CN106337048A - Transgenic chicken for expressing Mx-RNAis anti-influenza virus infection - Google Patents
Transgenic chicken for expressing Mx-RNAis anti-influenza virus infection Download PDFInfo
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Abstract
The invention provides a transgenic chicken for expressing Mx-RNAis anti-influenza virus infection, obtained through a lentivirus vector method. Experiments prove that exogenous gene carried by the transgenic chicken for expressing Mx-RNAis anti-influenza virus infection can be stably inherited, and the transgenic chicken can substantially inhibit in vivo copying of H5N1 bird flu viruses.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to a kind of expression mx-rnais resists
The transgenic chicken of influenza infection.
Background technology
Bird flu (avian influenza, ai) is the bird infectious diseases being caused by a type influenza virus.
Avian influenza virus belong to orthomyxovirus section, Influenza Virus, and its genome contains 8 sub-thread negative strand rnas, compile altogether
Code 11 kinds of virus proteins, wherein 8 be virion constituent (ha, na, np, m1, m2, pb1,
Pb2 and pa).Pa, pb1 and pb2 are three kinds of albumen of composition a type influenza virus polymerase, and they are with heterologous
Presented in tripolymer, it is the key of transcription and replication.Np is in the important feature egg within influenza virus
In vain, it is the primary protein component constituting virus nucleocapsid.Moreover it is possible to be formed with rna and pa, pb1 and pb2
Rnp, plays a role in viral transcription, duplication and packaging process.
The symptom of bird flu is mainly shown as respiratory tract, alimentary canal, reproductive system or nervous system abnormality, morbidity
Rate is higher, and highly pathogenic bird flu is almost 100% to the fatal rate of poultry, not only gives aviculture, animal husbandry band
Carry out huge economic loss, and publilc health is also constituted with serious threat.
A type influenza virus has very strong variation ability, and a kind of accumulation being because viral gene generation point mutation is drawn
The antigenic drift rising, another kind is producer weight due to two kinds of different one cells of influenza virus co-infection
Group, leads to the antigenicity of virus protein to change, the referred to as antigen because larger change virus antigenicity occur
Change.Antigenic drift and antigenic shift all can change virus surface proteins antigenicity, mainly affect hirst's hemagglutination
Based on element, next to that neuraminidase (vijaykrishna d et al., 2011).
Therefore those skilled in the art are devoted to developing the poultry kind to bird flu with stronger resistance, thus carrying
High resistance against diseases.
Content of the invention
It is an object of the invention to provide a kind of transgenosis cultivating expression mx-rnais resisiting influenza virus infection
The method of chicken and its application.
A kind of a first aspect of the present invention, there is provided expression cassette, described expression cassette includes mx gene and sirna
Coded sequence,
Wherein, the conserved genetic sequences of the selectively targeted a type different subtypes of poultry influenza virus of described sirna.
In another preference, described conserved genetic sequences are selected from the group:
Np gene order or its fragment, pa gene order or its fragment, pb2Gene order or its fragment or
A combination thereof.
In another preference, the sirna coded sequence targetting described np gene order includes seq id
Sequence shown in no.:4 and/or its complementary series.
In another preference, the sirna coded sequence targetting described pa gene order includes seq id
Sequence shown in no.:5 and/or its complementary series.
In another preference, target described pb2The sirna coded sequence of gene order includes seq id
Sequence shown in no.:6 and/or its complementary series.
In another preference, described mx gene is chicken mx gene.
In another preference, described chicken mx gene order is as shown in seq id no.:7.
In another preference, described expression cassette also includes promoter element, and described promoter element starts institute
State mx gene and the transcription of sirna coded sequence.
In another preference, described promoter element is cmv promoter or β-actin promoter.
In another preference, described expression cassette, from 5' end to 3' end, includes operability connection successively
Elements below:
(i) promoter element；
(ii) mx gene sequence；
(iii) sirna coded sequence；With
(iv) terminator codon.
In another preference, after described expression cassette is integrated into zooblast, render transgenic animal transcriptional is special
The sirna of the conserved genetic sequences of opposite sex targeting a type different subtypes of poultry influenza virus, and express mx albumen.
In another preference, described expression cassette also includes (v) and is optionally disposed in the connection sequence between each element
Row.
In another preference, described expression cassette also includes the neomycin selecting for cell, or puromycin resists
Property gene.
In another preference, before described marker gene can be located at element (i), element (i) and (ii) it
Between or element (iv) after.
In another preference, described animal is poultry, preferably chicken.
A kind of a second aspect of the present invention, there is provided carrier, described carrier contains first aspect present invention institute
The expression cassette stated.
In another preference, described carrier is slow virus carrier；Preferably, described slow virus carrier is with p201
Slow virus carrier is skeleton.
In another preference, contain antibiotic-screening in described carrier and mark.
A kind of a third aspect of the present invention, there is provided host cell, contains the present invention in described host cell
It is integrated with the expression cassette described in first aspect present invention in carrier described in second aspect or chromosome.
In another preference, described host cell is detached.
In another preference, described host cell is poultry embryonated egg, and described poultry includes but is limited to: chicken,
Duck, goose, turkey, ostrich.
A kind of a fourth aspect of the present invention, there is provided method of prepare transgenosis poultry, including step:
I () provides a carrier containing the expression cassette described in first aspect present invention or the basis from described carrier
The nucleic acid fragment of expression cassette described in invention first aspect；
(ii) carrier described in previous step or described nucleic acid fragment are proceeded in the embryonated egg of poultry, obtain
Obtain the embryonated egg through transfection；
(iii) embryonated egg through transfection that hatching previous step obtains, thus obtain described transgenic poultry.
In another preference, described poultry is chicken, duck, goose, turkey or ostrich.
In another preference, described transgenic poultry has the ability of anti influenza.
In another preference, described influenza is h5n1 influenza virus.
In another preference, methods described further comprises the steps of:
(vi) using the transgenic poultry obtaining in step (v) as f0 generation, hybridize with kind poultry with wild type,
Breed f1 generation, thus obtaining f1 for transgene mammal.
In another preference, methods described further comprises the steps of:
(vii) breed acquisition f2 for transgenic poultry using the f1 that step (vi) obtains for transgenic poultry.
In another preference, described step (ii) adopts equator fenestration, and described carrier is passed through micro- note
Penetrate pin and be expelled to embryonated egg subgerminal cavity, obtain the embryonated egg through transfection.
A fifth aspect of the present invention, there is provided described in first aspect present invention expression cassette, the present invention second
Carrier described in aspect or the host cell described in third aspect present invention, the purposes in reagent preparation,
It is characterized in that, described reagent is used for preparing non-human mammal.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and below (such as enforcement
Example) in can be combined with each other, thus constituting new or preferred skill between each technical characteristic of specifically describing
Art scheme.As space is limited, here is no longer tired out one by one and is stated.
Brief description
Fig. 1 shows the impact of specific mirna infected by influenza propagation in ha test；
Fig. 2 shows this level of relative transcript of each transfection group influenza virus pb2 gene；
The pcdna6.2emgfp-mx digestion qualification result of Fig. 3 display restructuring；
Fig. 4 shows ha analysis of experiments mx, rnais and the impact of mx-rnais infected by influenza propagation；
Fig. 5 is p201 (pprime-cmv-mx-sr) Vector map；
Fig. 6 transgenic chicken infects h5n1 avian influenza virus mortality results；
After Fig. 7 display influenza virus infection, extract wt chicken and tg chicken lungs virus, carry out titration of virus knot
Really；
After Fig. 8 display influenza virus infection, extract wt chicken and tg chicken nose swab virus, carry out titration of virus
Result.
Specific embodiment
The present inventor, by extensively in-depth study, obtains a kind of cultivation and has turning of notable bird flu resistance
The method of gene chicken, methods described transfects the construction of mx gene and rnai genomic constitution to chicken body,
Cultivate the ability that transgenic chicken has the opposing h5n1 avian influenza virus significantly improving.Test result indicate that,
The transgenic chicken of transfection mx gene and rnai gene and the chicken only transfecting mx gene or rnai gene simultaneously
Compare, there is the ability of significantly stronger anti-avian influenza virus it is shown that mx gene and rnai gene have
Significantly synergy.On this basis, complete the present invention.
Before describing the present invention it should be understood that the invention is not restricted to described concrete grammar and experiment condition,
Because this kind of method and condition can change.It should also be understood that its purpose of term used herein is only that description
Specific embodiments, and it is not intended to be restricted, the scope of the present invention is by only by appended claim
Book limits.
Unless otherwise defined, otherwise whole technology used herein and scientific terminology are respectively provided with as institute of the present invention
The identical meanings that the those of ordinary skill in genus field is generally understood that.As used herein, specifically enumerate mentioning
When using in numerical value, term " about " means that this value can change from the value enumerated and is not more than 1%.For example,
As used herein, statement " about 100 " include 99 and 101 and between whole values (for example, 99.1,99.2,
99.3rd, 99.4 etc.).
Although can use in the enforcement or test of the present invention and heretofore described similar or of equal value appointing
Where method and material, herein place enumerate preferred method and material.
Specifically, the invention provides a kind of anti-fowl of expression mx-rnais being obtained by slow virus carrier method
The transgenic chicken of influenza infection, this transgenic chicken is to be set using rna perturbation technique targets avian influenza virus
Meter sirna, is building up in p201 slow virus carrier with the mx gene tandem of chicken, builds recombinant slow virus and carries
Body p201 (pprime-cmv-mx-sr), and it is packaged into recombinant slow virus；Using equator fenestration, will recombinate
Slow virus is expelled to fertile egg subgerminal cavity by injection needle, the insemination egg normal condition through genetic manipulation
Hatch to going out shell, the transgenic chicken of mx-rnais gene is expressed in preparation.Using molecular biology and immunology
Method identification obtains f0 for positive transgenic chicken, mates with kind chicken with wild type, breeds f1 generation, screening
Go out f1 and breed f2 generation for after positive transgenic chicken, have been demonstrated that expression mx-rnais gene transgenic chicken is taken
The foreign gene of band can stablize heredity to f3 generation；H5n1 avian flu virus infection f1 tests for transgenic chicken
Show, this transgenic chicken can suppress the duplication of h5n1 avian influenza virus to a certain extent.This invention is
The transgenic chicken research that cultivation has anti-avian influenza characteristic is had laid a good foundation.
One of the present invention preferred embodiment in, first pass through and a type influenza virus different subtype protected
Keep gene sequencing, find its homologous region, target this homologous region and utilize rna perturbation technique to design sirna.
One of the present invention preferred embodiment in, a type influenza virus different subtype conserved genetic sequences
Including:
Np gene order, pa gene order, pb2Gene order or a combination thereof.
In another preference, the gene accession number of the ncbi of described np gene is 3654618.
In another preference, the gene accession number of the ncbi of described pa gene is 3655158.
In another preference, described pb2The gene accession number of the ncbi of gene is 3654615.
In another preference, the sirna coded sequence targetting described np gene order is as follows:
gctccgaagaaataagatccttgttttggccactgactgacaaggatcttttcttcggag
(seq id no.:4).
In another preference, the sirna coded sequence targetting described pa gene order is as follows:
gtcaggcactcctcaattgcttgttttggccactgactgacaagcaattggagtgcctga
(seq id no.:5).
In another preference, target described pb2The sirna coded sequence of gene order is as follows:
ggaatcagccttctagttgcttgttttggccactgactgacaagcaactaaggctgattc
(seq id no.:6).
One of the present invention preferred embodiment in, by the sirna of design and chicken mx gene or its piece
Duan Lianhe, starts amalgamation and expression mx-rnais by cmv promoter.
Preferably, the gene accession number of chicken mx gene ncbi is z23168.
Preferably, cmv promoter sequence is as follows:
gacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttc
cgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgt
atgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaaactgcccacttggc
agtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcc
cagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatg(seq id
no.:3).
Mx gene
Mx albumen is the dynamic superfamily protein gtp enzyme of inf induction, and molecular size range 70,000～80,000, has
Self-assembly is inclined to, and has stronger gtp hydrolysis properties.Mx albumen has inhibitory action to multiple negative strand rna viruses,
Thus there is the function of resisiting influenza virus.
Mx albumen is i type (α/β) and iii type (λ) interferon (ifn) induces produce to have cell itself sky
So crucial effector molecule of antiviral activity, is the label of instruction i type interferon and its acceptor correlation.
1962, lindenmann found when studying Inbred Mouse, with identical lethal dose influenza infection inbreeding
Mouse species a2g mouse and other panmixia small white mouse, only a2g mouse are resistant to survive.Grind further
Studying carefully discovery this is because there is a dominant gene on No. 16 chromosomes, a2g mouse opposing influenza virus can be made
Attack, therefore just by this unnamed gene for mx (myxovirus resistant) gene (lindenmann,
1962).Mx1 is the main intracellular resisiting influenza virus being produced by ifn mediation in mouse body and influenza virus-like disease
The effector of poison, the mx gene of other kind animals has similar function, and the expression of mx gene is subject to i type
Ifn (α/β) and the strict regulation and control of iii type ifn (γ).
Mx albumen has the antiviral activity of wide spectrum, and different animals kind mx albumen has different antiviral spies
The opposite sex, antiviral-mechanism is also different, and this is significant for human and animal's medical research.Mx albumen is disease-resistant
Poison specificity as the difference that positions in the cell and different, in nucleus, mx albumen can optionally press down
System is in the orthomyxovirus such as influenza virus (iv) of endoreduplication and the initial transcription of togavirus (thov), and cytoplasm
Interior mx albumen, in addition to the duplication that can suppress orthomyxovirus, may also suppress rhabdovirus, paramyxovirus and cloth Buddhist nun virus
Duplication (kane et al., 2012).
One of the present invention preferred embodiment in, described mx albumen is chicken mx albumen.Preferably, institute
The amino acid sequence stating chicken mx albumen is as follows:
mnnpwsnfssafgcpiqipkqnsnvppslpvpvgvfgvplrsgcsnqmafcapeltdrkpeheqkvskrl
ndreedkdeaaacsldnqydrkiqpcidlvdslrkldigndlmlpaiavigdrnsgkssvlealsgvalprdkg
vitrcplelklkkmtapqewkgviyyrnteiqlqnasevkkairkaqdivagtngsisgelisleiwspdvpdltl
idlpgiareavgnqpqdngqqiktllkkyigcketiivvvvpcnvdiattealkmaqevdptgertlgvltkpdl
vnegteetvlkiiqneviplrkgymivkcygqmdfcnelsftsaiqqereffethkhfstlldenkatiphlankl
tdelvgriiktlpaiekqvhdalqqakkelqkytqsthptvsdktiflvglikafnedisqtmhgkeswfgneirl
fpkirrefrtwgvkllessakveeivcsklpkyedqyrgrefpdfisywtfediikeqitkleepavamlnkviym
veekflqlankrfanfqnlnnaaqarigcisdrqattaknciltqfkmeriiycqdniyaddlkaaraegiskdt
kikdlafgcasrqcpsfalemvshvkayftgaskrlsnqipliilstvlhdfgnylqtsmlhllqgkeeinyllqe
Dheaanqqklltsrishlnkayqylvdfksl (seq id no.:2),
Preferably, its coding nucleotide sequence is as follows:
atgaacaatccatggtccaacttcagctcagcttttggatgtcccattcagatcccaaagcagaata
gtaatgtacccctttcctttaccagtacctgtaggagtttttggggtgcctctgcgatcaggctgcagcaat
cagatggctttctgtgcaccagaactgactgacagaaagcctgagcatgagcagaaagtgtccaagaggct
gaatgacagagaagaggacaaggatgaggcagcagcatgcagcttggacaaccaatatgacagaaagatc
cgaccttgcattgatcttgttgacagcctgagaaagcttgatataggaaacgacctgatgttgcctgcaatt
gcagtgattggagaccggaactctgggaaaagctctgtccttgaagctttgtctggtgttgctcttcctagg
gacaaaggtgtcattactcgctgtcctctggaacttaaactgaaaaaaatgacagctccgcaggaatggaa
aggggtaatttattaccgcaacacagaaatacagctccagaatgcatcagaggtgaagaaagcaataaga
aaagcccaagatatagtggctggcactaatggtagcattagtggagaactaatttcccttgaaatctggtc
tcctgacgtcccagacctgacactaattgatcttcctggaattgccagagaggccgtggggaaccagccac
aagataatggccaacagatcaaaacactacttaaaaaatatattggctgcaaagagacaatcattgtggta
gtggtaccatgtaatgtggatattgcaacaacagaagcgctgaaaatggctcaagaggtggatcccacag
gagaaaggacgcttggggtcctcactaaaccagacctagtgaacgaaggaactgaagagactgtccttaa
gatagtacaaaacgaggtcatcccactcagaaaaggttatatgattgtgaaatgttatgggcaaatggact
tctgcaacgaattgtccttcacctccgcaatccagcaagagagagaattctttgagactcacaaacatttca
gcactcttctggatgaaaataaggctactatcccacatctggcaaataagcttacagatgaacttgtggga
cgtattattaaaactttgcctgcaatagagaagcaagtacatgatgcactgcaacaagcaaagaaggaact
acaaaagtacacacaaagcacacacccaactgtcagcgataagacaattttccttgtggggttgatcaaag
cgtttaatgaagacatctctcagacaatgcatggaaaggaatcctggtttggaaacgaaatcagactgttt
ccaaaaatccgcagagagtttcggacatggggagtaaagctcctggagagctctgccaaagttgaagaaa
tcgtatgcagtaaattgcccaaatatgaagaccagtaccgtggacgggagttcccagactttatcagctac
tggacatttgaggacattataaaagagcaaattacgaagctggaggagccagctgttgcaatgctgaaca
aagtgatctatatggttgaagagaaatttttgcagctggctaacaagcgttttgctaattttcagaacttaa
acaacgctgctcaggtcagaattggttgcattagtgacagacaagcaacgactgcgaaaaattgcatcctg
actcaatttaaaatggagagaattatatactgccaggataacatctacacagatgatttaaaagctgccag
ggcagaaggcatcagcaaagatacaaaaatcaaagaccttgcttttggatgtgcttcacgtcaatgtccca
gctttgccctggaaatggtttctcacgtaaaggcctatttcactggagcaaataaacgcctgagcaatcag
attcctctgatcatcctctctactgtccttcatgactttggaaattatttgcagacctcaatgttgcatctct
tgcaaggaaaagaagaaataaactatttactccaggaagatcatgaagctgctaaccagcagaagttact
gaccagcagaattagtcacctcaacaaagcctaccaatacctggtagactttaagtctctgtag(seq id
no.:1).
sirna
Little interference rna (small interfering rna, sirna): it is a kind of little rna molecule (about 21-25nt),
Processed by dicer (there is in rnaase family specific enzyme to double-strand rna).Sirna is sirisc
Major Members, there is selective degradation in the intracellular mrna of mediation, thus leading to the expression silencing of target gene,
Produce the phenomenon of corresponding function phenotype disappearance, become rna interference (rna interference, rnai).
shrna
Bob folder rna (short hairpin rna, shrna) includes two short inverted repeats (e.g., seq id
No.:4,5,6 and its shrna of respective inverted repeats composition), centre is by a stem ring (loop) sequence
Separate, form hairpin structure, shrna sequence is cloned in plasmid vector, in animal body this hairpin quilt
Express, form one " double-strand rna " (shrna), and processed by rnai passage, form " little interference
rna”(sirna).
" expression cassette " or " construct " refers to a kind of inclusion target nucleic acid sequence or gene order, can express conjunction
Suitable albumen, is connected with the target nucleic acid sequence composition operation providing suitable transcription and translation in host cell.Described one-tenth
Divide and potentially include promoter, secretory signal sequence, tailing signal, intron sequences, insulator sequence and the present invention
Described other compositions." expression cassette " or " construct " may also include " carrier sequence "." carrier sequence "
Refer to be beneficial to clone and the expression of expression construct with the nucleotide sequence of present invention restructuring dna technique construction.Represent
The expression vector type of property includes but is not limited to plasmid, clay, phage vector, viral vectors, and yeast people
Work chromosome.
" bicistronic construct " refers to any constructs that can express two independent translation protein products.Both
Product may be from a single gene bicistronic construct or from two independent mrna translations, each
Independent mrna is encoded by same bicistronic construct." poly- cistron structure " refers to any be provided that two
The construct of above independent translation protein product expression.
" being operatively connected " refers to a target nucleic acid sequence and one or more regulating and controlling sequence (for example, promoter)
Connect on object, so as to the polypeptide in host cell inner expression target nucleic acid sequence coding.
" burst " refers to one section of nucleotide sequence, when the nucleotide sequence being introduced in coded polypeptide, can instruct
The cell expressing this polypeptide secretes this polypeptide (as butyrylcholine esterase and/or glycosyl transferase).Burst is best
Positioned at nucleic acid coding sequence 5' end, the polypeptide of such burst is located at the n- end of translation polypeptide." signal peptide "
Refer to the peptide sequence of burst translation.
" host cell " refers to the cell having transfected through one or more expression construct of the present invention, including the food in one's mouth
Newborn animal cultured cell in vitro and internal cell.
" transfection " refers to introduce the implementation process of one or more expression construct of the present invention in host cell,
Including (but being not limited to): microinjection, electroporation, liposome mediated transfection, calcium phosphate mediation transfection or virus
Mediated transfection etc..If expression construct of the present invention has transfected to host cell, it is called " transfection "." wink
When transfectional cell " refer to such host cell, introduce expression construct in this host cell but not permanent
Be incorporated into the genome of host cell or its offspring, thus this expression construct of over time can by host cell or
Its offspring loses." stable transfectional cell " refers to that the expression construct introducing host cell has been integrated into host
Cell and its genome of offspring.
" transgenosis " refers to that any one has been integrated into the expression construct of the present invention of transfection host cell genome
Structure.Host cell containing this kind of transgenosis is referred to as " transgenic cell ".This by partly or entirely turning base
Animal because of host cell composition is " transgenic animals ".First-selected transgenic animals are for transgene mammal (such as
Rodent or ruminant or domestic animal)." chimera " is referred to as by the animal that partial transgenic host cell forms
Or " chimeric animal ".
Term " promoter " used herein or " promoter unit (domain) " refer to that a kind of effective initial gene turns
The nucleotide sequence of recording function and the nucleotide sequence that can affect this gene transcription level, guiding gene nucleotide sequence is transcribed
For rna, it is typically found in the upstream of genes of interest coded sequence (5' end), usually, promoter or promoter
Region provides rna polymerase and correctly initiates other factors necessary to transcription and can transcribe water by controlling gene
The recognition site of flat correlation factor.
Herein, the variant of described promoter or promoter region (domain) inclusion promoter, promoter variants are permissible
By inserting or deleting nucleotide sequence, carry out random or rite-directed mutagenesis etc. to obtain.
In view of the teachings of the present invention and prior art, although it will be recognized by one of ordinary skill in the art that the present invention
The promoter sequence providing in embodiment is as shown in seq id no.:3, but the present invention also should be included with the present invention's
Promoter sequence (seq id no:3) have 50% or more (preferably more than 60%, more than 70%, more than 80%, more
Preferably more than 90%, more preferably more than 95%, most preferably more than 98%, such as 99%) nucleic acid of homology, as long as institute
State the function that nucleic acid also has initial gene transcription in cell." homology " refers to according to position identical percentage
Similar level (i.e. sequence similarity or homogeneity) between two or more pieces nucleic acid for the ratio.
" genes of interest " used herein refer to any and described promoter be directly or indirectly connected such that it is able to
Initiateed the gene of transcription by described promoter.
The promoter of the present invention can be operationally connected with genes of interest, and this genes of interest can with respect to promoter
To be derived from same species it is also possible to be derived from different species.Genes of interest as herein described does not especially limit
System can be the gene of rna or encodes gene or a combination thereof with specific function albumen.
The representative example of described genes of interest includes but is not limited to: metabolism, adjusting function related gene, resists
Property gene, tolerance related gene, fluorescence protein gene, and rna gene.
Described metabolism, adjusting function related gene be for example: glycolytic pathway, tricarboxylic acid cycle, aspartic acid,
The amino acid synthesis pathway such as threonine, isoleucine, lysine, methionine, and multiple may be with threonine
Synthesis or the degraded directly or indirectly pathway protein of correlation and its regulatory protein gene.
Described resistant gene is selected from the group: chloramphenicol, ampicillin, tetracycline, hygromycin, neomycin
Deng antibiotics resistance gene, antiviral gene etc..
Tolerance related gene is such as: gene related to environmental resistance such as high temperature resistance, high salt etc..
Fluorescence protein gene is such as: the fluorescin such as red fluorescent protein, green fluorescent protein, yellow fluorescence protein
Gene.
Rna gene is for example: sirna gene, rnai gene, microrna etc..
Present invention also offers a kind of expression casette, described expression cassette is from 5'-3' including but not limited to following unit
Part: the promoter of the present invention and objective gene sequence.
Present invention also offers a kind of recombinant vector, it comprises the promoter of the present invention and/or expression casette.?
In preferred embodiment, the promoter downstream of described recombinant vector comprises MCS or at least one digestion position
Point.When needing to express genes of interest, genes of interest is connected into suitable MCS or restriction enzyme site, thus
Be operably connected genes of interest and promoter.
In another preferred embodiment, described recombinant vector includes on 5' to 3' direction: promoter, mesh
Gene and terminator.If necessary, described recombinant vector can also include elements below: protein purification label；
3' polymerized nucleoside is acidified signal；Untranslated nucleotide sequence；Transhipment and targeting nucleotide sequence；Selected marker (antibiotic
Resistant gene, fluorescin etc.)；Enhancer；Or operator.
Method for Prepare restructuring carrier is well known to those of ordinary skill in the art.Expression vector can be
Bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other carriers.Always
It, as long as it can replicate and stable in host's body, any plasmid and carrier can be used, and preferably adopt
Use slow virus carrier.
Those of ordinary skill in the art can adopt known to method build contain promoter of the present invention and/or purpose
The carrier of gene order.These methods include vitro recombination dna technology, dna synthetic technology, In vivo recombination technology
Deng.
The promoter of the present invention, expression cassette or carrier, can be used for converting suitable host cell, so that host
Transcription purpose rna and/or express express target protein matter.Host cell is preferably mammalian cell.This area is general
Technical staff is aware that how to select suitable carrier and host cell.Can use this with restructuring dna transformed host cell
Routine techniques known to skilled person is carried out.
Term " being operatively connected " refers to the genes of interest of preparation transcriptional expression with a kind of routine side of this area
Formula is connected to its control sequence with transcribed and/or expression.
Material and method general introduction
It is an object of the invention to provide prepared by one kind mx-rnais gene can be expressed and there is suppression bird flu
The method of the transgenic chicken of virus replication, general introduction the method is as follows:
1st, target influenza virus gene np, pa and pb2, separately design synthesized single-gene and dual-gene,
The mirna of three gene tandem, is cloned into pcdnatm6.2-gw/emgfp-mir carrier, builds mirna
Expression vector pcdna6.2/np pcdna6.2/pa, pcdna6.2/pb, pcdna6.2/pa+np and
Pcdna6.2/pa+np+pb, after transfection mdck cell, infects h5n1 avian influenza virus, preferably described dry
Disturb gene.
2nd, build the eukaryon expression plasmid pcdna of recombination chicken mx genetm6.2-gw/emgfp-mx, pcr change
Make amplification and obtain the mx gene dna fragment that 5 ' and 3 ' ends are respectively provided with sali and xholi, by this piece
Section is inserted in the MCS of eukaryon expression plasmid pcdna6.2/emgfp, proves through digestion, sequencing
Obtain pcdna6.2/emgfp-mx expression vector.
3rd, pcdna6.2/pa+np+pb, pcdna6.2/mx and pcdna6.2/mx-rnais are turned respectively
After dye mdck cell, infect h5n1 influenza virus, evaluate mx, rnais and mx-rnais suppression h5n1
The efficiency of influenza virus.
4th, with pcdna6.2/pa+np+pb2 for template amplification Mutiple Targets mirnas, product with xho and
Ecor double digestion, orientation insertion slow virus carrier p201 (pprime-cmv-gfp-ff3), recombinated
Slow virus intermediate carrier p201-mirnas；Then with pcdnatm6.2-gw/emgfp-mx is template amplification
Chicken mx gene, pcr product is inserted into p201-mirnas carrier with after nhe and not double digestion, obtains
Slow virus intermediate carrier p201-mx-mirnas to restructuring；Finally by pcaggs-flpe carrier
β-actin promoter recovery purifying after sal and bgl double digestion is inserted into p201-mx-mirnas and carries
Body, is named as p201 (pprime-cmv-mx-sr) carrier, with the method identification weight of pcr, digestion and sequencing
Group plasmid, packs slow virus.
5th, preparation expression mx-rnais gene transgenic chicken, by equator fenestration, by the recombinant lentiviral of packaging
Virus injection, to insemination egg subgerminal cavity, obtains f0 for transgenic chicken.
6th, the cultivation of transgenic chicken, using molecular biology and immunologic method, is carried out for chicken to f0
Identification, after filtering out positive transgenic chicken, in f1 generation and f2 generation, turn with same kind wild type chicken crossbreed
Gene chicken is it was demonstrated that foreign gene can be integrated and express, and determines the integration site of transgenic chicken.
7th, evaluate its anti-h5n1 avian flu cytotoxic activity with f1 for transgenic chicken, result shows system of the present invention
Standby transgenic chicken has the ability that suppression h5n1 avian influenza virus replicate.
Main advantages of the present invention are:
(1) disclose the mx gene and sirna gene synergy in terms of resisiting influenza virus first；
(2) the excellent resistance to avian influenza virus is had using the transgenic chicken that the method for the present invention is cultivated.
(3) carrier using the present invention to build, cultivates transgenic chicken and has higher transgenosis success rate.
With reference to specific embodiment, the old present invention in detail further.It should be understood that these embodiments are only used for
The bright present invention rather than restriction the scope of the present invention.The experiment side of unreceipted detailed conditions in the following example
Method, generally writes institute in " Molecular Cloning: A Laboratory room guide " according to normal condition such as U.S. sambrook.j etc.
The condition stated, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and number
Calculate by weight.In following examples, experiment material used and reagent if no special instructions all can be from commercially available canals
Road obtains.
Experiment material source used in the embodiment of the present invention is as follows:
Pcdna6.2-emgfp-mir carrier, purchased from invitrogen company；
Pcdna6.2/emgfp eukaryon expression plasmid, purchased from invitrogen company；
Slow virus carrier p201 (pprime-cmv-gfp-ff3), purchased from addgene company；
Pcaggs-flpe carrier, purchased from open biosystems；
293ft incasing cells, purchased from invitrogen company.
The structure of embodiment 1.mirna expressing in series plasmid vector
The design of 1.1mirna sequence and synthesis
The present invention is the maximization that disturbance target point selected by realization utilizes, it is to avoid the sequence between because of different strains is poor
Different and limit rnai effect, therefore, the target sequence of sirna try one's best select more conservative between different strains
Region.Select for different influenza virus sub-strain conservative gene np, pa, pb2, design mirna coding
Sequence, is shown in Table 1.The sirna coded sequence targetting described np gene order includes seq id no.:7 institute
Reverse complementary sequence shown in the sequence shown and seq id no.:8；Target the sirna of described pa gene order
Coded sequence includes the sequence shown in seq id no.:9 and the reverse complemental sequence shown in seq id no.:10
Row；Target described pb2The sirna coded sequence of gene order includes the sequence shown in seq id no.:11
And the reverse complementary sequence shown in seq id no.:12；
Table 1
The structure of 1.2mirna expressing in series plasmid vector
Double-strand dna fragment after annealing is cloned into pcdna6.2-emgfp-mir carrier (be purchased from
Invitrogen company) in, it is named as pcdna6.2/np pcdna6.2/pa pcdna6.2/pb2.Send
Sequencing.With pcdna6.2/np as skeleton, through bgl and xho digestion, with pa mirna coded sequence
For Insert Fragment by pcdna6.2/pa through sal and bgl digestion, build np and pa expressing in series and carry
Body, names pcdna6.2/pa+np, builds pcdna6.2/pa+np+pb2 in the same way.By series connection weight
Group plasmid is identified through xho and bamh double digestion, correct after send sequencing, result shows to become in the present embodiment
Work(constructs pcdna6.2/pa+np+pb2 plasmid.
1.3 cellular levels evaluate each disturbance target point to vector expression gene inhibition
Mirna expression plasmid is transfected to mdck cell, 103Influenza infection, with ha test and
Real-time pcr testing inspection inhibition.7 groups of experiment point, mirna transfection group and negative plasmid turn
Dye group and blank transfection group.In experiment, each pair mirna is all provided with 3 holes, results averaged.
Above-mentioned test result indicate that pcdna6.2/np pcdna6.2/pa pcdna6.2/pb2 and
Pcdna6.2/pa+np group is 48 64 48 in the rush hour (72h) of virus titer, its ha value
32, show that they can suppress influenza virus to replicate in mdck cell to a certain extent, its virus suppression
Rate processed is respectively 62.5% 50% 62.5% and 75%.And its inhibiting rate of pcdna6.2/pa+np+pb2 group
For 81%.This show multiple mirna be together in series expression more single mirna have preferable inhibition (figure
1).Real-time result show pcdna6.2/np pcdna6.2/pa pcdna6.2/pb2,
Pcdna6.2/pa+np and pcdna6.2/pa+np+pb2 group viral copy number reduces by 3.6 3.2 respectively
4.6 6.3 and 9.1 times (Fig. 2).Wherein the suppression efficiency of pcdna6.2/pa+np is compared with other single mirna
The suppression efficiency of expression plasmid is high, and pcdna6.2/pa+np+pb2 in each transfection group, suppression efficiency is
High.This shows that multiple mirna are together in series expression, and more single mirna has preferable inhibition.
The carrier for expression of eukaryon pcdna of embodiment 2. recombination chicken mx genetmThe structure of 6.2-gw/emgfp-mx
Build
Pcr transforms amplification and obtains the mx gene dna piece that 5 ' and 3 ' ends are respectively provided with sal i and xhol i
Section (seq id no.:9), this fragment is inserted into eukaryon expression plasmid pcdna6.2/emgfp and (is purchased from
Invitrogen company) MCS in, transformed competence colibacillus cell, choose bacterium, extract recombinant plasmid,
Through sal i, xhol i digestion, 2 bands in result, and 1 is slightly larger than 2kb, the purpose bar with insertion
Band size is consistent；Another and 5.6kb, be that (Fig. 3, swimming lane 1 is pcdna6.2/emgfp belt carrier
Pcdna6.2/emgfp-mx sal i single endonuclease digestion product, swimming lane 2 are pcdna6.2/emgfp-mx xho i
Single endonuclease digestion product, swimming lane 3 are pcdna6.2/emgfp-mx sal i+xho i double digestion product, swimming lane 4
For pcdna6.2/emgfp-mx plasmid).Insertion genes of interest is sequenced, sequencing result shows to transform
Gene afterwards is consistent with desired design sequence, is named as pcdna6.2/emgfp-mx.
Embodiment 3. compares the efficiency that mx, rnais and mx-rnais suppress h5n1 influenza virus
Pcdna6.2/pa+np+pb, pcdna6.2/mx and pcdna6.2/mx-rnais are transfected respectively mdck
Cell, with 103tcid50H5n1 influenza infection, suppresses the effect of influenza virus with ha analysis of experiments,
With negative plasmid transfection group and blank transfection group for comparison.In experiment, every group is all provided with 3 holes, and result is averaged
Value.
Test result indicate that pcdna6.2/pa+np+pb, pcdna6.2/mx and pcdna6.2/mx-rnais
In 48h, its ha value is 68 and 2 to group, and its viral suppression is respectively 81% 75% and 93.75% (figure
4).This shows that rnais and mx are together in series expression, and more single expression has collaborative inhibition.
The structure of embodiment 4.p201-mx-mirnas carrier
With pcdna6.2/pa+np+pb2 for template amplification Mutiple Targets mirnas, product xho and ecor
Double digestion, orientation insertion slow virus carrier p201 (pprime-cmv-gfp-ff3) is (public purchased from addgene
Department), obtain the slow virus intermediate carrier p201-mirnas recombinating；Then with pcdna6.2-gw/emgfp-mx
For template amplification chicken mx gene, pcr product is inserted into p201-mirnas with after nhe and not double digestion
Carrier, obtains the slow virus intermediate carrier p201-mx-mirnas recombinating；Finally by pcaggs-flpe carrier
β-actin promoter in (purchased from open biosystems company) is with after sal and bgl double digestion
Recovery purifying is inserted into p201-mx-mirnas carrier, is named as p201 (pprime-cmv-mx-sr) carrier
(Fig. 5), identify recombinant plasmid with the method for pcr, digestion and sequencing.
The preparation of embodiment 5. transgenic chicken
The packaging of lv-mx-mirnas that recombinant slow virus build and concentration are according to invitrogen slow virus
Packaging system and liposome 2000 transfection explanation, p201 (pprime-cmv-mx-sr) slow virus are expressed and carry
Body and viralpowertmpacking mix (purchased from invitrogen company) cotransfection reach 70% to degrees of fusion
293ft incasing cells (purchased from invitrogen company), by real-time pcr method measure virus
Titre.Result shows that recombinant slow virus lv-mx-mirnas titre is: 1 × 2e+8=2.0e+8tu/ml.
Using equator fenestration, p201- β-mx-mirnas recombinant slow virus are injected by injection needle
To newborn fertile egg subgerminal cavity, the insemination egg normal condition through genetic manipulation is hatched to going out shell, successfully prepares
Proceed to the transgenic chicken (success rate is 16.4%) of mx-rnais gene.
(each condition below is the present invention preferably condition, such for the injection of fertile egg blastodisc and hatching
Under the conditions of injection fertile egg can have higher incubation rate, the chicken positive rate hatched is also higher)
(1) fresh fertile egg, after 75% alcohol surface sterilization, is flat on mark peak, room temperature in egg support
Overnight make blastodisc float to fix.
(2) mark dental burr open length of side 3mm about window, under cold light source find embryo
Disk, once finding blastodisc, drawing 2-3 μ l recombinant slow virus concentrate with injection needle and being expelled to blastodisc
Cavity of resorption, when blastodisc central, clear area reddens, illustrates to inject successfully.
(3) with sealed membrane, closing at opening is fixed.
(4) fertile egg that injecting virus and opening are not had injecting virus is all positioned over 37.8 DEG C of temperature, wet
In the incubator of degree 50%, it is transferred into after 18d in hatching device, 37.5 DEG C of temperature, humidity 50% is incubated
To going out shell.
The identification of embodiment 6. transgenic chicken is analyzed with integration site
The identification of 6.1 transgenic chickens
Gather the blood of transgenic chicken by wing venous, extract blood dna, carry out pcr using specific primer
Amplification, shows that f0 has the integration of foreign gene for transgenic chicken；Choose f0 for transgenic positive cock with
Wild type hen mates kind, breeds f1 generation, extracts f1 for the dna of chicken, rna, albumen, design mxjc,
Rnai two to primer, by method validations such as pcr, rt-pcr, southern, western, demonstrate,prove by result
Bright f1 is for the integration of the purposeful foreign gene of transgenic chicken and expression.
6.2f0 for integration site analysis
Extract f0 and represent the dna reaching mx-rnais gene transgenic chicken as template, special using design
Property primer form primers with six degenerate primers respectively and combine, the upstream and downstream flanking sequence to integration site
Carry out tail-pcr amplification, take the product of 15 μ l third time pcr amplifications, with 1% agarose gel electrophoresis
Detect the result obtaining.
Glue reclaim third round tail-pcr amplification in 750-2000bp about specific purpose fragment, company t
Carry, conversion, clone, sequencing；By the sequence that obtains of sequencing pass through on ncbi website blast program with
In genbank, the known nucleic acid sequence of chicken and constructed carrier sequence compare, and result shows mesh
Mark sequence is incorporated on No. 2 chromosomes of chicken that (about 2/5 transgenic chicken has 4 in 10 transgenic chickens
The integration site of chicken is in same position), apart from this site 5 ' end, 386bp is als2-c-terminal like
Sequence, 3 ' end 19526bp are cross-film inner ear protein sequence.
This shows, the transgene carrier that the present invention builds can be incorporated in the genome of transgenic chicken, and has
There is certain tendentiousness.
6.3f0 for integration site result verification
Between sequencing result sequence 44-749bp (chicken genome should be included and include hiv carrier again)
Design specificity verification primer, the stripe size with transgenic chicken genome as template amplification and expected results one
Cause.
Glue reclaim purpose band, even t carrier, conversion, clone, sequencing；By the sequence obtaining that is sequenced in ncbi
Entered with the known nucleic acid sequence of chicken in genbank and constructed carrier sequence by blast program on website
Row compares analysis, and result shows that target sequence is incorporated on No. 2 chromosomes of chicken, acquired results and above
Cause, show that checking is correct.
6.4f1 for integration site checking
With f0 for transgenic chicken integration site checking primer, to f1 for transgenic chicken genome as mould
Plate, carries out pcr amplification；The stripe size of amplification is consistent with expected results.Glue reclaim purpose band, even t
Carrier, conversion, clone, sequencing；The sequence that sequencing is obtained passes through blast program on ncbi website
Compare with the known nucleic acid sequence of chicken in genbank and constructed carrier sequence, result shows
Target sequence is incorporated on No. 2 chromosomes of chicken, consistent with the result in f0 generation.
The suppression h5n1 avian influenza virus replication activity research of embodiment 7. transgenic chicken
In P3 laboratory, with 104eid50The h5n1 subtype avian influenza virus of/ml infect respectively
F1 for transgenic positive chicken with each 12 with strain wild type chicken, connect the daily state of mind observing chicken after poison,
Morbidity and death condition, gather throat swab daily, and after infection 3,6 days every group cut open and kill 3, collection
Lung tissue, analysis throat swab and lung tissue virus titer, evaluate transgenosis suppression avian influenza virus replication activity.
Result shows, tg chicken postponed than the death time of wt chicken, all death in the 8th day after infection of wt chicken,
And all death (Fig. 6) in 11 days after infection of tg chicken；Tg infected group throat swab virus titer is after infection
3rd, it is below within 6 days wt infected group, significant difference (p ＜ 0.05) (Fig. 7)；The lung tissue virus of tg group
Titre was below wt group at 3,6 days, the 6th day significant difference (p ＜ 0.05) (Fig. 8).
The above results show, the transgenic chicken of the present invention can suppress the duplication of avian influenza virus effectively, and
Highly effective can delay morbidity and the death condition of transgenic chicken.For example, at the 3-5 days, tg chicken group dead
Rate of dying is only the 50% of wt chicken group, and when the 6th day, the death rate of tg chicken group was only the 1/4 of wt chicken group,
In addition median survival time extended to 7.5 days from 5.5 days (improving about 35%), and the longest life span was from 8 days
Extend to 11 days (improving 37.5%).
The all documents referring in the present invention are all incorporated as reference in this application, just as each document
It is individually recited as with reference to like that.In addition, it is to be understood that after the above-mentioned instruction content having read the present invention,
Those skilled in the art can make various changes or modifications to the present invention, and these equivalent form of values equally fall within this Shen
Please appended claims limited range.
Claims (10)
1. a kind of expression cassette is it is characterised in that described expression cassette includes mx gene and sirna coded sequence,
Wherein, the conserved genetic sequences of the selectively targeted a type different subtypes of poultry influenza virus of described sirna.
2. expression cassette as claimed in claim 1 is it is characterised in that described conserved genetic sequences are selected from down
Group:
Np gene order or its fragment, pa gene order or its fragment, pb2Gene order or its fragment or
A combination thereof；
Preferably, the sirna coded sequence targetting described np gene order is included shown in seq id no.:4
Sequence and/or its complementary series；
Target described pa gene order sirna coded sequence include the sequence shown in seq id no.:5 and
/ or its complementary series；And/or
Target described pb2The sirna coded sequence of gene order include the sequence shown in seq id no.:6 and
/ or its complementary series.
3. expression cassette as claimed in claim 1 is it is characterised in that described mx gene is chicken mx gene；
Preferably, described chicken mx gene order is as shown in seq id no.:1.
4. expression cassette as claimed in claim 1 is it is characterised in that described expression cassette also includes promoter
Element, described promoter element starts the transcription of described mx gene and sirna coded sequence.
5. expression cassette as claimed in claim 1 is it is characterised in that described promoter element is β-actin
Promoter or cmv promoter.
6. expression cassette as claimed in claim 1 is it is characterised in that described expression cassette is from 5' end to 3' end,
Include successively operability connection elements below:
(i) promoter element；
(ii) mx gene sequence；
(iii) sirna coded sequence；With
(iv) terminator codon.
7. expression cassette as claimed in claim 1 is it is characterised in that described animal is poultry, preferably
Chicken.
8. a kind of carrier is it is characterised in that described carrier contains expression cassette described in claim 1.
9. a kind of host cell is it is characterised in that containing described in claim 8 in described host cell
Carrier or chromosome in be integrated with expression cassette described in claim 1.
10. a kind of method of prepare transgenosis poultry is it is characterised in that include step:
I () provides a carrier containing expression cassette described in claim 1 or wants containing right from described carrier
Seek the nucleic acid fragment of expression cassette described in 1；
(ii) carrier described in previous step or described nucleic acid fragment are proceeded in the embryonated egg of poultry, obtain
Obtain the embryonated egg through transfection；
(iii) embryonated egg through transfection that hatching previous step obtains, thus obtain described transgenic poultry.
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| CN114807227A (en) * | 2022-05-09 | 2022-07-29 | 陕西理工大学 | Chicken fibroblast line with antiviral protein Mx gene knocked out and construction method thereof |
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