CN111676247A - Infectious clone construction, rescue and application of porcine reproductive and respiratory syndrome virus type 1 isolate - Google Patents
Infectious clone construction, rescue and application of porcine reproductive and respiratory syndrome virus type 1 isolate Download PDFInfo
- Publication number
- CN111676247A CN111676247A CN202010611744.2A CN202010611744A CN111676247A CN 111676247 A CN111676247 A CN 111676247A CN 202010611744 A CN202010611744 A CN 202010611744A CN 111676247 A CN111676247 A CN 111676247A
- Authority
- CN
- China
- Prior art keywords
- prrsv1
- strain
- infectious clone
- cells
- respiratory syndrome
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/10011—Arteriviridae
- C12N2770/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/10011—Arteriviridae
- C12N2770/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/10011—Arteriviridae
- C12N2770/10051—Methods of production or purification of viral material
- C12N2770/10052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Abstract
The invention provides an infectious clone virus of a porcine reproductive and respiratory syndrome virus type 1 (PRRSV1) wild strain, and provides a key genetic operation platform for researching Marc-145 cell adaptability determinants of PRRSV 1. Meanwhile, aiming at the actual problem that the PRRSV1 vaccine is lacked in China at present, the PRRSV1 wild strain infectious cloning platform can be used for developing a safer and more efficient novel PRRSV1 genetic engineering vaccine.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to infectious clone construction, rescue, modification and application of a porcine reproductive and respiratory syndrome virus type 1 strain.
Background
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is widely prevalent in swine countries worldwide. PRRSV infection mainly causes abortion in sows and respiratory symptoms throughout the herd. PRRSV can be divided into two genotypes (PRRSV1 and PRRSV2) and multiple gene subtypes. In 1995, PRRSV2 first appeared and spread rapidly across the country in our country. In 2006, "swine high fever disease" caused by outbreak of a highly pathogenic PRRSV2 variant strain (HP-PRRSV) in China. In addition, in recent years, the PRRSV1 wild strain is also separated in hong Kong, inner Mongolia, Heilongjiang, Liaoning, Fujian, Guizhou province and other provinces and cities of China.
PRRSV is a single-stranded positive-strand RNA virus belonging to the family arterivirus of the order nested viruses. The PRRSV genome is approximately 15kb in size, has a cap structure at the 5 'end, a Poly (A) tail at the 3' end, and 10 Open Reading Frames (ORFs) in the middle. ORF1a and ORF1b encode at least 16 nonstructural proteins that play important roles in viral replication and transcription. ORF2-7 encodes 8 structural proteins to constitute infectious virions.
The PRRSV1 and 2 isolates exist in China at the same time, and the PRRSV1 and 2 strains can be divided into a plurality of subtypes. The commercial vaccine developed based on a certain gene subtype strain can effectively prevent and control breeding disorder and respiratory symptoms caused by infection of homologous PRRSV wild strain, but has poor cross protection. Despite the continuous isolation of PRRSV1 in chinese swinery in recent years, the existing chinese PRRS vaccines are all type 2 PRRS vaccines. Therefore, there is an urgent need to develop new specific vaccines against PRRSV1 infection in our country. However, the existing PRRSV1 isolate in China can only be cultured on primary alveolar macrophage PAM and cannot be cultured on passage Marc-145 cells. Becomes a great problem for developing PRRSV1 passage live vaccine.
The RNA virus infectious clone is obtained by constructing virus cDNA clone in vitro, manually operating virus genes on a DNA level and rescuing infectious virus particles in vitro. Successful construction and genetic manipulation of PRRSV1 infectious clones can be used for developing novel genetic engineering vaccines. Although a PRRSV reverse genetic operation platform has been established in previous researches, the reverse genetic operation platform based on the PRRSV1 wild strain in China has not been reported so far.
Disclosure of Invention
Aiming at the scientific problem that the wild strain of PRRSV1 in China is difficult to culture on passage Marc-145 cells in vitro, the invention provides an infectious clone virus of the wild strain of porcine reproductive and respiratory syndrome virus type 1 (PRRSV1), and provides a key genetic operation platform for researching Marc-145 cell adaptability determinant factors of PRRSV 1. Meanwhile, aiming at the actual problem that the PRRSV1 vaccine is lacked in China at present, the PRRSV1 wild strain infectious cloning platform can be used for developing a safer and more efficient novel PRRSV1 genetic engineering vaccine.
The invention is realized by the following technical scheme:
in one aspect of the invention, a recombinant vector is provided, comprising a full-length cDNA sequence of PRRSV1 genome, wherein the 5 'end of the full-length cDNA sequence is added with Cytomegalovirus (CMV) eukaryotic promoter sequence, the 3' end is added with Hepatitis D virus ribozyme (Hepatitis D virus ribozyme) sequence and Bovine Growth Hormone polyadenylation signal (BGH) transcription termination sequence at the downstream of Poly (A) tail.
Preferred vectors of the invention are pACYC177 low copy vectors. Five single restriction sites (SgsI, Pfl23II, BglII, Bsp1407I and XbaI) are inserted into the pACYC177 vector by methods of gene synthesis, homologous recombination and the like for infectious clone construction of PRRSV1 attenuated strain HLJB 1.
In another aspect of the invention, a method for the simple preparation of an infectious clone of PRRSV1 is provided. The recombinant vector and the high-efficiency transfection reagent are mixed and then can directly transfect BHK-21 cells and then infect PAM cells so as to save and obtain HLJB1 infectious clone viruses.
In another aspect of the invention, the application of the artificially cloned PRRSV1 wild strain in researching the adaptability of PRRSV1 strain Marc-145 cells is also provided. By utilizing an infectious cloning platform, the PRRSV wild strain incapable of adapting to Marc-145 and the PRRSV1 vaccine strain capable of adapting to Marc-145 cells are subjected to corresponding gene fragment replacement, and the related gene and the key locus which determine the adaptability of the Marc-145 cells of the PRRSV1 wild strain are analyzed.
In another aspect of the invention, an artificially constructed PRRSV1 attenuated vaccine candidate strain is an infectious clonal virus of PRRSV1 attenuated strain HLJB 1. The artificially constructed attenuated vaccine candidate strain can be applied to preparation of a novel PRRSV1 genetic engineering vaccine.
The invention relates to establishment of a PRRSV1 low virulent strain infectious cloning platform, which can be used for subsequent development of a safer and more efficient novel PRRSV1 genetic engineering live vaccine. Provides key technical support and alternative prevention strategies for effectively preventing and controlling PRRS epidemic situations. The development of the novel safe and efficient PRRSV1 gene engineering live vaccine has great economic value and good market prospect. The remarkable features of the invention include:
1. the constructed infectious clone virus is based on the PRRSV1 low virulent strain HLJB1 in China.
2. The constructed infectious cloning method is simple and convenient, DNA transfection is directly carried out, transfection of in vitro transcribed virus RNA is not needed, and the defects that the RNA is unstable in vitro and is easy to degrade and the like are overcome. In addition, the infectious clone of the 3' end of the HP-PRRSV genome is precisely obtained by utilizing the self-cutting function of the hepatitis delta virus ribozyme. Is a rapid, simple and accurate PRRSV genetic operation platform.
3. The obtained PRRSV1 infectious clone platform can be applied to in vitro Marc-145 cell adaptability mechanism research of PRRSV1 wild strain.
4. The constructed PRRSV1 low virulent strain infectious clone virus can be used for developing a safer and more efficient novel PRRSV1 genetic engineering vaccine, and is beneficial to the prevention and control of PRRSV in China.
Drawings
FIG. 1 is a schematic diagram of the modification of pACYC177 vector in example 1 of the present invention;
FIG. 2 is a diagram showing the PCR amplification results of the whole gene fragment of the HLJB1 strain in example 1 (M: DNA Marker; 1-4: HLJB 1F 1, F2, F3 and F4 fragments);
FIG. 3 is a schematic diagram of the construction of the genome full-length cDNA connection of PRRSV1 low virulent strain HLJB1 in example 1 of the present invention;
FIG. 4 is a graph showing the results of restriction enzyme identification of the infectious clone virus rHLJB1 in example 1 of the present invention (M: DNA Marker; 1: rHLJB1 plasmid did not cut enzyme; 2: rHLJB1 plasmid XbaI single cut enzyme; 3: rHLJB1 plasmid SgsI and Pfl23II double cut enzyme; 4: rHLJB1 plasmid Pfl23II and BglII double cut enzyme; 5: rHLJB1 plasmid BglII and Bsp1407I double cut enzyme; 6: rHLJB1 plasmid Bsp1407I and XbaI double cut enzyme);
FIG. 5 is a graph showing the results of detection of rescued rHLJB1 infectious clonal virus using an indirect immunofluorescence assay against a PRRSV 1N protein monoclonal antibody in example 1 of the present invention.
Detailed Description
For routine experimentation in the following examples, see "molecular cloning experiments, south" third edition (Beijing: science publishers, 2002) written by Sambrook et al, the use of the instrument is described in reference to the instructions for the operation of the instrument.
In the examples of the present invention, the virus has an isolate of HLJB 1. The cells include BHK-21 cell line and primary alveolar macrophage PAM.
In the examples of the present invention, plasmids and strains: the pACYC177 plasmid was purchased from Youbao, the pDsRed-Express-C1 plasmid was stored in the laboratory, and the Trans1-T1 competent cells were purchased from Beijing Quanjin biology, Inc.
In the present examples, RNase Free H2O, pancreatin cell digest (phenol red) from Solambio, TRIpure Reagent total RNA Extraction from Eldelley, PrimeScript1st Strand cDNA Synthesis Kit, 2 × PrimeStar MAX DNA polymerase from TAKARA, Fastpure Plasmid Mini Kit from Novemedium Biotech, DMEM medium from Hyclone Biochemical Co., Ltd, fetal bovine serum from Sigma, DyLight, Goat-Mouse IgG (H + L) Secondary Antibody from Invitrogen, DNA Marker from Bo Biotech Co., Ltd, Zhejiang Tag Plus MasterMix from Kingkun Biotech, Kingson Biotech from Kingson Biotech, 2 from Kingson DNA Biotechnology Co., LtdTM3000Transfection Reagent was purchased from Invitrogen.
Example 1 construction and rescue of the full-Length cDNA clone of the genome of the porcine reproductive and respiratory syndrome Virus type 1 attenuated Strain HLJB1
1.1 pACYC177-New vector construction
By referring to the infectious clone construction method reported by people before and improving on the original basis, a method for constructing an HLJB1 infectious clone platform is designed. HLJB1 full genome sequences are analyzed by using DNAMAN software for comparison, and SgsI, Pfl23II, BglII, Bsp1407I and XbaI5 single enzyme cutting sites are selected for segmented insertion of the full length of genes during construction of the HLJB1 infectious cloning platform. As shown in FIG. 1, the vector modification scheme is that low copy plasmid pACYC177 is selected as the original vector, and a 911bp fragment containing CMV promoter, corresponding 4 cleavage sites (SgsI, Pfl23II, BglII, Bsp1407I and XbaI) and Bovine Growth Hormone (BGH) polyadenylation signal is inserted into the vector pACYC177 after its restriction site BamH I. The recombinant vector was synthesized by Kinzea virens, Suzhou and was designated pACYC177-New after sequence verification.
1.2 primer design
Amplification primers (see Table 1 below) comprising the PRRSV complete gene were designed 4 pairs using Primer5.0 based on the complete gene sequence of HLJB1 in GenBank (GenBank accession No.: KT224385), wherein primers HLJB1-1R4, HLJB1-XbaI-2R4 introduce hepatitis delta virus (HDV Ribozyme) sequence.
TABLE 1 primers for amplification of PRRSV whole gene in construction of infectious clones of the invention
1.3 construction of infectious clone of HLJB1
1.3.1 HLJB1 Whole Gene segmentation amplification
HLJB1 is taken as a template, and HLJB1-SgsI-F1 and HLJB1-Pfl23II-R1 are respectively taken as templates; HLJB1-Pfl23II-F2 and HLJB 1-BglII-R2; and HLJB1-BglII-F3 and HLJB1-Bsp1407I-R3 pairs to amplify F1, F2 and F3 fragments. Amplification of the F4 fragment was performed in two steps, first using HLJB1-Bsp1407I-F4 and HLJB1-1R4 for paired amplification of the F4-1 fragment, followed by F4-1 amplification product as template, and HLJB1-Bsp1407I-F4 and HLJB1-XbaI-2R4 for paired amplification of the F4 fragment for addition of hepatitis D virus (HDV Ribozyme) sequences. Taking HLJB1 whole gene segmented amplification as an example, the specific operation is as follows:
the PCR reaction system comprises 2 μ L of HLJB1 cDNA template, 1 μ L of upstream and downstream primer pairs (10 μ M) respectively, 20 μ L of 2 × PrimeStarMAX DNA Polymearse, and RNase Free H2And O is supplemented to 40 mu L.
The PCR reaction program is: 10s at 98 ℃, 30s at 60 ℃ and 5min at 72 ℃ for 35 cycles.
The HLJB1 whole gene was amplified in segments according to the method described above. As shown in FIG. 2, 5. mu.L of the PCR reaction product was electrophoresed on 0.9% agarose gel to obtain four gene fragments with fragment sizes of 4371bp,5230bp, 3758bp and 2001bp, respectively.
1.3.2 linking of fragments of HLJB1
And (3) purifying the amplification product by using a PCR product purification kit, carrying out double enzyme digestion on the purified product and the PACYC177-New plasmid, sequentially connecting to the PACYC177-New vector, and constructing a strategy shown in figure 3. The specific operation steps are as follows:
firstly, carrying out double digestion on PACYC177-New plasmid and HLJB 1F 1 amplification products by using SgsI and Pfl23II, wherein the PACYC177-New enzyme digestion system is as follows: 20 μ L of PACYC177-New plasmid (100 ng/. mu.L), 2 μ L of SgsI, 2 μ L of Pfl23II 2, 4 μ L of 10 × CutSmart Buffer, and 40 μ L of RNase Free H2O. The HLJB 1F 1 enzyme cutting system is as follows: the purified product of HLJB 1F 1 (200 ng/. mu.L) was 10. mu.L, SgsI 2. mu.L, Pfl23II 2. mu.L, 10 × CutSmart Buffer 4. mu.L, RNase FreeH2O made up to 40. mu.L. Reaction conditions are as follows: water bath at 37 ℃ for 30 minutes.
Separating the enzyme-digested band by agarose gel electrophoresis, cutting the target band and carrying out gel recovery. The linear vector pACYC177-New recovered from the gel and the enzyme digestion product HLJB 1F 1 are connected by T4 DNA ligase according to the molar ratio of 1:8, and then Trans1-T1 competent cells are transformed, independent colonies are picked for pure culture, and detection primers are used for carrying out PCR detection on bacteria liquid. Selecting PCR positive bacteria for overnight enrichment culture, extracting plasmid DNA, carrying out double enzyme digestion identification by using SgsI and Pfl23II, carrying out 0.8% agarose gel electrophoresis, and selecting the plasmid with the correct enzyme digestion size for next-step fragment ligation.
The connection method of HLJB 1F 2, F3 and F4 fragments is similar to the connection method of HLJB 1F 1, and the infectious clone plasmid rHLJB1 containing HLJB1 whole genes is obtained by connecting according to a construction strategy diagram shown in FIG. 3. The double-enzyme digestion identification of the rHLJB1 recombinant plasmid is shown in FIG. 4.
1.4 rescue of infectious clonal Virus of rHLJB1
BHK-21 cells were previously cultured in DMEM medium containing 10% FBS as 2 × 105The cells/well density was inoculated into 24-well cell culture plates, and the plates were incubated at 37 ℃ in a 5% CO2 incubator until the cell density reached about 80%. According to LipofectamineTM3000Transfection Reagent indicated cell Transfection, the procedure was as follows:
first, plasmid premix was prepared in an EP tube: 500ng of infectious clone plasmid, P30001 mu L and DMEM 25 mu L; in another EP tube a Lip3000 premix was placed: lip30001.5. mu.L, DMEM 25. mu.L; and finally, mixing the premixed solution obtained in the two steps, standing at room temperature for 15min, and adding the cells to be transfected. 36-48h after cell transfection, whole transfected BHK21 cell culture plates were frozen at-80 ℃ for use.
Using RPMI +1:50 streptomycin +1:500 piperacillin +1:500 gentamycin culture medium containing 2% fetal bovine serum to lay PAM cells, taking primary PAM frozen in liquid nitrogen to lay 24-well plates, each well being 1 × 105A cell. The transfected BHK-21 frozen-thawed product was then centrifuged at 10000rpm for 5 minutes and the supernatant was added to the plated PAM. Daily observations were made and double antibiotics (1:50 streptomycin, 1:500 gentamicin and piperacillin) were added, cultured for 3-4 days, and cell supernatants were carefully collected for IFA assays. As shown in FIG. 5, after the PAM cells are inoculated with the rescued virus and detected by using PRRSV anti-N protein monoclonal antibody 15A1 after 90hpi, the rescued virus (rHLJB1) and the parent strain (HLJB1) both show specific red fluorescence, and no fluorescence is generated in a negative control group, so that the rescued survival of the rHLJB1 infectious clone virus is confirmed.
Sequence listing
<110> Yangzhou university
<120> infectious clone construction, rescue and application of porcine reproductive and respiratory syndrome virus type 1 isolate
<160>9
<170>SIPOSequenceListing 1.0
<210>1
<211>34
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
agctcgggcg cgcctacatg atgtgtaggg tatt 34
<210>2
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
atggcctgac taggatcgta cggaacggca accaccgt 38
<210>3
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
acggtggttg ccgttccgta cgatcctagt caggccat 38
<210>4
<211>39
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
agcacagcat cgagaggaga tctaccagcc ccaaccggt 39
<210>5
<211>39
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
accggttggg gctggtagat ctcctctcga tgctgtgct 39
<210>6
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
ataagcaagt ccgcgttgta caaatacgat tcatcggt 38
<210>7
<211>38
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
accgatgaat cgtatttgta caacgcggac ttgcttat 38
<210>8
<211>69
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
agcgaggagg ctgggaccat gccggccttt tttttttttt ttttttttaa tttcggtcac 60
atggttcct 69
<210>9
<211>87
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
acagtctaga gtcccattcg ccattaccga ggggacggtc ccctcggaat gttgcccagc 60
cggcgccagc gaggaggctg ggaccat 87
Claims (4)
1. Infectious clone construction of a porcine reproductive and respiratory syndrome virus type 1 isolate is characterized by providing a recombinant vector, which comprises a PRRSV1 genome full-length cDNA sequence, wherein the 5 'end of the full-length cDNA sequence is added with a cytomegalovirus eukaryotic promoter sequence, the 3' end Poly (A) downstream is added with a hepatitis D virus ribozyme sequence and a bovine growth hormone polyadenylation signal transcription termination sequence;
the vector is a pACYC177 low-copy vector, five single restriction sites SgsI, Pfl23II, BglII, Bsp1407I and XbaI are inserted into the pACYC177 vector through gene synthesis and homologous recombination, and the vector is used for infectious clone construction of PRRSV1 attenuated strain HLJB 1.
2. The infectious clone rescue of the porcine reproductive and respiratory syndrome virus type 1 isolate according to claim 1, which is characterized in that the recombinant vector and the high-efficiency transfection reagent are mixed and then can directly transfect BHK-21 cells and then infect PAM cells so as to obtain HLJB1 infectious clone virus through rescue.
3. The use of the porcine reproductive and respiratory syndrome virus type 1 isolate of claim 1, wherein the use of an artificially cloned PRRSV1 field strain for studying the adaptability of PRRSV1 strain Marc-145 cells is provided: by utilizing an infectious cloning platform, the PRRSV wild strain incapable of adapting to Marc-145 and the PRRSV1 vaccine strain capable of adapting to Marc-145 cells are subjected to corresponding gene fragment replacement, and the related genes and key sites for determining the adaptability of the Marc-145 cells of the PRRSV1 wild strain are analyzed.
4. The application of the porcine reproductive and respiratory syndrome virus type 1 isolate as claimed in claim 1, which is characterized in that an artificially modified PRRSV1 attenuated vaccine candidate strain is provided, the artificially modified attenuated vaccine candidate strain is an infectious clone virus of PRRSV1 attenuated strain HLJB1, and the artificially modified attenuated vaccine candidate strain can be applied to preparation of a novel PRRSV1 genetic engineering vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010611744.2A CN111676247A (en) | 2020-06-30 | 2020-06-30 | Infectious clone construction, rescue and application of porcine reproductive and respiratory syndrome virus type 1 isolate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010611744.2A CN111676247A (en) | 2020-06-30 | 2020-06-30 | Infectious clone construction, rescue and application of porcine reproductive and respiratory syndrome virus type 1 isolate |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111676247A true CN111676247A (en) | 2020-09-18 |
Family
ID=72437492
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010611744.2A Pending CN111676247A (en) | 2020-06-30 | 2020-06-30 | Infectious clone construction, rescue and application of porcine reproductive and respiratory syndrome virus type 1 isolate |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111676247A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662885A (en) * | 2020-06-22 | 2020-09-15 | 扬州大学 | Construction, rescue and application of infectious clone of virulent and attenuated strains of two highly homologous genome porcine reproductive and respiratory syndrome viruses |
| CN114317574A (en) * | 2021-12-28 | 2022-04-12 | 扬州大学 | Cultivation and application of Marc-145 cell adaptive type 1 porcine reproductive and respiratory syndrome virus |
| CN114395583A (en) * | 2021-11-01 | 2022-04-26 | 扬州大学 | cDNA clone of porcine reproductive and respiratory syndrome virus expressing secreted luciferase and construction method and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130142824A1 (en) * | 2010-06-02 | 2013-06-06 | Virginia Tech Intellectual Properties, Inc. | Novel modified live-attenuated vaccines (mlv) created by dna shuffling against porcine reproductive and respiratory syndrome virus (prrsv) |
| CN103305638A (en) * | 2013-02-21 | 2013-09-18 | 北京世纪元亨动物防疫技术有限公司 | Dual real-time fluorescence PCR (Polymerase Chain Reaction) detection primer pair, probes, kit and detection method for type 1 and type 2 porcine circovirus |
| JP2014507151A (en) * | 2011-02-17 | 2014-03-27 | ベーリンガー インゲルハイム フェトメディカ ゲゼルシャフト ミット ベシュレンクテル ハフツング | New European PRRSV strain |
| CN103695465A (en) * | 2013-12-16 | 2014-04-02 | 中国农业科学院哈尔滨兽医研究所 | CDNA (Complementary Deoxyribonucleic Acid) infectious clone of porcine reproductive and respiratory syndrome virus vaccine strain and application thereof |
| JP2017501985A (en) * | 2013-12-03 | 2017-01-19 | インターベット インターナショナル ベー． フェー． | Swine vaccine against PRRS and Lawsonia intracellularis |
| CN109762792A (en) * | 2019-01-18 | 2019-05-17 | 南京农业大学 | A kind of porcine reproductive and respiratory syndrome virus is fitted into strain and its application |
| KR20200069603A (en) * | 2018-12-07 | 2020-06-17 | 대한민국(농림축산식품부 농림축산검역본부장) | PNA probe for detecting Porcine reproductive and respiratory syndrome virus genetic type and a method for detecting Porcine reproductive and respiratory syndrome virus genetic type using the same |
-
2020
- 2020-06-30 CN CN202010611744.2A patent/CN111676247A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130142824A1 (en) * | 2010-06-02 | 2013-06-06 | Virginia Tech Intellectual Properties, Inc. | Novel modified live-attenuated vaccines (mlv) created by dna shuffling against porcine reproductive and respiratory syndrome virus (prrsv) |
| JP2014507151A (en) * | 2011-02-17 | 2014-03-27 | ベーリンガー インゲルハイム フェトメディカ ゲゼルシャフト ミット ベシュレンクテル ハフツング | New European PRRSV strain |
| CN103305638A (en) * | 2013-02-21 | 2013-09-18 | 北京世纪元亨动物防疫技术有限公司 | Dual real-time fluorescence PCR (Polymerase Chain Reaction) detection primer pair, probes, kit and detection method for type 1 and type 2 porcine circovirus |
| JP2017501985A (en) * | 2013-12-03 | 2017-01-19 | インターベット インターナショナル ベー． フェー． | Swine vaccine against PRRS and Lawsonia intracellularis |
| CN103695465A (en) * | 2013-12-16 | 2014-04-02 | 中国农业科学院哈尔滨兽医研究所 | CDNA (Complementary Deoxyribonucleic Acid) infectious clone of porcine reproductive and respiratory syndrome virus vaccine strain and application thereof |
| KR20200069603A (en) * | 2018-12-07 | 2020-06-17 | 대한민국(농림축산식품부 농림축산검역본부장) | PNA probe for detecting Porcine reproductive and respiratory syndrome virus genetic type and a method for detecting Porcine reproductive and respiratory syndrome virus genetic type using the same |
| CN109762792A (en) * | 2019-01-18 | 2019-05-17 | 南京农业大学 | A kind of porcine reproductive and respiratory syndrome virus is fitted into strain and its application |
Non-Patent Citations (3)
| Title |
|---|
| NANHUA CHEN等: "Identification of Two Porcine Reproductive and Respiratory Syndrome Virus Variants Sharing High Genomic Homology but with Distinct Virulence", 《VIRUSES》 * |
| 李子娟等: "PRRSV疫苗的研究进展及前景展望", 《黑龙江畜牧兽医》 * |
| 王星晨: "猪繁殖与呼吸综合征病毒基因1型毒株GZ11-G1的致病性及其分子基础", 《中国博士学位论文全文数据库（电子期刊）农业科技辑》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662885A (en) * | 2020-06-22 | 2020-09-15 | 扬州大学 | Construction, rescue and application of infectious clone of virulent and attenuated strains of two highly homologous genome porcine reproductive and respiratory syndrome viruses |
| CN114395583A (en) * | 2021-11-01 | 2022-04-26 | 扬州大学 | cDNA clone of porcine reproductive and respiratory syndrome virus expressing secreted luciferase and construction method and application thereof |
| CN114395583B (en) * | 2021-11-01 | 2024-03-29 | 扬州大学 | cDNA clone of porcine reproductive and respiratory syndrome virus for expressing secretion type luciferase, construction method and application thereof |
| CN114317574A (en) * | 2021-12-28 | 2022-04-12 | 扬州大学 | Cultivation and application of Marc-145 cell adaptive type 1 porcine reproductive and respiratory syndrome virus |
| CN114317574B (en) * | 2021-12-28 | 2023-12-08 | 扬州大学 | Marc-145 cell-adaptive type 1 pig breeding and respiratory syndrome virus cultivation and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111676247A (en) | Infectious clone construction, rescue and application of porcine reproductive and respiratory syndrome virus type 1 isolate | |
| CN113215192B (en) | Construction method of double-fluorescence-marker gene recombinant strain of porcine reproductive and respiratory syndrome virus | |
| CN113061586B (en) | Recombinant serum 4 type avian adenovirus for expressing serum 8 type avian adenovirus fiber protein and preparation method thereof | |
| CN111662885A (en) | Construction, rescue and application of infectious clone of virulent and attenuated strains of two highly homologous genome porcine reproductive and respiratory syndrome viruses | |
| CN110628817A (en) | Construction method and application of recombinant porcine reproductive and respiratory syndrome virus for expressing African swine fever virus p30 protein | |
| CN111996174A (en) | Porcine reproductive and respiratory syndrome virus, cloning vector thereof and gene insertion method | |
| CN110468155B (en) | System, method and application for rescuing porcine intestinal tract type A coronavirus | |
| CN111676198A (en) | Method for quickly constructing duck tembusu virus reverse genetic strain | |
| CN110904055A (en) | Porcine reproductive and respiratory syndrome virus recombinant vaccine strain PRRSV-SP and preparation method and application thereof | |
| US20080131459A1 (en) | Full-Length Infectious Cdna Clone for Porcine Reproductive and Respiratory Syndrome Virus(Prrsv) and Uses Thereof | |
| CN113046329B (en) | Porcine reproductive and respiratory syndrome virus chimeric recombinant PRRSV DIVA vaccine strain cDY56 | |
| CN116463297A (en) | Recombinant serum type 4 avian adenovirus expressing chicken infectious anemia virus VP1 protein and preparation method thereof | |
| CN113736799B (en) | Construction method and application of goat parainfluenza virus 3-type infectious cDNA clone | |
| CN115584352A (en) | Porcine Epidemic Diarrhea Virus (PEDV) ORF3 and E protein trans-complementary single-round infection system and application | |
| CN114395583B (en) | cDNA clone of porcine reproductive and respiratory syndrome virus for expressing secretion type luciferase, construction method and application thereof | |
| CN113584080A (en) | Construction and application of Nluc-labeled recombinant porcine delta coronavirus infectious clone plasmid | |
| Zhao et al. | Successful establishment of a reverse genetic system for QX-type infectious bronchitis virus and technical improvement of the rescue procedure | |
| CN111235114A (en) | EV71 replication-defective virus and preparation method and application thereof | |
| CN112522204A (en) | Recombinant MARC-145 cell and construction method and application thereof | |
| CN117925707A (en) | Infectious clone virus of virulent strain of NADC30-like porcine reproductive and respiratory syndrome virus and application thereof | |
| CN104694561A (en) | Construction method of PRRSV recombinant plasmids of expressing sea pansy or firefly luciferase gene and application thereof | |
| CN113584063B (en) | Reverse genetic operation method based on transcriptional regulatory sequence redistribution and application thereof | |
| CN115992100A (en) | Construction and application of NADC34-like porcine reproductive and respiratory syndrome virus reverse genetic operation platform | |
| CN114606207B (en) | Porcine epidemic diarrhea virus variant strain independent of pancreatin and construction and application thereof | |
| CN117187300A (en) | Preparation method and application of porcine reproductive and respiratory syndrome chimeric virus for expressing African swine fever virus related antigen protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200918 |