaagg tcccgctcgg tgatgtgaaa attggcagtc 2460

acataattaa agacacatgc gaggtgcctt cagatctttg tgccctactt gctgccaaac 2520

ccgaactgga aggaggcctt tccacagttc aacttctgtg tgtgtttttc ctcctgtgga 2580

gaatgatggg gcatgcttgg acgcccttgg ttgctgtggg gtttttcatc ctgaatgaga 2640

ttctcccagc tgtcctggtc cggagtgttt tctcctttgg gatgtttgtg ctatcttggc 2700

tcacaccatg gtctgcgcaa gtcctgatga tcaggcttct gacagcagcc cttaacagaa 2760

acagatggtc tcttggtttt tatagccttg gtgcagtaac cagttttgtc gcagatcttg 2820

cggtaactca agggcatccg ttacaggtgg taatgaactt aagcacctat gccttcctgc 2880

ccc 2883

Claims

1. A recombinant PRRS virus, the cDNA sequence corresponding to the genome of the recombinant PRRS virus is a sequence obtained by deleting the cDNA sequence corresponding to the genome of the wild type PRRS virus from the DNA molecule shown in the 2833-3036 th site at the 5' end;

the cDNA sequence corresponding to the genome of the wild type PRRS virus is shown as a sequence 1 in a sequence table.

2. Use of the recombinant PRRS virus of claim 1 for the preparation of a PRRS vaccine;

or, the use of the recombinant PRRS virus of claim 1 for the preparation of a PRRS attenuated vaccine.

3. Use of the recombinant PRRS virus of claim 1 in the manufacture of a medicament for the prevention of porcine reproductive and respiratory syndrome.

The DNA fragment is obtained by deleting the DNA molecule shown in the 2833-3036 th site from the 5' end of the sequence 1 in the sequence table.

5. Use of the DNA fragment of claim 4 for the preparation of the recombinant PRRS virus of claim 1;

or, the use of the DNA fragment of claim 4 for the preparation of a PRRS vaccine;

or, the use of the DNA fragment of claim 4 for the preparation of a PRRS attenuated vaccine.

6. A recombinant plasmid, a recombinant bacterium or a recombinant cell comprising the DNA fragment of claim 5.

7. The recombinant plasmid according to claim 6, wherein: the recombinant plasmid is a sequence obtained by deleting DNA molecules which correspond to the 2833-3036 th site at the 5' tail end in the sequence 1 in the plasmid pcDNA3.1-HV.

8. Use of the recombinant plasmid, recombinant bacterium or recombinant cell of claim 6 in the preparation of the recombinant PRRS virus of claim 1.

9. Use of the recombinant plasmid, recombinant bacterium or recombinant cell of claim 6 in the preparation of a PRRS vaccine.

10. Use of the recombinant plasmid, recombinant bacterium or recombinant cell of claim 6 in the preparation of a PRRS attenuated vaccine.