Construction and application of a reverse genetic manipulation platform for nadc34-like porcine reproductive and respiratory syndrome virus

Abstract

The present application relates to a virus of reverse genetic operation construction of NADC34-like PRRSV2. Specifically, the full gene sequence of NADC34-like PRRSV2 BJ1805-2 isolated strain is segmented and connected to the vector by using PCR amplification, enzyme digestion and other methods with pACY177 plasmid as the carrier, and the BJ1805-2 full-length infectious clone recombinant plasmid (rBJ1805-2) is obtained. The infectious clone virus rBJ1805-2 is rescued in vitro, and the first NADC34-like PRRSV2 strain infectious clone platform in China is successfully built. The present application also relates to a modification method for constructing NADC34-like PRRSV2 strain which can adapt to Marc-145 cell passage culture. The infectious clone virus constructed by the present application has good in vitro and in vivo proliferation efficiency, and can cause specific viremia after inoculation in pigs; it has good safety, does not cause fever after inoculation in pigs, and will not cause piglet death. The modified strain can be used as a candidate strain for developing the first NADC34-like PRRSV2 specific vaccine in China, and is conducive to the prevention and control of PRRSV epidemic in China.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, and in particular to a strain of NADC34-like porcine reproductive and respiratory syndrome virus infectious clone virus and a strain of modified virus with Marc-145 cell tropism edited and produced based on the strain. BACKGROUND

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that seriously endangers the healthy development of the pig industry. PRRSV infection can cause abortion in sows and respiratory syndrome in piglets. Recent studies have classified PRRSV into two species: PRRSV1 and PRRSV2. Among them, high pathogenic PRRSV2 (HP-PRRSV2), NADC30-like PRRSV2 and NADC34-like PRRSV2 are dominant.

[0003] PRRSV is a single-stranded positive-sense RNA virus belonging to the Arteriviridae family, with a genome size of about 15 kb, containing 10 open reading frames (ORFs). ORF1a and ORF1b genes encode at least 16 non-structural proteins, and ORF2-7 genes encode 8 structural proteins. Among them, the three small envelope proteins (GP2a, GP3 and GP4) encoded by ORF2-4 genes form a heterotrimer through non-covalent bonds and play a key role in PRRSV infection of target cells. Studies have shown that GP2a, GP3 and GP4 are key proteins that determine the cell tropism of PRRSV.

[0004] Vaccine immunization is the main means of preventing and controlling PRRS in China. However, the existing commercial PRRS attenuated vaccine has poor cross-protection effect, and a specific attenuated vaccine for NADC34-like PRRSV2 strain has not yet been developed. In addition, the NADC34-like PRRSV2 isolate does not have Marc-145 cell tropism, which is an important obstacle to the development of its specific vaccine.

[0005] Reverse genetic manipulation technology has become an important means for the development of new genetic engineering vaccines. However, there is currently no report on the construction of a NADC34-like PRRSV2 reverse genetic manipulation platform, and there is no report on the modification of Marc-145 cell tropism virus based on NADC34-like PRRSV2. SUMMARY

[0006] The present application is aimed at the problems existing in the prior art, and provides a construction method of a NADC34-like PRRSV2 infectious clone virus for a NADC34-like PRRSV2 strain that has appeared in China in recent years and is widely prevalent, and a NADC34-like PRRSV2 modified strain that can be adapted to Marc-145 cell in vitro passage culture is obtained on the basis thereof. The modified strain can be used as a candidate strain for the development of the first NADC34-like PRRSV2 specific vaccine in China.

[0007] The object of the present application is achieved by the following technical solutions.

[0008] A strain of NADC34-like porcine reproductive and respiratory syndrome virus, the virus is porcine reproductive and respiratory syndrome virus (PRRSV) rBJ1805-2; preserved in China Center for Type Culture Collection, located in Wuhan University in Wuhan, China, with a preservation number of CCTCC NO: V202250, and a preservation date of 2022.6.29.

[0009] A recombinant vector for constructing a NADC34-like porcine reproductive and respiratory syndrome virus reverse genetic manipulation platform, the recombinant vector is pACYC177-rBJ1805-2, the recombinant vector takes pACY177 plasmid as a carrier, wherein the full gene sequence of the NADC34-like PRRSV2 BJ1805-2 isolate is contained, and the full gene sequence is shown as SEQ ID NO: 1.

[0010] A construction method of a NADC34-like porcine reproductive and respiratory syndrome virus reverse genetic manipulation platform, the method is as follows:

[0011] RNA of the NADC34-like PRRSV2 isolate BJ1805-2 is extracted, and the viral RNA is reverse transcribed into cDNA using a reverse transcription kit; the full gene of the NADC34-like PRRSV2 isolate BJ1805-2 is amplified by designing primers and using a segment amplification method with the cDNA as a template, and then each fragment generated by amplification is sequenced, and finally the full gene sequence of the NADC34-like PRRSV2 BJ1805-2 isolate is obtained by splicing through sequence comparison software;

[0012] The full gene sequence of the NADC34-like PRRSV2 BJ1805-2 isolate is segmented and connected to the vector by using PCR amplification and enzyme digestion and ligation methods with pACY177 plasmid as the carrier, and a BJ1805-2 full-length infectious clone recombinant plasmid is obtained;

[0013] Then the NADC34-like porcine reproductive and respiratory syndrome virus is rescued by the method of infectious clone, and the virus obtained by the rescue is NADC34-like PRRSV2 infectious clone virus rBJ1805-2.

[0014] Preferably, in the method, the primer set for amplifying the full gene of the NADC34-like PRRSV2 isolate BJ1805-2 is as follows:

[0015]

[0016]

[0017] Preferably, in the method, the method for obtaining the BJ1805-2 full-length infectious clone recombinant plasmid is as follows:

[0018] (1) BJ1805-2 full gene fragment amplification

[0019] The BJ1805-2 full genome is divided into three fragments for amplification;

[0020] (2) BJ1805-2 fragment connection

[0021] The above amplification products are purified using a product purification kit, the purified products are double-digested with pACYC177 plasmid, and sequentially connected to the pACYC177 vector, to finally obtain the infectious clone plasmid pACYC177-rBJ1805-2 containing the full genome sequence.

[0022] Preferably, in the step (1) BJ1805-2 full gene fragment amplification, a PacI single enzyme digestion site is introduced at the 5' end of the BJ1805-2 genome, a hepatitis D virus (HDV Ribozyme) sequence is added at the 3' end, and the following primer set is used:

[0023]

[0024] Preferably, in the step (1) BJ1805-2 full gene fragment amplification, the following method is used:

[0025] First, the primers BJ1805-2-PacI-F1 and BJ1805-2-Bsu36I-R1; BJ1805-2-Bsu36I-F2 and BJ1805-2-XbaI-R2 are combined to amplify BJ1805-2-F1 and BJ1805-2-F2 fragments;

[0026] Secondly, the amplification of BJ1805-2-F3 needs two steps; first, using the primer pair of BJ1805-2-XbaI-F3 and BJ1805-2-1R3, F3-1 is obtained; then, using F3-1 as a template, the primer combination of BJ1805-2-XbaI-F3 and BJ1805-2-AscI-2R4, F3-2 is obtained.

[0027] Preferably, the method for connecting each fragment of BJ1805-2 in step (2) is as follows:

[0028] The connection method of BJ1805-2-F1: using PacI and Bsu36I to double enzyme cut pACYC177-New plasmid and BJ1805-2-F1 amplification product, the enzyme cutting system of pACYC177-New is: pACYC177-New plasmid 15 μL, PacI 3 μL, Bsu36I 3 μL, 10 × CutSmart Buffer 4 μL, RNase Free H2O is supplemented to 40 μL; the enzyme cutting system of BJ1805-2-F1 is: BJ1805-2-F1 purified product 10 μL, PacI 2 μL, Bsu36I 2 μL, 10 × CutSmart Buffer 4 μL, RNase Free H2O is supplemented to 40 μL; the reaction condition is: 37℃ water bath for 30 minutes;

[0029] The connection method of BJ1805-2-F2 and BJ1805-2-F3-2: same as the connection method of BJ1805-2-F1 above.

[0030] The application of the NADC34-like porcine reproductive and respiratory syndrome virus reverse genetic manipulation platform constructed by the method, the application is to use the reverse genetic manipulation platform to modify, construct and rescue NADC34-like PRRSV2 strain rBJ1805-2-ORF2-4-CON suitable for Marc-145 cell in vitro culture, and the specific method is as follows:

[0031] Based on the reverse genetic manipulation platform, the sequence of the small envelope protein coding gene ORF2-4-CON obtained by synthesis is used to replace the corresponding fragment in the NADC34-like PRRSV2 infectious clone virus rBJ1805-2 by using the homologous recombination method.

[0032] Preferably, the sequence of the small envelope protein coding gene ORF2-4-CON is as shown in SEQ ID NO: 38.

[0033] In one aspect of the present application, the full gene sequence of NADC34-like PRRSV2 isolate BJ1805-2 is provided. Specifically, the full gene of BJ1805-2 is amplified by designing primers for segmental amplification, and then each fragment amplified is sequenced respectively. Finally, the full gene sequence of NADC34-like PRRSV2 BJ1805-2 isolate is obtained by sequence alignment software splicing.

[0034] In another aspect of the present application, a virus of NADC34-like PRRSV2 constructed by reverse genetic manipulation is provided. Specifically, the full gene sequence of NADC34-like PRRSV2 BJ1805-2 isolate is segmented and connected to the vector by using PCR amplification, enzyme digestion and other methods, with pACY177 plasmid as the carrier, to obtain a full-length infectious clone recombinant plasmid (rBJ1805-2) of BJ1805-2. The infectious clone virus rBJ1805-2 (CCTCC NO: V202250; preservation date: 2022.6.29) is rescued in vitro, and a first NADC34-like PRRSV2 strain infectious clone platform in China is successfully built.

[0035] In another aspect of the present application, a method for constructing a modified NADC34-like PRRSV2 strain adapted to Marc-145 cell passage culture is provided. Specifically, the synthetic small envelope protein coding gene ORF2-4-CON consensus sequence is replaced with the corresponding fragment in the infectious clone virus rBJ1805-2 of NADC34-like PRRSV2 by using homologous recombination and other methods, based on the reverse genetic manipulation platform (rBJ1805-2) of NADC34-like PRRSV2 in the present application. The modified NADC34-like PRRSV2 strain (rBJ1805-2-ORF2-4-CON) adapted to Marc-145 cells is constructed and rescued.

[0036] The present application has the following technical effects: 1. A NADC34-like PRRSV2 wild strain which has been prevalent in China in recent years is isolated, and the whole gene sequence thereof is determined; 2. The NADC34-like PRRSV2 isolated strain is used as a parent virus to construct a reverse genetic operation platform; 3. The constructed infectious cloned virus has good in-vivo and in-vitro proliferation efficiency, and can cause specific viremia after inoculation in pigs; 4. The constructed infectious cloned virus has good safety, and does not cause fever (in-vitro <40℃), and even less causes death of piglets after inoculation in pigs; 5. A NADC34-like PRRSV2 modified strain which can adapt to Marc-145 cells is obtained based on the NADC34-like PRRSV2 reverse genetic operation platform; 6. The constructed modified virus can cause cytopathic effect (CPE) after inoculation in Marc-145 passage cells; 7. The constructed reverse genetic operation platform and the modified virus which can adapt to Marc-145 cells can be used for developing the first NADC34-like PRRSV2 live vaccine in China, and are beneficial to the prevention and control of PRRSV epidemic in China. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a schematic diagram of the construction strategy of the rBJ1805-2 infectious clone in Example 1;

[0038] Figure 2 is the PCR amplification result of the three fragments of the genome of BJ1805-2 in Example 1;

[0039] Figure 3 is the indirect immunofluorescence identification result of BJ1805-2 and rBJ1805-2 detected by the indirect immunofluorescence method using the monoclonal antibody 6A1 against PRRSV-N protein in Example 1;

[0040] Figure 4 is the dynamic proliferation result of BJ1805-2 and its infectious cloned virus rBJ1805-2 in primary PAM cells detected by the fluorescence quantitative PCR method in Example 1;

[0041] Figure 5 is the viremia produced by BJ1805-2 and its infectious cloned virus rBJ1805-2 in pigs in Example 1;

[0042] Figure 6 is the body temperature dynamic change of the pigs inoculated with BJ1805-2 and its infectious cloned virus rBJ1805-2 in Example 1;

[0043] Figure 7 is the body weight change of the pigs inoculated with BJ1805-2 and its infectious cloned virus rBJ1805-2 in Example 1;

[0044] Figure 8 is a schematic diagram of the construction strategy of rBJ1805-2-ORF2-4-CON chimeric virus in Example 2;

[0045] Figure 9 is the result of indirect immunofluorescence detection of rBJ1805-2-ORF2-4-CON chimeric virus replicating on Marc-145 cells using monoclonal antibody 6A1 against PRRSV-N protein at 2dpi, 5dpi, 8dpi after rescue on Marc-145 cells in Example 2;

[0046] Figure 10 is the dynamic growth result of rBJ1805-2-ORF2-4-CON chimeric virus on Marc-145 cells in Example 2;

[0047] Figure 11 is the plaque result of rBJ1805-2-ORF2-4-CON chimeric virus produced on Marc-145 cells in Example 2.

[0048] Deposit information

[0049] Porcine reproductive and respiratory syndrome virus (PRRSV) rBJ1805-2, deposited in China Center for Type Culture Collection, located in Wuhan University, Wuhan, China, with the accession number CCTCC NO:V202250, and the deposit date is June 29, 2022. DETAILED DESCRIPTION

[0050] The following routine experimental methods in the examples refer to Sambrook et al. Molecular Cloning: A Laboratory Manual, 3rd ed. (Beijing: Science Press, 2002), and the use of instruments refers to the instrument operation manual.

[0051] In the examples of the present application, the virus is the BJ1805-2 isolate. The cells are the BHK-21 cell line, the Marc-145 cell line and the porcine primary alveolar macrophage PAM.

[0052] In the examples of the present application, the plasmids and strains: the pACYC177 plasmid is purchased from Yubao Biological, the Trans1-T1 competent cells are purchased from Beijing Quanshijin Biological Co., Ltd., and the pUC57-Synthesis-ORF2-4-CON is synthesized in Suzhou Jinweizhi Company.

[0053] In the embodiments of the present application, other reagents used: RNase Free H2O, trypsin cell digestion solution (phenol red) were purchased from Solarbio Company; TRIpure Reagent total RNA extraction reagent was purchased from Aidley Biological Company; PrimeScript 1stStrand cDNA Synthesis Kit, 2xPrimeSTAR MAX DNA Polymearse were purchased from TAKARA Company; FastPure Plasmid Mini Kit was purchased from Novozyme Biological Technology Co., Ltd.; homologous recombination reagent was purchased from Novozyme Biological Technology Co., Ltd.; DMEM culture medium was purchased from HyClone Biological Chemical Products Co., Ltd.; fetal bovine serum was purchased from Sigma Company; DyLight 594, Goat anti-Mouse IgG (H+L) Secondary Antibody was purchased from Invitrogen; DNA Marker was purchased from Zhejiang Bolijin Technology Co., Ltd.; 2xBioGold Tag Plus PCR MasterMix was purchased from Zhejiang Bolijin Technology Co., Ltd.; Gel Extraction Kit was purchased from Beijing Kangwei Century Biological Technology Co., Ltd.; DNA restriction endonuclease was purchased from Thermo Fisher Company; T4 DNA Ligase, Lipofectamine TM 3000Transfection Reagent was purchased from Invitrogen.

[0054] Example 1 Construction of reverse genetic manipulation platform of NADC34-like PRRSV2 BJ1805-2 strain

[0055] 1.1, Whole genome sequencing of NADC34-like PRRSV2 BJ1805-2

[0056] First, 200 μl of cell supernatant was used for total RNA extraction using TRIpure Reagent (Aidley), and the extracted total RNA was stored at -20℃ or long-term stored at -80℃. The viral RNA was reverse transcribed into cDNA using the reverse transcription kit (PrimeScriptTM1ststrand cDNA Synthesis Kit, TAKARA), and the obtained cDNA was stored at -20℃ for standby. The reaction system and procedure are shown in Table 1 and Table 2.

[0057] Table 1, BJ1805-2 total RNA pretreatment system and procedure

[0058]

[0059] Table 2. BJ1805-2 total RNA reverse transcription system and procedure

[0060]

[0061] The BJ1805-2 genome was amplified in segments using PCR amplification. First, using BJ1805-2 cDNA as a template, the full BJ1805-2 genome sequence was amplified using a combination of universal primers for PRRSV full-genome amplification (Table 3, numbers 2-21) and some BJ1805-2-specific primers (Table 3, numbers 22-30). Specific amplification primer information is shown in Table 3.

[0062] Table 3. Primers for PRRSV full gene amplification

[0063]

[0064]

[0065] The system and procedure of PCR amplification are shown in Tables 4 and 5.

[0066] Table 4. Reaction system

[0067]

[0068] Table 5. Reaction Procedure

[0069]

[0070] The amplified PCR products were then electrophoresed on a 1% gel and sequenced using the Sanger sequencing service of GeneWeichi. The sequences of the individual fragments were then spliced ​​together to obtain the complete genome sequence of BJ1805-2 (SEQ ID NO: 1).

[0071] 1.2. Primer design for reverse genetics platform construction

[0072] according to Figure 1 Following the construction strategy shown, primers for the three BJ1805-2 fragments were designed using Primer 5.0. A PacI restriction site was introduced at the 5' end of the BJ1805-2 genome, and a hepatitis D virus (HDV) Ribozyme sequence was added to the 3' end. Detailed primer information for the BJ1805-2 infectious clone construction is shown in Table 6.

[0073] Table 6. Primers for constructing the BJ1805-2 infectious clone

[0074]

[0075]

[0076] 1.3 Construction of BJ1805-2 infectious clone

[0077] 1.3.1. Segmental amplification of the BJ1805-2 full gene fragment

[0078] like Figure 1 As shown, the whole genome of BJ1805-2 was divided into three fragments for amplification. First, the primers BJ1805-2-PacI-F1 and BJ1805-2-Bsu36I-R1; BJ1805-2-Bsu36I-F2 and BJ1805-2-XbaI-R2 in Table 6 were combined to amplify the BJ1805-2-F1 and BJ1805-2-F2 fragments. Secondly, the amplification of BJ1805-2-F3 needs to be carried out in two steps. First, the BJ1805-2-XbaI-F3 and BJ1805-2-1R3 primer pairs were used to amplify F3-1. Then, using F3-1 as a template, the BJ1805-2-XbaI-F3 and BJ1805-2-AscI-2R4 primer combinations were used to amplify F3-2. The specific reaction system and reaction procedure are shown in Tables 7 and 8.

[0079] Table 7. Reaction system

[0080]

[0081] Table 8. Reaction Procedure

[0082]

[0083] After the PCR reaction is completed, take 2 μL of the reaction product and perform electrophoresis on a 0.9% agarose gel. Figure 2 As shown, the obtained gene fragments F1 were 5127 bp in size, F2 were 4146 bp in size, and F3-2 were 6061 bp in size.

[0084] 1.3.2. Connection of BJ1805-2 fragments

[0085] The amplified product was purified using a product purification kit. The purified product was double-digested with the pACYC177 plasmid and subsequently ligated into the pACYC177 vector. The construction strategy is as follows: Figure 1 The specific steps are as follows:

[0086] Firstly, the pACYC177-New plasmid and the BJ1805-2-F1 amplified product were double enzyme cut by PacI and Bsu36I. The enzyme cutting system of pACYC177-New was as follows: pACYC177-New plasmid (200 ng / μL) 15 μL, PacI 3 μL, Bsu36I 3 μL, 10 × CutSmart Buffer 4 μL, RNase Free H2O supplemented to 40 μL. The enzyme cutting system of BJ1805-2-F1 was as follows: BJ1805-2-F1 purified product (200 ng / μL) 10 μL, PacI 2 μL, Bsu36I 2 μL, 10 × CutSmart Buffer 4 μL, RNase Free H2O supplemented to 40 μL. The reaction condition was 37 °C water bath for 30 min. The specific information is shown in Table 9 and Table 10.

[0087] Table 9, double enzyme cutting system of pACYC177 plasmid

[0088] Name pACYC177 plasmid Pac I Bsu36 I 10x CutSmart Buffer RNase Free H2O Dose 3000 ng 3 μL 3 μL 4 μL To 40 μL

[0089] Table 10, double enzyme cutting system of BJ1805-2-F1 fragment

[0090] Name Anheal2-F1 fragment Pac I Bsu36 I 10x CutSmart Buffer RNase Free H2O Dose 2000 ng 2 μL 2 μL 4 μL To 40 μL

[0091] The enzyme cutting bands were separated by agarose gel electrophoresis, and the target bands were cut for gel recovery. The linear carrier pACYC177-New and the enzyme cutting product BJ1805-2-F1 were connected by T4 DNA ligase at a molar ratio of 1:8, and the Trans1-T1 competent cells were transformed. Independent colonies were picked and cultured for PCR detection using detection primers. The PCR positive bacteria were selected for overnight culture, and the plasmid DNA was extracted and identified by double enzyme cutting with PacI and Bsu36I. The correct size of the plasmid was selected by 1% agarose gel electrophoresis, and then sequencing verification was performed. The next fragment connection was continued. The connection method of F2 and F3-2 fragments was similar to that of F1, and the connection was performed according to the construction strategy shown in Table 11. Figure 1 The infectious clone plasmid pACYC177-rBJ1805-2 containing the whole genome sequence was finally obtained.

[0092] 1.4, rescue of rBJ1805-2 infectious clone

[0093] The BHK-21 cells were inoculated in a 12-well cell culture plate at a density of 5 × 10 5 cells / well in DMEM medium containing 10% FBS, and cultured in a 37 °C, 5% CO2 incubator until the cell density reached about 90%. The Lipofectamine 2000 transfection reagent was used to transfect the rBJ1805-2 infectious clone plasmid into the BHK-21 cells according to the manufacturer's instructions. TM3000Transfection Reagent instructions for cell transfection are as follows:

[0094] First, configure the plasmid premix solution in an EP tube: infectious clone plasmid pACYC177-rBJ1805-2 20 μg, Lip3000 4 μL, Optin-MEM 50 μL; configure the Lip3000 premix solution in another EP tube: Lip3000 3 μL, Optin-MEM 50 μL; finally, mix the premix solutions obtained in the above two steps, stand at room temperature for 15 min, and then add the cells to be transfected. 36-48 h after cell transfection, seal the 12-well plate with a sealing bag and freeze-thaw twice at -80°C for standby. The specific information is shown in Table 11.

[0095] Table 11, transfection system

[0096] Category \ Name Plasmid Lip3000 P3000 DMEM Plasmid premix 2000 ng - 3 μL 50 μL Lip3000 premix - 4 μL - 50 μL

[0097] Use 2 mL of RPMI (1640) medium containing 2% FBS to inoculate 2 x 10 5 cells / well of primary PAM cells into a 12-well cell culture plate in a 37°C, 5% CO2 incubator. Take 500 μL of BHK-21 cell transfection supernatant and overlay it on the primary PAM cells. Incubate for 2 h, then discard the supernatant and add RPMI (1640) medium containing 2% FBS for continued culture. In addition, infect primary PAM cells with BJ1805-2 prototoxin as a positive control. After 3-4 days of culture, collect the cell supernatant and leave the cells for IFA identification. As shown in Figure 3 , PRRSV anti-N protein monoclonal antibody 6A1 detection, specific red fluorescence was observed for rescued virus (rBJ1805-2) and parent virus (BJ1805-2), and no specific fluorescence was observed in the negative control group, confirming that the rBJ1805-2 infectious clone virus was successfully rescued on primary PAM cells.

[0098] 1.5, rBJ1805-2 and BJ1805-2 proliferation activity assay

[0099] Use 2 mL of RPMI (1640) medium containing 2% FBS to inoculate 2 x 10 5rBJ1805-2 and BJ1805-2 viruses were inoculated into 4 pigs at 6 weeks of age, respectively. The pigs were inoculated with 2 mL of rBJ1805-2 virus containing 10 5 TCID 50 of rBJ1805-2 virus, and 2 mL of BJ1805-2 virus containing 10 5 TCID 50 of BJ1805-2 virus. Another 2 pigs were inoculated with 2 mL of DMEM medium. The serum samples were collected at 3, 5, 7, 10, 12, 14 dpi (day post infection) and the viral load was detected by qRT-PCR. The body temperature of the pigs was monitored daily and the body weight of the pigs was monitored weekly. As shown in Table 14, the rBJ1805-2 and BJ1805-2 viruses can proliferate in the pigs to produce viremia; as shown in Table 15, the rBJ1805-2 and BJ1805-2 viruses do not cause the body temperature to rise; and as shown in Table 16, the rBJ1805-2 and BJ1805-2 viruses do not cause the body weight to decrease.

[0100] Table 12, reaction system

[0101] Name cDNA 2x EX Taq Primer (10 μM) Probe (10 μM) RNase Free H2O Dose 1 μL 20 μL 0.5 μL 0.4 μL To 20 μL

[0102] Table 13, reaction procedure

[0103]

[0104] The dynamic proliferation of the rBJ1805-2 infectious clone virus in the primary PAM cells is shown in Table 17, and the growth curve of the virus is similar to that of the parent virus BJ1805-2. Figure 4

[0105] 1.6, pig inoculation test of rBJ1805-2 and BJ1805-2

[0106] Ten pigs negative for PRRSV, PRV (pseudorabies virus), PCV (porcine circovirus), PEDV (porcine epidemic diarrhea virus) and other important pathogens were selected, and 4 pigs were inoculated with 2 mL of rBJ1805-2 virus containing 10 5 TCID 50 of rBJ1805-2 virus, and 4 pigs were inoculated with 2 mL of BJ1805-2 virus containing 10 5 TCID 50 of BJ1805-2 virus. Another 2 pigs were inoculated with 2 mL of DMEM medium. The serum samples were collected at 3, 5, 7, 10, 12, 14 dpi (day post infection) and the viral load was detected by qRT-PCR. The body temperature of the pigs was monitored daily and the body weight of the pigs was monitored weekly. As shown in Table 14, the rBJ1805-2 and BJ1805-2 viruses can proliferate in the pigs to produce viremia; as shown in Table 15, the rBJ1805-2 and BJ1805-2 viruses do not cause the body temperature to rise; and as shown in Table 16, the rBJ1805-2 and BJ1805-2 viruses do not cause the body weight to decrease. Figure 5 Figure 6 Figure 7As shown in the results, pigs inoculated with rBJ1805-2 and BJ1805-2 viruses experienced slight weight loss, but did not cause obvious clinical symptoms or death, preliminarily indicating that both the infectious cloned virus and the parental virus are attenuated strains with good safety.

[0107] Example 2: Transformation and construction of NADC34-like PRRSV2 that can be adapted to Marc-145 cell culture in vitro

[0108] Build and rescue

[0109] 2.1. Synthesis of pUC57-Synthesis-ORF2-4CON plasmid

[0110] Thirty representative Chinese PRRSV isolates were collected from GenBank.

[0111] The ORF2-4 gene sequences encoding envelope proteins were aligned using DNAMAN 6.0. A consensus ORF2-4 sequence (ORF2-4-CON) was generated using DNAMAN 6.0. The ORF2-4-CON sequence was compared with the corresponding region of the JSTZ1712-12 strain (MK906026), and frameshift mutations were manually corrected to ensure correct expression of all envelope proteins. The ORF2-4-CON sequence was synthesized by GENEWIZ, Suzhou, China. The ORF2-4-CON sequence is shown in SEQ ID NO: 38.

[0112] 2.2 Primer Design

[0113] According to Figure 8 The construction strategy shown was to design primers for amplifying ORF2-4-CON, the front segment of BJ1805-2-F3, and the back segment of BJ1805-2-F3 using Primer 5.0, with at least 20 bp of homology arms between the primers.

[0114] The specific information of the primers is shown in Table 14.

[0115] Table 14. Primers for the construction of rBJ1805-2-ORF2-4-CON

[0116]

[0117]

[0118] 2.3 Construction and rescue of the modified virus rBJ1805-2-ORF2-4-CON

[0119] 2.3.1、ORF2-4-CON, rBJ1805-2-F3 front and rBJ1805-2-F3 rear amplification The primers rBJ1805-2-ORF2-CON-F and rBJ1805-2-ORF4-CON-R; rBJ1805-2-XbaI-F3 and rBJ1805-2-ORF2-CON-R; rBJ1805-2-ORF4-CON-F and JXA1-RNOTI2fu-1 in Table 14 were combined into primer pairs respectively for amplification of ORF2-4-CON, rBJ1805-2-F3 front and rBJ1805-2-F3 rear respectively. The specific procedures of the reaction are shown in Table 15 and Table 16.

[0120] Table 15, reaction system

[0121]

[0122] Table 16, reaction procedure

[0123]

[0124] After the PCR reaction was completed, 2 μL of the reaction product was taken and electrophoresed using a 1% agarose gel. The obtained fragment ORF2-4-CON was 2624 bp in size, the fragment rBJ1805-2-F3 front was 1748 bp in size, and the fragment rBJ1805-2-F3 rear was 1802 bp in size.

[0125] 2.3.2, rBJ1805-2-ORF2-4-CON each fragment homologous recombination

[0126] pACYC177-BJ1805-2 was double digested with Xba I and Asc I to obtain the pACYC177-BJ1805-2-F1+F2 linearized vector. The specific operation method was similar to that in Example 1. For specific information, see Table 17.

[0127] Table 17, pACYC177-BJ1805-2 plasmid double digestion system

[0128]

[0129] The digested bands were separated by agarose gel electrophoresis, and the target bands were cut off for gel recovery. After gel recovery, the concentration was determined using NanoDrop for standby. The ORF2-4-CON, BJ1805-2-F3 front and BJ1805-2-F3 rear were connected to the pACYC177-BJ1805-2-F1+F2 linearized vector using the homologous recombination method. The specific operation of homologous recombination is as follows:

[0130] The homologous recombination system is: 52 ng ORF2-4-CON, 34 ng BJ1805-2-F3 front section, and 36 ng BJ1805-2-F3 rear section, 200 ng pACYC177-BJ1805-2-F1+F2 linearized intermediate vector, 4 μl 5×CE MultiS Buffer, and 2 μl Exanse MultiS. The reaction condition is 37°C for 30 min. See Table 18 for details.

[0131] Table 18, homologous recombination system

[0132]

[0133] The homologous recombination product is transformed into Trans1-T1 competent cells, and independent colonies are picked for pure culture, and bacterial liquid PCR detection is performed using detection primers. The PCR positive bacteria are selected for overnight culture, plasmid DNA is extracted, double enzyme digestion is performed using Xba I and Asc I, 0.9% agarose gel electrophoresis is performed, and the plasmid with correct enzyme digestion size is reserved.

[0134] According to the above method, the construction and identification of the rBJ1805-2-ORF2-4-CON modified virus are completed.

[0135] 2.3.3, rescue of rBJ1805-2-ORF2-4-CON modified virus

[0136] The BHK21 cells are inoculated in a 24-well cell culture plate at a density of 2.5×10 5 cells / well using DMEM medium containing 10% FBS, and are cultured in a 37°C, 5% CO2 incubator until the cell density reaches about 80%. Cell transfection is performed according to the Lipofectamine TM 3000 Transfection Reagent instructions. 36-48 h after cell transfection, the 12-well plate is sealed and frozen at -80°C, and after repeated freezing and thawing twice, the entire cell suspension is taken, centrifuged at 10,000×g for 4 min, and the supernatant is collected. The transfection method is similar to that of Example 1. See Table 19 for details.

[0137] Table 19, transfection system

[0138] Category \ Name Plasmid Lip3000 P3000 DMEM Plasmid premix 2000 ng - 3 μL 50 μL Lip3000 premix - 4 μL - 50 μL

[0139] The Marc-145 cells are inoculated in a 12-well cell culture plate at a density of 2×10 5 cells / well using DMEM medium containing 10% FBS, and are cultured in a 37°C, 5% CO2 incubator until the cell density reaches about 80%, and then DMEM medium containing 2% FBS is used.

[0140] Take all supernatant to Marc-145 cells, continue to culture. At the same time, rBJ1805-2 and BJ1805-2 rescued successfully in Example 1 are inoculated into Marc-145 cells, and the cell condition is observed every day. As shown in Figure 9 , 120hpi after Marc-145 cells are inoculated with the modified virus rBJ1805-2-ORF2-4-CON, CPE phenomena such as cell shrinkage and aggregation can be observed, while rBJ1805-2 and BJ1805-2 cannot infect Marc-145 cells. The obtained rBJ1805-2-ORF2-4-CON is continuously passaged, and the fourth generation of the modified virus is inoculated into Marc-145, and 144hpi is detected using PRRSV anti-N protein monoclonal antibody 6A1, as shown in Figure 9 , specific red fluorescence of the N protein of the modified virus can be observed, and no fluorescence is produced in the negative control group, proving that the virus is successfully rescued.

[0141] 2.4, rBJ1805-2-ORF2-4-CON replication characteristics on Marc-145 cells

[0142] Marc-145 cells are inoculated into 24-well cell culture plates at a density of 1×10 5 cells / well using DMEM medium containing 10% FBS, and are cultured in a 37°C, 5% CO2 incubator until the cell density reaches about 80%, and then DMEM medium containing 2% FBS is used.

[0143] The fourth generation of rBJ1805-2-ORF2-4-CON virus liquid is inoculated into Marc-145 cells in a 6-well plate. Six time points of 24hpi, 48hpi, 72hpi, 96hpi, 120hpi and 144hpi are taken for multi-step growth curve determination. The specific operation method is the same as in Example 1. The multi-step growth curve determination results are shown in Figure 10 , rBJ1805-2-ORF2-4CON successfully replicates on Marc-145 cells.

[0144] 2.5, rBJ1805-2-ORF2-4-CON plaque test

[0145] Marc-145 cells are inoculated into 6-well cell culture plates at a density of 3×10 5 cells / well using DMEM medium containing 10% FBS, and are cultured in a 37°C, 5% CO2 incubator until the cell density reaches about 80%, and then DMEM medium containing 2% FBS is used. The cell density is observed the next day, and the next step is performed when the cells reach a dense state.

[0146] The 4th passage virus stocks of BJ1805-2, rBJ1805-2 and rBJ1805-2-ORF2-4-CON were used to infect Marc-145 cells for 1.5 hours. After 1.5 hours, the virus solution was discarded and the cells were gently washed 1 to 2 times with PBS and then overlaid with 0.7% low-melt agarose medium (30 mL medium formula: 15 mL DMEM + 600 uL FBS + 300 uL penicillin-streptomycin + 15 mL 1.4% low-melt agarose solution) until the medium cooled to about 37°C. The 6-well plates were then inverted and incubated for 6 days. After the cytopathic effect was observed, the cells were fixed with 4% paraformaldehyde for 1 hour. After the cells were fixed, they were stained with crystal violet for 1 hour. After the staining was complete, the cells were washed with water and observed. The results of the plaque assay are shown in Figure 11 Figure 6, where rBJ1805-2-ORF2-4-CON was able to form plaques on Marc-145 cells, while rBJ1805-2 and BJ1805-2 were not able to produce plaques.

Claims

1. A NADC34-like porcine reproductive and respiratory syndrome virus, characterized in that: The virus is porcine reproductive and respiratory syndrome virus (PRRSV) rBJ1805-2; it is deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University in Wuhan, China, the deposit number is CCTCC NO: V202250, and the deposit date is 2022.6.

29.

2. The NADC34-like porcine reproductive and respiratory syndrome virus according to claim 1, characterized in that: The RNA of the NADC34-like PRRSV2 isolate BJ1805-2 was extracted, and the viral RNA was reverse transcribed into cDNA using a reverse transcription kit; the cDNA was used as a template, and the whole gene of the NADC34-like PRRSV2 isolate BJ1805-2 was amplified by designing primers for segmented amplification, and each amplified fragment was sequenced separately, and finally the whole gene sequence of the NADC34-like PRRSV2 BJ1805-2 isolate was obtained by splicing using sequence alignment software, and the whole gene sequence was shown in SEQ ID NO: 1; using the pACYC177 plasmid as a vector, the whole gene sequence of the NADC34-like PRRSV2 BJ1805-2 isolate was segmented and connected to the vector by PCR amplification and enzyme digestion and ligation methods to obtain the BJ1805-2 full-length infectious clone recombinant plasmid; then the NADC34-like porcine reproductive and respiratory syndrome virus was rescued by the infectious cloning method, and the rescued virus was NADC34-like PRRSV2 infectious clone virus rBJ1805-2.

3. The NADC34-like porcine reproductive and respiratory syndrome virus according to claim 2, characterized in that: The primer set used to amplify the full genome of the NADC34-like PRRSV2 isolate BJ1805-2 is as follows: 。 4. The NADC34-like porcine reproductive and respiratory syndrome virus according to claim 2, characterized in that: The method for obtaining the BJ1805-2 full-length infectious clone recombinant plasmid is as follows: (1) Segmental amplification of the entire BJ1805-2 gene The whole genome of BJ1805-2 was divided into three fragments for amplification; (2) Connection of BJ1805-2 fragments The amplified product was purified using a product purification kit, and the purified product was double-digested with the pACYC177 plasmid and sequentially ligated into the pACYC177 vector to finally obtain the infectious clone plasmid pACYC177-rBJ1805-2 containing the full genome sequence.

5. The NADC34-like porcine reproductive and respiratory syndrome virus according to claim 4, characterized in that: In the step (1) of segmented amplification of the BJ1805-2 full gene fragment, a PacI single restriction site was introduced at the 5' end of the BJ1805-2 genome, and a hepatitis D virus HDV Ribozyme sequence was added at the 3' end, and the following primer set was used: 。 6. Use of the NADC34-like porcine reproductive and respiratory syndrome virus according to any one of claims 1 to 5 in constructing and rescuing the NADC34-like PRRSV2 strain rBJ1805-2-ORF2-4-CON suitable for in vitro culture of Marc-145 cells, characterized in that: The synthesized small envelope protein encoding gene ORF2-4-CON sequence was replaced with the corresponding ORF2-4 gene fragment in the NADC34-like PRRSV2 infectious clone virus rBJ1805-2 by homologous recombination; the small envelope protein encoding gene ORF2-4-CON sequence is shown in SEQ ID NO: 38.

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