CN117384965A - Method for constructing bovine nodular skin disease virus TK gene deletion strain LSDV-△TK - Google Patents

Abstract

The invention provides a method for constructing a bovine nodular skin disease virus TK gene-deleted strain LSDV-ΔTK, which includes the following steps: constructing pUC19T-LoxP-pmH5-EGFP-LoxP-ΔTK transfer vector with a deleted TK gene; and constructing a green fluorescent transfer vector Mark the strain LSDV‑LoxP‑pmH5‑EGFP‑LoxP‑△TK; use the Cre‑LoxP recombinase system to construct the TK gene deletion strain LSDV‑△TK. The present invention uses homologous recombination technology to directionally insert the LoxP‑pmH5‑EGFP‑LoxP gene expression frame into the TK gene of the viral genome of the LSDV strain to construct the green fluorescent labeled strain LSDV‑LoxP‑pmH5‑EGFP‑LoxP‑△TK. Cre-Loxp recombinase system excises the EGFP tag to construct LSDV-ΔTK.

Description

Method for constructing bovine nodular skin disease virus TK gene deletion strain LSDV-△TK

Technical field

The present invention relates to a method for constructing a bovine nodular skin disease virus TK gene-deleted strain LSDV-ΔTK.

Background technique

Lumpy skin disease (LSD), also commonly known as bovine nodular rash, bovine nodular dermatitis or bovine bumpy skin disease, is caused by bovine nodular skin disease, a member of the genus Capripoxvirus in the family Poxviridae. An acute and subacute infectious disease of cattle caused by Lumpy skindisease virus (LSDV). The disease first appeared in the Republic of Zambia in 1929, and then spread rapidly to the Middle East, Eastern Europe, and Central Asia. On August 12, 2019, a bovine nodular skin epidemic occurred in Yili Prefecture, Xinjiang Uygur Autonomous Region. This was the first confirmed LSD epidemic in China. The disease has caused significant economic losses to my country's cattle industry and seriously affected the healthy development of the animal husbandry industry. At present, there is still a lack of specific vaccines and better treatments for this disease.

The insertion position of foreign genes has a great influence on the rescue of recombinant viruses. Current research shows that proteins related to capripox virus virulence mainly include three categories: envelope proteins, proteins related to nucleotide metabolism, and optional capsid proteins. There are three non-essential regions for replication in the capripox virus genome: the thymidine kinase (TK) gene, the γ-interferon receptor (γ-IFNR) gene and the ribonucleic acid reductase ( Theribonucleotide reductase (RR) gene. The virulence of LSDV is affected by its genes and encoded proteins. The deletion of virulence-related genes can weaken the virulence and reduce the pathogenicity of the virus, laying the foundation for the creation of new genetically engineered attenuated vaccines.

The Cre-LoxP system is a recombinase system that can perform deletions, insertions, translocations and inversions at specific DNA sites. It is a widely used gene editing tool. The Cre-Loxp recombinase system has the advantages of high efficiency, strong specificity, wide range of applications, and expression driven by a type II promoter. Taking advantage of these characteristics of the Cre-Loxp recombinase system, researchers can modify the Loxp site sequence as needed when constructing vectors for specific gene mutation or repair, increasing the application scope of the system.

Contents of the invention

In order to solve the problems existing in the prior art, the present invention provides a method for constructing a bovine nodular skin disease virus TK gene-deleted strain LSDV-ΔTK. The present invention uses homologous recombination and the Cre-Loxp system to directionally insert the gene expression boxes (LoxP-pmH5-EGFP-LoxP) of two LoxP sites in the same direction into the LSDV/FJ/CHA/2021 strain (Genbank accession number: OP752701) at the TK gene of the viral genome, a green fluorescent labeled strain (LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK) was constructed. In order to ensure subsequent vaccine research and ensure that no tag protein remains in the viral genome, MDBK-Cre cells that stably express Cre recombinase activity are used to provide exogenous Cre recombinase and target two Loxp sites in the same direction in the viral genome. , specifically excise the fluorescent protein tag (EGFP) in the green fluorescent-labeled virus genome, and construct an LSDV strain lacking the TK gene, named LSDV-△TK.

The invention provides a method for constructing bovine nodular dermatosis virus TK gene deletion strain LSDV-ΔTK, which includes the following steps:

(1) Construct the pUC19T-LoxP-pmH5-EGFP-LoxP-ΔTK transfer vector lacking the TK gene;

(2) Construct a green fluorescent labeled strain LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK;

(3) Use the Cre-LoxP recombinase system to construct the TK gene-deleted strain LSDV-ΔTK.

Preferably, in step (1), the method of constructing the pUC19T-LoxP-pmH5-EGFP-LoxP-ΔTK transfer vector lacking the TK gene is: cloning the upstream and downstream 1000bp homology arms and EGFP expression cassette of the LSDV TK gene respectively. , using the overlapping PCR method, the three DNA fragments were fused, and the homologous recombination method was used to connect the pUC19T empty vector that had been double digested with BamHI and EcoR I to construct the pUC19T-LoxP-pmH5-EGFP-LoxP-ΔTK transfer vector.

Preferably, in step (2), the method for constructing the green fluorescent labeled strain LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK is: transfecting the pUC19T-LoxP-pmH5-EGFP-LoxP-ΔTK transfer vector Vero cells were subsequently infected with wild-type LSDV strain.

Preferably, in step (3), the method of using the Cre-LoxP recombinase system to construct the TK gene-deleted strain LSDV-ΔTK is: first construct MDBK cells stably expressing Cre recombinase, and then label the virus with green fluorescence. MDBK-Cre cells were infected with strain LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK.

Preferably, in step (3), the method for constructing MDBK cells stably expressing Cre recombinase is:

S1, using the cDNA of plasmid pcDNA3.1-Cre-Flag as a template, use primers to amplify the Cre gene to obtain the Cre recombinase gene;

S2, after recovering the amplification product from step S1, use homologous recombination technology to connect it to the lentiviral shuttle plasmid pLV-Puro double-digested by EcoR I and BamH I to obtain the lentiviral packaging plasmid pLv-Puro-Cre;

S3, transfect the lentiviral packaging plasmid pLv-Puro-Cre into 293T cells for lentiviral packaging. After culture, collect the cell supernatant and centrifuge. Infect MDBK cells with the centrifuged supernatant, conduct screening and subcloning, and obtain stable MDBK cell line MDBK-Cre expressing cyclic recombinase.

Preferably, in step (1), the primer is:

pLv-Cre-Puro-For:TTTCAGGTGTCGTGAGGATCCGCCACCATGTCCAATTTACTGACCGTACACCAAAATTTG;

pLv-Cre-Puro-Rev: GCGGCCGCCCTCGAGGAATTCTCATTATTTATCGTCATCGTCTTTGTAGTCGCTTCCTCCTCCATCGCCATCTTCCAGCAG.

Preferably, in step (3), 10% FBSDMEM medium containing 1.25 μg/mL puromycin is used for screening.

Preferably, after obtaining MDBK-Cre, the method further includes the following steps: cultivating MDBK-Cre cells using a medium containing 10% FBSDMEM, and culturing at 37°C and 5% carbon dioxide.

The invention also provides a bovine nodular skin disease virus TK gene-deleted strain LSDV-ΔTK, which is prepared by applying the above method.

The present invention also provides the application of the above-mentioned bovine nodular skin disease virus TK gene-deleted strain LSDV-ΔTK in constructing bovine nodular skin disease virus live vector vaccine.

The TK gene is a widely studied virulence gene in the poxvirus genome. This gene belongs to a non-essential region for virus replication. Many studies have used this non-essential region to insert foreign genes to construct live poxvirus vector vaccines.

The present invention uses homologous recombination technology to directionally insert the LoxP-pmH5-EGFP-LoxP gene expression frame into the TK gene of the viral genome of the LSDV/FJ/CHA/2021 strain to construct a green fluorescent marker strain (LSDV-LoxP-pmH5-EGFP -LoxP-△TK), the Cre-Loxp recombinase system was used to remove the EGFP tag between LoxP sites with the same orientation in the viral genome, and a bovine nodular skin virus virus strain lacking the TK gene (LSDV-△TK ).

Using homologous recombination and the Cre-Loxp system, the present invention successfully constructed a TK gene-deleted LSDV strain (LSDV-△TK) without fluorescent marker protein (EGFP), which provides a basis for subsequent attenuated bovine nodular skin disease virus vaccines and The development of live vector vaccines provides candidate strains and viral vector platforms. The invention greatly expands the application scope of the Cre-Loxp system and provides important technical and theoretical support for the research and development of genetic engineering vaccines such as bovine nodular dermatosis virus.

Description of the drawings

The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. In the attached picture:

Figure 1 is a schematic diagram of the Cre-Loxp system applied to the construction of LSDV TK gene-deficient strains.

Figure 2 is a map of the transfer vector pUC19T-LoxP-pLEO-EGFP-LoxP.

Figure 3 is a map of the transfer vector pUC19T-LoxP--pmH5-EGFP-LoxP.

Figure 4 is a map of the transfer vector pUC19T-LoxP-pSS-EGFP-LoxP.

Figure 5 is a map of the transfer vector pUC19T-LoxP-p7.5-EGFP-LoxP.

Figure 6. Screening evaluation of effective poxvirus promoters.

Figure 7 shows the rescue and purification of the LSDV strain lacking the green fluorescent marker of the TK gene (LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK).

Figure 8 is a map of plasmid pcDNA3.1-Cre.

Figure 9 shows the PCR amplification results of Cre recombinase gene.

Figure 10 shows that MDBK-Cre cells have high catalytic recombination activity.

Figure 11 shows the rescue and purification of the LSDV-ΔTK strain by removing the green fluorescent tag.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative experiments in the following examples were repeated three times, and the results were averaged.

Example 1

1 Cre-LoxP recombinase system is used to construct green fluorescent labeled strains

Screening of non-essential regions for LSDV replication is a prerequisite for constructing deletion recombinant viruses. The positions of the TK genes of poxviruses of different genera in the family Poxviridae vary, but the positions of the TK genes are all in the middle region of the viral genome and are highly conserved. The TK gene is a replication non-essential gene of a widely studied poxvirus. Many studies have used this non-essential region to insert foreign genes to construct live vector vaccines. In the present invention, the TK gene is selected as the insertion position of the exogenous green fluorescent marker protein EGFP. As shown in Figure 1, based on the genome sequence of the LSDV/FJ/CHA/2021 strain (GenBank accession number: OP752701), the Loxp site with the same direction was constructed into the gene expression frame of the transfer vector using homologous recombination for subsequent use. Excision of fluorescent protein tags.

Figure 1 is a schematic diagram of the Cre-Loxp system applied to the construction of LSDV TK gene-deficient strains.

Cre recombinase (Cyclization Recombination Enzyme, Cre) is encoded by the Cre gene of E. coli phage P1 and is a 38kDa protein composed of 343 amino acids. It not only has catalytic activity, but also is similar to a restriction enzyme and can specifically recognize the LoxP (Locus of X-overP1) site. The site is 34 bp long, including two 13 bp inverted repeats and an 8 bp spacer region. Among them, the inverted repeat sequence is the specific recognition site of Cre recombinase, and the spacer region determines the direction of the LoxP site. Based on the position and orientation of the LoxP site, the Cre-LoxP system has multiple ways to induce recombination. When the LoxP sites are located on the same DNA strand and in the same direction, Cre recombinase binds to the inverted repeat sequence regions at both ends of the LoxP site to form a dimer. This dimer combines with dimers at other LoxP sites to form a tetramer. Subsequently, the DNA between the LoxP sites is cleaved by Cre recombinase, and the nicks are relinked by DNA ligase.

2 Screening evaluation of four effective poxvirus promoters

The strength of the promoter is one of the main factors that determines gene expression, whether it is regulating the reporter gene or the effective exogenous expression of the target antigen gene. Strong promoters have higher efficiency in initiating the synthesis of mRNAs, while weak promoters have lower efficiency. In order to ensure the subsequent effective and successful construction of LSDV deletion recombinant strains, we first screened and verified the promoter activities of four (pLEO, pmH5, pSS and p7.5) poxviruses (Table 1). Using homologous recombination technology and overlapping PCR method, pUC19T-LoxP-pLEO-EGFP-LoxP, pUC19T-LoxP--pmH5-EGFP-LoxP, pUC19T-LoxP-pSS-EGFP-LoxP and pUC19T-LoxP-p7.5- were constructed. EGFP-LoxP four transfer vectors (Figure 2-Figure 5), the four successfully constructed promoter transfer vectors, using Poly plus transfection reagent, according to the transfection reagent ratio plasmid concentration is 1:2, the transfection cloth is in 6 Vero cells in the plate wells (the confluence rate reached 80%-100%) were transfected for 18 hours and then infected with 5 μL of 6.23lg TCID ₅₀ wild-type LSDV strain (LSDV/FJ/CHA/2021). After infection for 24h.pi (hour post-infection, hpi), the 6-well plate was placed under an inverted fluorescence microscope for observation and photography. As shown in Figure 2, the intensity of green fluorescence shows that the efficiency and activity of poxvirus promoter 3 (Promoter-3, pmH5) is significantly higher than the other three promoters.

Figure 2 is a map of the transfer vector pUC19T-LoxP-pLEO-EGFP-LoxP.

Figure 3 is a map of the transfer vector pUC19T-LoxP--pmH5-EGFP-LoxP.

Figure 4 is a map of the transfer vector pUC19T-LoxP-pSS-EGFP-LoxP.

Figure 5 is a map of the transfer vector pUC19T-LoxP-p7.5-EGFP-LoxP.

Figure 6. Screening evaluation of effective poxvirus promoters.

Table 1 Poxvirus promoter sequences

3 Construction, rescue and purification of green fluorescent labeled LSDV strain lacking TK gene (LSDV-LoxP-pmH5-EGFP-LoxP-△TK)

In the present invention, we simultaneously used homologous recombination technology and the Cre-LoxP system to construct an LSDV strain lacking the green fluorescent marker of the TK gene (LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK), and used the methods in Table 2 Two pairs of PCR primers were used to verify the purification of the recombinant virus.

According to the genome sequence of LSDV/FJ/CHA/2021 (NCBI database GeneBank accession number OP752701), 1000 bp were selected upstream and downstream of the TK gene (ORF68), and primers were designed to amplify them as homology arms. Clone the EGFP expression cassette gene sequence. Using the fusion PCR method, the three DNA fragments of the upstream homology arm, EGFP expression box (LoxP-pmH5-EGFP-LoxP-SV40 poly(A)) and the downstream homology arm were fused in sequence, and then linked to by homologous recombination. On the pUC19T empty vector digested by BamHI and EcoRI, a pUC19T-LoxP-pmH5-EGFP-LoxP-ΔTK transfer vector lacking the TK gene was constructed.

Among them, the nucleotide sequence of the upstream homology arm is as follows:

ATCAATGAAACTTAATGGAGTTTATGGTTTTACGTATAAAAATGAACTAAGAAAACTTAGTTCAGATAAAGAAATTGATGAATATAGCAACAAGCCATTACAAGAACCAGTTAGATTAAATGACTTTATTGGACTGTTTGATTGTGTAAAGAAAAATATACCACTAACAAATATTCCAATTATGGAATAAAATATCATCAGTTAAATGGAGAAAAATAACTCAAAAAACATTTATTTTACTCCTGTATTTATAGAACCTACTATAAAACATTCCCTTTTG CAATCTTACAAATATACATATATCATAATATTTGAAATTATAACAGTGATGGTATTATTATTTTATTTTTTAAGTCAGAAATCCACATGTTGTTTAAATTCAAACAACAAAAAGTTATCAGTCCGATAGACAAATTTTCAAAAACCACTTTATACTGTAAAGAAAATAAGCTTTTTATTAGTGGGTTACCTAATACTATGTACTCAAAGGAAGCACTATCATTAAATAGACAACCGATAACATATAAATATTGTAATGATCTTTTACAATCAAATAAATGGATC ACAGCAAGTATTTATTAACGATATTCTTAGAAAATGATGACTCCTTTTTTAAATACTTATCAGAACAAGATGATGAAACAGCTATGTCTGATATCGAAACTATTGTAACATATTTAAATTTTTTATTGTCATTGTTAATTAGATCAAAGGATAAATTAGAGTCGATAGGTTATTATTATGAACCACTGTCTGAAGAATGTAAAACATTAGTTGATTTTTCCAATATGAAAAATTTTAGGATATTATTAAAAGATTCCTATAAATATACTAAAT AAACAAATAACTGTAAATAAAGGGTACTTATCAGATTTTGTTACGACATTGATGAGATTAAAAAAAGAACTTTTTTTAGAATCACCAGAGCCGATAACATATATAGACCCTAGAAAAGATCCAACATTTTTAAACATTTTATCAATATTGCACGAAAATAA

The nucleotide sequence of the downstream homology arm is as follows:

TAAATATTAATGAAAAAAAATCAAAAAAAAGTGATCTATTTACTTATTAAACTATATAGTTAATAAGTAAAATGGGTATCAGACACGAGTTAGATATTTTGCTTGTTTCTGAAAATCTCGCACTGAAGAATGTTGAACTTCTTAAAGGTGATAGTTATGGATGTACTATTAATATAAAAGTTAATCAACAAAAAATTGGATTTTATTATTATTATTACGGCCCGATTGGACAGAGGTAAGGAATGTTAAAAAAAAAAAATGGTTAGTAACGGTGT TGTTATTGATACAACACTAATTAAAAAATCTTTTTACGAAGAAGTATATTCATCATCTGTAACAGTTTTTCAAAATACTACCGTTGAATTTTTTAGTGATACTAGTAAGAAATATAAAGAAGAATATCCCATTGTTAACATAAACACCATAAAAGCGTTATTACGAAATAAAAGATTCAAAAATGACATGTATAAATTTTGAATCACCTATAAGTGATTATGATCAAGTAAATTATTTAAAAGATTACATAAATATAAGTGATGATTATTATCT GTATGACGCATGCGATGATTGTATCATTAGTAGCGATGATGATGACGATAATGATAATGCGGACGATGACGATGAGGACGACGAGGTTAATGATATAGAGGATGACTATGAATGATTTTTTTGAAAACTAATGATTAATATATTTAAATATTAACAAAATTTACTTTAAAAATGGAAGCAGTATCTATGGATAAACCATTTATGTATTTTGACGAAATAGATAACGAATTAGAATATGACCCTAAAACTTCCGAAGAAAAGCCAAAAAA ATTACCATATCAAGGTCAATTAAAATTACTTCTTTGTGAATTATTTTTTCTGAGTAAGTTACAAAGACATGGAATATTGGATGGATGCACAATAGTATATGTTGGATCAGCTCCAGGAACACATATTAAATACTTAAGAGATCATTTTTTATCTATGGGATTAGTTATAAGATGGATATTG

The specific construction method of the green fluorescent labeled strain (LSDV-LoxP-pmH5-EGFP-LoxP-△TK), as well as the specific methods of rescue and purification are as follows: use Poly plus transfection reagent, pUC19T-LoxP-pmH5-EGFP-LoxP -△TK transfer vector (1:2), transfect Vero cells distributed in 6 plate wells (confluence rate reaches 80%-100%), 18 hours after transfection, and then infected with 5μL 6.23lg TCID ₅₀ wild-type LSDV strain (LSDV/FJ/CHA/2021). 24h.pi after infection, the 6-well plate was placed under an inverted fluorescence microscope for observation and photography. The virus strain was continuously passaged three times in wild-type MDBK cells (10% FBSDMEM), using 10-fold gradient dilution (10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 and 10 ^-6 ). After subcloning three times in a 96-well plate, the fluorescently labeled strain (LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK) was positively selected.

Table 2 PCR primers

The prepared green fluorescent labeled strain (LSDV-LoxP-pmH5-EGFP-LoxP-△TK) was subjected to subsequent purification verification experiments. The experimental process was as follows: As shown in Figures 7A and 7B, the LSDV TK gene was specifically designed to The PCR results of two pairs of primers (Primer 1 and Primer 2) (Table 2) showed that, compared with the positive control (LSDV group) and the negative control (ddH ₂ O group), subclone #11 was a homozygous fluorescently labeled strain ( LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK). The sequencing results for “#11” also further proved that we have successfully obtained a homozygous fluorescently labeled LSDV strain lacking the TK gene (LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK) (Figure 7C). The fluorescently labeled strain (LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK, #11) can grow well on Wild-type MDBK and produce obvious cytopathological changes.

Figure 7 shows the rescue and purification of the LSDV strain lacking the green fluorescent marker of the TK gene (LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK). Among them, (A) PCR detection results of EGFP gene in LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK strain using primer 1 in Table 2; (B) PCR detection results of TK in LSDV field strain using primer 2 in Table 2 Gene PCR detection results; (C) "#11" clone Sanger sequencing analysis results; (D) Pictures of typical pathological changes produced by the LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK strain in MDBK cells. Note: 20×, 10× and 2× indicate magnification.

As can be seen from Figure 7, the PCR results of primer 1 and primer 2 (Table 2) show that clone #11 is a pure green fluorescent labeled strain missing strain (shown by the red dotted line). The sequencing results of clone #11 also showed that the green fluorescent marker deleted strain was homozygous (Figure 7C). At the same time, the green fluorescent marker-deficient strain (LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK, #11) can also produce obvious cytopathic effect (CPE) in MDBK cells (shown by green fluorescence in Figure 7D) .

4 Construction of LSDV-△TK recombinant strain using Cre-LoxP recombinase system

In order to remove the EGFP tag in the viral genome and ensure the development of subsequent vaccines, the present invention uses a lentiviral packaging system to construct MDBK cells (MDBK-Cre) that stably express Cre recombinase for "exogenous" supply of Cre recombinase.

4.1 The preparation method of MDBK-Cre cell line is as follows:

4.1.1 Molecular cloning of Cre recombinase gene

Cre recombinase is produced by Escherichia coli phage P1 and belongs to the λInt enzyme supergene family. The full length of the Cre gene coding region sequence is 1029bp, encoding a 38kDa protein consisting of 343 amino acids. Based on the Cre sequence in the Pubmed database (NCBI database accession number: Extension time 75 s), the Cre gene was amplified using a specific primer pair (Table 3), and detected by 1% agarose gel electrophoresis (Figure 9). The results showed that the Cre recombinase gene had been successfully cloned. Then, the product was recovered from the gel and connected to the EcoRI and BamHI double-digested lentiviral shuttle plasmid pLV-Puro (purchased from addgene) using homologous recombination technology. The correct positive plasmid was sequenced and named: pLv-Puro- Cre.

Figure 8 is a map of plasmid pcDNA3.1-Cre.

Figure 9 shows the PCR amplification results of Cre recombinase gene. Among them, Marker: DNA molecular mass (DL5000); Cre: Cre gene PCR amplification product.

Table 3 PCR primers

4.1.2 Lentiviral transduction of MDBK cells and puromycin drug screening

The lentiviral packaging plasmids pLV-Puro-Cre, VSV-G and pxPAX2 were transfected into 293T cells (approximately 80% confluence) at a ratio of 2:1:2 for lentiviral packaging. After culturing for 24 hours, collect the cell supernatant, centrifuge the resulting supernatant at 1500 rpm for 5 min, and filter out the cell residues with a 0.45 μm filter. Use 100 μL of successfully packaged centrifuged lentivirus culture supernatant to infect bovine kidney cells (Madin-Darby Bovine Kidney, MDBK), and set up a negative cell control. Cultivate in 10% FBSDMEM medium containing puromycin at a concentration of 1.25 μg/mL. After drug screening for 2 days, replace the culture medium with a new one and continue drug screening for about 5 days. After all negative control cells die, the surviving cells are replaced into a 6-well cell plate to continue drug screening and culture. The cells that have undergone three drug screenings are subcloned in a 96-well plate. After the cells grow into a single clonal cell line, they are expanded, cultured and frozen for subsequent verification. In this experiment, a positive clone cell line that stably and efficiently expressed Cre recombinase was successfully screened through subcloning, named MDBK-Cre.

4.1.3 Culture of MDBK-Cre cells

MDBK-Cre cells were cultured in DMEM medium containing 10% FBS, placed in a 37°C, 5% carbon dioxide incubator, and subcultured. The growth characteristics of this cell line were good.

4.2 Construction of LSDV strain lacking TK gene (LSDV-△TK)

Infect MDBK-Cre cells with the green fluorescent marker deleted strain LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK prepared in step 3. The specific method is: Plate 1×10 ⁶ MDBK-Cre cells in a 6-well plate. After the cells have adhered and the growth confluence rate is 80%-100%, they are infected with 5 μL LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK fluorescence. Mark strains. When the LSDV deletion strain carrying the LoxP site in the same direction (LSDV-LoxP-pmH5-EGFP-LoxP-△TK) is infected and replicated in MDBK-Cre cells, the Cre recombinase will bind to the LoxP site, thereby converting The EGFP tag located at two LoxP sites in the viral genome was excised. Subsequently, LSDV-ΔTK was obtained using plaque purification and 96-well subcloning methods, and then through reverse purification (as shown in Figure 10A).

Figure 10 shows that MDBK-Cre cells have high catalytic recombination activity. Among them, (A) Schematic diagram of MDBK-Cre excluding the green fluorescent labeled LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK EGFP tag in the genome; (B) Typical LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK in the wild Cytopathic effect (CPE) in Wild-type MDBK cells (Wild-type MDBK) and MDBK-Cre cells.

As shown in Figure 10B, compared with wild-type MDBK cells (as shown by the white arrow, left in Figure 10B), LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK was observed after 48 h.p.i. The fluorescence in the typical cytopathic lesions (CPE) of infected MDBK-Cre cells "disappears" (as shown by the red arrow), and the fluorescence around the CPE exhibits a "shrunk" appearance (as shown by the green arrow), indicating that the Cre recombinase is It has high catalytic recombination activity and can effectively recognize and cut the EGFP tag between two Loxp sites in the same direction in the LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK genome. It has successfully removed the EGFP tag in the genome of the fluorescently labeled virus strain. EGFP tag to quench its fluorescence.

Subsequently, the LSDV-LoxP-pmH5-EGFP-LoxP-ΔTK strain was continuously passaged three times in MDBK-Cre cells (10% FBS DMEM), using 10-fold gradient dilution (10 ^-1 , 10 ^-2 , 10 ^{- 3} , 10 ^-4 , 10 ^-5 and 10 ^-6 ). After subcloning twice in a 96-well plate, the non-fluorescent strain (LSDV-△TK) was reverse purified. We successfully obtained the purified LSDV-△ TK strain. Use PCR amplification and sequencing to verify the purified virus. The primers used in PCR amplification are shown in Table 4, and the verification results are shown in Figure 11.

Table 4 PCR primers

Figure 11 shows the rescue and purification of the LSDV-ΔTK strain by removing the green fluorescent tag. Among them, (A) PCR electrophoresis verification results; (B) LSDV-ΔTK clone #1 genome sequencing results.

As shown in Figure 11A, the PCR results of two pairs of primers (Primer1 and Primer 2) (Table 3) specifically set for the LSDV TK gene showed that compared with the positive control (LSDV group) and the negative control (ddH ₂ 0 group) , four subclones (#1, #2, #3 and #4) have used the Cre-Loxp recombinase system to completely remove the EGFP tag in the green fluorescent labeled strain (LSDV-EGFP-ΔTK). The sequencing results for “#1” also further proved that we have successfully obtained the LSDV strain lacking the TK gene (LSDV-ΔTK) (Figure 11B).

Finally, it should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it is still The technical solutions described in the foregoing embodiments may be modified, or some of the technical features may be equivalently replaced. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. The construction method of Niu Jiejie skin disease virus TK gene deletion strain LSDV-delta TK is characterized by comprising the following steps: the method comprises the following steps:

(1) Constructing a pUC19T-LoxP-pmH 5-EGFP-LoxP-delta TK transfer vector deleted of the TK gene;

(2) Constructing a green fluorescent marker strain LSDV-LoxP-pmH 5-EGFP-LoxP-delta TK;

(3) The Cre-LoxP recombinase system is utilized to construct a TK gene deletion strain LSDV-delta TK.

2. The construction method according to claim 1, wherein: in the step (1), the method for constructing the pUC19T-LoxP-pmH 5-EGFP-LoxP-delta TK transfer vector deleted of the TK gene comprises the following steps: and respectively cloning 1000bp homology arms and EGFP expression frames at the upstream and downstream of the LSDV TK gene, fusing three DNA fragments by using an overlap PCR method, and connecting the three DNA fragments with a pUC19T empty vector subjected to BamH I and EcoR I double enzyme digestion by using a homologous recombination method to construct a pUC19T-LoxP-pmH 5-EGFP-LoxP-delta TK transfer vector.

3. The construction method according to claim 1, wherein: in the step (2), the method for constructing the green fluorescent marker strain LSDV-LoxP-pmH 5-EGFP-LoxP-delta TK is as follows: vero cells were transfected with pUC19T-LoxP-pmH 5-EGFP-LoxP-DeltaTK transfer vector, followed by infection with wild-type LSDV strain.

4. The construction method according to claim 1, wherein: in the step (3), the method for constructing the TK gene deletion strain LSDV-delta TK by using the Cre-LoxP recombinase system comprises the following steps: MDBK cells stably expressing Cre recombinase are first constructed, and then the MDBK-Cre cells are infected with a green fluorescent marker strain LSDV-LoxP-pmH 5-EGFP-LoxP-delta TK.

5. The construction method according to claim 4, wherein: in the step (3), the method for constructing MDBK cells stably expressing Cre recombinase comprises the following steps:

s1, using cDNA of a plasmid pcDNA3.1-Cre-Flag as a template, and amplifying a Cre gene by using a primer to obtain a Cre recombinase gene;

s2, after the amplification product glue obtained in the step S1 is recovered, connecting the amplification product glue to a lentivirus shuttle plasmid pLV-Puro cut by EcoR I and BamH I by using a homologous recombination technology to obtain a lentivirus packaging plasmid pLv-Puro-Cre;

s3, transfecting a lentiviral packaging plasmid pLv-Puro-Cre into 293T cells for lentiviral packaging, culturing, collecting cell supernatant, centrifuging, infecting MDBK cells with the supernatant after centrifuging, screening and subcloning to obtain an MDBK cell line MDBK-Cre stably expressing cyclase.

6. The construction method according to claim 5, wherein: in the step (1), the primer is:

pLv-Cre-Puro-For：TTTCAGGTGTCGTGAGGATCCGCCACCATGTCCAATTTACTGACCG TACACCAAAATTTG；

pLv-Cre-Puro-Rev：GCGGCCGCCCTCGAGGAATTCTCATTATTTATCGTCATCGTCTTTGT AGTCGCTTCCTCCTCCATCGCCATCTTCCAGCAG。

7. the construction method according to claim 5, wherein: in step (3), screening was performed with 10% FBSDMEM medium containing puromycin at 1.25. Mu.g/mL.

8. The construction method according to claim 5, wherein: after obtaining MDBK-Cre, the method further comprises the following steps: MDBK-Cre cells were cultured using a medium containing 10% FBSDMEM, and cultured at 37℃under 5% carbon dioxide.

9. Niu Jiejie strain LSDV-delta TK deleted of the TK gene of the skin disease virus is prepared by the method of any one of claims 1-5.

10. Use of the bovine nodular skin disease virus TK gene deleted strain LSDV- Δtk of claim 9 in the construction of a bovine nodular skin disease virus live vector vaccine.