CN105671051B - Application of the duck BCL2L15 gene in the anti-AIV virus of livestock and poultry - Google Patents
Application of the duck BCL2L15 gene in the anti-AIV virus of livestock and poultry Download PDFInfo
- Publication number
- CN105671051B CN105671051B CN201610084095.9A CN201610084095A CN105671051B CN 105671051 B CN105671051 B CN 105671051B CN 201610084095 A CN201610084095 A CN 201610084095A CN 105671051 B CN105671051 B CN 105671051B
- Authority
- CN
- China
- Prior art keywords
- gene
- bcl2l15
- cell
- virus
- duck
- 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.)
- Expired - Fee Related
Links
- 101150075099 Bcl2l15 gene Proteins 0.000 title claims abstract description 56
- 241000272525 Anas platyrhynchos Species 0.000 title abstract description 30
- 241000700605 Viruses Species 0.000 title abstract description 26
- 244000144977 poultry Species 0.000 title abstract description 8
- 244000144972 livestock Species 0.000 title abstract description 7
- 230000014509 gene expression Effects 0.000 claims abstract description 23
- 230000009261 transgenic effect Effects 0.000 claims abstract description 8
- 230000029812 viral genome replication Effects 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 52
- 241001473385 H5N1 subtype Species 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 5
- 210000002950 fibroblast Anatomy 0.000 claims description 3
- 210000003837 chick embryo Anatomy 0.000 claims description 2
- 101150099105 alien gene Proteins 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 31
- 101000971075 Homo sapiens Bcl-2-like protein 15 Proteins 0.000 abstract description 21
- 102100021896 Bcl-2-like protein 15 Human genes 0.000 abstract description 17
- 238000000034 method Methods 0.000 abstract description 13
- 241001465754 Metazoa Species 0.000 abstract description 11
- 238000002474 experimental method Methods 0.000 abstract description 9
- 206010064097 avian influenza Diseases 0.000 abstract description 8
- 238000005516 engineering process Methods 0.000 abstract description 6
- 208000002979 Influenza in Birds Diseases 0.000 abstract description 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 4
- 150000001413 amino acids Chemical class 0.000 abstract description 3
- 230000000840 anti-viral effect Effects 0.000 abstract description 2
- 238000010362 genome editing Methods 0.000 abstract description 2
- 230000005764 inhibitory process Effects 0.000 abstract description 2
- 241000712461 unidentified influenza virus Species 0.000 description 33
- 239000013642 negative control Substances 0.000 description 13
- 206010022000 influenza Diseases 0.000 description 12
- 230000003612 virological effect Effects 0.000 description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 201000010099 disease Diseases 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 241000287828 Gallus gallus Species 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000001717 pathogenic effect Effects 0.000 description 4
- 231100000614 poison Toxicity 0.000 description 4
- 239000002574 poison Substances 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 3
- 101150112014 Gapdh gene Proteins 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 230000007918 pathogenicity Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000272522 Anas Species 0.000 description 2
- 241000272814 Anser sp. Species 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 241001500351 Influenzavirus A Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 108091034135 Vault RNA Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 210000005013 brain tissue Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000003208 gene overexpression Methods 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 231100000518 lethal Toxicity 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 231100000816 toxic dose Toxicity 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 241000271566 Aves Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000702626 Infectious bursal disease virus Species 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 108010015268 Integration Host Factors Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241000713869 Moloney murine leukemia virus Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 231100000645 Reed–Muench method Toxicity 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 101150003160 X gene Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000009361 aviculture Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010307 cell transformation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002498 deadly effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 208000037797 influenza A Diseases 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000001617 migratory effect Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 229960004854 viral vaccine Drugs 0.000 description 1
- 206010048282 zoonosis Diseases 0.000 description 1
Classifications
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Environmental Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Toxicology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to antiviral gene fields, specifically disclose application of the duck BCL2L15 gene in the anti-AIV virus of livestock and poultry.The present invention provides a new gene BCL2L15 relevant to anti-avian influenza, and utilizes the method for molecular biology combination cell biological experiment for the first time, it was demonstrated that BCL2L15 has the function of inhibition AIV virus replication.Analysis shows, relative to no cell for being overexpressed BCL2L15 gene, the cell for being overexpressed BCL2L15 gene can significantly inhibit the duplication of AIV virus, therefore can carry out deep functional study for duck BCL2L15 gene, so that it is determined that the key protein structural domain or amino acid of its anti-AIV virus.Using the gene editings method such as transgenic technology, the transgenosis livestock and poultry of inducibility height expression duck or other animal BCL2L15 genes are obtained, the transgenosis agricultural animal excellent variety of the multiple types such as anti-AIV virus is cultivated.
Description
Technical field
The present invention relates to antiviral gene fields, specifically, being related to duck BCL2L15 gene in the anti-AIV virus of livestock and poultry
Application.
Background technique
Influenza virus is the minus-stranded rna virus containing 8 rna gene group segments.Highly pathogenic bird flu (Highly
Pathogenic Avian Influenza, HPAI) it is as caused by orthomyxovirus section Influenzavirus A influenza virus with fowl
Deadly infectious disease based on class.Since bird flu occurs for Italian report in 1878, two hypotype strains of H5 and H7 continue
Influenza Outbreak is caused in family chicken all over the world or turkey, seriously threatens the development of aviculture.Recently research have indicated that last century 4
Have in secondary worldwide flu outbreak 3 times, i.e. spanish influenza in 1918, (H2N2) the hypotype Asia influenza of the first of nineteen fifty-seven 2 and
First 3 (H3N2) hypotype Mao flu of nineteen sixty-eight, it is closely related with avian influenza virus.This 3 worldwide flu outbreaks are to people
Class brings serious disaster, causes upheaval and huge economic loss to society.World Organization for Animal Health (OIE) is by bird flu
(AIV) it is classified as the zoonosis that must be reported, China is classified as a kind of animal epidemic.
Aquatic bird includes the natural host that a duck is influenza virus, and the hypotype of the various combination of all 16 kinds of HA and 9 kinds of NA is all
It can be separated in aquatic bird.Influenza virus is always maintained at the feature of low pathogenicity to aquatic bird, and aquatic bird virus infection is not fallen ill, but
It can outwardly toxin expelling.The low strain of certain pairs of aquatic bird pathogenicities then shows as chicken or other hosts highly pathogenic.However,
With the continuous evolution of influenza virus, the equilibrium state of aquatic bird and influenza virus is broken.As being reported in Hong Kong for the first time within 2002
There are the H5N1 hypotype strain of lethal aquatic bird, the 2005 lethal migratory bird events of Qinghai lake large-scale outbreak H5N1 subtype influenza virus
Deng.Popular new strain has various ways in crowd: avian influenza virus or other influenza viruses and human influenza virus send out
Raw gene rearrangement generates epidemic strain, avian flu that popular strain, the avian influenza virus of infection people generate after adapting in pig body
Poison travels to the popular strain of people's generation and the old strain of human influenza virus is popular again after the several years, in addition, there are also the stream of peoples
The antigenic drift etc. of Influenza Virus itself.
In short, the maximum feature of influenza A is that host is extensive, hypotype is numerous, and variant form multiplicity, morbidity is unexpected,
Popular, pathogenic strong, easily induction severe complication, harm is huge.Therefore, scientists the variation to study flu virus,
It evolves, the popular and regularity of distribution is also dedicated to identify the immune base of new resisiting influenza virus while improving prevention and control influenza ability
Cause, parsing host influence the molecular mechanism research of influenza virus infectious, pathogenicity and immune response, to improve the anti-of host
Characteristic of disease energy and the exploitation for promoting prevention and control influenza virus new tool.
Summary of the invention
In order to solve the problems in the existing technology, it is anti-in livestock and poultry that the object of the present invention is to provide duck BCL2L15 genes
Application in AIV virus.
In order to achieve the object of the present invention, technical scheme is as follows:
It is SEQ ID that the present invention provides BCL2L15 genes influencing the application in virus replication, the BCL2L15 gene
Nucleotide sequence shown in nucleotide sequence shown in NO.1 or SEQ ID No.1 is substituted, it is one or several to be deleted and/or added
The nucleotide nucleotide sequence obtained as derived from SEQ ID No.1 with same function.The ammonia of its protein expressed
Base acid sequence is as shown in SEQ ID NO.2.
Specifically, the application includes the overexpression by BCL2L15 gene in cell to inhibit virus in cell
In duplication, and viral duplication dramatically increases in the cell for knocking out BCL2L15.
The virus can be AIV, NDV, IBDV etc..
Preferably, the virus is avian influenza virus.BCL2L15 gene crosses table in the cell of infection avian influenza virus
It reaches, the duplication of avian influenza virus can be significantly inhibited.
Further, the application the present invention also provides BCL2L15 gene in anti-AIV virus.It should be noted that this hair
The bright application that the gene is claimed in anti-AIV virus inhibits since anti-AIV virus not equivalent to treats bird flu
The animal that AIV virus is also not equivalent to infected in the duplication of AIV virus can be cured, therefore, the claimed skill of the present invention
Art scheme is not related to the diagnostic and therapeutic method of disease.
More specifically, the present invention provides a kind of specific method, by the CDS sequence construct of BCL2L15 gene to can
In the carrier of efficiently expressing exogenous gene, recombinant vector is obtained, and recombinant vector is imported in birds DF1 cell.
Preferably, the carrier is the carrier for expression of eukaryon containing strong promoter, to realize the table excessively of BCL2L15 gene
It reaches.
More preferably, in order to preferably realize the overexpression of BCL2L15 gene, nucleotide sequence such as SEQ ID
Shown in NO.3.The carrier starts target gene high expression in eukaryocyte by CAG promoter, and have independent GFP and
The bis- selection markers of Neo, using in Transposon System high effective integration cellular genome.
When using above-mentioned vector construction recombinant vector, the CDS sequence of BCL2L15 gene is inserted into SEQ ID NO.3 institute
Show at the 2828bp of sequence.
The present invention also provides a kind of transgenic cells containing BCL2L15 gene.
Preferably, the transgenic cell is chick embryo fibroblast immortalized cell line DF1.
The present invention also provides a kind of vehicles cells, the vehicles cells are the cell for being overexpressed BCL2L15 gene.It is described
Cell does not have the function of totipotential differentiation.
The present invention also provides a kind of vehicles cells for production of vaccine, the vehicles cells are to have knocked out BCL2L15
The cell of gene.The cell does not have the function of totipotential differentiation.
The present invention also provides a kind of construction methods of transgenic animals, and the method includes being transferred to this hair in animal body
The bright duck BCL2L15 gene.
The beneficial effects of the present invention are:
The prior art only discloses BCL2L15 gene and participates in apoptosis process, with cell transformation and gastroenteric tumor
Occur related.And the present invention provides the theoretical foundations that the viral transgenes animal such as anti-AIV is prepared using BCL2L15 gene.
The present invention analyzes to obtain BCL2L15 gene mRNA sequencing fragment according to transcript profile data result, respectively at two plants
H5N1 strain (A/duck/Hubei/49/05, DK/49, height cause a disease) and (A/goose/Hubei/65/05, GS/65, low cause
Disease), the expression in the 1st, the 2nd and the 3rd day spleen, lung and brain tissue after infecting 4 week old Shaoxing ducks is significantly increased (see Fig. 1),
Confirm that duck BCL2L15 gene is resistant to the important candidate gene of influenza virus.
The present invention provides a new gene BCL2L15s relevant to anti-avian influenza, and utilize molecular biology knot for the first time
The method for closing Cell Biology Experiment, it was demonstrated that BCL2L15 has the function of inhibition AIV virus replication.
BCL2L15 gene of the present invention can be used for preparing the transgenic animals of resisiting influenza virus, in addition, knocking out
The cell line of BCL2L15 gene can be used as the vehicles cells of the viral vaccines such as AIV production.
BCL2L15 gene provided by the present invention can be used for inhibiting influenza virus, can especially significantly inhibit AIV virus
Duplication, therefore deep functional study can be carried out for duck BCL2L15 gene, so that it is determined that the key of its anti-AIV virus
Protein structure domain or amino acid.Using the gene editings method such as transgenic technology, obtains inducibility height and express BCL2L15 gene
Transgenosis livestock and poultry, cultivate the transgenosis agricultural animal excellent variety of the multiple types such as anti-AIV virus.
Detailed description of the invention
Fig. 1 is the duck BCL2L15 gene mRNA sequence that the present invention is analyzed using transcript profile data, respectively at two plants
H5N1 strain (A/duck/Hubei/49/05, DK/49, height cause a disease) and (A/goose/Hubei/65/05, GS/65, low cause
Disease), the expression pattern in the 1st, the 2nd and the 3rd day spleen, lung and brain tissue after infecting 4 week old Shaoxing ducks；When abscissa represents
Between point, ordinate represents relative expression quantity.
Fig. 2 is the map of carrier PiggyBac described in the embodiment of the present invention 2, and wherein X gene is BCL2L15.
Fig. 3 is after transiently transfecting BCL2L15 and negative control plasmids in the embodiment of the present invention 2 for 24 hours under the mirror of DF1 cell
Observe result.
Fig. 4 is after transiently transfecting BCL2L15 and negative control plasmids 48h in the embodiment of the present invention 2, to collect the total egg of cell
The Western blot of the overexpression effect of BCL2L15 gene is verified after white.Experiment process group, which is followed successively by, is overexpressed BCL2L15
Group (OE), negative control group (Mock), using GAPDH gene as internal reference.
Fig. 5 is to be overexpressed BCL2L15 gene in the embodiment of the present invention 3 in DF1 cell, inhibits influenza disease in embodiment 6
The duplication of malicious DK/49 (left side) and GS/65 (right side), abscissa indicate the time point that cell supernatant is collected after infecting, ordinate table
Show the logarithm of EID50.
Fig. 6 is endogenous BCL2L15 base in the embodiment of the present invention 3 after DF1 cell infection influenza virus A IV (DK/49) 48h
Because the expression of mRNA changes, using GAPDH gene as internal reference.
Fig. 7 is that cell is overexpressed before and after BCL2L15 gene in the embodiment of the present invention 3, influenza virus infection AIV (DK/49)
Afterwards, relative expression's variation of viral different form RNA, using GAPDH gene as internal reference.
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field
Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Include in the present embodiment experimental implementation:
1) extraction of total serum IgE and reverse transcription reaction
The Trizol reagent of the extraction and application Invitrogen company of tissue or cell total rna production, and in strict accordance with production
Product specification is operated；Reverse transcription reaction using Promega company MMLV Reverse Transcriptase Reagents, and in strict accordance with saying
Bright book is operated.
2) gene cloning and vector construction
The cDNA obtained using reverse transcription carries out PCR amplification as template, using special primer.Product connects after purification by gel
It is connected on pEasy-Blunting simple carrier, picking monoclonal colonies, extracts plasmid after sequencing identification is correct.Plasmid is double
After digestion, target gene is connected on corresponding carrier using T4 ligase.
3) cell culture and transfection
Chicken embryo fibroblasts system (DF1) is frozen early period by this laboratory.Cell recovery culture strict aseptic technique, adds
Complete medium (DMEM+10%FBS) is cultivated, and 37 DEG C, 5%CO2Under the conditions of cultivate, change a not good liquor within every two days.Cell turns
Contaminate referenceThe operation of HD Transfection Reagent (Promega) specification.
4)Western blot
The stringent reference standard experimental method of Western blot carries out, and primary antibody is that the Anti-flag label of Abcam company is anti-
Body, secondary antibody are the Goat anti-mouse antibodies that the HRP of Beijing Zhong Shan Golden Bridge is marked；GAPDH internal reference primary antibody is purchased from green skies biotechnology
Research institute.
5) H5N1 subtype influenza virus challenge viral dosage
GS65 and DK49 challenge viral dosage is completed in the Harbin laboratory veterinary institute P3, and viral growth curves measurement is chosen
12h, for 24 hours, 36h, 48h, 60h and 72h amount to 6 time points, attacking toxic dose is MOI=0.001, and virus titer measuring method is
Chicken embryo median infective dose measures (EID50), and calculates EID50 value according to Reed-Muench method.
6) real time RT-PCR technology detection host factor and the expression of influenza virus gene are utilized
Fluorescence real-time quantitative PCR instrument is the ABI 7500 of ABI company production, and quantitative reagent is Germany Roche's480SYBR Green I Master, and operated according to product description.Utilize 2-ΔΔCtMethod will be former
Beginning Ct value is converted to opposite gene expression amount, using GAPDH as reference gene.
Embodiment 1 obtains duck BCL2L15 full length gene coding region sequence using experimental methods of molecular biology
Splice sequence referring to the Duck genome reference sequences on the website Ensemble, and according to transcript profile, designs duck
BCL2L15 full length gene CDS area cloning primer BF/BR, sequence are as follows:
BF:5'-CAATGACAACGTTTGAGGAACAGAC-3',
BR:5'-GTAGTAAAACACTTCCTCTCAGTCA-3'.
Using duck spleen tissue cDNA as template, PCR amplification is carried out using the Q5 exo+ polymerase of NEB company, amplification produces
Object is connected in carrier T after glue recycles and is sequenced.It is found after sequence alignment analysis: duck BCL2L15 full length gene code area
For 483bp (SEQ ID NO.1), 160 amino acid (SEQ ID NO.2) are encoded altogether.
The present embodiment successful clone obtains duck BCL2L15 full length gene coding region sequence.
Embodiment 2 is instantaneously overexpressed BCL2L15 gene using cytologic experiment method in cell
1, the building of duck BCL2L15 gene overexpression carrier
According to duck BCL2L15 coding sequence, its carrier for expression of eukaryon primer eBF/eBR, upstream and downstream primer are designed
All it is separately introduced into Mlu I and Pme I restriction enzyme site (underscore mark)；In addition, upstream primer is before initiation codon ATG
Kozak (box mark) sequence is introduced, introduces flag label (box mark) in target gene C-terminal.Primer sequence is as follows:
EBF:5'-CGACGCGT ATGACAACGTTTGAGGAACAGACGA-3',
EBR:5'-GGGTTTAAACTCA GTCATCCAAGTTCTC
CCATCCTCCA-3'。
The CDS sequence (as shown in sequence table CDS) for the BCL2L15 gene that amplification is obtained imports initial carrier
PiggyBac-X gene (Vector map is as shown in Figure 2), building obtain PiggyBac-BCL2L15 carrier.Due to PiggyBac
Empty carrier has CAG strong promoter, can make to import BCL2L15 gene therein effectively high expression.
2, BCL2L15 gene is instantaneously overexpressed in cell
The PiggyBac-X gene of PiggyBac-BCL2L15 plasmid and not connected duck target gene CDS region sequence is empty
Vector plasmid transfects chicken DF1 cell line using Fugene HD (Promega) respectively, while transfection reagent will only be added, not add
The processing group for entering any plasmid is also set as negative control group (DF1).Microscopically observation cell state and transfection effect after transfection for 24 hours
Rate (as shown in Figure 3).
3, the verifying of duck BCL2L15 gene overexpression carrier
The above transfection is had to the cell of PiggyBac-BCL2L15 and PiggyBac-X gene empty carrier, in CO2Culture
48h is cultivated in case, extracts the total protein of cell, carries out Western blot detection using Anti-flag tag antibody.
Western blot is the results show that PiggyBac-BCL2L15-C-Flag carrier can the effectively high expression in DF1 cell
BCL2L15 albumen (as shown in Figure 4).Above transfection group cell (being overexpressed target genome, negative control NC and DF1 group) can be with
Challenge viral dosage for next step.
Embodiment 3 is infected using the anti-AIV of cytologic experiment method validation duck BCL2L15 gene
1, duck BCL2L15 gene inhibits influenza virus duplication
The good transfection PiggyBac-BCL2L15 of growth conditions, PiggyBac-X gene empty carrier (NC) and DF1 is thin
Born of the same parents are with 2 × 105The density of a/ml is inoculated in 12 porocyte culture plates, carries out attacking poison after cytotostatic is adherent.It attacks malicious real
Testing and selecting strain is 2 plants of H5N1 subtype avian influenza virus, i.e. DK/49 and GS/65, and attacking toxic dose is MOI=0.001, and experiment sets 3
Hole independently repeats.Collect respectively 12h after attacking poison, for 24 hours, 36h, 48h, 60h and 72h cell supernatant, for EID50 detect.Carefully
Born of the same parents' challenge viral dosage is completed in the Harbin laboratory veterinary institute P3.
Attack poison as the result is shown: compared with negative control group (NC), duck BCL2L15 gene inhibits answering for DK/49 influenza virus
System, and reach significant difference (P < 0.05) in 36h, 48h and 72h reach extremely significant difference (P < 0.01, Fig. 5)；Meanwhile duck
BCL2L15 effectively inhibits the duplication of GS/65 influenza virus, is reaching extremely significant difference (P < 0.01) with 60h for 24 hours, in 36h and
48h reaches significant difference (P < 0.05, Fig. 5).Wherein negative control group (NC) is consistent with DF1 group viral growth curves trend, no
There are significant differences.
2, the expression of BCL2L15 gene is dramatically increased after cell infection AIV
The present invention is using Real time RT-PCR technology detection DF1 cell after infecting AIV (DK/49) virus-4 8h
The relative expression quantity of BCL2L15 gene mRNA, as the result is shown: for DF1 cell after infection AIV virus, extremely significant increase is endogenous
The expression quantity (P < 0.01, Fig. 6) of BCL2L15 gene mRNA.
3, duck BCL2L15 gene significantly inhibits the expression of AIV virus various forms RNA
The present invention further utilizes Real time RT-PCR technology detection cell before and after being overexpressed BCL2L15 gene,
After infecting AIV (DK/49) virus-4 8h, the expression of influenza virus M gene vRNA, cRNA and mRNA change, as the result is shown: cell
After infecting AIV, it is overexpressed BCL2L15 gene, significantly inhibits the expression quantity (P < 0.05, Fig. 7) of influenza virus M gene cRNA,
The extremely significant expression quantity (P < 0.01, Fig. 7) for inhibiting influenza virus M gene vRNA, the expression of infected by influenza M gene mRNA do not have
It influences.
In conclusion the present inventor is by comparing the DF1 cell and negative control (NC) cell for being overexpressed BCL2L15 gene
The viral level of middle AIV, to analyze the influence that BCL2L15 gene replicates AIV virus in DF1 cell.Finally obtain
Conclusion: duck BCL2L15 gene is able to suppress the duplication of AIV virus.
The gene of coding BCL2L15 albumen of the present invention can be used for preparing the viral transgenes animal such as anti-AIV, be
The broad-spectrum disease resistance breeding of poultry, fowl provides a kind of new tool, and application prospect is very wide.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (4)
1.BCL2L15 gene is influencing the application in virus replication, which is characterized in that through BCL2L15 gene in cell
It is overexpressed to inhibit duplication of the H5N1 virus in cell, or make answering for H5N1 virus in cell by knocking out BCL2L15 gene
System dramatically increases；The BCL2L15 gene is nucleotide sequence shown in SEQ ID NO.1.
2. application according to claim 1, which is characterized in that by the CDS sequence construct of BCL2L15 gene to can be efficient
In the carrier of expression alien gene, recombinant vector is obtained, and recombinant vector is imported in birds DF1 cell.
3. application according to claim 2, which is characterized in that the carrier is that the eukaryotic expression containing strong promoter carries
Body.
4. a kind of transgenic cell containing BCL2L15 gene, which is characterized in that the transgenic cell be chick embryo fibroblast forever
OEG cell system DF1, the BCL2L15 gene are nucleotide sequence shown in SEQ ID NO.1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610084095.9A CN105671051B (en) | 2016-02-06 | 2016-02-06 | Application of the duck BCL2L15 gene in the anti-AIV virus of livestock and poultry |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610084095.9A CN105671051B (en) | 2016-02-06 | 2016-02-06 | Application of the duck BCL2L15 gene in the anti-AIV virus of livestock and poultry |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105671051A CN105671051A (en) | 2016-06-15 |
| CN105671051B true CN105671051B (en) | 2019-01-18 |
Family
ID=56302871
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610084095.9A Expired - Fee Related CN105671051B (en) | 2016-02-06 | 2016-02-06 | Application of the duck BCL2L15 gene in the anti-AIV virus of livestock and poultry |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105671051B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103881981A (en) * | 2013-08-26 | 2014-06-25 | 华中农业大学 | Living-vector vaccine of H5N1 subtype of avian influenza virus and duck enteritis virus |
-
2016
- 2016-02-06 CN CN201610084095.9A patent/CN105671051B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103881981A (en) * | 2013-08-26 | 2014-06-25 | 华中农业大学 | Living-vector vaccine of H5N1 subtype of avian influenza virus and duck enteritis virus |
Non-Patent Citations (5)
| Title |
|---|
| BCL2L15 (BCL2-like 15);Maria-Angeliki S Pavlou et al.;《Atlas Genet Cytogenet Oncol Haematol.》;20110930;第16卷(第2期);第117页Function |
| Effect of avian influenza A H5N1 infection on the expression of microRNA-141 in human respiratory epithelial cells;Wai-Yip Lam et al.;《BMC Microbiology》;20130510;第13卷(第104期);摘要，表2 |
| Expression of pro-apoptotic Bfk isoforms reduces during malignant transformation in the human gastrointestinal tract;Clare E. Dempsey et al.;《FEBS Letters》;20050608;第579卷;摘要, 第3648页左栏最后一段 |
| Thirty years of BCL-2: translating cell death discoveries into novel cancer therapies;Alex R. D. Delbridge et al.;《NATURE REVIEWS》;20160129;第16卷;第99-109页 |
| XM_005011727.2;GenBank;《NCBI》;20150709;ORIGIN |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105671051A (en) | 2016-06-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kim et al. | Pathobiological features of a novel, highly pathogenic avian influenza A (H5N8) virus | |
| CN104974989B (en) | Attenuated vaccine strain of VII type NDV L gene mutations and preparation method thereof | |
| US20190203170A1 (en) | Avian Cells for Improved Virus Production | |
| CN110305898A (en) | The rescue of the non-susceptible H9N2 hypotype acclimatization to cold avian influenza virus of mammalian cell | |
| CN104962581A (en) | Recombined new castle disease virus vaccine strain for expressing African swine fever virus p72 proteins | |
| CN104232594A (en) | Recombinant homologous avian H1N1 influenza virus inactivated vaccine strain (JS40/PR8) as well as preparation method and application of inactivated vaccine strain | |
| CN103468651A (en) | Recombination Newcastle vaccine strain rAI4-S1 for expressing infectious bronchitis virus S1 protein and generating method thereof | |
| US20220370594A1 (en) | Whole avian-origin reverse genetic system and its use in producing h7n9 subtype avian influenza vaccine | |
| CN105121634B (en) | The cell line and its production method of virus production ability with raising | |
| CN104073470A (en) | Spinner-flask culture method for H9N2 subtype of avian influenza virus | |
| CN105671051B (en) | Application of the duck BCL2L15 gene in the anti-AIV virus of livestock and poultry | |
| CN101560503A (en) | Influenza A virus Vero cell adapted strain and application thereof | |
| EA018438B1 (en) | Medium supplement for virus production | |
| CN103333916A (en) | Construction method and application of BHK cell line suitable for newcastle disease virus proliferation | |
| CN109266623A (en) | One strain vaccine strain rSHA- △ 200 and its construction method and application | |
| CN105567701B (en) | DCSTAMP gene and its application | |
| CN105543232B (en) | ZC3HAV1 gene and its application | |
| CN103215267B (en) | Suppress siRNA and its application of influenza virus related gene | |
| CN100467591C (en) | Recombinant fowl influenza virus strain, its preparation method and the vaccine obtained therefrom | |
| Dai et al. | Inhibition of ERK/MAPK suppresses avian leukosis virus subgroup A and B replication | |
| CN103898066B (en) | A kind of influenza A virus Vero cell acclimatization to cold strain and application thereof | |
| CN102397540B (en) | Recombinant phage vaccine for avian influenza A and construction method for recombinant phage vaccine | |
| CN111454908A (en) | Tpl2 defective MDCK cell strain and construction method and application thereof | |
| CN1746298B (en) | Artificial recombinant H7 subinfluenza virus and use thereof | |
| CN109529032A (en) | A kind of Pseudorabies virus membrane glycoprotein gD gene mRNA vaccine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190118 Termination date: 20210206 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |