CN115992100A - Construction and application of NADC34-like porcine reproductive and respiratory syndrome virus reverse genetic operation platform - Google Patents

Abstract

The invention relates to a virus constructed by reverse genetic manipulation of NADC34-like PRRSV2. Specifically, pACY177 plasmid is used as a carrier, and the whole gene sequence of the NADC34-like PRRSV2BJ1805-2 isolate is connected to the carrier in a segmented way by using methods of PCR amplification, enzyme digestion connection and the like to obtain the BJ1805-2 full-length infectious clone recombinant plasmid (rBJ 1805-2). The infectious clone virus rBJ1805-2 is saved in vitro, and the first NADC34-like PRRSV2 strain infectious clone platform in China is successfully built. The invention also relates to a transformation method for constructing the NADC34-like PRRSV2 strain which can adapt to Marc-145 cell subculture. The constructed infectious clone virus has good in-vitro and in-vivo proliferation efficacy, and can cause specific viremia after being inoculated into a pig body; has good safety, does not cause fever after the pig body is inoculated, and does not cause death of piglets. The modified strain can be used as a candidate strain for developing a first NADC34-like PRRSV2 specific vaccine in China, and is beneficial to the prevention and control of PRRSV epidemic situation in China.

Description

Construction and application of NADC34-like porcine reproductive and respiratory syndrome virus reverse genetic operation platform

Technical Field

The invention relates to the technical field of bioengineering, in particular to a NADC34-like porcine reproductive and respiratory syndrome virus infectious clone virus and an engineered virus with Marc-145 cell tropism based on the strain editing.

Background

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is an important pathogen that severely jeopardizes the healthy development of the pig industry in China. PRRSV infection can cause abortion in sows and respiratory syndrome in piglets. Recent studies split PRRSV into two species (species): PRRSV1 and PRRSV2. The main epidemic of China is PRRSV2, wherein, the high pathogenicity PRRSV2 (HP-PRRSV 2), NADC30-like PRRSV2 and NADC34-like PRRSV2 are mainly used. Among them, NADC34-like PRRSV2 strain in China was reported earlier than 2017 and spread epidemic all over the country rapidly. The strain has strong pig adaptability and large virulence change, and causes great economic loss to pig industry in China.

PRRSV is a single-stranded positive strand RNA virus belonging to the family arterividae, whose genome is approximately 15kb in size, comprising 10 Open Reading Frames (ORFs). The ORF1a and ORF1b genes encode at least 16 nonstructural proteins and the ORF2-7 gene encodes 8 structural proteins. Among them, three kinds of small envelope proteins (GP 2a, GP3 and GP 4) encoded by ORF2-4 genes play a key role in PRRSV infection of target cells by forming heterotrimers through non-covalent bonds. Studies have shown that GP2a, GP3 and GP4 are key proteins that determine PRRSV cell tropism.

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

Reverse genetic manipulation techniques have become an important tool in the development of novel genetically engineered vaccines. However, no report exists on construction of a reverse genetic operation platform of NADC34-like PRRSV2, and no report on obtaining Marc-145 cell tropic virus based on NADC34-like PRRSV2 modification exists.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention provides a construction method of NADC34-like PRRSV2 infectious clone virus, and simultaneously obtains a NADC34-like PRRSV2 modified strain which can adapt to Marc-145 cell in-vitro subculture, aiming at NADC34-like PRRSV2 strain which appears in recent years and is widely popular in China. The modified strain can be used as a candidate strain for developing a first NADC34-like PRRSV2 specific vaccine in China.

The aim of the invention is realized by the following technical scheme:

a strain of NADC34-like Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) rBJ1805-2; the special culture medium is preserved in China center for type culture Collection, the preservation address is the university of Wuhan in Wuhan, the preservation number is CCTCC NO: V202250, and the preservation date is 2022.6.29.

The recombinant vector is pACYC177-rBJ1805-2, takes pACY177 plasmid as a vector, and contains the whole gene sequence of NADC34-like PRRSV2BJ1805-2 isolate, wherein the whole gene sequence is shown in SEQ ID NO. 1.

A construction method of a reverse genetic operation platform of NADC34-like porcine reproductive and respiratory syndrome virus comprises the following steps:

extracting RNA of NADC34-like PRRSV2 isolate BJ1805-2, and reversely transcribing the viral RNA into cDNA by using a reverse transcription kit; amplifying NADC34-like PRRSV2 isolate BJ1805-2 whole genes by taking the cDNA as a template and designing a primer segmentation amplification method, sequencing each amplified fragment respectively, and finally splicing by sequence comparison software to obtain the whole gene sequence of the NADC34-like PRRSV2BJ1805-2 isolate;

the pACY177 plasmid is used as a carrier, and a PCR amplification and enzyme digestion connection method is utilized to connect the complete gene sequence of the NADC34-like PRRSV2BJ1805-2 isolate to the carrier in a segmented manner, so as to obtain a BJ1805-2 full-length infectious clone recombinant plasmid;

the NADC34-like porcine reproductive and respiratory syndrome virus is then rescued by an infectious cloning method, and the virus obtained by the rescue is NADC34-like PRRSV2 infectious clone virus rBJ1805-2.

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

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preferably, in the method, the method for obtaining the BJ1805-2 full-length infectious clone recombinant plasmid is as follows:

(1) BJ1805-2 whole gene fragment segmented amplification

The BJ1805-2 whole genome is divided into three fragments for amplification;

(2) BJ1805-2 fragment ligation

And purifying the amplified product by using a product purification kit, carrying out double digestion on the purified product and the pACYC177 plasmid, and sequentially connecting the purified product and the pACYC177 plasmid to the pACYC177 vector to finally obtain the infectious clone plasmid pACYC177-rBJ1805-2 containing the complete genome sequence.

Preferably, in the step (1) of the segmented amplification of the BJ1805-2 whole gene fragment, a PacI single cleavage site is introduced at the 5 'end of the BJ1805-2 genome, a hepatitis delta virus (HDV Ribozyme) sequence is added at the 3' end, and the following primer set is adopted:

preferably, in the step (1) of the segmented amplification of the BJ1805-2 whole gene fragment, the following method is adopted:

first, primers BJ1805-2-PacI-F1 and BJ1805-2-Bsu36I-R1; BJ1805-2-Bsu36I-F2 and BJ1805-2-XbaI-R2, and amplifying to obtain BJ1805-2-F1 and BJ1805-2-F2 fragments;

secondly, the amplification of BJ1805-2-F3 is required to be performed in two steps; firstly, amplifying by using a BJ1805-2-XbaI-F3 primer pair and a BJ1805-2-1R3 primer pair to obtain F3-1; then, F3-2 was amplified using F3-1 as a template, and a combination of BJ1805-2-XbaI-F3 and BJ1805-2-AscI-2R4 primers.

Preferably, the method for ligating the fragments of the BJ1805-2 in the step (2) is as follows:

the connection method of BJ1805-2-F1 comprises the following steps: pacI and Bsu36I were used to double cleave the pACYC177-New plasmid and BJ1805-2-F1 amplification product, the pACYC177-New cleavage system was: pACYC177-New plasmid 15. Mu.L, pacI 3. Mu.L, bsu36I 3. Mu.L, 10 XCutSmart Buffer 4. Mu.L, RNase Free H ₂ O was added to 40. Mu.L; the BJ1805-2-F1 enzyme digestion system is as follows: BJ1805-2-F1 purified product 10. Mu.L, pacI 2. Mu.L, bsu36I 2. Mu.L, 10×CutSmart Buffer 4. Mu.L, RNase Free H2O to 40. Mu.L; reaction conditions: water bath at 37 ℃ for 30 minutes;

BJ1805-2-F2 and BJ1805-2-F3-2 are connected by: the method for connecting BJ1805-2-F1 is the same as that described above.

The application of the NADC34-like porcine reproductive and respiratory syndrome virus reverse genetic operation platform constructed by the method is that the NADC34-like PRRSV2 strain rBJ1805-2-ORF2-4-CON which is suitable for Marc-145 cell in-vitro culture is reconstructed, constructed and saved by utilizing the reverse genetic operation platform, and the specific method is as follows:

based on the reverse genetic operation platform, the corresponding fragment in the NADC34-like PRRSV2 infectious clone virus rBJ1805-2 is replaced by the ORF2-4-CON sequence of the small envelope protein coding gene obtained by synthesis by utilizing a homologous recombination method.

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

In one aspect of the invention, there is provided the complete gene sequence of NADC34-like PRRSV2 isolate BJ1805-2. Specifically, BJ1805-2 whole genes are amplified by a method of designing primer segmentation amplification, then all fragments generated by the amplification are sequenced respectively, and finally, the whole gene sequence of NADC34-like PRRSV2BJ1805-2 isolate is obtained by sequence comparison software splicing.

In another aspect of the invention, a virus constructed by reverse genetic manipulation of NADC34-like PRRSV2 is provided. Specifically, pACY177 plasmid is used as a carrier, and the whole gene sequence of the NADC34-like PRRSV2BJ1805-2 isolate is connected to the carrier in a segmented way by using methods of PCR amplification, enzyme digestion connection and the like to obtain the BJ1805-2 full-length infectious clone recombinant plasmid (rBJ 1805-2). The infectious clone virus rBJ1805-2 (preservation number: CCTCC NO: V202250; preservation day: 2022.6.29) is saved in vitro, and the first NADC34-like PRRSV2 strain infectious clone platform in China is successfully built.

In another aspect of the invention, a method for engineering a construction of a NADC34-like PRRSV2 strain adaptable to Marc-145 cell subculture is provided. Specifically comprises the steps of replacing corresponding fragments in the NADC34-like PRRSV2 infectious clone virus rBJ1805-2 with a small envelope protein coding gene ORF2-4-CON consensus sequence obtained by synthesis by using methods such as homologous recombination based on a NADC34-like PRRSV2 reverse genetic operating platform (rBJ 1805-2). Construction and rescue of NADC34-like PRRSV2 engineered strain (rBJ 1805-2-ORF 2-4-CON) that was compatible with Marc-145 cells.

The invention has the following technical effects: 1. a NADC34-like PRRSV2 wild strain popular in China in recent years is separated and the whole gene sequence thereof is determined; 2. constructing a reverse genetic operation platform by taking a NADC34-like PRRSV2 isolate as a parent virus; 3. the constructed infectious clone virus has good in-vitro and in-vivo proliferation efficacy, and specific viremia can be caused by inoculating pig bodies; 4. the constructed infectious clone virus has good safety, does not cause fever (in vitro <40 ℃) after the pig body is inoculated, and does not cause death of piglets; 5. the NADC34-like PRRSV2 modified strain which can adapt to Marc-145 cells is obtained based on the NADC34-like PRRSV2 reverse genetic operation platform modification; 6. the constructed engineered virus inoculated Marc-145 passage cells can cause Cytopathy (CPE); 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 is beneficial to the prevention and control of PRRSV epidemic situation in China.

Drawings

FIG. 1 is a schematic diagram of the construction strategy of the infectious clone rBJ1805-2 in example 1;

FIG. 2 shows the PCR amplification results of 3 fragments of the BJ1805-2 genome in example 1;

FIG. 3 is an indirect immunofluorescence assay of example 1 for detecting BJ1805-2 and r BJ1805-2 using monoclonal antibody 6A1 against PRRSV-N protein;

FIG. 4 shows the dynamic proliferation results of BJ1805-2 and its infectious clone virus rBJ1805-2 on primary PAM cells using the fluorescent quantitative PCR method in example 1;

FIG. 5 shows viremia of BJ1805-2 and its infectious clone virus rBJ1805-2 in example 1 in pigs;

FIG. 6 shows the dynamic change in body temperature of BJ1805-2 and its infectious clone virus rBJ1805-2 vaccinated pigs in example 1;

FIG. 7 shows the weight change of BJ1805-2 and its infectious clone virus rBJ1805-2 vaccinated pigs in example 1;

FIG. 8 is a schematic diagram of the construction strategy of the rBJ1805-2-ORF2-4-CON modified virus in example 2;

FIG. 9 is a cytopathic plot of 2dpi, 5dpi, 8dpi after rescue of rBJ1805-2-ORF 2-4-CON-modified virus on Marc-145 cells in example 2 and results of indirect immunofluorescence assay of rBJ1805-2-ORF 2-4-CON-modified virus replicated on Marc-145 cells using monoclonal antibody 6A1 directed against PRRSV-N protein;

FIG. 10 is the dynamic proliferation of the rBJ1805-2-ORF2-4-CON chimeric virus on Marc-145 cells in example 2;

FIG. 11 shows plaque results on Marc-145 cells of the rBJ1805-2-ORF2-4-CON chimeric virus of example 2.

Preservation information

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) rBJ1805-2 is preserved in China center for type culture Collection, with a preservation address of university of Wuhan in Wuhan China, a preservation number of CCTCC NO: V202250, and a preservation date of 2022.6.29.

Detailed Description

For routine experimentation in the following examples, see the third edition of the molecular cloning Experimental guidelines written by Sambrook et al (Beijing: science Press, 2002), the use of the apparatus is referred to the instruction manual of the apparatus.

In an embodiment of the invention, the virus has the BJ1805-2 isolate. Cells include BHK-21 cell line, marc-145 cell line and porcine primary alveolar macrophage PAM.

In the examples of the present invention, plasmids and strains: pACYC177 plasmid was purchased from Youbao organism, trans1-T1 competent cells from Beijing full gold organism Co., ltd, pUC57-Synthesis-ORF2-4-CON was synthesized in Suzhou Jin Wei Intelligent.

In embodiments of the invention, other reagents are used: RNase Free H ₂ O, pancreatin cell digests (phenol red) were purchased from Solarbio company; the TRIpure Reagent total RNA extraction Reagent was purchased from ideley biosystems; primeScript1st Strand cDNA Synthesis Kit, 2X PrimeSTAR MAX DNA Polymearse, available from TAKARA corporation; fastPure Plasmid Mini Kit from Norvigilant Biotechnology Co., ltd; homologous recombination reagents were purchased from novinaan biotechnology limited; DMEM medium was purchased from HyClone biochemicals inc; fetal bovine serum was purchased from Sigma; dylight 594, coat anti-Mouse IgG (H+L) Secondary Antibody from Invitrogen; DNA markers were purchased from Bo and gold technologies Inc. Zhejiang; 2X BioGold Tag Plus PCR MasterMix from Bogiu technology Co., ltd; gel Extraction Kit from Beijing kang is a century biotechnology Co., ltd; DNA restriction endonucleases were purchased from Thermo Fisher company; t4 DNA Ligase and Lipofectamine ^TM 3000Transfection Reagent from Invitrogen.

EXAMPLE 1 construction of NADC34-like PRRSV2BJ1805-2 Strain reverse genetic manipulation platform

1.1 Whole genome sequencing of NADC34-like PRRSV2BJ1805-2

First, 200. Mu.l of the cell supernatant was extracted with TRIPURE Reagent (Edley), and the extracted total RNA was stored at-20℃or at-80℃for a long period of time. The viral RNA was reverse transcribed into cDNA using a reverse transcription kit (PrimeScript 1st strand cDNA Synthesis Kit, TAKARA), and the resulting cDNA was stored at-20℃for use. The reaction system and procedure are detailed in tables 1 and 2.

Table 1, BJ1805-2 Total RNA pretreatment System and procedure

Table 2, BJ1805-2 Total RNA reverse transcription System and procedure

The whole genome of BJ1805-2 was amplified by PCR amplification. First, the cDNA of BJ1805-2 is used as a template, and then the general primers (No. 2-21 of Table 3) for PRRSV whole gene amplification and the primers (No. 22-30 of Table 3) specific to part of BJ1805-2 are used for combined amplification of BJ1805-2 whole gene sequence. Specific amplification primer information is shown in Table 3.

TABLE 3 PRRSV complete gene amplification primers

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The PCR amplification system and the procedure are shown in tables 4 and 5.

TABLE 4 reaction System

TABLE 5 reaction procedure

Then, the amplified PCR product was electrophoresed on a 1% gel, and then the successfully amplified fragment was sequenced using Sanger sequencing service from Jin Weizhi. Then, the sequences of the fragments were spliced to obtain the whole genome sequence of BJ1805-2 (SEQ ID NO: 1).

1.2 primer design constructed by reverse genetic manipulation platform

Primers were designed to amplify three fragments of BJ1805-2 according to the construction strategy shown in FIG. 1 using Primer 5.0. A PacI single cleavage site is introduced at the 5 'end of the BJ1805-2 genome, and a hepatitis delta virus (HDVRibozyme) sequence is added at the 3' end. Specific information on the construction of the primers for the infectious clone of BJ1805-2 is shown in Table 6.

Table 6, BJ1805-2 infectious clone construction primer

1.3 construction of BJ1805-2 infectious clone

1.3.1, BJ1805-2 Total Gene fragment segmented amplification

As shown in FIG. 1, the BJ1805-2 whole genome is divided into three fragments for amplification. First, primers BJ1805-2-PacI-F1 and BJ1805-2-Bsu36I-R1 in Table 6 were prepared; BJ1805-2-Bsu36I-F2 and BJ1805-2-XbaI-R2, and the BJ1805-2-F1 and BJ1805-2-F2 fragments. Second, amplification of BJ1805-2-F3 is performed in two steps. F3-1 was amplified using BJ1805-2-XbaI-F3 and BJ1805-2-1R3 primer pairs. Then, F3-2 was amplified using F3-1 as a template, and a combination of BJ1805-2-XbaI-F3 and BJ1805-2-AscI-2R4 primers. The specific reaction system and the reaction procedure are shown in tables 7 and 8.

TABLE 7 reaction System

TABLE 8 reaction procedure

After completion of the PCR reaction, 2. Mu.L of the reaction product was taken and subjected to electrophoresis using an agarose gel having a concentration of 0.9%. As shown in FIG. 2, the size of the obtained gene fragment F1 was 5127bp, the size of F2 was 4146bp, and the size of F3-2 was 6061bp.

1.3.2 ligation of fragments BJ1805-2

The amplification product described above was purified using a product purification kit. The purified product was digested with pACYC177 plasmid, which was in turn ligated into pACYC177 vector, and the construction strategy is shown in FIG. 1. The specific operation steps are as follows:

first, pacI and Bsu36I double digestion pACYC177-New plasmid and BJ1805-2-F1 amplification products are used, and the pACYC177-New digestion system is as follows: pACYC177-New plasmid (200 ng/. Mu.L) 15. Mu.L, pacI 3. Mu.L, bsu36I 3. Mu.L, 10 XCutSmart Buffer 4. Mu.L, RNase Free H ₂ O was added to 40. Mu.L. The BJ1805-2-F1 enzyme digestion system is as follows: BJ1805-2-F1 purified product (200 ng/. Mu.L) 10. Mu.L, pacI 2. Mu.L, bsu36I 2. Mu.L, 10 XCutSmart Buffer 4. Mu.L, RNase Free H ₂ O was added to 40. Mu.L. Reaction conditions: water bath at 37 ℃ for 30 minutes. Specific information is shown in tables 9 and 10.

Table 9, pACYC177 plasmid double enzyme cutting system

Name of the name

pACYC177 plasmid

PacI

Bsu36I

10×CutSmart Buffer

RNase Free H 2 O

Dosage of

3000ng

3μL

3μL

4μL

Is added to 40 mu L

Table 10, BJ1805-2-F1 fragment double cleavage System

Name of the name

Anhel 2-F1 fragment

PacI

Bsu36I

10×CutSmart Buffer

RNase Free H 2 O

Dosage of

2000ng

2μL

2μL

4μL

Is added to 40 mu L

The enzyme-cut bands were separated by agarose gel electrophoresis, and the target band was cut off for gel recovery. The linear vector pACYC177-New recovered by the gel and the enzyme-cleaved product BJ1805-2-F1 are connected according to the molar ratio of 1:8, and are connected by T4 DNA ligase to transform the Trans1-T1 competent cells, and independent bacterial colonies are selected for pure culture, and bacterial liquid PCR detection is carried out by using detection primers. Selecting PCR positive bacteria for overnight enrichment culture, extracting plasmid DNA, carrying out double enzyme digestion identification by using PacI and Bsu36I, carrying out 1% agarose gel electrophoresis, selecting plasmids with correct enzyme digestion size for sequencing verification, and then continuing to carry out the connection of the next fragment. The method for connecting F2 and F3-2 fragments is similar to the method for connecting F1, and the infectious clone plasmid pACYC177-rBJ1805-2 containing the whole genome sequence is finally obtained by connecting according to the construction strategy diagram shown in FIG. 1.

Rescue of 1.4, rBJ1805-2 infectious clones

BHK-21 cells were pre-treated with DMEM medium containing 10% FBS at 5X 10 ⁵ cell/well density was seeded on 12 well cell culture plates at 37℃in 5% CO ₂ Is cultured until the cell density reaches about 90%. According to Lipofectamine ^TM 3000Transfection Reagent illustrates the cell transfection performed as follows:

first, a plasmid premix was prepared in an EP tube: infectious clone plasmid pACYC177-rBJ1805-2 2000ng, P3000. Mu.L, optin-MEM 50. Mu.L; in another EP tube, lip3000 premix was prepared: lip 3000. Mu.L, optin-MEM 50. Mu.L; finally, the premixed solution obtained in the two steps is mixed, and after standing for 15min at room temperature, the cells to be transfected are added. 36-48h after cell transfection, sealing the 12-hole plate by a sealing bag, and freezing and thawing for 2 times at-80 ℃ for standby. The specific information is shown in table 11.

TABLE 11 transfection System

Kind/name	Plasmid(s)	Lip3000	P3000	DMEM
					Plasmid premix	2000ng	-	3μL	50μL
Lip3000 premix	-	4μL	-	50μL

Primary PAM cells were plated at 2X 10 using 2mL of RPMI (1640) medium containing 2% FBS ⁵ cell/well density was inoculated into 12-well cell culture plates and placed at 37℃in 5% CO ₂ Is a culture of (a) a strain. 500. Mu.L of BHK-21 cell transfection supernatant was overlaid on primary PAM cells, and after incubation for 2 hours, the supernatant was discarded, and the culture was continued by adding RPMI (1640) medium containing 2% FBS. In addition, BJ1805-2 primary toxin was used as a positive control to infect primary PAM cells. After 3-4 days of culture, the cell supernatant was collected, and the cells were left for IFA identification. As shown in FIG. 3, PRRSV anti-N protein monoclonalAntibody 6A1 detection, the rescue virus (rBJ 1805-2) and the parent virus (BJ 1805-2) can see specific red fluorescence, the negative control group has no specific fluorescence, and the rBJ1805-2 infectious clone virus is successfully rescued on primary PAM cells.

1.5, rBJ1805-2 and BJ1805-2 value-added Activity assay

Primary PAM cells were plated at 2X 10 using RPMI (1640) medium containing 2mL of 2% FBS ⁵ cell/well density was inoculated into 12-well cell culture plates and placed at 37℃in 5% CO ₂ After the culture is completely adhered, the culture is carried out by a culture box with the weight ratio of 1:2000 (1. Mu.L) of virus solution was added thereto, and supernatants were collected at 2hpi (hours post infection), 24hpi,48hpi,72hpi,96hpi, respectively, and the supernatant viral RNA was extracted using a TRIPURE Reagent. mu.L of the obtained cDNA was used for real-time PCR detection of PRRSV1 ORF7 gene. The specific reaction system and amplification procedure are shown in tables 12 and 13.

TABLE 12 reaction System

Name of the name

cDNA

2×EX Taq

Primer (10 mu M)

Probe (10 mu M)

RNase Free H 2 O

Dosage of

1μL

20μL

0.5μL

0.4μL

Is added to 20 mu L

TABLE 13 reaction procedure

The dynamic proliferation of rBJ1805-2 infectious cloned virus on primary PAM cells is shown in fig. 4, which shows a growth curve similar to that of parent virus BJ1805-2.

Pig vaccination test with 1.6, rBJ1805-2 and BJ1805-2

10 important pathogenic negative pigs such as PRRSV, PRV (pseudorabies virus), PCV (circovirus), PEDV (porcine epidemic diarrhea virus) and the like are selected, and 2mL of the important pathogenic negative pigs contain 10 ⁵ TCID(s) ₅₀ rBJ1805-2 virus liquid of (A) is inoculated with 4-head 6-week-old piglets, and 2mL of the liquid contains 10 ⁵ TCID(s) ₅₀ 4 piglets were vaccinated with BJ1805-2 virus solution. Another 2 piglets were inoculated with 2mL DMEM medium. Serum was collected at 3, 5, 7, 10, 12, 14, dpi (day postinfection) and tested for virus content by qRT-PCR. At the same time, the body temperature change condition of the piglets is monitored every day, and the body weight change condition of the piglets is monitored every week. As shown in fig. 5, rBJ1805-2 and BJ1805-2 viruses can proliferate in pigs to produce viremia; as shown in fig. 6, rBJ1805-2 and BJ1805-2 viruses do not cause an increase in body temperature; as shown in FIG. 7, there was a slight weight loss in the pigs vaccinated with rBJ1805-2 and BJ1805-2. But does not cause obvious clinical symptoms or death, and the primary indication shows that the infectious clone virus and the parent virus are both attenuated strains and have better safety.

Example 2 transformation and construction of NADC34-like PRRSV2, which is amenable to in vitro culture of Marc-145 cells

Building and rescuing

2.1, pUC57-Synthesis-ORF2-4CON plasmid Synthesis

30 representative Chinese PRRSV isolates were collected from GenBank. By ClustalX

2.0 alignment of ORF2-4 gene sequences encoding envelope proteins. The DNAMAN 6.0 procedure was used to generate a consensus ORF2-4 sequence (ORF 2-4-CON). The ORF2-4-CON sequence was compared with the corresponding region of the JSTZ1712-12 strain (MK 906026), and the frameshift mutations were manually modified to ensure correct expression of all envelope proteins. ORF2-4-CON sequence was synthesized by GENEWIZ, perilla, china. The sequence of ORF2-4-CON is shown as SEQ ID NO. 38.

2.2 primer design

Primers for amplifying the front and rear segments of ORF2-4-CON, BJ1805-2-F3 and BJ1805-2-F3 were designed using Primer 5.0 according to the construction strategy shown in FIG. 8, and the primers contained homology arms of at least 20bp therebetween.

The specific primer information is shown in Table 14.

Construction primers of Table 14, rBJ1805-2-ORF2-4-CON

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2.3 construction and rescue of the modified Virus rBJ1805-2-ORF2-4-CON

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

TABLE 15 reaction System

TABLE 16 reaction procedure

After completion of the PCR reaction, 2. Mu.L of the reaction product was taken and subjected to electrophoresis using an agarose gel having a concentration of 1%. The obtained fragment ORF2-4-CON has 2624bp, the front segment of fragment rBJ1805-2-F3 has 1748bp, and the rear segment of fragment rBJ1805-2-F3 has 1802bp.

2.3.2, rBJ1805-2-ORF2-4-CON fragments homologous recombination

The pACYC177-BJ1805-2 was digested with XbaI and AscI to obtain pACYC177-BJ1805-2-F1+F2 linearized vector. The specific procedure was similar to that in example 1. Specific information is shown in Table 17.

Table 17, pACYC177-BJ1805-2 plasmid double enzyme digestion system

The enzyme-cut bands were separated by agarose gel electrophoresis, and the target band was cut off for gel recovery. After the gel was recovered, the concentration was measured using NanoDrop and ready for use. ORF2-4-CON, BJ1805-2-F3 front and BJ1805-2-F3 rear were ligated to pACYC177-BJ1805-2-F1+F2 linearized vector by homologous recombination. The homologous recombination is specifically performed as follows:

the homologous recombination system is as follows: 52ng of ORF2-4-CON, 34ng of BJ1805-2-F3 front and 36ng of BJ1805-2-F3 rear, 200ng of pACYC177-BJ1805-2-F1+F2 linearization intermediate vector, 4. Mu.l of 5 XCE MultiS Buffer and 2. Mu.l of Exanse MultiS. The reaction conditions were 37℃for 30min. See table 18 for details.

TABLE 18 homologous recombination System

The homologous recombination product is transformed into Trans1-T1 competent cells, independent colonies are picked for pure culture, and bacterial liquid PCR detection is carried out by using detection primers. Selecting PCR positive bacteria, culturing overnight, extracting plasmid DNA, performing double enzyme digestion identification by using XbaI and AscI, performing 0.9% agarose gel electrophoresis, and selecting plasmids with correct enzyme digestion size for reservation.

Construction and identification of rBJ1805-2-ORF2-4-CON modified virus were completed according to the above method.

2.3.3 rescue of the rBJ1805-2-ORF2-4-CON engineered Virus

BHK21 cells were prepared at 2.5X10 s using DMEM medium containing 10% FBS ⁵ cell/well density was seeded on 24 well cell culture plates at 37℃with 5% CO ₂ Is cultured until the cell density reaches about 80%. According to Lipofectamine ^TM 3000Transfection Reagent illustrates the transfection of cells. 36-48h after cell transfection, sealing a 12-hole plate, freezing at-80 ℃, repeatedly freezing and thawing twice, taking all cell suspensions, centrifuging at 10000 Xg for 4min, and collecting supernatant. The transfection procedure was similar to that of example 1. Details are shown in Table 19.

TABLE 19 transfection System

Kind/name	Plasmid(s)	Lip3000	P3000	DMEM
					Plasmid premix	2000ng	-	3μL	50μL
Lip3000 premix	-	4μL	-	50μL

Marc-145 cells were pre-treated with DMEM medium containing 10% FBS at 2X 10 ⁵ cell/well density was seeded on 12 well cell culture plates at 37℃in 5% CO ₂ To a cell density of about 80%, and a DMEM medium containing 2% fbs was used instead.

Adding Marc-145 cells into the whole supernatant, and continuously culturing. Meanwhile, marc-145 cells were inoculated with rBJ1805-2 and BJ1805-2, which were successfully rescued in example 1, and the cells were observed every day. As shown in FIG. 9, the modified virus rBJ1805-2-ORF2-4-CON showed CPE phenomena such as shrinkage and aggregation in 120hpi cells after Marc-145 cells were inoculated, while rBJ1805-2 and BJ1805-2 could not infect Marc-145 cells. The obtained rBJ1805-2-ORF2-4-CON is continuously passaged, the 4 th generation modified virus is inoculated with Marc-145,144hpi and then detected by using PRRSV anti-N protein monoclonal antibody 6A1, as shown in figure 9, the modified virus N protein has specific red fluorescence, and a negative control group has no fluorescence, thus proving that the virus rescue is successful.

Value-added characterization of 2.4, rBJ1805-2-ORF2-4-CON on Marc-145 cells

Marc-145 cells were pre-conditioned 1X 10 with DMEM medium containing 10% FBS ⁵ cell/well density was seeded on 24 well cell culture plates at 37℃with 5% CO ₂ To a cell density of about 80%, and a DMEM medium containing 2% fbs was used instead.

The rBJ1805-2-ORF2-4-CON 4 th generation virus liquid stock solution is inoculated on Marc-145 cells of a 6-well plate. Six time points, 24hpi,48hpi,72hpi,96hpi, 120hpi and 144hpi, were taken for the multi-step growth curve assay. The specific operation method was the same as in example 1. The results of the multi-step growth curve assay are shown in FIG. 10, rBJ1805-2-ORF2-4CON was successfully replicated on Marc-145 cells.

2.5, rBJ1805-2-ORF2-4-CON plaque assay

Marc-145 cells were pre-conditioned at 3X 10 using DMEM medium containing 10% FBS ⁵ cell/well density was seeded on 6 well cell culture plates at 37℃in 5% CO ₂ To a cell density of about 80%, and a DMEM medium containing 2% fbs was used instead. The next day the cell density was observed until the cells reached a dense state for the next step.

Marc-145 cells were infected with the 4 th generation viral stocks of BJ1805-2, rBJ1805-2 and rBJ1805-2-ORF2-4-CON for 1.5h. After 1.5h, the virus solution was discarded, and the solution was gently washed with PBS for 1 to 2 times, and 0.7% low-melting agarose culture solution (30 mL medium formulation: 15mL DMEM+600uL FBS+300uL Green streptomycin+15 mL 1.4% low-melting agarose solution was finally poured) was prepared, and after cooling to about 37℃the single-layer cells were directly covered. The 6-well plate was then cultured upside down and observed after 6 days. After cytopathic formation, the medium was covered with 4% paraformaldehyde and left to stand overnight for 1h after crystallization violet staining. After completion of the dyeing, the mixture was rinsed with water and observed. Plaque assay results are shown in FIG. 11, rBJ1805-2-ORF2-4-CON was able to form plaques on Marc-145 cells, whereas rBJ1805-2 and BJ1805-2 were unable to produce plaques.

Claims (10)

1. A strain of NADC34-like porcine reproductive and respiratory syndrome virus, characterized in that the virus is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) rBJ1805-2; the special culture medium is preserved in China center for type culture Collection, the preservation address is the university of Wuhan in Wuhan, the preservation number is CCTCC NO: V202250, and the preservation date is 2022.6.29.

2. The recombinant vector for constructing the reverse genetic operation platform of the NADC34-like porcine reproductive and respiratory syndrome virus is characterized in that the recombinant vector is pACYC177-rBJ1805-2, the recombinant vector takes pACY177 plasmid as a vector, and the recombinant vector contains the whole gene sequence of an NADC34-like PRRSV2BJ1805-2 isolate, and the whole gene sequence is shown in SEQ ID NO. 1.

3. The method for constructing a reverse genetic manipulation platform of NADC34-like porcine reproductive and respiratory syndrome virus according to claim 1, which is characterized in that the method comprises the following steps:

extracting RNA of NADC34-like PRRSV2 isolate BJ1805-2, and reversely transcribing the viral RNA into cDNA by using a reverse transcription kit; amplifying NADC34-like PRRSV2 isolate BJ1805-2 whole genes by taking the cDNA as a template and designing a primer segmentation amplification method, sequencing each amplified fragment respectively, and finally splicing by sequence comparison software to obtain the whole gene sequence of the NADC34-like PRRSV2BJ1805-2 isolate;

the pACY177 plasmid is used as a carrier, and a PCR amplification and enzyme digestion connection method is utilized to connect the complete gene sequence of the NADC34-like PRRSV2BJ1805-2 isolate to the carrier in a segmented manner, so as to obtain a BJ1805-2 full-length infectious clone recombinant plasmid;

the NADC34-like porcine reproductive and respiratory syndrome virus is then rescued by an infectious cloning method, and the virus obtained by the rescue is NADC34-like PRRSV2 infectious clone virus rBJ1805-2.

4. The method for constructing a reverse genetic manipulation platform of a NADC34-like porcine reproductive and respiratory syndrome virus according to claim 3, wherein the primer set for amplifying the whole gene of the NADC34-like PRRSV2 isolate BJ1805-2 is as follows:

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5. the method for constructing a reverse genetic manipulation platform of NADC34-like porcine reproductive and respiratory syndrome virus according to claim 3, wherein the method for obtaining BJ1805-2 full-length infectious clone recombinant plasmid is as follows:

(1) BJ1805-2 whole gene fragment segmented amplification

The BJ1805-2 whole genome is divided into three fragments for amplification;

(2) BJ1805-2 fragment ligation

And purifying the amplified product by using a product purification kit, carrying out double digestion on the purified product and the pACYC177 plasmid, and sequentially connecting the purified product and the pACYC177 plasmid to the pACYC177 vector to finally obtain the infectious clone plasmid pACYC177-rBJ1805-2 containing the complete genome sequence.

6. The method for constructing a reverse genetic manipulation platform of NADC34-like porcine reproductive and respiratory syndrome virus according to claim 5, wherein in the step (1) of BJ1805-2 whole gene fragment segmented amplification, pacI single enzyme cleavage site is introduced at the 5 'end of BJ1805-2 genome, and hepatitis delta virus (HDV Ribozyme) sequence is added at the 3' end, and the following primer group is adopted:

7. the method for constructing a reverse genetic manipulation platform of NADC34-like porcine reproductive and respiratory syndrome virus according to claim 6, wherein the following method is adopted in the step (1) of segment amplification of BJ1805-2 whole gene segment:

first, primers BJ1805-2-PacI-F1 and BJ1805-2-Bsu36I-R1; BJ1805-2-Bsu36I-F2 and BJ1805-2-XbaI-R2, and amplifying to obtain BJ1805-2-F1 and BJ1805-2-F2 fragments;

secondly, the amplification of BJ1805-2-F3 is required to be performed in two steps; firstly, amplifying by using a BJ1805-2-XbaI-F3 primer pair and a BJ1805-2-1R3 primer pair to obtain F3-1; then, F3-2 was amplified using F3-1 as a template, and a combination of BJ1805-2-XbaI-F3 and BJ1805-2-AscI-2R4 primers.

8. The method for constructing a reverse genetic manipulation platform of a NADC34-like porcine reproductive and respiratory syndrome virus according to claim 7, wherein the method for ligating the fragments of the BJ1805-2 in the step (2) is as follows:

the connection method of BJ1805-2-F1 comprises the following steps: pacI and Bsu36I were used to double cleave the pACYC177-New plasmid and BJ1805-2-F1 amplification product, the pACYC177-New cleavage system was: pACYC177-New plasmid 15. Mu.L, pacI 3. Mu.L, bsu36I 3. Mu.L, 10 XCutSmart Buffer 4. Mu.L, RNase Free H ₂ O was added to 40. Mu.L; the BJ1805-2-F1 enzyme digestion system is as follows: BJ1805-2-F1 purified product 10. Mu.L, pacI 2. Mu.L, bsu36I 2. Mu.L, 10×CutSmart Buffer 4. Mu.L, RNase Free H ₂ O was added to 40. Mu.L; reaction conditions: water bath at 37 ℃ for 30 minutes;

BJ1805-2-F2 and BJ1805-2-F3-2 are connected by: the method for connecting BJ1805-2-F1 is the same as that described above.

9. The use of the reverse genetic manipulation platform of the NADC34-like porcine reproductive and respiratory syndrome virus constructed by the method of any one of claims 3 to 8, wherein the use is to reconstruct, construct and rescue a NADC34-like PRRSV2 strain rBJ1805-2-ORF2-4-CON suitable for Marc-145 cell in vitro culture by using the reverse genetic manipulation platform, and the specific method is as follows:

based on the reverse genetic operation platform, the corresponding fragment in the NADC34-like PRRSV2 infectious clone virus rBJ1805-2 is replaced by the ORF2-4-CON sequence of the small envelope protein coding gene obtained by synthesis by utilizing a homologous recombination method.

10. The use according to claim 9, wherein the sequence of ORF2-4-CON of the gene encoding the small envelope protein is shown in SEQ ID NO. 38.

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