CN111996213A

CN111996213A - Construction method of porcine reproductive and respiratory syndrome virus double-fluorescence labeling gene recombinant strain

Info

Publication number: CN111996213A
Application number: CN202010081916.XA
Authority: CN
Inventors: 韦祖樟; 王豪; 谢欣; 任同伟; 王玉旭; 贺微; 陈樱; 欧阳康; 黄伟坚
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2020-02-06
Filing date: 2020-02-06
Publication date: 2020-11-27
Also published as: CN113215192B; CN113215192A

Abstract

The invention discloses a construction method of a dual-fluorescence-labeled gene recombinant strain of porcine reproductive and respiratory syndrome virus, which comprises the steps of taking a porcine reproductive and respiratory syndrome virus passage attenuated strain Gxnn1396-P96 as a parent strain, deleting 465 nucleotides in an NSP2 region by using a genetic engineering method, inserting a red fluorescent protein gene in a deletion position, and inserting a green fluorescent protein gene between ORF1b and ORF2a on the basis to obtain the dual-fluorescence-labeled recombinant PRRSV virus. Sequencing shows that the recombinant virus is stable in heredity, and the fluorescence abundance of the marker gene which is not inserted is reduced when the marker gene continuously passes to 20 generations in cells. In conclusion, the invention establishes a set of live porcine reproductive and respiratory syndrome virus vector system, can insert a plurality of exogenous genes simultaneously, is more efficient than a common live virus vector system, has stable heredity, and lays a foundation for developing recombinant porcine reproductive and respiratory syndrome virus gene engineering vaccines.

Description

Construction method of porcine reproductive and respiratory syndrome virus double-fluorescence labeling gene recombinant strain

Technical Field

The invention belongs to the technical field of porcine reproductive and respiratory syndrome virus, and particularly relates to a construction method of a dual-fluorescence-labeled gene recombinant strain of porcine reproductive and respiratory syndrome virus.

Background

Porcine Reproductive and Respiratory Syndrome (PRRS), also known as porcine reproductive and respiratory syndrome, is a highly contagious disease of pigs caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), characterized by sow reproductive disorders and piglet respiratory disorders, pigs being the sole infected host, since PRRSV infection was first discovered in the united states in 1987 to date, PRRSV has experienced multiple outbreaks, causing significant economic losses to the world's swine industry.

The PRRSV belongs to a member of the genus arterivirus, family arteriviridae, genus arterivirus, the genome of which is single-stranded positive-strand RNA, the total length of which is about 15.6kb and contains at least 10 open reading frames, the 5 'end of which contains a cap structure and the 3' end of which contains PolyA. ORF1a and ORF1b occupy approximately genome 3/4 and encode viral replicase proteins pp1a and pp1 b. pp1a and pp1b replicase proteins form 14 non-structural proteins by self-cleavage. ORF2a, ORF2b, ORF3, ORF4, ORF5, ORF5a, ORF6 and ORF7 encode structural proteins of the virus, wherein ORF2a, ORF2b, ORF3, ORF4 and ORF5a encode the minor structural proteins GP2a, 2b, GP3, GP4 and GP5a of the virus, and ORF5, ORF6 and ORF7 encode the major structural proteins GP5, M and N of the virus.

The rapid development of the PRRSV reverse genetics system lays a solid foundation for the development of PRRSV as a live viral vector vaccine. The NSP2 is the most varied of all proteins coded by PRRSV, and amino acid deletion and insertion often occur in a hypervariable region (HVR) of the NSP2, until now, many scholars at home and abroad use an HVR region of NSP2 as a target point for inserting a foreign gene, but the obtained recombinant virus is unstable in heredity, and most of the inserted foreign genes are gradually lost in the process of continuous passage. In addition, there is another strategy to express foreign genes by inserting additional independent transcriptional units to generate additional subgenomic mrma (sg mrna). Due to the cross-overlapping of most of the PRRSV open reading frames, the strategy has limited selectable targets. To date, ORF1b and ORF2a, ORF7 and 3' UTR are the major foreign gene insertion sites studied. According to the strategy, a Transcription Regulatory Sequence (TRS) is inserted behind an exogenous gene, the sg mRNA of the inserted exogenous gene is regulated and synthesized by an upstream genome TRS of the exogenous gene, and the artificially inserted TRS regulates the generation of the sg mRNA of a downstream virus structural protein.

So far, the research of expressing the exogenous gene by PRRSV is mainly a single site, and the heredity of part of recombinant viruses is unstable, and there is no report that a plurality of sites stably express the exogenous gene at the same time.

Disclosure of Invention

The invention aims to solve the technical problem of providing a construction method of a dual-fluorescence labeled gene recombinant strain of porcine reproductive and respiratory syndrome virus.

In order to solve the technical problems, the invention adopts the following technical scheme:

a construction method of a porcine reproductive and respiratory syndrome virus double-marker gene recombinant strain comprises the steps of taking the porcine reproductive and respiratory syndrome virus Gxnn1396-P96 as a parent strain, deleting 465 nucleotides (3049-3513nt) in an NSP2 region by using a genetic engineering method, inserting a marker gene into the deletion position, and inserting another marker gene between ORF1b and ORF2a on the basis to obtain the recombinant PRRSV with the inserted double-marker.

A construction method of a dual-fluorescence-labeled gene recombinant strain of porcine reproductive and respiratory syndrome virus comprises the steps of taking a porcine reproductive and respiratory syndrome virus passage attenuated strain Gxnn1396-P96 as a parent strain, deleting 465 nucleotides (3049-3513nt) in an NSP2 region by using a genetic engineering method, inserting a red fluorescent protein gene at the deletion position, and inserting a green fluorescent protein gene between ORF1b and ORF2a on the basis to obtain the dual-fluorescence-labeled recombinant PRRSV virus.

The recombinant PRRSV virus is rGX-RFP-GFP.

The construction method comprises the following steps:

(1) construction of PRRSV NSP2 deletion mutant pGX-NSP2-RFP expressing red fluorescent protein

In PRRSV infectious clone, an SOE-PCR method is adopted to delete the 624 th amino acid-778 th amino acid site region of NSP2 hypervariable region, two enzyme cutting sites of BstZ17I and SbfI are introduced into the deleted region, firstly NSP2155aa deletion mutant pGX-NSP2-D465 is constructed, then Red Fluorescent Protein (RFP) gene is inserted, and the inserted RFP recombinant clone pGX-NSP2-RFP in NSP2 is obtained.

(2) Construction of recombinant pGX-12BS-GFP expressing a Green fluorescent clone between ORF1b and ORF2a of PRRSV

In the cloning of PRRSV infectivity, two restriction sites Bstb I and Sbf I were introduced between ORF1b and ORF2a by SOE-PCR method, and SOE-PCR product was cloned to Zero

Obtaining pTOPO-12BS on the PCR vector; cloning a transcription regulatory sequence TRS (sequence table SEQ. NO. ID.16) to an Sbf I enzyme cutting site by using mutation PCR to obtain pTOPO-12BS-TRS, cloning a corresponding fragment containing the enzyme cutting site and the TRS into pGXAM through Asc I and Mlu I to obtain a recombinant clone pGX-12 BS-TRS; on this basis, the GFP was cloned into pGX-12BS-TRS using the artificially introduced cleavage sites Bstb I and Sbf I to obtain the recombinant plasmid pGX-12 BS-GFP.

(3) Construction of recombinant plasmid pGX-RFP-GFP for simultaneously expressing red fluorescent protein gene and green fluorescent protein gene

And simultaneously carrying out enzyme digestion on pGX-12BS-GFP and pGX-NSP2-RFP by using two restriction enzyme digestion sites of Asc I and Mlu I, recovering digestion products, and then carrying out ligation by using T4 enzyme to obtain pGX-RFP-GFP.

The step (1) is specifically operated as follows: using full-length infectious clone pGXAM of PRRSV as a template, amplifying by using JX451F and JX6463R, and cloning the PCR product to Zero

Carrying out PCR vector to obtain a shuttle plasmid pTOPO-P2; PCR products GF-D155R-BstZ17IF and D155F-JR-SbfI are obtained by using pTOPO-P2 as a template and using mutation primers NSP 2D 155 BstZ17IF and NSP 2D 155 SbfI R and outer primers GX2350F and GX3921R for amplification; performing a second round of fusion PCR amplification by using PCR products GF-D155R-BstZ17IF and D155F-JR-SbfI as templates and using outer primers GX2350F and GX3921R, fusing the PCR products GF-D155R-BstZ17IF and D155F-JR-SbfI, digesting the fused PCR products and pTOPO-P2 by using Afe I and Bbs I restriction enzyme sites at the same time, obtaining a deletion 155aa after T4 is connected, introducing Bstz17I and Sbf I restriction enzyme sites pTOPO-P2-D465, and using Spe I and Afl II to simultaneously digest pTOPO-P2-D465 and pGXAM, and performing equal-segment replacement of corresponding regions to obtain pGX-NSP 2-D465; on the basis of this, the red fluorescent protein gene was amplified using primers RFP BstZ17I F and RFP SbfI R, and pGX-NSP2-D465 was digested with BstZ17I and SbfI to obtain a recombinant clone pGX-NSP2-RFP in which the RFP gene was inserted in the NSP2 deletion region.

The step (2) is specifically operated as follows: using full-length infectious clone pGXAM of PRRSV as a template, amplifying two PCR products containing BstbI and Sbf I restriction enzyme cutting sites by using mutation primers 12BS F and 12BS R and outer primers GF11706 and JX14402Mlu I, using the two PCR products as templates, performing second-round fusion PCR amplification by using outer primers GF11706 and JX14402Mlu I, and cloning the fusion PCR products to Zero

PCR vector to obtain shuttlePlasmid pTOPO-12 BS; using pTOPO-12BS as a template, using primers SbfI-TRS-F and JX14402MluI to obtain a PCR product containing TRS by using mutation PCR, simultaneously carrying out enzyme digestion on the PCR product and pTOPO-12BS by using SbfI and Mlu I, and carrying out T4 ligation to obtain pTOPO-12 BS-TRS; then, simultaneously carrying out enzyme digestion on pTOPO-12BS-TRS and pGXAM by using Asc I and Mlu I, and inserting an enzyme digestion fragment containing an enzyme digestion site and TRS into pGXAM to obtain a recombinant plasmid pGX-12 BS-TRS; the GFP gene was amplified using GFP-Bstb I-F and GFP-Sbf I-R amplification primers for the GFP gene, and then the PCR product and pGX-12BS-TRS were digested simultaneously using Bstb I and Sbf I, and the GFP gene was inserted into pGX-12BS-TRS to obtain a recombinant plasmid pGX-12 BS-GFP.

The step (3) is specifically operated as follows: and simultaneously digesting pGX-12BS-GFP and pGX-NSP2-RFP by using two restriction enzyme sites of Asc I and Mlu I, and performing equal-segment replacement on corresponding regions to obtain the recombinant plasmid pGX-RFP-GFP.

The virus Gxnn1396-P96 is a virus with a preservation number of CCTCC NO: v202020, classified and named as porcine reproductive and respiratory syndrome virus Gxnn 1396-P96.

The porcine reproductive and respiratory syndrome virus double-fluorescence labeled gene recombinant strain obtained by the method.

The recombinant strain is applied to the preparation of genetic engineering vaccines.

NSP2 is a nonstructural protein with the largest change in PRRSV, a hypervariable region of the nonstructural protein is frequently deleted, and the inventor finds that an NSP2 region of a PRRSV strain naturally deletes 465 nucleotides in clinical detection. Accordingly, the inventor establishes a construction method of a dual-fluorescence-labeled gene recombinant strain of the porcine reproductive and respiratory syndrome virus, takes a porcine reproductive and respiratory syndrome virus passage attenuated strain Gxnn1396-P96 as a parent strain, deletes 465 nucleotides in an NSP2 region by using a genetic engineering method and inserts a red fluorescence protein gene into a deletion position, and inserts a green fluorescence protein gene between ORF1b and ORF2a on the basis to obtain the dual-fluorescence-protein gene-labeled recombinant PRRSV. Sequencing shows that the recombinant virus is stable in heredity, and the fluorescence abundance of the marker gene which is not inserted is reduced when the marker gene continuously passes to 20 generations in cells. In conclusion, the invention establishes a set of live porcine reproductive and respiratory syndrome virus vector system, can insert a plurality of exogenous genes simultaneously, is more efficient than a common live virus vector system, has stable heredity, and lays a foundation for developing recombinant porcine reproductive and respiratory syndrome virus gene engineering vaccines.

Drawings

FIG. 1 is a schematic diagram of construction of PRRSV infectious clone pGXAM by taking parental strain Gxnn1396-P96 as a template

FIG. 2 is a schematic diagram of the construction of recombinant plasmids rGX-NSP2-RFP, rGX-12BS-GFP and rGX-RFP-GFP.

FIG. 3 is a 6 th generation map of recombinant viruses rGX-NSP2-D465, rGX-NSP2-RFP, rGX-12BS-TRS, rGX-12BS-GFP and parental strain rGXAM, in which: a1-a4 are rGXAM, rGX-NSP2-D465, rGX-NSP2-RFP and Mock, and b1-b4 are rGXAM, rGX-12BS-TRS, rGX-12BS-GFP and Mock.

FIG. 4 is a fluorescence plot of rGX-NSP2-RFP, rGX-12BS-GFP, rGX-RFP-GFP at different time points, in which: c rGX-NSP2-RFP, d rGX-12BS-GFP and g rGX-RFP-GFP are respectively 12h, 24h, 48h and 72h from left to right.

FIG. 5 is an indirect immunofluorescence map of recombinant viruses rGX-NSP2-D465, rGX-NSP2-RFP, rGX-12BS-TRS, rGX-12BS-GFP and parental strain rGXAM, in which: e1-e4 are rGXAM, rGX-NSP2-D465, rGX-NSP2-RFP and Mock, and f1-f4 are rGXAM, rGX-12BS-TRS, rGX-12BS-GFP and Mock.

Figure 6 is a graph of the growth curves of recombinant virus and parental strain rGXAM, in which: the recombinant virus A is rGX-NSP2-D465, rGX-NSP2-RFP and parental strain rGXAM, the recombinant virus B is rGX-12BS-TRS, rGX-12BS-GFP and parental strain, and the recombinant virus C is rGX-RFP-GFP and parental strain rGXAM.

FIG. 7 is a confocal image of recombinant virus rGX-RFP-GFP.

Description of preservation information

The porcine reproductive and respiratory syndrome virus Gxnn1396-P96 with the preservation number of CCTCC NO: v202020, date of deposit: 16.01.2019, the preservation address is as follows: wuhan university, post code 430072, storage unit: china center for type culture Collection.

Detailed Description

The following are examples in which TOPO vectors for clone sequencing and intermediate cloning construction were constructed from invitrogen and vectors containing GFP and RFP genes were stored in the laboratory of the applicant.

Primer sequences used in the following examples:

(1) SOE-PCR deleted the NSP2 region 155aa and introduced two restriction sites Bstz17I and Sbf I.

GX2530F TTCCCCGCCGAGCGCTGCGGACGCTTC (sequence listing SEQ. NO. ID.1)

GX3921R CACACGCCGAGAAGACCCAGAAAATA (sequence listing SEQ. NO. ID.2)

NSP 2D 155 BstZ17I F AATGACACCAACCCTGCAGTATACAAACCTGCAGGAGCGCCCTCCAAGGGAGAA (SEQ. NO. ID.3 of the sequence Listing)

NSP 2D 155 SbfI R TTCTCCCTTGGAGGGCGCTCCTGCAGGTTTGTATACTGCAGGGTTGGTGTCATT (SEQ. NO. ID.4 of the sequence Listing)

(2) Amplification of the RFP Gene

RFP BstZ17I F GACACCAACCCTGCAGTATACATGGTGAGCAAGGGCGAGG (SEQ. NO. ID.5)

RFP Sbf I-R TCCCTTGGAGGGCGCTCCTGCAGGCTAGTTTCCGGACTTGTACAGCTCG (SEQ. NO. ID.6)

(3) SOE-PCR inserts two restriction sites Bstb I and Sbf I between ORF1b and ORF2a

GF11706 gcgtttcgggcgcgccagaaagg (sequence listing SEQ. NO. ID.7)

12BS R AGGCTTTGCATAGACCCCATTTCATCCTGCAGGTACC TTCGAATTCAATTCAGGCCTAAAGTTGTTC (sequence listing SEQ. NO. ID.8)

12BS F GAACCAACTTAGGCCTGAATTGAATTCGAAGGTACCTGCAGGATGAAATGGGTCTATGCAAAGCCT (sequence listing SEQ. NO. ID.9)

JX14402Mlu I acgccggacaaaacgcgtggttatca (sequence table SEQ. NO. ID.10)

(4) Amplification of TRS Gene

Sbf I-TRS-F ACGCCTGCAGGATGGTTCCGCGGCAACCCCTTTAACCAGAGTTTCAGCGGAACAATATGAAATGGGGTCTATGCAAAGCCTCT (SEQ. NO. ID.11)

(5) Amplification of GFP Gene

GFP-Bstb I-F GAGTTCGAAATGCCCGCCATGAAGATC (sequence listing SEQ. NO. ID.12)

GFP-Sbf I-R ATATCCTGCAGGCTAGGCGAATGCGATCGGGGTCTTGAA (SEQ. NO. ID.13 of the sequence Listing)

(6) Amplification of PRRSV P2 fragment

JX451F tgcacgaatgactagtgaaaacc (SEQ. NO. ID.14 of the sequence Listing)

JX6463R gggcggccgcgaaggcataggtgcttaagtt (sequence listing SEQ. NO. ID.15)

Example 1 construction of PRRSV NSP2 deletion mutant pGX-NSP2-RFP expressing Red fluorescent protein

Using full-length infectious clone pGXAM of PRRSV as a template, amplifying by using JX451F and JX6463R, and cloning the PCR product to Zero

Carrying out PCR vector to obtain a shuttle plasmid pTOPO-P2; PCR products GF-D155R-BstZ17IF and D155F-JR-SbfI are obtained by using pTOPO-P2 as a template and using mutation primers NSP 2D 155 BstZ17IF and NSP 2D 155 SbfI R and outer primers GX2350F and GX3921R for amplification; performing a second round of fusion PCR amplification by using PCR products GF-D155R-BstZ17IF and D155F-JR-SbfI as templates and using outer primers GX2350F and GX3921R, fusing the PCR products GF-D155R-BstZ17IF and D155F-JR-SbfI, digesting the fused PCR products and pTOPO-P2 by using Afe I and Bbs I restriction enzyme sites at the same time, obtaining a deletion 155aa after T4 is connected, introducing Bstz17I and Bbf I restriction enzyme sites pTOPO-P2-D465, and using Spe I and Afl II to simultaneously digest pTOPO-P2-D465 and XApGM, and performing equal-segment replacement of corresponding regions to obtain X-NSP 2-D465; on the basis, the red fluorescent protein gene (SEQ. NO. ID.18) was amplified using primers RFP BstZ17I F and RFP SbfI R, and pGX-NSP2-D465 was digested with BstZ17I and SbfI to obtain a recombinant clone pGX-NSP2-RFP in which the RFP gene was inserted in the NSP2 deletion region.

Example 2 construction of expression of the Green fluorescent recombinant clone pGX-12BS-GFP between PRRSV ORF1b and ORF2a

Using full-length infectious clone pGXAM of PRRSV as a template, amplifying two PCR products containing BstbI and Sbf I restriction enzyme cutting sites by using mutation primers 12BS F and 12BS R and outer primers GF11706 and JX14402Mlu I, using the two PCR products as templates, performing second-round fusion PCR amplification by using outer primers GF11706 and JX14402Mlu I, and performing second-round fusion PCR amplification on the fusion PCR productsCloning of the object into Zero

Carrying out PCR vector to obtain a shuttle plasmid pTOPO-12 BS; using pTOPO-12BS as a template, using primers SbfI-TRS-F and JX14402MluI to obtain a PCR product containing TRS by using mutation PCR, simultaneously carrying out enzyme digestion on the PCR product and pTOPO-12BS by using SbfI and Mlu I, and carrying out T4 ligation to obtain pTOPO-12 BS-TRS; then, simultaneously carrying out enzyme digestion on pTOPO-12BS-TRS and pGXAM by using Asc I and Mlu I, and inserting an enzyme digestion fragment containing an enzyme digestion site and TRS into pGXAM to obtain a recombinant plasmid pGX-12 BS-TRS; the GFP gene was amplified using amplification primers GFP-Bstb I-F and GFP-Sbf I-R for the GFP gene (SEQ. NO. ID.17), and then the PCR product and pGX-12BS-TRS were digested simultaneously using Bstb I and Sbf I, and the GFP gene was inserted into pGX-12BS-TRS to obtain a recombinant plasmid pGX-12 BS-GFP.

Example 3 construction of recombinant plasmid pGX-RFP-GFP simultaneously expressing Red fluorescent protein Gene and Green fluorescent protein Gene

EXAMPLE 4 rescue of recombinant viruses

The constructed recombinant plasmid was transfected into a 6-well BHK-21 (milk hamster kidney cell) cell plate of good growth at a transfection dose of 2. mu.g according to the instructions of Lipfectamine 2000, the 6-well plate was replaced with MEM maintenance solution containing 2% fetal bovine serum, and the transfected cell plate was placed in a medium containing 5% CO₂Culturing in a 37 deg.C incubator for 48h, collecting supernatant, inoculating 300 μ L of the collected supernatant to Marc-145 (Vero cell) cells, placing 6-well plate in a container containing 5% CO₂Culturing in an incubator at 37 ℃ for 12 hours and observing cytopathy once. Obvious cytopathic effect is observed at 24h, and supernatant is collected when 60% of cells are shed and stored at-80 ℃.

Continuous passage of recombinant viruses

And (3) diluting the rescued virus by 100 times, inoculating 300 mu L of the rescued virus to Marc-145 cells with good growth, collecting cell supernatant when the cells fall off by 60 percent, inoculating the collected supernatant to the Marc-145 cells with good growth again by the same method, circulating the cells in sequence, continuously carrying out passage for 10 generations, and completely storing the collected cell supernatant at-80 ℃.

Identification of recombinant viruses

(1) Indirect Immunofluorescence (IFA) identification of recombinant viruses

The cell supernatant from the P3 passage was diluted 100-fold. And (3) inoculating 300 mu L of the cell culture medium on a Marc-145 well-grown cell 6-well plate, culturing the cell plate in an incubator with 37 ℃ and 5% carbon dioxide for 24h, discarding the supernatant, washing the cell plate for 3 times by using PBS, and fixing the cell by using glacial methanol at 4 ℃ for 30 min. After the fixation is finished, washing by PBS for 3 times, adding 500 mu L of 1.0% BSA into each hole, sealing at room temperature for 30min, after the sealing is finished, washing by PBS for 3 times, adding 500 mu L of PRRSV monoclonal antibody SDOW (1:2000 dilution) into each hole, incubating at room temperature for 2h, after the incubation is finished, washing by PBS for 3 times, adding 500 mu L of 1:500 diluted secondary antibody into each hole, incubating at room temperature in a dark place for 1h, after the completion, washing by PBS for 3 times, and placing under a fluorescence inverted microscope to observe fluorescence. Significant red or green fluorescence was observed under a fluorescence inverted microscope.

(2) Genetic stability of recombinant viruses

After the obtained recombinant virus is continuously passaged on Marc-145 cells for 10 generations, both green fluorescence and red fluorescence can be stably expressed, cell supernatants collected by P6 generation are identified through PCR, and virus sequences of inserted GFP genes, inserted RFP genes and insertion sites can be detected, which shows that the recombinant PRRSV viruses rGX-NSP2-RFP, rGX-12BS-GFP and rGX-RFP-GFP obtained in the experiment are stable within at least 6 generations and the sign of reduced fluorescence abundance is still observed when P10 generation is not observed.

(3) Characterization of the proliferation Properties of recombinant viruses

The rescued recombinant virus and parental virus were infected with Marc-145 cells in a 6-well plate at an MOI of 0.01 and cell supernatants were harvested 12h, 24h, 48h, 72h, 96h and 120h after infection, and TCID50 of the cell supernatants harvested at each time point was determined, based on the TCID determined₅₀A multi-step growth curve was plotted (fig. 5). The obtained recombinant viruses rGX-NSP2-RFP, rGX-12BS-GFP possesses similar viral propagation properties as the parental strain rGXAM, but the viral propagation properties of rGX-RFP-GFP are slightly weaker than the parental strain rGXAM.

Sequence listing

<110> Guangxi university

Construction method of <120> porcine reproductive and respiratory syndrome virus double-fluorescence labeling gene recombinant strain

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ctagccaaca cacaggcaac ttca 24

<210> 2

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cacacgccga gaagacccag aaaata 26

<210> 3

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aatgacacca accctgcagt atacaaacct gcaggagcgc cctccaaggg agaa 54

<210> 4

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ttctcccttg gagggcgctc ctgcaggttt gtatactgca gggttggtgt catt 54

<210> 5

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gacaccaacc ctgcagtata catggtgagc aagggcgagg 40

<210> 6

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tcccttggag ggcgctcctg caggctagtt tccggacttg tacagctcg 49

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gcgtttcggg cgcgccagaa agg 23

<210> 8

<211> 67

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aggctttgca tagaccccat ttcatcctgc aggtaccttc gaattcaatt caggcctaaa 60

gttgttc 67

<210> 9

<211> 66

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gaaccaactt aggcctgaat tgaattcgaa ggtacctgca ggatgaaatg ggtctatgca 60

aagcct 66

<210> 10

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

acgccggacg acaaacgcgt ggttatca 28

<210> 11

<211> 83

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

acgcctgcag gatggttccg cggcaacccc tttaaccaga gtttcagcgg aacaatatga 60

aatggggtct atgcaaagcc tct 83

<210> 12

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gagttcgaaa tgcccgccat gaagatc 27

<210> 13

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atatcctgca ggctaggcga atgcgatcgg ggtcttgaa 39

<210> 14

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tgcacgaatg actagtgaaa acc 23

<210> 15

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gggcggccgc gaaggcatag gtgcttaagt t 31

<210> 16

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atggttccgc ggcaacccct ttaaccagag tttcagcgga acaatatgaa 50

<210> 17

<211> 666

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atgcccgcca tgaagatcga gtgccgcatc accggcaccc tgaacggcgt ggagttcgag 60

ctggtgggcg gcggagaggg cacccccgag cagggccgca tgaccaacaa gatgaagagc 120

accaagggcg ccctgacctt cagcccctac ctgctgagcc acgtgatggg ctacggcttc 180

taccacttcg gcacctaccc cagcggctac gagaacccct tcctgcacgc catcaacaac 240

ggcggctaca ccaacacccg catcgagaag tacgaggacg gcggcgtgct gcacgtgagc 300

ttcagctacc gctacgaggc cggccgcgtg atcggcgact tcaaggtggt gggcaccggc 360

ttccccgagg acagcgtgat cttcaccgac aagatcatcc gcagcaacgc caccgtggag 420

cacctgcacc ccatgggcga taacgtgctg gtgggcagct tcgcccgcac cttcagcctg 480

cgcgacggcg gctactacag cttcgtggtg gacagccaca tgcacttcaa gagcgccatc 540

caccccagca tcctgcagaa cgggggcccc atgttcgcct tccgccgcgt ggaggagctg 600

cacagcaaca ccgagctggg catcgtggag taccagcacg ccttcaagac cccgatcgca 660

ttcgcc 666

<210> 18

<211> 720

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60

gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120

cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180

ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240

cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300

gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360

ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420

atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480

gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540

gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600

aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660

cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagtc cggaaactag 720

Claims

1. A method for constructing a porcine reproductive and respiratory syndrome virus double-marker gene recombinant strain is characterized by comprising the following steps: the porcine reproductive and respiratory syndrome virus Gxnn1396-P96 is used as a parent strain, a genetic engineering method is utilized to delete 465 nucleotides in an NSP2 region and insert a marker gene at the deletion position, and on the basis, another marker gene is inserted between ORF1b and ORF2a to obtain the recombinant PRRSV inserted with double gene markers.

2. A method for constructing a dual-fluorescence labeled gene recombinant strain of porcine reproductive and respiratory syndrome virus is characterized by comprising the following steps: the porcine reproductive and respiratory syndrome virus passage weakening strain Gxnn1396-P96Gxnn is used as a parent strain, an NSP2 region 465 nucleotides are deleted by a genetic engineering method, a red fluorescent protein gene is inserted into the deletion position, and on the basis, a green fluorescent protein gene is inserted between ORF1b and ORF2a to obtain the recombinant PRRSV inserted with a double fluorescent protein gene marker.

3. The construction method according to claim 2, wherein: the recombinant PRRSV virus is rGX-RFP-GFP.

4. The building method according to claim 2, characterized by comprising the steps of:

In PRRSV infectious cloning, an SOE-PCR method is adopted to delete 624 th amino acid-778 th amino acid site region of NSP2 hypervariable region, two enzyme cutting sites of BstZ17I and SbfI are introduced into the deleted region, deletion mutant pGX-NSP2-D465 of pGXAM is firstly constructed, and then red fluorescent protein RFP gene is inserted to obtain recombinant plasmid pGX-NSP 2-RFP.

(2) Construction of recombinant pGX-12BS-GFP, a green fluorescent recombinant clone expressed between PRRSV ORF1b and ORF2 a.

Obtaining a shuttle plasmid pTOPO-12BS on the PCR vector; cloning a transcription regulation sequence TRS to an Sbf I enzyme cutting site by utilizing mutation PCR to obtain pTOPO-12BS-TRS, and cloning corresponding fragments obtained by cutting the pTOPO-12BS-TRS by Asc I and Mlu I into pGXAM to obtain a recombinant clone pGX-12 BS-TRS; on the basis, GFP is inserted into pGX-12BS-TRS through the artificially introduced restriction enzyme sites Bstb I and Sbf I to obtain the recombinant plasmid pGX-12 BS-GFP.

And simultaneously carrying out enzyme digestion on pGX-12BS-GFP and pGX-NSP2-RFP by using two restriction enzyme digestion sites of Asc I and Mlu I, recovering corresponding enzyme digestion products, and then carrying out ligation by using T4 enzyme to obtain the recombinant plasmid pGX-RFP-GFP.

5. The construction method according to claim 4, characterized in that step (1) is specifically operated as follows: using full-length infectious clone pGXAM of PRRSV as a template, amplifying by using JX451F and JX6463R, and cloning the PCR product to Zero

Carrying out PCR vector to obtain a shuttle plasmid pTOPO-P2; PCR products GF-D155R-BstZ17IF and D155F-JR-SbfI are obtained by using pTOPO-P2 as a template and using mutation primers NSP 2D 155 BstZ17IF and NSP 2D 155 SbfI R and outer primers GX2350F and GX3921R for amplification; performing a second round of fusion PCR amplification by using PCR products GF-D155R-BstZ17IF and D155F-JR-SbfI as templates and using outer primers GX2350F and GX3921R, fusing the PCR products GF-D155R-BstZ17IF and D155F-JR-SbfI, performing enzyme digestion on the fused PCR product and pTOPO-P2 by using Afe I and Bbs I restriction enzyme sites at the same time, obtaining a clone pTOPO-P2-D465 deleting 155aa and introducing Bstz17I and Sbf I restriction enzyme sites after T4 is connected, performing equal-segment replacement of corresponding regions by using SpeI and Afl II to perform enzyme digestion on pTOPO-P2-D465 and XApGM at the same time, and obtaining X-NSP 2-D465; on the basis, the red fluorescent protein gene was amplified using primers RFP BstZ17I F and RFP SbfI R, and pGX-NSP2-D465 and the PCR amplification product were simultaneously digested with BstZ17I and SbfI to insert the RFP gene into pGX-NSP2-D465, thereby obtaining a recombinant clone pGX-NSP2-RFP in which the RFP gene was inserted into the NSP2 deletion region.

6. The construction method according to claim 4, wherein the step (2) is specifically operated as follows: using full-length infectious clone pGXAM of PRRSV as a template, amplifying two PCR products containing BstbI and Sbf I restriction enzyme cutting sites by using mutation primers 12BS F and 12BS R and outer primers GF11706 and JX14402Mlu I, and using the two PCR products as templates and outer primers GF11706 and JX14402Mlu I to perform PCRSecond round of fusion PCR amplification, cloning of fusion PCR products to Zero

Carrying out PCR vector to obtain a shuttle plasmid pTOPO-12 BS; using pTOPO-12BS as a template, using primers SbfI-TRS-F and JX14402MluI to obtain a PCR product containing TRS by using mutation PCR, simultaneously carrying out enzyme digestion on the PCR product and pTOPO-12BS by using SbfI and Mlu I, and carrying out T4 ligation to obtain pTOPO-12 BS-TRS; then, simultaneously carrying out enzyme digestion on pTOPO-12BS-TRS and pGXAM by using Asc I and Mlu I, and inserting an enzyme digestion fragment containing an enzyme digestion site and TRS into pGXAM to obtain a recombinant plasmid pGX-12 BS-TRS; the GFP gene was amplified using GFP-Bstb I-F and GFP-Sbf I-R amplification primers for the GFP gene, and then the PCR product and pGX-12BS-TRS were digested simultaneously using Bstb I and Sbf I, and the GFP gene was inserted into pGX-12BS-TRS to obtain a recombinant plasmid pGX-12 BS-GFP.

7. The construction method according to claim 4, characterized in that step (3) is specifically operated as follows: and simultaneously digesting pGX-12BS-GFP and pGX-NSP2-RFP by using two restriction enzyme sites of Asc I and Mlu I, and performing equal-segment replacement on corresponding regions to obtain the recombinant plasmid pGX-RFP-GFP.

8. The process of any of claims 2 to 7, characterized in that: the virus Gxnn1396-P96 is a virus with a preservation number of CCTCC NO: v202020, classified and named as porcine reproductive and respiratory syndrome virus Gxnn 1396-P96.

9. The porcine reproductive and respiratory syndrome virus double-fluorescent-labeled gene recombinant strain obtained by the method of any one of claims 2 to 8.

10. The use of the recombinant strain of claim 8 in the preparation of genetically engineered vaccines.