CN113151354B - Vector for conditional knockout of target gene and method for conditional knockout of target gene - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a conditional knockout fragment and a plasmid for conditional knockout of a target gene on a virus or cell genome, and a method for conditional knockout of the target gene, wherein the conditional knockout fragment comprises a left homologous recombination arm LA of the target gene, a screening marker gene expression element, a lac repressor gene expression element, a lac operator gene expression element, and a right homologous recombination arm RA of the target gene; simultaneously constructing a plasmid containing the conditional knockout fragment; and provides a method for conditional knockout of a target gene by using the plasmid. Compared with the prior art, the conditional knockout of the target gene is realized only by one conditional knockout fragment or plasmid containing the conditional knockout, and the method has the advantages of simple operation process, high knockout efficiency, convenient screening process and the like.

Description

Vector for conditional knockout of target gene and method for conditional knockout of target gene

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a carrier for conditionally knocking out a target gene and a method for conditionally knocking out the target gene.

Background

The gene knockout is one of gene targeting technologies, and refers to an exogenous DNA introduction technology in which a DNA fragment containing a certain known sequence is subjected to homologous recombination with a gene having the same or similar sequence in a receptor cell genome, and is integrated into the receptor cell genome to obtain expression. The gene knockout technology is an irreplaceable component in the construction of transgenic animals because the gene of an organism is changed aiming at a sequence with a known but unknown function, and the function of a specific gene is lost, so that partial functions are shielded, the organism can be further influenced, and the biological function of the gene can be further presumed.

Methods commonly used for gene knockout at present mainly include CRISPR/Cas9 gene editing technology and homologous recombination technology. The CRISPR/Cas9 system works on the principle that crRNA (CRISPR-derived RNA) is combined with tracrRNA (trans-activating RNA) through base pairing to form a tracrRNA/crRNA complex, and the complex guides nuclease Cas9 protein to cut double-stranded DNA with sequence target sites paired with the crRNA; therefore, by artificially designing the two RNAs, sgRNA (single-guide RNA) with a guiding function can be transformed and formed to guide the site-specific cleavage of the Cas9 on the DNA, so that the method has the advantages of easy operation, high efficiency and the like, but has the technical defect of easy off-target. Whereas Homologous Recombination techniques rely strictly on homology between DNA molecules, Recombination between 100% recombined DNA molecules is common between non-sister chromosomes, known as Homologous Recombination, while Recombination between or within DNA molecules with less than 100% homology is known as homologus Recombination; the latter can be edited by proteins responsible for base mispairing such as MutS in prokaryotic cells or MSH2-3 in eukaryotic cells.

In the preparation process of the virus vaccine, because homologous recombination or CRISPR/Cas9 technology is adopted to delete individual virus genes, the virus replication can be weakened while the attenuation is obtained; if it is an essential gene for virus survival, the above method cannot be used to knock out the virus. A conditional knockout refers to a way to repress the expression of a gene of interest under specific induction conditions by controlling the expression of the gene of interest through conditional induction. Currently, the conditional knockout of a target gene is generally realized by using an escherichia coli lac repression operation system, taking a virus strain BA71V as an example, the specific method comprises the following steps: (1) a transfer vector obtained by cloning the coding sequences of lacI gene and reporter gene gusA downstream of the ASFV U104L early gene promoter; (2) inserting the transfer vector into a nonessential TK gene of a virus strain BA71V, constructing, purifying and screening to obtain a recombinant virus vGUSTREP expressing lac repressor protein; (3) constructing a recombinant expression vector containing lacZ gene fused with a viral promoter p72 and lac operator gene fused with a viral promoter p 72.4; (4) and then inserting the recombinant expression vector into the upstream of the target gene in the recombinant virus vGUSTRAP, and screening for the second time to obtain the recombinant virus with the target gene subjected to conditional knockout. The recombinant Virus inhibits the Expression of lac I in the absence of IPTG (. beta. -D-thiogalactoside), prevents lac operon Expression, and thus inhibits the Expression of a target Gene, thereby achieving a conditional knock-out of the target Gene (references, rodi gu, Nogal M L, M Redrejo-Rodr I gu, et al. the African Swine Virus membrane Protein pE248R is required for Virus infection and an early position [ J ]. Journal of Virology,2009, 83(23):12290 + 12300; or Ram n Garc a-Escherichia, G Andr é, F Almaz n, general. Expression [ 31 ] Expression of Protein of fire Virus [ 76, J ]: flower of Protein of fire family ] 1998). However, the process of two-step vector construction is complicated, and the final conversion power is significantly reduced through two-step conversion, which greatly increases the screening time and the screening cost. However, no method for achieving conditional knockout of a target gene by one-step vector construction has been reported yet.

The invention constructs an expression vector for target gene knockout, which comprises upstream and downstream gene sequences of a target gene initiation codon, a lacI gene sequence for coding a lac repressor and a lacO gene sequence for coding a lac operator; compared with the prior art, the conditional knockout of the target gene is realized only by one conditional knockout fragment or a vector containing the conditional knockout fragment, and the method has the advantages of simple operation process, high knockout efficiency, convenient screening process and the like.

Disclosure of Invention

In order to solve the problems, the invention constructs an expression vector for target gene knockout, wherein the expression vector comprises upstream and downstream gene sequences of a target gene initiation codon, a lacI gene sequence for coding a lac repressor and a lacO gene sequence for coding a lac operator; and the conditional knockout of the target gene is realized through the expression vector, and the method has the advantages of simple operation, high knockout efficiency and the like. The method specifically comprises the following steps:

in a first aspect, the present invention provides a conditional knock-out fragment for conditional knock-out of a gene of interest in the genome of a virus or cell, said conditional knock-out fragment having from 5 'to 3' a structure according to formula (I):

LA-T1-lacI-lacO-RA

formula (I);

wherein, the LA is a left homologous recombination arm sequence of a target gene, the LA comprises a whole or partial sequence of a target gene promoter and/or a part or whole sequence of an upstream gene of the target gene, and the end point of the 3' end of the LA sequence is selected from a sequence between an initiation codon of the target gene and a stop codon of the upstream gene of the target gene;

the T1 is a screening marker gene expression element;

the lacI is a lac repressor gene expression element;

the lacO is a lac operon gene expression element;

the RA is a right homologous recombination arm sequence of a target gene, comprises all or part of a gene sequence of the target gene, and the 3' end of the RA is a target gene initiation codon.

The LA sequence is not coincident with the RA sequence.

Preferably, said T1 comprises a selectable marker gene and its promoter; the lacI comprises a lac repressor gene and a promoter thereof; the lacO includes a lac operator gene and its promoter.

Preferably, the lac repressor gene sequence is shown as SEQ ID NO. 1; the lac operon gene sequence is shown in SEQ ID NO. 2.

Preferably, the promoter in T1, lacI and lacO is any one of eukaryotic promoters, respectively;

preferably, the selectable marker is any of the fluorescent proteins including, but not limited to eGFP, mcherry, GFP, YFP.

Preferably, the T1 comprises eGFP gene and p72 promoter; the lacI comprises a lac repressor gene and a U104L promoter; the lacO includes the lac operator gene and the p72 promoter.

Preferably, the gene sequence of the lacI is shown as SEQ ID NO. 3; the gene sequence of lacO is shown in SEQ ID NO. 4; the gene sequence of T1 is shown in SEQ ID NO. 5.

Preferably, the sequences of LA and RA are 1.0-1.5kb in length.

In a second aspect, the present invention provides a use of the conditional knockout fragment of the first aspect in preparing a gene-conditional knockout cell line of interest, or a recombinant virus, or a recombinant vaccine.

In a third aspect, the present invention provides a plasmid comprising a conditional knock-out fragment according to the first aspect above.

Preferably, the plasmid is: the conditional knock-out fragment of the first aspect is ligated by genetic engineering means to an original plasmid selected from any one of pUC18, pUC19, pUC57, pUC118, pUC119, pCA, pCK, pCC1 or variants thereof, but not limited to any one of pUC18, pUC19, pUC57, pUC118, pUC119, pCA, pCK, pCC1 or variants thereof.

In a fourth aspect, the present invention provides a use of the plasmid of the third aspect in the preparation of a gene conditional knockout cell line of interest, or a recombinant virus, or a recombinant vaccine.

In a fifth aspect, the present invention provides a method for conditional knock-out of a gene of interest, the method comprising the steps of:

(1) constructing a plasmid according to the third aspect;

(2) transfecting the plasmid in the step (1) to a host cell containing a target gene, and culturing and screening to obtain a host cell with the target gene knocked out; or transfecting the plasmid in the step (1) and the virus containing the target gene into a host cell, and culturing and screening to obtain the target gene knockout virus or vaccine.

In a sixth aspect, the present invention provides a conditional knockout cell line of a gene of interest, or a recombinant virus, or a recombinant vaccine, prepared according to the method of the fifth aspect.

The invention has the beneficial effects that:

the invention constructs a conditional knockout fragment for conditional knockout of a target gene on a viral or cellular genome, wherein the conditional knockout fragment comprises a left homologous recombination arm LA of the target gene, a screening marker gene expression element, a lac repressor gene expression element, a lac operator gene expression element and a right homologous recombination arm RA of the target gene;

meanwhile, the invention constructs a plasmid containing the conditional knockout fragment and provides a method for carrying out conditional knockout on a target gene by using the plasmid;

the method realizes conditional knockout of a target gene only by one conditional knockout fragment or plasmid containing the conditional knockout fragment, and has the advantages of simple operation process, high knockout efficiency, convenient screening process and the like.

Drawings

FIG. 1 is a schematic diagram of a strategy for constructing a recombinant ASFV virus strain with conditional knockout of E248R gene;

FIG. 2E248R shows the result of cytofluorescence detection of P11 generation recombinant virus of attenuated African swine fever virus strain with conditional knockout of gene under the condition of adding IPTG;

FIG. 3 shows the results of gene knock-out of each strain in the presence or absence of IPTG;

FIG. 4 shows the results of detecting the expression of E248R gene in recombinant viruses in the presence and absence of IPTG.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments. The scope of the invention is not limited to the examples described below.

Experimental cells, viral sources as described in the examples below:

other reagents in the experiment are common commercial reagents unless otherwise specified; the procedures in the experiments are those known in the art unless otherwise specified.

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.

Biological safety approval and African swine fever laboratory activity approval: according to the related requirements of biosafety of a biosafety level 3 laboratory (BSL-3) and related biological safety of African swine fever, the Lanzhou veterinary research institute of the Chinese agricultural academy of sciences, the biological safety committee of the laboratory animal ethics, the biological safety committee of the Chinese agricultural academy of sciences, the Experimental animal ethics of the Lanzhou veterinary research institute of the Chinese agricultural academy of sciences, and the biological safety committee of the Lanzhou veterinary research institute of the Chinese agricultural academy of sciences, the permission of developing the highly pathogenic ASFV pathogen and animal research is obtained by step and reported by step by the agricultural department, and the permission is recorded in the agricultural rural department and meets the requirements of the national biological safety level.

Sources of materials used in the examples:

cell and virus: primary bone marrow macrophages (BMDM) used in the invention are all separated from healthy pigs (purchased from animal center of Lanzhou veterinary research institute of Chinese academy of agricultural sciences) of 2-4 months age, red blood cells are removed by using a red blood cell lysate (purchased from Biosharp company) after the cells are collected aseptically, the red blood cells are centrifuged at low speed, the supernatant is discarded, the cells are resuspended in an RPMI1640 complete culture medium (purchased from Gibco company) containing 10% FBS, and the RPMI1640 complete culture medium is placed at 37 ℃ and 5% CO₂Culturing in an incubator. BMDM cell culture was supplemented with additional 10ng/mL final concentration of recombinant porcine GM-CSF (purchased from R) in RPMI1640 complete medium&D Systems Co.), 5% CO at 37 deg.C₂And (3) inducing in an incubator, washing once every 2-3 days, centrifuging the cells which are not attached to the wall, adding the cells into a new cell bottle again, changing the liquid for continuous induction, and freezing and storing or using the cells after 3-7 days. ASFV is amplified by PAM cells, and the virus titer is determined, and BMDM cells are used for plasmid transfection and virus recombination experiments.

The type II African swine fever virus strain ASFV CN/GS/2018 is from the national African swine fever regional laboratory (Lanzhou), belongs to genotype II, and has a virus titer of 5 × 10⁷TCID₅₀The strain/mL is the 4 th generation strain after PAM cell propagation, is preserved in China center for type culture Collection in 12 months and 21 days in 2020, and has the preservation number of CCTCC NO: v202096; and (4) storage address: wuhan, Wuhan university, China; and (4) contacting the telephone: 027-68752319.

The following examples demonstrate the effect of the vector and the method for knocking out the target gene of the invention by using the E248R gene in the African swine fever virus ASFV CN/GS/2018 isolate as the target gene, but the invention is not limited to the African swine fever virus E248R gene, nor to the African swine fever virus ASFV CN/GS/2018 isolate, and the strategy of the invention is applicable to knocking out all target genes.

In the following examples, for the convenience of subsequent virus purification, an eGFP gene selection marker element for promoting expression of p72 promoter was synthesized simultaneously with the synthesis of regulatory sequences, but the invention is not limited to eGFP gene selection markers, and other selection marker genes used in the field of genetic engineering are all suitable, and the selection marker is preferably fluorescent protein.

Although the implementation selects specific homologous recombination arm sequences (LA, shown as SEQ ID NO.6 and RA, shown as SEQ ID NO. 7) from the upstream and downstream sequences of the E248R gene initiation codon, the homologous recombination arm sequences can be selected on the basis of not knocking out the upstream and downstream genes of the target gene and not damaging the integrity of the target gene sequence (including the initiation codon of the target gene), wherein the selection of the homologous recombination arm sequences can be realized according to the following aspects: the LA is selected from continuous fragments at the upstream of the initiation codon of the target gene, but does not comprise the initiation codon of the target gene, and the end point of the 3' end of the LA sequence is positioned between the initiation codon of the target gene and the terminator of the upstream gene of the target gene; the RA is selected from a contiguous segment downstream of the start codon of the gene of interest, including the start codon of the gene of interest.

An operon is a generic term for a promoter, an operator, and a series of closely linked structural genes, and is a functional unit of transcription. The lac operon, namely the lactose operon, is an operon responsible for the transport and metabolism of lactose in Escherichia coli and other bacteria of the family Enterobacteriaceae.

A repressor belongs to a regulatory gene, and refers to a gene fragment that, in an inducible expression system, when an expression product thereof binds to an operator gene, a ribonucleic acid polymerase cannot pass through the operator gene, thereby, synthesis of ribonucleic acid is believed to be hindered, and the synthetic expression of the enzyme is prevented; when the expression product of the lac repressor is combined with the lactose operon of escherichia coli, the ribonucleic acid polymerase cannot pass through the operator gene, so that the synthesis of ribonucleic acid is hindered, and the synthetic expression of the gene is prevented; wherein the inducer (such as IPTG) can combine with the lac repressor to inactivate the lac repressor, inhibit the combination of the inducer and the lac operon gene, release the inhibition of the lac operon and enable the target gene to be normally synthesized and expressed.

Homologous recombination arms generally refer to flanking sequences on both sides of the foreign sequence to be inserted on the targeting vector that are identical to the genomic sequence and serve to identify and bring about the region of recombination.

Plasmids are small circular DNA molecules that can replicate autonomously outside the cell chromosome. The plasmid containing the conditional knockout segment constructed by the invention can be recombined with a virus genome after being transfected into cells, and can realize the knockout of a target gene by regulating and controlling the expression of the target gene through condition induction.

Example 1

1. Construction of target gene knockout homologous recombination transfer vector

(1) Gene synthesis

Synthesizing a gene sequence for coding a lac repressor and a gene sequence for coding a lac operator, wherein the gene sequence of the lac repressor is shown as SEQ ID NO. 1; the gene sequence of the lac operon is shown as SEQ ID NO. 2; simultaneously synthesizing a lac repressor gene element lacI started by a U104L promoter and a lac operator gene element lacO started by a p72 promoter; the sequence of the gene element lacI is shown as SEQ ID NO. 3; the gene sequence of the gene element lacO is shown in SEQ ID NO. 4.

(2) Screening expression cassette construction

In order to facilitate subsequent virus purification, the p72 promoter is synthesized to promote the expression of the eGFP gene screening marker element at the same time of synthesizing the regulatory sequence, and the sequence of the eGFP gene screening marker element for promoting the expression of the p72 promoter is shown as SEQ ID NO. 5.

(3) Construction of homologous recombination transfer vector

Cutting the pUC118 vector by EcoRI and HindIII restriction enzymes, and recovering a skeleton fragment; then, the lac repressor gene expression element lacI, the lac operon gene expression element lacO and the eGFP screening marker element synthesized in the step (1) are simultaneously connected to a skeleton fragment of pUC118 to obtain a pUC118-ASFV IPTG vector; the vector comprises a p72 promoter (p72 promoter), an eGFP gene, a U104L promoter (U104L promoter), a lac repressor expression gene (lacI), a p72 promoter (p72 promoter) and a lac operator expression gene (lac operator) from left to right;

the upstream and downstream sequences of the initiation codon of the E248R gene are designed as homologous recombination arms (LA, the sequence is shown in SEQ ID NO. 6; and RA, the sequence is shown in SEQ ID NO. 7), and two homologous arms are cloned to the left side of an eGFP screening marker element and the right side of a lac operator gene expression element lacO in the pUC118-ASFV IPTG vector by a Gibson connection method, so that a homologous recombination transfer vector pUC118-LR-iE248R-eGFP-lacI for conditionally knocking out the E248R gene is obtained.

A specific construction strategy is shown in FIG. 1, wherein lacI is a lac repressor expression gene.

Although the implementation selects specific homologous recombination arm sequences (LA, shown in SEQ ID NO.6 and RA, shown in SEQ ID NO. 7) from the upstream and downstream sequences of the E248R gene start codon, the homologous recombination arm sequences can be selected on the basis of not knocking out the upstream and downstream genes of the target gene and not damaging the integrity of the target gene sequence, wherein the selection of the homologous recombination arm sequences can be realized according to the following aspects: the LA comprises all or partial sequences of a target gene promoter and/or all or partial sequences of an upstream gene of a target gene, and the end point of the 3' end of the LA sequence is selected from a sequence between an initiation codon of the target gene and a stop codon of the upstream gene of the target gene; the RA is a right homologous recombination arm sequence of a target gene, comprises all or part of a gene sequence of the target gene, and the 3' end of the RA is a target gene initiation codon.

2. Cell transfection and recombinant virus screening

The BMDM cells were recovered and plated in 6-well plates (cell number about 10)⁶One/hole), using

The DNA transfection reagent was used to transfect the homologous recombinant transfer vector pUC118-LR-iE248R-eGFP-lacI prepared in 1 above. Add 500. mu.l buffer, 2. mu.g recombinant plasmid, 6. mu.l transfection reagent into EP tube, mix well, stand for 10min, then add into six-well plate.

After 6h of transfection, the complete culture solution for transfection is discarded, the BMDM cells are directly infected by an ASFV CN/GS/2018 virus strain (MOI ═ 1), the solution is not changed after infection, the number of fluorescent cells is observed by a fluorescence microscope after 48h and pictures are taken, and after the complete culture medium containing IPTG is cultured, the expression of a large amount of fluorescence can be observed, which indicates that the suspected recombinant virus successfully infects the cells (figure 2, GFP, IPTG +); after cell digestion, picking all the fluorescent cells in the single well, blowing off the fluorescent cells in a new culture dish with care, settling for 1 hour, picking all the single fluorescent cells in the infected well, repeatedly freezing and thawing, inoculating the cells into a 96-well BMDM cell which is paved in advance and cultured by a complete culture medium containing IPTG (IPTG final concentration in the culture medium is 1.25mM), observing the cells with fluorescence every 12 hours, marking, and continuously observing for 72 hours. The results showed that the proportion of the number of fluorescent cells in a portion of the wells reached 100%, indicating the success of recombinant virus construction (FIG. 2, TRANS, IPTG +), which was named ASFV iE 248R-eGFP-lacI.

Similarly, the fluorescent cells were picked up, replaced with a complete medium containing no IPTG, and after 48 hours of culture, the number of fluorescent cells was observed using a fluorescence microscope and photographed, and after culture in a complete medium containing no IPTG, no expression of fluorescence was observed (FIG. 2, GFP, IPTG-); after cell digestion, selecting all the fluorescent cells in the single wells, blowing off the fluorescent cells in a new culture dish with care, settling for 1 hour, selecting all the single cells in the infected wells, repeatedly freezing and thawing, inoculating the cells into the BMDM cells which are paved in advance and cultured by a complete culture medium without IPTG, observing the cells with fluorescence every 12 hours, and after marking, continuously observing for 72 hours. The results showed that no fluorescent cells were still observed in the 96-well plate (fig. 2, TRANS, IPTG); the recombinant virus ASFV iE248R-eGFP-lacI is shown to cause E248R not to be expressed under the IPTG-free condition, namely, the recombinant virus ASFV iE248R-eGFP-lacI realizes conditional knockout of E248R under the IPTG-free condition.

3. Identification of Gene knockout results

The recombinant virus was obtained by performing 10 limiting dilution and scale-up cultures of 100% of the positive wells, during which wild ASFV and recombinant ASFV genomic DNAs were extracted using a viral genome extraction kit (purchased from Beijing Tiangen Biotech Co., Ltd.), and their purities were identified by PCR using primers for ASFV E248R (ASFV E248R sur-F: aatatttccgctgtttgcgcctac; ASFV E248R sur-R: ttaacgcaacttcttggtctttt).

As shown in FIG. 3, the result of the purity detection shows that the E248R related band can be detected by using the purity detection primer to detect the genome under the condition of IPTG (IPTG +); and under the condition without IPTG (IPTG-), the genome is detected by using a purity detection primer, and a related band of E248R cannot be detected, so that the construction and the purification success of the recombinant virus ASFViE 248R-eGFP-lacI with IPTG conditionally induced E248R deletion are proved.

In addition, as shown in fig. 4, the result of the E248R gene expression test is that under the condition of IPTG (IPTG +), the E248R gene is normally expressed, i.e. under the condition of IPTG, the recombinant virus ASFV iE248R-eGFP-lacI constructed by the invention can be normally replicated without affecting the replication of the virus; under the condition without IPTG (IPTG-), the E248R gene is hardly expressed, further showing that the constructed recombinant virus ASFViE 248R-eGFP-lacI realizes the conditional knockout of the E248R gene under the condition without IPTG, and obtains the recombinant virus with the E248R conditional gene knockout.

Although only the African swine fever E248R gene is taken as an example, the invention constructs the recombinant virus with E248R conditional gene knockout. The core of the invention is the construction of conditional knockout fragments (LA-T1-lacI-lacO-RA, wherein LA is the left homologous recombination arm sequence of a target gene, LA comprises all or part of the promoter sequence of the target gene and/or all or part of the upstream gene sequence of the target gene, and the end point of the 3 'end of the LA sequence is selected from the sequence between the start codon of the target gene and the stop codon of the upstream gene of the target gene, T1 is a selection marker gene expression element, lacI is a lac repressor gene expression element, lacO is a lac operator gene expression element, RA is the right homologous recombination arm sequence of the target gene, comprises all or part of the gene sequence of the target gene, and the 3' end of RA is the start codon of the target gene). Therefore, other genes are selected as target knockout genes, LA sequences and RA sequences are designed according to the design principle of the conditional knockout fragment of the invention by using upstream and downstream gene sequences of the target knockout genes, and the purpose of conditional knockout of the target genes on genomes of other viruses or cells can be realized by constructing the conditional knockout fragment of the invention or preparing plasmids containing the conditional knockout fragment.

The above embodiments are merely preferred embodiments of the present invention, and not intended to limit the scope of the invention, so that equivalent changes or modifications made based on the structure, characteristics and principles of the invention should be included in the claims of the present invention.

Sequence listing

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tctgccagca agagcgtgtc aataatttta agctgatcgt taattaattt ttggtttaac 120

tctttgttat tatcaagatc cttcgcataa accgccatat ttaataaaaa caataaatta 180

tttttataac attatatatc cggatggtga gcaagggcga ggagctgttc accggggtgg 240

tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg 300

agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca 360

agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca 420

gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct 480

acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg 540

tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg 600

aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata 660

tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg 720

aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc 780

ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca 840

acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg 900

gcatggacga gctgtacaag taagtccccg gggacaaaaa aaaaa 945

<210> 6

<211> 1000

<212> DNA

<213> African swine fever (African swine farm virus)

<400> 6

attaattaaa aacgtattta taacgctgtt gcagttgaaa ttttggtata ggtcggaaat 60

attgcccgag cctccgtatt ctgcaatgtt ctgacatatg gtgagtccgg aggggcactg 120

cttgttggtc aaaatatttc tttgctccgt tgttttatag gcatttttat ttccattaca 180

cggagcaaac gcacattcag cccatagggt gccggagttc acacaggcac aatactggct 240

atacgcatac tcatcctttg agcacaatcc ctgtttatcg catatgctcc caataatatt 300

gtcatcctcc gccgtttgtt gatttgtatg cgagcgtaaa atagcggccc aggccttggg 360

ctcctttttt tgcagctcgg aaatcgaagg gcctgtacag ctaaagtcga cccaaatatc 420

attgcatttc gtggaaactg gcatgcaaga cataattgaa ataattaata agtatatatc 480

atggcaacaa atttttttat tcaacctatc accgaagaag ctgaagcata ctacccacct 540

tccgtgataa cgaataaacg gaaggacctg ggggtagacg tatactgttg ctccgaccta 600

gtgcttcaac ctggactaaa tattgttcgc ctgcatatta aagtagcatg cgaacacatg 660

ggcaaaaaat gcggttttaa aatcatggcg agaagcagta tgtgcaccca tgaacggctg 720

ctcatccttg caaacggaat tggtttaata gacccgggtt atgtgggcga gctcatgctc 780

aagatcatta atcttggcga caccccggtc caaatatggg ccaaagaatg tttggtgcag 840

ttggtggccc aaggtgacca tgtgcctgac catatcaaca tcctaaaaag aaaccaaata 900

tttccgctgt ttgcgcctac cccaagaggc gagggtagat ttgggagcac gggcgaggcc 960

gggattatga gaacttaatt ttattttttt tcttaacata 1000

<210> 7

<211> 1000

<212> DNA

<213> African swine fever (African swine farm virus)

<400> 7

atgggaggct ctacaagcaa aaattccttt aaaaatacga ccaacattat cagcaattcc 60

attttcaatc agatgcaaag ttgtatttcc atgttggatg gcaaaaatta cataggcgta 120

ttcggtgatg gaaatatttt aaaccacgtt ttccaggatt taaacttatc attaaacaca 180

agttgcgtgc aaaagcacgt aaacgaggaa aatttcatta caaatctttc gaaccaaatt 240

actcaaaatt taaaagacca agaagttgcg ttaacccaat ggatggacgc aggaactcac 300

gatcagaaaa cggatataga agaaaatata aaggtaaact taacaaccac acttattcaa 360

aactgcgttt catccctgtc gggtatgaac gtgctggtgg tgaaggggaa tggcaacatt 420

gttgaaaacg caactcagaa gcagtcgcag caaatcatct ctaactgctt gcaggggagc 480

aagcaggcca tagacaccac aaccggcatc actaacacgg taaatcagta ctcacactac 540

acctcaaaaa acttttttga cttcattgca gacgcaattt cggctgtttt taaaaacatc 600

atggtcgcgg ctgtagttat cgttctaatc atcgtagggt ttatagccgt cttttacttt 660

ttgcattcac ggcaccgcca tgaggaggaa gaagaagctg aaccactcat aagcaacaag 720

gtattaaaaa atgctgccgt ttcgtaataa tttaattaaa agtaaaaaaa aaaggtattg 780

ttatagtgat ggcagatttt aattctccaa tccagtattt gaaagaagat tcgagggacc 840

ggacctctat aggttctcta gaatacgatg aaaatgccga cacgatgata ccgagcttcg 900

cagcaggctt ggaagagttt gaacccattc ccgactatga ccctaccaca tcaacttccc 960

tgtattcaca attgacccac aacatggaaa aaatcgcaga 1000

Claims (12)

1. A conditional knock-out fragment for conditional knock-out of a gene of interest in the genome of a virus or cell, said conditional knock-out fragment having from the 5 '-3' end a structure according to formula (i):

LA-T1-lacI-lacO-RA

formula (I);

wherein, the LA is a left homologous recombination arm sequence of the target gene, the LA is selected from a continuous fragment at the upstream of an initiation codon of the target gene but does not comprise the initiation codon of the target gene, and the end point of the 3' end of the LA sequence is selected from a sequence between the initiation codon of the target gene and a stop codon of the upstream gene of the target gene;

the T1 is a screening marker gene expression element, and comprises a screening marker gene and a promoter thereof;

the lacI is a lac repressor gene expression element and comprises a lac repressor gene and a promoter thereof;

the lacO is a lac operator gene expression element and comprises a lac operator gene and a promoter thereof;

the RA is a right homologous recombination arm sequence of the target gene, is selected from a continuous segment at the downstream of an initiation codon of the target gene and comprises the initiation codon of the target gene, and the 3' end of the RA is the initiation codon of the target gene.

2. The conditional knock-out fragment of claim 1, wherein the lac repressor gene sequence is set forth in SEQ ID No. 1; the lac operon gene sequence is shown in SEQ ID NO. 2.

3. The conditional knock-out fragment of claim 2, wherein the promoter in T1, lacI, and lacO is any one of eukaryotic promoters; the screening marker is any one of fluorescent proteins.

4. The conditional knock-out fragment of claim 3, wherein T1 comprises an eGFP gene and a p72 promoter; the lacI comprises a lac repressor gene and a U104L promoter; the lacO includes the lac operator gene and the p72 promoter.

5. The conditional knock-out fragment of claim 4, wherein the lacI gene sequence is set forth in SEQ ID No. 3; the gene sequence of lacO is shown in SEQ ID NO. 4; the gene sequence of T1 is shown in SEQ ID NO. 5.

6. The conditional knock-out fragment of claim 1, wherein the sequences of LA and RA are 1.0-1.5kb in length.

7. Use of a conditional knock-out fragment according to any of claims 1 to 6 for the preparation of a gene of interest conditional knock-out cell line, or a recombinant virus, or a recombinant vaccine.

8. A plasmid comprising a conditional knock-out fragment according to any of claims 1 to 6.

9. The plasmid of claim 8 wherein the conditional knock-out fragment of any one of claims 1 to 6 is ligated by genetic engineering means to an original plasmid comprising any one of pUC18, pUC19, pUC57, pUC118, pUC119, pCA, pCK, pCC1 or a variant thereof.

10. Use of the plasmid according to claim 8 or 9 for the preparation of a gene conditional knock-out cell line of interest, or a recombinant virus, or a recombinant vaccine.

11. A method for conditional knock-out of a gene of interest, comprising the steps of:

(1) constructing the plasmid of claim 8 or 9;

(2) transfecting the plasmid in the step (1) to a host cell containing a target gene, and culturing and screening to obtain a host cell with the target gene knocked out; or transfecting the plasmid in the step (1) and the virus containing the target gene into a host cell, and culturing and screening to obtain the target gene knockout virus or vaccine.

12. The conditional knockout cell line of the target gene, or the recombinant virus, or the recombinant vaccine prepared by the method of claim 11.

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