CN114958757B - Recombinant ST cell capable of stably expressing TreT, construction method and application thereof - Google Patents
Recombinant ST cell capable of stably expressing TreT, construction method and application thereof Download PDFInfo
- Publication number
- CN114958757B CN114958757B CN202011529269.0A CN202011529269A CN114958757B CN 114958757 B CN114958757 B CN 114958757B CN 202011529269 A CN202011529269 A CN 202011529269A CN 114958757 B CN114958757 B CN 114958757B
- Authority
- CN
- China
- Prior art keywords
- tret
- cells
- virus
- recombinant
- sus
- 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.)
- Active
Links
- 238000010276 construction Methods 0.000 title claims abstract description 12
- 241000700605 Viruses Species 0.000 claims abstract description 70
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 35
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 35
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 5
- 230000035755 proliferation Effects 0.000 claims description 25
- 238000012216 screening Methods 0.000 claims description 19
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 claims description 17
- 241000710777 Classical swine fever virus Species 0.000 claims description 16
- 238000001890 transfection Methods 0.000 claims description 11
- 239000006285 cell suspension Substances 0.000 claims description 10
- 108091026890 Coding region Proteins 0.000 claims description 7
- 238000004321 preservation Methods 0.000 claims description 7
- 239000013598 vector Substances 0.000 claims description 5
- 238000004113 cell culture Methods 0.000 claims description 2
- 238000009629 microbiological culture Methods 0.000 claims description 2
- 206010012735 Diarrhoea Diseases 0.000 claims 1
- LXWYCLOUQZZDBD-LIYNQYRNSA-N csfv Chemical compound C([C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)[C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O)C1=CC=C(O)C=C1 LXWYCLOUQZZDBD-LIYNQYRNSA-N 0.000 claims 1
- 108010078791 Carrier Proteins Proteins 0.000 abstract description 12
- 102000014914 Carrier Proteins Human genes 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 5
- 230000004083 survival effect Effects 0.000 abstract description 5
- 238000009825 accumulation Methods 0.000 abstract description 4
- 238000007710 freezing Methods 0.000 abstract description 3
- 230000008014 freezing Effects 0.000 abstract description 3
- 238000012423 maintenance Methods 0.000 abstract description 3
- 230000001902 propagating effect Effects 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 117
- 241001135549 Porcine epidemic diarrhea virus Species 0.000 description 13
- 238000003860 storage Methods 0.000 description 10
- 238000004114 suspension culture Methods 0.000 description 10
- 230000003612 virological effect Effects 0.000 description 9
- 230000005727 virus proliferation Effects 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 239000007788 liquid Substances 0.000 description 6
- 238000003776 cleavage reaction Methods 0.000 description 5
- 238000003306 harvesting Methods 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 230000001464 adherent effect Effects 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 241000256135 Chironomus thummi Species 0.000 description 2
- 108700010070 Codon Usage Proteins 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241000282898 Sus scrofa Species 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000012809 post-inoculation Methods 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 230000000644 propagated effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 241000256128 Chironomus <genus> Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 241001331386 Polypedilum vanderplanki Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000013373 clone screening Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002840 non-reducing disaccharides Chemical class 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 230000002381 testicular Effects 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 101150100915 treT gene Proteins 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0683—Cells of the male genital tract, e.g. prostate, epididymis; Non-germinal cells from testis, e.g. Leydig cells, Sertoli cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
-
- 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
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- 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
-
- 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
-
- 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
- C12N2510/00—Genetically modified cells
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16751—Methods of production or purification of viral material
-
- 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
- C12N2770/20011—Coronaviridae
- C12N2770/20051—Methods of production or purification of viral material
-
- 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
- C12N2770/24011—Flaviviridae
- C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
- C12N2770/24351—Methods of production or purification of viral material
-
- 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
-
- 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
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Reproductive Health (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Insects & Arthropods (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of biology, and particularly relates to a recombinant ST cell for stably expressing trehalose transport protein, and a construction method and application thereof. Recombinant ST cells capable of stably expressing trehalose transport proteins are obtained through a bioengineering means, and then ST cells with trehalose accumulation capacity are obtained through a way of adding trehalose into a culture environment. The cell can protect the virus in the process of propagating the progeny virus, and experiments prove that the recombinant ST cell propagates the virus, so that the survival rate of the virus is obviously improved at 37 ℃, and the obvious maintenance effect on the survival rate of the virus is shown for different freezing conditions.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a recombinant ST cell for stably expressing trehalose transport protein, and a construction method and application thereof.
Background
Trehalose is a non-reducing disaccharide, and is formed by connecting two molecules of glucopyranose through alpha, alpha-1, 1 glycosidic bonds. Has stable chemical property. Trehalose can provide effective protection for biological macromolecules in extreme environments such as high temperature, low temperature, high osmotic pressure, drying and the like.
The midge (Polypedilum vanderplanki) larvae can accumulate a large amount of trehalose under extreme environments such as desiccation or salt stress, so that the activity is recovered after the larvae are separated from the extreme environments. Thus, the aquatic midge is also known as "dehydrated organism". The reason for this is that the chironomus aquatic larvae can express trehalose transport proteins (Trehalose transporter, treT) in extreme environments, thereby transporting trehalose into the body and achieving accumulation of trehalose.
ST cells, porcine testicular cells (swovestis), are widely used in the proliferation of enveloped viruses.
The ST cells with the capacity of synthesizing or transporting trehalose are obtained through biotechnology means, so that the survival rate of progeny viruses in a low-temperature freezing environment is improved, and the ST cells have positive effects and important significance for products based on viruses.
Thus, finding an ST cell capable of synthesizing or transporting trehalose is a research hotspot for those skilled in the art. However, there is no report on the synthesis and transport of trehalose by mammals, and this is a difficulty for those skilled in the art.
Disclosure of Invention
Aiming at the report about the synthesis and transport of trehalose in mammals at present, the invention discloses a recombinant ST cell capable of stably expressing trehalose transport protein, a construction method of the cell and application of the cell in virus proliferation, thereby being expected to realize the transport and accumulation of trehalose in a culture environment into cells by animal cells.
Specifically, the invention discloses a recombinant ST cell (ST-Sus-Tret-1) capable of stably expressing trehalose transport protein, which is preserved in China general microbiological culture Collection center (CGMCC) at 11/6/2020, with the preservation number of CGMCC No.21010 and the preservation address of China academy of microorganisms at 3 of North Chenxi Lu No.1 in the Korean region of Beijing.
The recombinant ST cells contain a trehalose transport protein coding sequence Tret-1 shown in SEQ ID 1, wherein the trehalose transport protein coding sequence Tret-1 is a recombinant nucleotide sequence optimized according to pig codon preference.
Meanwhile, the invention also discloses a construction method of the recombinant ST cells, which comprises the following steps:
(1) Constructing a vector pCMV-Tret-1 containing a trehalose transporter coding sequence;
(2) The transfection complexes were added drop wise to ST cell culture broth, transfected, and G418 was used to screen for stable and continuously growing recombinant ST-Sus-Tret-1 cells in resistance.
Wherein, the ST cells are ST-Sus cells grown in single cell suspension.
Preferably, in the step (2), two rounds of screening are performed using G418, wherein the screening concentration in the first round is 500ug/ml, and the screening concentration in the second round is 1000ug/ml. Preferably, 20 successive rounds of screening are used.
Furthermore, the invention also discloses the application of the recombinant ST cells (ST-Sus-Tret-1) in proliferating viruses.
Specifically, in the invention, the target virus to be proliferated is further subjected to single cell clone screening on recombinant ST cells (ST-Sus-Tret-1), so that single cell clones aiming at the target virus are obtained, and the target virus is used for proliferation of the virus.
In a preferred embodiment, the recombinant ST cells ST-Sus-Tret-1 are used for the proliferation of Porcine Epidemic Diarrhea Virus (PEDV).
Furthermore, the invention also discloses a monoclonal ST-Sus-Tret-1-9E2 for proliferation of Porcine Epidemic Diarrhea Virus (PEDV).
In a preferred embodiment, the recombinant ST cells (ST-Sus-Tret-1) are used for the proliferation of Classical Swine Fever Virus (CSFV).
Furthermore, the invention also discloses a single cell clone ST-Sus-Tret-1-4C7 for proliferating Classical Swine Fever Virus (CSFV).
In a preferred embodiment, the recombinant ST cells (ST-Sus-Tret-1) are used for the proliferation of pseudorabies virus (PRV).
Furthermore, the invention also discloses a single cell clone ST-Sus-Tret-1-3D3 for propagating pseudorabies virus (PRV).
The invention obtains the recombinant ST cells capable of stably expressing the trehalose transport protein through a bioengineering means, and further obtains ST cells with trehalose accumulation capacity through adding trehalose into a culture environment. The cell can protect the virus in the process of propagating the progeny virus, and experiments prove that the recombinant ST cell propagates the virus, not only can the survival rate of the virus be obviously improved under the condition of 37 ℃, but also the survival rate of the virus can be obviously maintained under different freezing conditions.
Drawings
FIG. 1 is an electrophoresis chart of the enzyme digestion of a Tret-1 recombinant expression vector;
wherein M: DNA marker,1:pCMV-Tret-1 was digested with NheI and NotI.
FIG. 2 shows the detection of recombinant expression of Tret-1 by a cell clone recovering adherent growth after FACS sorting of ST-Sus-Tret-1 cells.
Detailed Description
For a better understanding of the present invention, we will further describe the present invention with reference to specific examples.
EXAMPLE 1 construction of ST-Sus-Tret-1 cells
1.1 vector construction
The nucleotide sequence codon of the trehalose transport protein (Tret) is optimized according to the pig codon preference, and the optimized recombinant nucleotide sequence Tret-1 is shown as SEQ ID NO. 1. The sequence was cloned into pUC57 vector to give pUC-Tret-1 vector.
The fragment is amplified from pUC-Tret-1 plasmid containing Tret-1 coding sequence by using upstream and downstream primers with NheI and NotI cleavage sites, and cloned into eukaryotic expression vector of same double cleavage after double cleavage, so as to construct eukaryotic expression vector pCMV-Tret-1 of Tret-1 for transfection operation.
Specifically, the primers p1, p2 used in this example are as follows:
p1:GCCGGTACCATGGAGCTGAACAACAAGGAGGAC (NheI cleavage site at the streak)
p2:GCCGCGGCCGCTCACTTGTCATCGTCGTCCTTG (NotI cleavage site at the streaked position)
The pUC-Tret-1 is used as a template, the coding sequence of the Tret-1 is amplified by PCR reaction, and a flag label is added at the C end. The target fragment amplified by PCR and the pCMV plasmid are treated by NheI and NotI double enzyme digestion, and a recombinant eukaryotic expression vector pCMV-Tret-1 is constructed, and the enzyme digestion identification is shown in figure 1. The plasmid for transfection with higher quality is obtained by extraction with Qiagen company plasmid big extraction kit.
1.2 production of ST-Sus-Tret-1 cells
The pCMV-Tret-1 is transfected into ST-Sus cells grown in single cell suspension, and the recombinant ST cells with resistance to G418 drugs and capable of stably expressing the Tret-1 protein are obtained by two rounds of screening with the G418 drugs.
Specifically, ST-Sus cells were first grown at an initial cell density of 5X 10 5 cells/ml were inoculated into 50ml Falcon tubes at 15ml,180rpm,37℃and 5% CO 2 After overnight incubation, used for transfection.
The medium was replaced with Opti-MEM medium prior to transfection and incubated in the incubator for 10 minutes.
The transfection complex was then formulated according to the instructions of the 25kDa branched PEI transfection reagent and incubated for 5 minutes at room temperature. After incubation is completed, the transfection complex is added drop-wise to ST-Sus cells, placed in an incubator for 6-8 hours, during which the cells are intermittently agitated.
After the transfection was completed, the culture supernatant containing the remaining transfection complex was removed and replaced with a normal medium for suspension culture.
ST-Sus cells 24-72 hours after transfection were re-inoculated in a 1:3-1:5 ratio into 50ml Falcon tubes at 180rpm,37℃at 5% CO 2 Recombinant cell selection was performed in G418 medium containing 500 ug/ml. G418 was performed at a frequency of 3 days for the primary fluidThe screening was continued for a first round of screening time of approximately 2 to 3 weeks (20 consecutive generations). Recombinant ST-Sus-Tret-1 cells capable of sustained proliferation under G418 selection pressure were selected for continued culture. In the second round of screening, the working concentration of G418 screening is increased to 1000ug/ml, and the pressure screening is continued for 20 generations according to the screening method of the first round, so that the recombinant ST-Sus-Tret-1 cells which can stably and continuously grow in the G418 screening resistance are finally obtained.
EXAMPLE 2 preparation of monoclonal ST-Sus-Tret-1-9E2, monoclonal ST-Sus-Tret-1-4C7 and monoclonal ST-Sus-Tret-1-3D3
Screening recombinant ST-Sus-Tret-1 cells which stably and continuously grow in the resistance obtained in the example 1, sorting the cells into a 96-well plate by a flow cytometer to form single cell clones in each well, and preparing cell clone double plates after the cells recover to adherent growth to form 173 cell clones which effectively adherent grow, wherein the detection of the recombinant expression of the Tret-1 of the cell clones is shown in figure 2.
Aiming at PEDV, CSFV, PRV proliferation, screening single cell clones by adopting PEDV, CSFV and PRV, screening cell clones which are optimal for proliferation of the three viruses from 173 cell clones, and naming the cell clones respectively to obtain single cell clone ST-Sus-Tret-1-9E2 which is high-efficiency proliferation of the PEDV, single cell clone ST-Sus-Tret-1-4C7 which is high-efficiency proliferation of the CSFV and single cell clone ST-Sus-Tret-1-3D3 which is high-efficiency proliferation of the PRV.
EXAMPLE 3 use of single cell suspension grown ST-Sus-Tret-1-9E2 cells in proliferation of PEDV
ST-Sus-Tret-1-9E2 cells were plated at 1X 10 6 Single cell suspension culture was performed at 180rpm at 37℃under 5% CO at the initial culture density of cells/ml 2 And serum-free suspension growth medium for ST-Sus cells.
To the viable cell density of 4.5X10 6 The cells/ml and PEDV receives virus according to MOI=0.1, the virus proliferation period is 24-36 hours, and the virus titer reaches 8.75-9.0 lgTCID 50 /ml。
Example 4 comparison of the effects of ST-Sus-Tret-1-9E2 cells with Primary ST-Sus cells in PEDV proliferation applications
4.1 ST-Sus-Tret-1-9E2 cells were expressed as 1X 10 6 Single cell suspension culture was performed at 180rpm at 37℃under 5% CO at the initial culture density of cells/ml 2 And serum-free suspension growth medium for ST-Sus cells. To achieve a viable cell density of 10×10 6 PEDV was inoculated at a rate of moi=0.3, and the virus growth was carried out by increasing the volume of the virus growth maintenance solution (containing trehalose 0.1 to 80 um) by 1 time, with the growth cycle being 24 to 36 hours.
The living cells of ST-Sus-Tret-1-9E2 grown by single cell suspension are reduced to 0.1-1.2X10 6 Virus harvest can be performed at cells/ml.
The harvested virus can be preserved at 4-70 ℃ for 1 year. The proliferation potency of PEDV can reach 8.75-9.0 lgTCID 50 Per ml, the loss of viral potency after one year of storage at-20 ℃ is less than 0.5lgTCID 50 Per ml, the loss of viral potency after one year of storage at-70 ℃ is less than 0.2lgTCID 50 /ml。
4.2 the original ST-Sus cells were grown at 1X 10 6 Single cell suspension culture was performed at 180rpm at 37℃under 5% CO at the initial culture density of cells/ml 2 And serum-free suspension growth medium for ST-Sus cells. To achieve a viable cell density of 10×10 6 PEDV was inoculated at a rate of moi=0.3, and the virus growth was carried out by increasing the volume of the virus growth maintenance solution (containing trehalose 0.1 to 80 um) by 1 time, with the growth cycle being 24 to 36 hours.
The living cells of ST-Sus grown in single cell suspension are reduced to 0.1-1.2X10 6 Virus harvest can be performed at cells/ml.
The proliferation potency of the original ST-Sus cells is 7.33-7.77 lgTCID 50 Per ml, the loss of viral potency after one year of storage at-20℃is greater than 2.5lgTCID 50 Per ml, the loss of viral potency after one year of storage at-70 ℃ is greater than 1.5lgTCID 50 /ml。
EXAMPLE 5 use of single cell suspension grown ST-Sus-Tret-1-4C7 cells in the proliferation of CSFV
ST-Sus-Tret-1-4C7 cells were plated at 1X 10 6 Single cell suspension culture is carried out by taking cells/ml as initial culture density, and the density of living cells reaches 2.5X10 6 The cells/ml, CSFV receives virus according to 2% (volume ratio), the virus proliferation period is 13-15 days, the virus liquid is harvested in 3-5 batches, and the virus titer reaches 200-250 ten thousand RID/ml.
Example 6 effect comparison of ST-Sus-Tret-1-4C7 cells with Primary ST-Sus cells in CSFV proliferation application
6.1 ST-Sus-Tret-1-4C7 cells were expressed as 1X 10 6 Single cell suspension culture was performed at 180rpm at 37℃under 5% CO at the initial culture density of cells/ml 2 And serum-free suspension growth medium for ST-Sus cells. To achieve a viable cell density of 2.5X10 6 The cells/ml, CSFV receives the virus according to 2% (volume ratio, seed virus titer is at least 20 ten thousand RID/ml), the virus multiplication of CSFV is carried out by increasing 1 time volume of virus multiplication maintaining liquid (containing trehalose 0.1 um-80 um), the virus multiplication period is 13-15 days, the virus liquid is harvested in 3-5 batches, each batch of virus culture liquid is harvested except the last generation, and fresh virus multiplication maintaining liquid is used for supplementing to the original volume of culture. Specific post-inoculation 2/3 volumes of virus solution were harvested on day 4, 2/3 volumes of virus solution on day 7, 1/2 volumes of virus solution on day 10, 1/2 volumes of virus solution on day 13, and all virus cultures on day 15. The harvested virus can be preserved at 4 to-70 ℃.
The CSFV proliferation potency can reach 200-250 ten thousand RID/ml (different virus potency of different virus harvest generations are different), the virus potency loss is less than 50 ten thousand RID/ml after preservation for one year at 20 ℃, and the virus potency loss is less than 20 ten thousand RID/ml after preservation for one year at 70 ℃.
6.2 the original ST-Sus cells were grown at 1X 10 6 Single cell suspension culture was performed at 180rpm at 37℃under 5% CO at the initial culture density of cells/ml 2 And serum-free suspension growth medium for ST-Sus cells. To achieve a viable cell density of 2.5X10 6 CSFV is inoculated with 2% virus (volume ratio, seed virus titer is at least 20 thousand RID/ml), virus proliferation of CSFV is carried out by adding 1 time volume of virus proliferation maintaining solution (containing trehalose 0.1-80 um), virus proliferation period is 13-15 days, virus solution is harvested in 3-5 batches, each batch of virus culture solution is harvested except the last generation, and fresh virus proliferation is maintainedThe liquid is complemented to the original volume of the culture. Specific post-inoculation 2/3 volumes of virus solution were harvested on day 4, 2/3 volumes of virus solution on day 7, 1/2 volumes of virus solution on day 10, 1/2 volumes of virus solution on day 13, and all virus cultures on day 15.
The original ST-Sus cell proliferation potency is 50-100 ten thousand RID/ml (the virus potency of different virus collection generations are different), the virus potency loss is greater than 100 ten thousand RID/ml after one-year preservation at 20 ℃, and the virus potency loss is greater than 50 ten thousand RID/ml after one-year preservation at 70 ℃.
EXAMPLE 7 use of single cell suspension grown ST-Sus-Tret-1-3D3 cells in proliferation of PRV
ST-Sus-Tret-1-3D3 cells were grown at 1X 10 6 Single cell suspension culture is carried out by taking cells/ml as initial culture density, and the density of living cells reaches 3.0x10 6 The cells/ml and PRV are inoculated according to MOI=0.05, the virus proliferation period is 48-66 hours, and the virus titer reaches 9.0-9.5 lgTCID 50 /ml。
Example 8 comparison of the effects of ST-Sus-Tret-1-3D3 cells with Primary ST-Sus cells in PRV proliferation applications
8.1 ST-Sus-Tret-1-3D3 cells were expressed as 1X 10 6 Single cell suspension culture was performed at 180rpm at 37℃under 5% CO at the initial culture density of cells/ml 2 And serum-free suspension growth medium for ST-Sus cells. To achieve a viable cell density of 3.0X10 6 PRV was inoculated at a rate of mol=0.05, and PRV virus was propagated by increasing the volume of virus growth maintaining solution (containing trehalose 0.1 to 80 um) by 1 time, with a period of 48 to 66 hours. The living cells of ST-Sus-Tret-1-3D3 grown by single cell suspension are reduced to 0.1-1.0X10 6 Virus harvest can be performed at cells/ml. The harvested virus can be preserved at 4-70 ℃ for 1 year.
PRV proliferation potency can reach 9.0-9.5 lgTCID 50 Per ml, less than 0.5lgTCID after 3 months of storage at 4 DEG C 50 Per ml, the loss of viral potency after one year of storage at-20 ℃ is less than 1.0lgTCID 50 Per ml, the loss of viral potency after one year of storage at-70 ℃ is less than 0.2lgTCID 50 /ml。
9.2 willPrimitive ST-Sus cells were grown at 1X 10 6 Single cell suspension culture was performed at 180rpm at 37℃under 5% CO at the initial culture density of cells/ml 2 And serum-free suspension growth medium for ST-Sus cells. To achieve a viable cell density of 3.0X10 6 PRV was inoculated at a rate of mol=0.05, and PRV virus was propagated by increasing the volume of virus growth maintaining solution (containing trehalose 0.1 to 80 um) by 1 time, with a period of 48 to 66 hours. The living cells of ST-Sus grown in single cell suspension are reduced to 0.1-1.0X10 6 Virus harvest can be performed at cells/ml.
The proliferation potency of the original ST-Sus cells is 8.0-8.5 lgTCID 50 Per ml, the loss of viral titer after 3 months of storage at 4℃is greater than 1.5lgTCID 50 Per ml, the loss of viral potency after one year of storage at-20℃is greater than 4.5lgTCID 50 Per ml, the loss of viral potency after one year of storage at-70 ℃ is greater than 2.0lgTCID 50 /ml。
What has been described above is a specific embodiment of the present invention. It should be noted that modifications and adaptations to the invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.
Sequence listing
<110> academy of agricultural sciences in Jiangsu province
<120> recombinant ST cells capable of stably expressing TreT, construction method and application thereof
<130> 202010095
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1539
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
atggagctga acaacaagga ggacagcccg cggcacacag ttccttttgt gcgccaaatc 60
acagaagatg ggaaggcgaa gctggaaatt tatcggccga ctaccaaccc catttatatc 120
tacacccaga ttctggcggc catcgctgtg agcatggggt ctatggtggt gggctttgcc 180
tctgcctaca catctcctgc cctggtatcc atgcagaaca ccacaattac ttcttttaaa 240
gtgaccgagc aggaggcctc ctgggtggga ggtatcatgc cactggcggg gctggcgggg 300
ggcatcgctg gaggaccatt tattgagtac ctcgggcgta aaaataccat acttgccacg 360
gctgtgccct tcatcgttgc ctggttactt attgctttcg ccaactccat ctggatggtg 420
ctggctggaa gggccctctc aggattctgc gtgggtatag cgtcgctttc gctgccagtg 480
tacttggggg agaccgtgca gcccgaggtc cggggaaccc ttgggctgct acctacagct 540
tttggaaaca tcggaatcct tatctgcttc gtagcaggca aatatgtgaa ctggagtgga 600
ctcgctttta tcgggagcat tttgcccatt cctttcatgg ttctgaccct gctgatccca 660
gagactccac gctggtttgt gacgcgcggg cgagaagaaa gagccagaaa agcactgcag 720
tggctccgtg gcaagaaggc agatgtcgaa cccgaactga aaggtatagt gaagagccac 780
tgtgaagcag agcgccatgc ttcacagaat gccatctttg acctcatgaa gaggagcaat 840
ctgaaaccgc tccttattgc tttaggcttg atgtttttcc agcagctgag cggtattaat 900
gcagtgattt tttatacagt gagtatcttc aaggacgccg gcagtacaat cgatgagaac 960
ttatgcacca taatcgtcgg cgtagtcaat tttggggcca ctttctttgc aactgtgttg 1020
attgacagac tggggaggaa aattctcctc tacatctccg aagtggccat ggtcatcacg 1080
ttgttaactc tgggcacctt cttctactac aagaattcag gaaatgatgt tagtaacatt 1140
ggctggctcc ccctagcatc cttcgtcatc tatgtcattg ggttctcctc tggagttggc 1200
cccatcccct ggttgatgct gggagaaatc ctccctggaa agatccgggg ttctgctgca 1260
agtgtggcga cgggcttcaa ctggacctgt actttcattg tcacaaagac ttttgctgac 1320
atcgtggctg ccattggcaa tcatggcgcc ttctggttct ttggcgttat atgtctgatt 1380
ggcctcttct tcgtaatatt ttttgttcca gaaacacaag gtaaatccct ggaggagata 1440
gagaggaaga tgatgggccg agtcaggaga atgagtagcg tcgcaaacat gaaaccttta 1500
tcattcaata tggattacaa ggacgacgat gacaagtga 1539
Claims (7)
1. A recombinant ST cell (ST-Sus-TreT-1) capable of stably expressing a trehalose transporter (TreT), characterized in that: the cell is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.21010 in the year 11 and 6 of 2020.
2. The recombinant ST cell of claim 1, wherein: the recombinant ST cells contain trehalose transporter coding sequence Tret-1 shown in SEQ ID NO. 1.
3. The method for constructing a recombinant ST cell according to claim 1, comprising the steps of:
(1) Constructing a vector pCMV-Tret-1 containing a trehalose transporter coding sequence;
(2) The transfection complexes were added drop wise to ST cell culture broth, transfected, and the recombinant ST cells stably and continuously grown in resistance were selected using G418 (ST-Sus-Tret-1).
4. A method of construction according to claim 3, wherein: the ST cells are ST-Sus cells grown in single cell suspension.
5. The construction method according to claim 3 or 4, wherein: in the step (2), two rounds of screening are carried out by using G418, wherein the screening concentration of the first round is 500ug/ml, and the screening concentration of the second round is 1000ug/ml.
6. The construction method according to claim 5, wherein: each round used 20 successive rounds of screening.
7. Use of the recombinant ST cell (ST-Sus-Tret-1) of claim 1 for the proliferation of viruses, such as epidemic diarrhea virus (PEDV), proliferation of swine fever virus (CSFV), pseudorabies virus (PRV).
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011529269.0A CN114958757B (en) | 2020-12-22 | 2020-12-22 | Recombinant ST cell capable of stably expressing TreT, construction method and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011529269.0A CN114958757B (en) | 2020-12-22 | 2020-12-22 | Recombinant ST cell capable of stably expressing TreT, construction method and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114958757A CN114958757A (en) | 2022-08-30 |
CN114958757B true CN114958757B (en) | 2023-10-24 |
Family
ID=82954265
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011529269.0A Active CN114958757B (en) | 2020-12-22 | 2020-12-22 | Recombinant ST cell capable of stably expressing TreT, construction method and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114958757B (en) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105026561A (en) * | 2012-09-28 | 2015-11-04 | 加州大学评议会 | Trophic conversion of photoautotrophic bacteria for improved diurnal properties |
CN105112352A (en) * | 2015-07-13 | 2015-12-02 | 郑州爱科生物科技有限公司 | ST cell adapting to full-suspension culture, and application thereof, and vaccine virus culturing method |
CN109517800A (en) * | 2018-12-05 | 2019-03-26 | 江苏省农业科学院 | The reconstruct ST cell and its construction method and purposes that one plant of reinforcement cholesterol is endogenously synthesized |
CN110387354A (en) * | 2019-08-16 | 2019-10-29 | 江苏省农业科学院 | Pseudorabies virus Attenuation strain and its application |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004110372A2 (en) * | 2003-05-30 | 2004-12-23 | Stratatech Corporation | Room temperature storage of organs |
US7892789B2 (en) * | 2007-03-05 | 2011-02-22 | Takahiro Kikawada | Method of increasing cell permeability to trehalose by recombinantly producing a trehalose transporter |
-
2020
- 2020-12-22 CN CN202011529269.0A patent/CN114958757B/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105026561A (en) * | 2012-09-28 | 2015-11-04 | 加州大学评议会 | Trophic conversion of photoautotrophic bacteria for improved diurnal properties |
CN105112352A (en) * | 2015-07-13 | 2015-12-02 | 郑州爱科生物科技有限公司 | ST cell adapting to full-suspension culture, and application thereof, and vaccine virus culturing method |
CN109517800A (en) * | 2018-12-05 | 2019-03-26 | 江苏省农业科学院 | The reconstruct ST cell and its construction method and purposes that one plant of reinforcement cholesterol is endogenously synthesized |
CN110387354A (en) * | 2019-08-16 | 2019-10-29 | 江苏省农业科学院 | Pseudorabies virus Attenuation strain and its application |
Non-Patent Citations (3)
Title |
---|
Intracellular trehalose via transporter TRET1 as a method to cryoprotect CHO-K1 cells;Tsutomu Uchida等;《Cryobiology》;第77卷;第50-57页 * |
Trehalose uptake and dehydration effects on the cryoprotection of CHO–K1 cells expressing TRET1;Tsutomu Uchida等;《Cryobiology》;第90卷;第30-40页 * |
猪传染性胃肠炎病毒在ST细胞中增殖规律的研究;周燕, 王建超, 华平, 邹勇, 谭文松;中国兽医科技(第06期);第423-427页 * |
Also Published As
Publication number | Publication date |
---|---|
CN114958757A (en) | 2022-08-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112522173B (en) | Engineering bacterium for producing heterologous alkaline protease and construction method thereof | |
CN113604425A (en) | WAYNE293LVPRO cell adapted to serum-free medium environment and application thereof | |
CN102453716A (en) | Clone and application of pig skeletal muscle specificity expression gene alpha-actin promoters | |
CN114958757B (en) | Recombinant ST cell capable of stably expressing TreT, construction method and application thereof | |
CN111944810B (en) | sgRNA for targeted deletion of TNF alpha gene, TNF alpha gene-deleted porcine embryo fibroblast line and application thereof | |
CN103305504A (en) | Composition and method for site-specific recombination in hamster cells | |
CN116286931B (en) | Double-plasmid system for rapid gene editing of Ralstonia eutropha and application thereof | |
CN109295075B (en) | NfOCP1 drought-resistant gene, amino acid sequence coded by same and application thereof in improving plant drought resistance | |
CN108220216B (en) | Ammonium-resistant nitrogen-fixing microorganism for over-expression of glnR gene and construction method and application thereof | |
CN115948402A (en) | Recombinant Shewanella capable of producing 5-aminolevulinic acid and application thereof | |
JP2024502061A (en) | Recombinant Bacillus microorganism and method for producing breast milk oligosaccharides using the same | |
CN116024092A (en) | Chlorella pyrenoidosa improved variety resource with high protein yield and high genetic transformation efficiency and application thereof | |
CN107602675B (en) | Nostoc flagelliforme Nfcuspin 1 drought-resistant gene and amino acid sequence and application thereof | |
CN114478799A (en) | Artificial tandem polypeptide ADPs for improving nitrogen fixation efficiency of rhizobia, expression vector and construction and application of recombinant rhizobia | |
CN109207490B (en) | Nfgdh drought-resistant gene, amino acid sequence coded by same and application thereof in improving plant drought resistance | |
CN107603988B (en) | Nostoc flagelliforme NfcrtO drought-resistant gene and amino acid sequence and application thereof | |
CN111019873B (en) | Method for rapidly integrating large-fragment DNA on genome of clostridium acetobutylicum | |
CN115161287A (en) | Recombinant porcine reproductive and respiratory syndrome virus and construction method and application thereof | |
CN114774421B (en) | Mutant of endogenous promoter of zymomonas mobilis | |
CN107603989B (en) | Nostoc flagelliforme Nfgal E drought-resistant gene and amino acid sequence and application thereof | |
CN115976086B (en) | Method for editing bacteria CRISPR-Cas9 gene and application thereof | |
CN115851795B (en) | High-yield plasmid, construction method and application thereof | |
CN116042684B (en) | Coli and application thereof in catalytic synthesis of psicose | |
CN109593730B (en) | Duck plague virus Lorf5 gene traceless deletion strain DPV CHv-delta Lorf5 and construction method thereof | |
CN109337917B (en) | Nfdirp drought-resistant gene, coded amino acid sequence thereof and application thereof in improving plant drought resistance |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |