A recombinant porcine reproductive and respiratory syndrome virus and its construction and application

Abstract

The present invention belongs to the field of biotechnology, and relates to a recombinant porcine reproductive and respiratory syndrome virus and its construction and application. The present invention replaces the NSP2 gene sequence of the HP-PRRSV GD strain with the gene sequence shown in SEQ ID NO.1, and replaces the ORF5-7 and 3'UTR gene fragments with the gene sequence shown in SEQ ID NO.2, thereby rescuing the recombinant porcine reproductive and respiratory syndrome virus. The recombinant porcine reproductive and respiratory syndrome virus can infect MARC-145 cells and has a high virus titer; compared with HP-PRRSV GD, the recombinant porcine reproductive and respiratory syndrome virus can not only provide good protection against the attack of HP-PRRSV, but also provide good protection against the attack of NADC30-like PRRSV, and can be used to prepare PRRSV diagnostic reagents or new PRRSV vaccines.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant porcine reproductive and respiratory syndrome virus and a construction and application thereof. Background Art

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS) caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection is one of the most important viral infectious diseases of pigs besides African swine fever (ASF). It mainly causes reproductive dysfunction in sows and respiratory dysfunction in pigs of all ages.

[0003] PRRSV is a single-stranded positive-sense RNA virus of the order Nidovirales and the family Arteriviridae. It was first introduced into my country in 1995. In 1996, the first strain of PRRSV was isolated in my country and named CH1a. Since then, PRRSV has become more prevalent, and the PRRSV strains prevalent at this stage are customarily called classical PRRSV. In 2008, a new PRRSV strain was identified in the United States, which had a low genomic homology with the previous PRRSV strain and was named PRRSV NADC30 strain. Around 2013, a PRRSV strain with a genome highly similar to the American NADC30 PRRSV strain appeared in my country and quickly broke out nationwide. It was called NADC30-like PRRSV. Currently, NADC30-like PRRSV and HP-PRRSV are the dominant PRRSV strains in my country.

[0004] Vaccination is considered an important measure to prevent viral infection. However, the current commercial PRRSV vaccines in my country are all developed with HP-PRRSV or classical PRRSV as seed viruses, and the protection effect on NADC30-like PRRSV is poor. Reverse genetic manipulation technology can achieve the transformation and modification of viruses to produce recombinant viruses, and is an important technical means to develop new PRRSV vaccines. Using HP-PRRSV vaccine strains as the skeleton and chimeric key gene fragments of NADC30-like PRRSV to construct recombinant viruses provides a new solution for the development of new PRRSV vaccines that can provide cross-protection against NADC30-like PRRSV. However, there are currently a variety of commercial PRRSV attenuated vaccines in my country, and the immune effects of different vaccines vary. Therefore, there are the following key issues in the construction of recombinant PRRSV virus strains: ① It is necessary to select a suitable vaccine attenuated strain as the backbone. Different backbones will lead to significant differences in the immune protection efficiency of the prepared recombinant PRRSV virus strains; ② The genome variation of the NADC30-like PRRSV cluster is very high. In addition to its high genome differences with HP-PRRSV, classical PRRSV and other clusters, it is also reflected in the large genome differences within the NADC30-like PRRSV cluster. Therefore, it is necessary to select a suitable NADC30-like PRRSV strain; ③ The selection of gene fragments that need to be replaced is the key to constructing recombinant PRRSV virus strains. Even if the publicly available PRRSV gene sequence with immunogenic function is replaced, the cellular infectivity and immunogenicity of the replaced recombinant PRRSV virus strain cannot be expected; and replacing too many functional genes may change the cellular tropism and immunogenicity of the recombinant virus, which is not conducive to its development as a vaccine strain.

[0005] The present invention replaces the NSP2 gene sequence of the HP-PRRSV GD attenuated strain with the gene sequence described in SEQ ID NO.1 through reverse genetic manipulation technology, and replaces the ORF5-7 and 3'UTR genes with the gene sequence described in SEQ ID NO.2, thereby constructing a chimeric infectious clone, successfully rescuing and obtaining a PRRSV recombinant virus strain PRRSV rGD-SX-5U2. The obtained recombinant strain can give piglets good protection when facing the attack of HP-PRRSV or NADC30-like PRRSV, and can be used to prepare PRRSV diagnostic reagents or PRRSV vaccines. Summary of the invention

[0006] The present invention aims at the industry problem that the current commercial PRRSV vaccine cannot provide good protection against the dominant epidemic strain of NADC30-like PRRSV. By using reverse genetic manipulation technology, the genome of the HP-PRRSV GD attenuated strain is modified to obtain a recombinant porcine reproductive and respiratory syndrome virus. The recombinant porcine reproductive and respiratory syndrome virus can infect MARC-145 cells and has a high virus titer. Animal experiments show that the recombinant strain can give piglets good protection when facing the attack of HP-PRRSV or NADC30-like PRRSV, which specifically includes the following contents:

[0007] In a first aspect, the present invention provides a recombinant porcine reproductive and respiratory syndrome virus, wherein the recombinant porcine reproductive and respiratory syndrome virus is a recombinant virus strain obtained by replacing the NSP2 gene sequence of the HP-PRRSV GD strain with the gene sequence shown in SEQ ID NO.1, and replacing the ORF5-7 and 3'UTR gene fragments with the gene sequence shown in SEQ ID NO.2.

[0008] Preferably, the full-length cDNA sequence of the recombinant porcine reproductive and respiratory syndrome virus is as shown in SEQ ID NO.3.

[0009] In a second aspect, the present invention provides a use of the recombinant porcine reproductive and respiratory syndrome virus described in the first aspect in the preparation of a PRRSV diagnostic reagent or a PRRSV vaccine.

[0010] In a third aspect, the present invention provides a method for preparing a recombinant porcine reproductive and respiratory syndrome virus, the method comprising:

[0011] Construction of infectious clone: ​​The NSP2 gene sequence of HP-PRRSV GD strain was replaced with the gene sequence shown in SEQ ID NO.1 by genetic engineering means, and the ORF5-7 and 3'UTR gene fragments were replaced with the gene sequence shown in SEQ ID NO.2, and then connected into the plasmid to obtain the infectious clone plasmid;

[0012] Virus rescue: The obtained infectious clone plasmid is rescued to obtain recombinant porcine reproductive and respiratory syndrome virus.

[0013] Preferably, the method is:

[0014] (1) Construction of a full-genome infectious clone plasmid of the HP-PRRSV GD strain;

[0015] (2) replacing the NSP2 gene sequence in the infectious clone plasmid in step (1) with the gene sequence shown in SEQ ID NO.1, and replacing the ORF5-7 and 3'UTR gene fragments with the gene sequence shown in SEQ ID NO.2, to obtain a recombinant infectious clone plasmid;

[0016] (3) Transfecting the recombinant infectious clone plasmid described in step (2) into BHK21 cells and then transferring to MARC-145 cells for rescue to obtain the recombinant porcine reproductive and respiratory syndrome virus.

[0017] Preferably, the step (1) is:

[0018] Using the genomic cDNA of the HP-PRRSV GD strain as a template, fragment A2 was amplified using the primers shown in SEQ ID NO.4-5, and fragment B2 was amplified using the primers shown in SEQ ID NO.6-7. Fragments A2 and B2 were overlapped to obtain fragment A2+B2; fragment C2 was amplified using the primers shown in SEQ ID NO.8-9; fragment A2+B2 and fragment C2 were connected into the pBeloBac11 vector linearized with SfiⅠ and RsrⅡ restriction sites by homologous recombination technology to obtain pBAC-GD-SU plasmid;

[0019] Using the HP-PRRSV GD strain genomic cDNA as a template, using the primers shown in SEQ ID NO.10-11 to amplify fragment D2; using the primers shown in SEQ ID NO.12-13 to amplify fragment E2, fragments D2 and E2 are overlapped to obtain fragment PU; using fragment PU as a template, using SEQ ID NO.10 and SEQ ID NO.14 to amplify GD-PU-A with a marker, using SEQ ID NO.15 and SEQ ID NO.13 to amplify GD-PU-B with a marker;

[0020] GD-PU-A and GD-PU-B were simultaneously inserted into the pBAC-GD-SU plasmid linearized with PmeⅠHE RsrⅡ restriction sites by homologous recombination technology to construct the full genome infectious clone plasmid pBAC-GD of HP-PRRSV GD strain.

[0021] Preferably, the step (2) is:

[0022] Using the gene fragment shown in SEQ ID NO.2 as a template, the primers shown in SEQ ID NO.16-17 were used to amplify the SX-B fragment; using the infectious clone plasmid pBAC-GD as a template, the primers shown in SEQ ID NO.18-19 were used to amplify the GD-A fragment; using homologous recombination technology, the fragments GD-A and SX-B were simultaneously connected to the infectious clone plasmid pBAC-GD linearized with PmeⅠ and RsrⅡ restriction sites to obtain the infectious clone plasmid pBAC-GD-ORF5-U (SX);

[0023] Using the gene fragment shown in SEQ ID NO.1 as a template, the primers shown in SEQ ID NO.20-21 were used to amplify the fragment SX-D; using the infectious clone plasmid pBAC-GD as a template, the primers shown in SEQ ID NO.4 and 22 were used to amplify the GD-C fragment; the primers shown in SEQ ID NO.23-24 were used to amplify the fragment GD-E; using homologous recombination technology, the fragments GD-C, SX-D and GD-E were simultaneously connected to the infectious clone plasmid pBAC-GD-ORF5-U (SX) linearized with SfiⅠ and PmeⅠ restriction sites to obtain the recombinant infectious clone plasmid pBAC-GD-SX-5U2.

[0024] Preferably, the step (3) is:

[0025] The supernatant of BHK21 cells transfected with the recombinant infectious clone plasmid pBAC-GD-SX-5U2 was collected and transferred to MARC-145 cells. The cell supernatant was then replaced with DMEM medium containing 3% FBS until typical CPE appeared to obtain the recombinant porcine reproductive and respiratory syndrome virus.

[0026] In a fourth aspect, the present invention provides a recombinant porcine reproductive and respiratory syndrome virus prepared according to the method described in the third aspect.

[0027] In a fifth aspect, the present invention provides the use of the recombinant porcine reproductive and respiratory syndrome virus described in the fourth aspect in the preparation of a PRRSV diagnostic reagent or a PRRSV vaccine.

[0028] The beneficial effects of the present invention are as follows: based on the major industry problem that the current commercial PRRSV vaccine cannot provide good protection against NADC30-like PRRSV, the present invention utilizes reverse genetic manipulation technology, takes HP-PRRSV GD strain as the skeleton, replaces the NSP2 gene sequence of HP-PRRSV GD strain with the gene sequence shown in SEQ ID NO.1, and replaces the ORF5-7 and 3'UTR gene fragments with the gene sequence shown in SEQ ID NO.2, constructs and successfully rescues the recombinant porcine reproductive and respiratory syndrome virus rGD-SX-5U2; the recombinant strain rGD-SX-5U2 can infect MARC-145 cells and has a high virus titer, and animal experiments show that the recombinant strain can give piglets good protection when facing the attack of HP-PRRSV or NADC30-like PRRSV. It can be used to prepare PRRSV diagnostic reagents and / or vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, some of the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0030] Figure 1 HP-PRRSV GD strain infectious clone rGD rescue IFA identification diagram;

[0031] Figure 2 IFA identification diagram of rescued strain rGD-SX-5U2;

[0032] Figure 3 Figure 1 shows the sequencing results of the rescued strain rGD-SX-5U2;

[0033] Figure 4 WB identification of rescued strain rGD-SX-5U2 infecting MARC-145 cells;

[0034] Figure 5 Growth curve of rescued strain rGD-SX-5U2 on MARC-145 cells;

[0035] Figure 6 Diagram of measuring body temperature of piglets in animal experiments;

[0036] Figure 7 Figure 1 shows the determination of virus titer in the lung tissue of piglets in animal experiments;

[0037] Figure 8 Lung tissue damage in piglets in animal experiments. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The restriction endonucleases and T4 DNA ligase used in the present invention were purchased from NEB; the high-fidelity amplification enzymes used were GXL, HSDNA polymerase, cloning vector pMD19-T and competent cells HST08 were all purchased from TAKARA; seamless cloning kit ClonExpressMultiS One Step Cloning Kit was purchased from Nanjing Novogene Biotechnology Co., Ltd.; the cloning vector pEASY-Blunt (Simple), competent cells Trans5α, and fetal bovine serum used in cell culture were all purchased from Beijing Quanshijin Biotechnology Co., Ltd.; fluorescent secondary antibody, culture medium DMEM, and transfection reagent Lipofectamine 3000 were purchased from Thermo Fisher.

[0040] The PRRSV N protein monoclonal antibody (6D10) used in the present invention was prepared in the laboratory of the inventor, and the BHK21 and MARC-145 cells used were both stored in the laboratory of the inventor; the HP-PRRSV GD strain described in the present invention is a commercially available porcine reproductive and respiratory syndrome commercial vaccine strain, which can be obtained by technicians in this field commercially; the attack strains PRRSV SD-YL1712 and PRRSV SX-YL1806 were isolated and stored in the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.

[0041] The specific embodiments include the following:

[0042] Example 1 Construction and rescue of HP-PRRSV GD strain infectious clone

[0043] (1) Construction of infectious clone of HP-PRRSV GD strain

[0044] The genomic RNA of HP-PRRSV GD strain was extracted and reverse transcribed into cDNA, and the whole genome sequence was amplified using cDNA as a template; the whole genome of HP-PRRSV GD strain was divided into the upper half SU and the lower half PU using the restriction site PmeⅠ;

[0045] Construction of the upper half SU: Using the genomic cDNA of the PRRSV GD strain as a template, fragment A2 was amplified using the primers shown in SEQ ID NO.4-5; fragment B2 was amplified using the primers shown in SEQ ID NO.6-7; using fragments A2 and B2 as templates, overlap splicing was performed using the primers shown in SEQ ID NO.4 and 7 to obtain fragment A2+B2; continuing to use the genomic cDNA of the PRRSV GD strain as a template, fragment C2 was amplified using the primers shown in SEQ ID NO.8-9; the pBeloBac11 empty vector was linearized using the two restriction sites of SfiⅠ and RsrⅡ, and fragments A2+B2 and fragment C2 were connected to the linearized pBeloBac11 vector using homologous recombination technology to obtain the pBAC-GD-SU plasmid.

[0046] The lower half PU was constructed by using the genomic cDNA of the PRRSV GD strain as a template and amplifying the fragment D2 using the primers shown in SEQ ID NOs.10-11; amplifying the fragment E2 using the primers shown in SEQ ID NOs.12-13; using the fragments D2 and E2 as templates, overlapping the fragments PU using the primers shown in SEQ ID NOs.10 and 13; using the fragment PU as a template, amplifying the marker-tagged fragment GD-PU-A using the primers shown in SEQ ID NOs.10 and 14, and amplifying the marker-tagged fragment GD-PU-B using the primers shown in SEQ ID NOs.15 and 13.

[0047] The aforementioned pBAC-GD-SU plasmid was linearized using the two restriction sites PmeⅠ and RsrⅡ, and the above-mentioned fragments GD-PU-A and GD-PU-B were simultaneously embedded into the linearized pBAC-GD-SU plasmid using homologous recombination technology to obtain the full-length plasmid of pBAC-GD infectious clone.

[0048] (2) Rescue of HP-PRRSV GD strain infectious clones

[0049] The full-length plasmid of pBAC-GD infectious clone was transfected into BHK21 cells using lipo3000 transfection reagent for rescue. The supernatant was discarded 12 hours after transfection and washed three times with PBS, and replaced with DMEM medium containing 10% FBS. The cell supernatant was collected 48 hours after transfection, filtered through a 0.22μm filter, and then transferred to MARC-145 cells. The cells were cultured statically under appropriate conditions until typical CPE appeared, and the samples were collected for IFA identification.

[0050] (3) IFA identification of infectious clones of HP-PRRSV GD strain

[0051] The supernatant of BHK21 cells transfected with the full-length plasmid of pBAC-GD infectious clone was collected and transferred to MARC-145 cells. After 2 hours, the cell supernatant was replaced with DMEM medium containing 3% FBS. When typical CPE appeared, the cell supernatant was discarded, fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton-X-100 solution for 15 minutes, blocked with 1% BSA solution for 30 minutes, and incubated with the sample for 1 hour after the monoclonal antibody against PRRSV N protein was diluted 1:2500, and the goat anti-mouse antibody coupled to green fluorescent protein was diluted 1:2500 and incubated with the sample for 1 hour. The nuclear dye DAPI was diluted 1:5000 and incubated for 5 minutes, and the fluorescence was observed under a fluorescence microscope.

[0052] The results are as follows Figure 1 As shown, obvious green fluorescence was observed in both HP-PRRSV GD strain and rescued strain samples, indicating that the infectious clone pBAC-GD was successfully rescued, and the rescued strain was named rPRRSV.

[0053] Example 2 Construction and rescue of recombinant PRRSV strain rGD-SX-5U2 infectious clone

[0054] (1) Construction of the infectious clone of the recombinant PRRSV strain rGD-SX-5U2

[0055] The gene sequence shown in SEQ ID NO. 1-2 was synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0056] Using the gene described in SEQ ID NO.2 as a template, using the primers shown in SEQ ID NO.16-17 to amplify the fragment SX-B; using the pBAC-GD infectious clone full-length plasmid constructed in Example 1 as a template, using the primers shown in SEQ ID NO.18-19 to amplify the fragment GD-A; using the PmeⅠ and RsrⅡ restriction sites to linearize the pBAC-GD infectious clone full-length plasmid constructed in Example 1; using homologous recombination technology, the fragments GD-A and SX-B are simultaneously connected to the linearized pBAC-GD infectious clone full-length plasmid to obtain the pBAC-GD-ORF5-U (SX) infectious clone plasmid;

[0057] Using the gene described in SEQ ID NO.1 as a template, fragment SX-D was amplified using the primers shown in SEQ ID NOs.20-21; using the pBAC-GD infectious clone full-length plasmid constructed in Example 1 as a template, fragment GD-C was amplified using the primers shown in SEQ ID NOs.4 and 22; fragment GD-E was amplified using the primers shown in SEQ ID NOs.23-24; the above-mentioned pBAC-GD-ORF5-U (SX) infectious clone plasmid was linearized using two endonucleases, SfiⅠ and PmeⅠ; using homologous recombination technology, the above-mentioned fragments GD-C, SX-D and GD-E were simultaneously connected to the linearized pBAC-GD-ORF5-U (SX) infectious clone plasmid to obtain the recombinant infectious clone plasmid pBAC-GD-SX-5U2.

[0058] (2) Rescue of the infectious clone of the recombinant PRRSV strain rGD-SX-5U2

[0059] The same method as the rescue method of the HP-PRRSV GD strain infectious clone described in step (2) of Example 1 was used to rescue the recombinant infectious clone plasmid pBAC-GD-SX-5U2.

[0060] (3) IFA identification of the infectious clone of the recombinant PRRSV strain rGD-SX-5U2

[0061] The same identification method as described in step (3) of Example 1 was used to perform IFA identification of the recombinant infectious clone plasmid pBAC-GD-SX-5U2. Figure 3 As shown, obvious green fluorescence was visible, indicating that the recombinant infectious clone pBAC-GD-SX-5U2 prepared by the present invention was successfully rescued, and the rescued strain was named rGD-SX-5U2.

[0062] (4) Sequencing and Identification of Recombinant PRRSV Strain rGD-SX-5U2

[0063] To further verify the sequence authenticity of the rescued recombinant strain rGD-SX-5U2, the genomic RNA of the rescued strain rGD-SX-5U2 was extracted, and the chimeric position was sequenced after RT-PCR amplification and compared with the expected sequence of the constructed vector. Figure 2 As shown, it was shown that the sequence of the rescued recombinant strain rGD-SX-5U2 was consistent with expectations.

[0064] Example 3 Proliferation characteristics of recombinant PRRSV strain rGD-SX-5U2 on MARC-145 cells

[0065] (1) WB identification of MARC-145 cells infected with recombinant PRRSV rGD-SX-5U2

[0066] The recombinant PRRSV strain rGD-SX-5U2 rescued in Example 2 was transferred to MARC-145 cells and 48 hours later, cell samples were collected and identified by WB using the PRRSV-N specific monoclonal antibody 6D10. Figure 4 As shown, rGD-SX-5U2 and the positive control strain SD-YL1712 detected the target protein (14KDa) consistent with the expected size of N protein, while the NADC30-like PRRSV SX-YL1806 and the negative control sample did not detect the target band. This shows that the recombinant strain rGD-SX-5U2 was successfully rescued and has the infectivity to MARC-145 cells.

[0067] (2) Growth curve determination of recombinant PRRSV rGD-SX-5U2 on MARC-145 cells

[0068] The rescued recombinant strain rGD-SX-5U2 and the backbone strain rPRRSV rescued in Example 1 were inoculated into MARC-145 cells at an MOI of 0.01, and the growth curves were measured after sampling at different time points.

[0069] The results are as follows Figure 5 As shown in Figure 2, the viral load of the recombinant strain rGD-SX-5U2 in MARC-145 cells gradually increased over time, with a peak of 10 5.625 / mL, which appeared at 72hpi; before 72hpi, the replication rate of the rGD-SX-5U2 strain was slightly lower than that of the backbone strain rGD, but there was no significant difference between the two at 72hpi.

[0070] Example 4 Evaluation of the immune protection effect of the recombinant PRRSV strain rGD-SX-5U2

[0071] To further evaluate the protective effect of the recombinant PRRSV strain rGD-SX-5U2 against HP-PRRSV and NADC30-like PRRSV, an animal challenge protection experiment was designed as shown in Table 1, and various indicators were collected for analysis, where rGD-SX-5U2 was denoted as NSP2, HP-PRRSV SD-YL1712 was denoted as HP, and NADC30-like PRRSV SX-YL1806 was denoted as DC30.

[0072] Table 1 Animal challenge protection test

[0073]

[0074] (1)Body temperature

[0075] After the first immunization, the rectal temperature of piglets was measured daily to record the changes in body temperature.

[0076] The results are as follows Figure 6 As shown in the figure, after immunization with rGD-SX-5U2, the body temperature of piglets in the NSP2-HP and NSP2-DC30 groups increased transiently in a short period of time, with the NSP2-HP group showing an increase at 2dpi, 5dpi, 6dpi, 7dpi, 10dpi, etc., and the NSP2-DC30 group showing an increase at 6dpi, 7dpi, 9dpi, 10dpi, 11dpi, etc. After 16dpi, the body temperature of piglets in the NSP2-HP and NSP2-DC30 groups and the three unimmunized groups of piglets (NC-HP, NC-DC30 and NC) remained below 40°C, and no fever was observed. After being challenged with HP-PRRSV and NADC30-like PRRSV at 42dpi, the body temperatures of the two non-immunized direct challenge groups (NC-HP and NC-DC30) were significantly increased. The fever state of the piglets in the NC-HP group lasted for 11 days, with a peak temperature of 40.63°C at 53dpi; the fever state of the piglets in the NC-DC30 group lasted for 12 days, with a peak temperature of 40.9°C at 47dpi; after 58dpi, the body temperatures of the two groups of piglets gradually returned to normal. The body temperatures of the piglets in the two immunized challenge groups (NSP2-HP and NSP2-DC30) remained normal since the challenge at 42dpi, and no fever symptoms appeared. The body temperature of the piglets in the NC group showed no abnormalities throughout the whole process. It shows that the recombinant PRRSV strain rGD-SX-5U2 described in this application has a protective effect against HP-PRRSV and NADC30-like PRRSV.

[0077] (2) Virus titer in lung tissue

[0078] At the end of the experiment, all piglets were necropsied and the PRRSV viral load in the lung tissue of each group of piglets was measured.

[0079] The results are as follows Figure 7 As shown, PRRSV was detected in the lung tissues of all PRRSV challenged piglets, among which the NC-HP group and NC-DC30 group had higher viral loads in the lung tissues, while no PRRSV was detected in the negative control group. Compared with the NC-HP group, the viral load in the lung tissues of piglets in the NSP2-HP group was significantly reduced (P<0.001); similarly, compared with the NC-DC30 group, the viral load in the lung tissues of piglets in the NSP2-DC30 group was also significantly reduced (P<0.001), indicating that the recombinant PRRSV strain rGD-SX-5U2 described in this application has a protective effect on HP-PRRSV and NADC30-like PRRSV.

[0080] (3) Lung tissue damage

[0081] During autopsy, the lung condition was observed and lung tissues were collected for HE and IHC identification.

[0082] The results are as follows Figure 8 As shown, macroscopic lung tissue damage showed that the lungs of piglets in the non-immunized direct challenge group NC-HP and NC-DC30 groups showed obvious pathological characteristics, mainly including decreased lung elasticity, widened interstitial space, hard nodules and shrimp-like changes, etc., among which the NC-HP group was more serious than the NC-DC30 group. The lungs of piglets in the NSP2-HP group after immunization and challenge group showed significant improvement compared with the NC-HP group: lung elasticity was restored, hard nodules and shrimp-like changes were almost not seen, but the interstitial space was still slightly widened. The lungs of piglets in the NSP2-DC30 group after immunization and challenge group also showed significant improvement compared with the NC-DC30 group: lung elasticity was restored, hard nodules were not seen, but there was still a small amount of shrimp-like changes, the interstitial space was slightly widened, and there was no obvious abnormality in the NC group. The protective effect of the recombinant PRRSV strain rGD-SX-5U2 against HP-PRRSV and NADC30-like PRRSV.

[0083] In summary, the recombinant porcine reproductive and respiratory syndrome virus rGD-SX-5U2 prepared by the present invention can infect MARC-145 cells and has a higher virus titer; compared with HP-PRRSV GD, the rGD-SX-5U2 can not only provide good protection against the attack of HP-PRRSV, but also provide good protection against the attack of NADC30-like PRRSV, and can be used to prepare PRRSV diagnostic reagents or new PRRSV vaccines.

[0084] The disclosure of the above embodiments enables those skilled in the art to implement or use the present invention. The present invention will not be limited to the embodiments shown herein, but should be consistent with the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A recombinant porcine reproductive and respiratory syndrome virus, It is characterized in that The recombinant porcine reproductive and respiratory syndrome virus is a recombinant virus strain obtained by replacing the NSP2 gene sequence of the HP-PRRSV GD strain with the gene sequence shown in SEQ ID NO.1, and replacing the ORF5-7 and 3'UTR gene fragments with the gene sequence shown in SEQ ID NO.

2.

2. The recombinant porcine reproductive and respiratory syndrome virus according to claim 1, It is characterized in that The full-length cDNA sequence of the recombinant porcine reproductive and respiratory syndrome virus is shown in SEQ ID NO.

3.

3. Use of the recombinant porcine reproductive and respiratory syndrome virus as described in claim 1 or 2 in the preparation of a PRRSV diagnostic reagent or a PRRSV vaccine.

4. A method for preparing a recombinant porcine reproductive and respiratory syndrome virus, It is characterized in that The method comprises: Construction of infectious clone: ​​The NSP2 gene sequence of HP-PRRSV GD strain was replaced with the gene sequence shown in SEQ ID NO.1 by genetic engineering means, and the ORF5-7 and 3'UTR gene fragments were replaced with the gene sequence shown in SEQ ID NO.2, and then connected into the plasmid to obtain the infectious clone plasmid; Virus rescue: The obtained infectious clone plasmid is rescued to obtain recombinant porcine reproductive and respiratory syndrome virus.

5. The preparation method according to claim 4, It is characterized in that The method is: (1) Construction of a full-genome infectious clone plasmid of the HP-PRRSV GD strain; (2) replacing the NSP2 gene sequence in the infectious clone plasmid in step (1) with the gene sequence shown in SEQ ID NO.1, and replacing the ORF5-7 and 3'UTR gene fragments with the gene sequence shown in SEQ ID NO.2, to obtain a recombinant infectious clone plasmid; (3) Transfecting the recombinant infectious clone plasmid described in step (2) into BHK21 cells and then transferring to MARC-145 cells for rescue to obtain the recombinant porcine reproductive and respiratory syndrome virus.

6. The preparation method according to claim 5, It is characterized in that The step (1) is: Using the genomic cDNA of the HP-PRRSV GD strain as a template, fragment A2 was amplified using the primers shown in SEQ ID NO.4-5, and fragment B2 was amplified using the primers shown in SEQ ID NO.6-7. Fragments A2 and B2 were overlapped to obtain fragment A2+B2; fragment C2 was amplified using the primers shown in SEQ ID NO.8-9; fragment A2+B2 and fragment C2 were connected into the pBeloBac11 vector linearized with SfiⅠ and RsrⅡ restriction sites by homologous recombination technology to obtain pBAC-GD-SU plasmid; Using the HP-PRRSV GD strain genomic cDNA as a template, using the primers shown in SEQ ID NO.10-11 to amplify fragment D2; using the primers shown in SEQ ID NO.12-13 to amplify fragment E2, fragments D2 and E2 are overlapped to obtain fragment PU; using fragment PU as a template, using SEQ ID NO.10 and SEQ ID NO.14 to amplify GD-PU-A with a marker, using SEQ ID NO.15 and SEQ ID NO.13 to amplify GD-PU-B with a marker; GD-PU-A and GD-PU-B were simultaneously inserted into the pBAC-GD-SU plasmid linearized with PmeⅠHE RsrⅡ restriction sites by homologous recombination technology to construct the full genome infectious clone plasmid pBAC-GD of HP-PRRSV GD strain.

7. The preparation method according to claim 6, It is characterized in that The step (2) is: Using the gene fragment shown in SEQ ID NO.2 as a template, the primers shown in SEQ ID NO.16 and 17 were used to amplify the SX-B fragment; using the infectious clone plasmid pBAC-GD as a template, the primers shown in SEQ ID NO.18 and 19 were used to amplify the GD-A fragment; using homologous recombination technology, the fragments GD-A and SX-B were simultaneously connected to the infectious clone plasmid pBAC-GD linearized with PmeⅠ and RsrⅡ restriction sites to obtain the infectious clone plasmid pBAC-GD-ORF5-U (SX); Using the gene fragment shown in SEQ ID NO.1 as a template, the fragment SX-D was amplified using the primers shown in SEQ ID NOs.20 and 21; using the infectious clone plasmid pBAC-GD as a template, the GD-C fragment was amplified using the primers shown in SEQ ID NOs.4 and 22; the fragment GD-E was amplified using the primers shown in SEQ ID NOs.23 and 24; using homologous recombination technology, the fragments GD-C, SX-D and GD-E were simultaneously connected to the infectious clone plasmid pBAC-GD-ORF5-U (SX) linearized with SfiⅠ and PmeⅠ restriction sites to obtain the recombinant infectious clone plasmid pBAC-GD-SX-5U2.

8. The preparation method according to claim 7, It is characterized in that The step (3) is: The supernatant of BHK21 cells transfected with the recombinant infectious clone plasmid pBAC-GD-SX-5U2 was collected and transferred to MARC-145 cells. The cell supernatant was replaced with DMEM medium containing 3% FBS until typical CPE appeared to obtain the recombinant porcine reproductive and respiratory syndrome virus.

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