Application of proteoglycan gene in resisting African swine fever virus infection

Abstract

The invention provides application of a proteoglycan gene in resisting African swine fever virus infection. Specifically, the invention provides a novel target gene SDC2 for resisting the infection of the African swine fever virus, the expression of the SDC2 gene is inhibited by using an interference agent, antisense oligonucleotide, a small molecule compound, an antibody or a gene editing reagent and the like, the replication of the African swine fever virus can be remarkably reduced, and an effective means is provided for resisting the infection of the African swine fever virus. In practical application, the SDC2 gene can be used as a gene marker target for cultivating or preparing a new animal breeding material for resisting the African swine fever virus, can be used for preparing a genetic marker for providing a molecular marker for breeding, and has a wide application prospect in research and development of vaccines and medicines for resisting the infection of the African swine fever virus.

Description

Technical Field

[0001] The present application relates to the technical field of animal disease-resistant breeding, and in particular, to the application of a proteoglycan gene in resisting African swine fever virus infection. Background Art

[0002] African swine fever (ASF) is a highly lethal and virulent infectious disease caused by the African swine fever virus (ASFV). The infection rate and mortality rate of its virulent strains are as high as 100%, and it is classified as a Class I animal disease in China. Due to the complex structure and rapid mutation rate of the African swine fever virus, and the unclear immune escape mechanism and protective response mechanism, there are currently no effective vaccines and drugs for the prevention and control of African swine fever except for biosafety measures. Therefore, breeding or preparing African swine fever-resistant pigs and improving the disease resistance of pig herds from a genetic perspective have become one of the important ways to ensure the quality and safety of breeding pigs under the normalization of African swine fever.

[0003] However, the necessary prerequisite for breeding or preparing African swine fever-resistant pigs is the identification of host target genes. Since the host infection mechanism of African swine fever virus is complicated and has not yet been fully elucidated, the host disease resistance target genes screened at the current stage are extremely limited, which has become a bottleneck problem that needs to be solved in the process of breeding or preparing African swine fever-resistant pigs. Summary of the invention

[0004] Based on the defects of the prior art, the applicant of this application successfully identified the target genes that regulate the invasion and replication of African swine fever virus in the target cells infected with African swine fever virus - porcine alveolar macrophages (PAMs), providing new targets for the breeding or preparation of animals resistant to African swine fever virus, which is of great significance to improving the level of disease-resistant breeding research.

[0005] Specifically, this application involves the following aspects:

[0006] 1. An SDC2 gene interfering agent, comprising miRNA, siRNA, dsRNA or shRNA; or comprising a plasmid or viral vector containing the miRNA, siRNA, dsRNA or shRNA, the nucleotide sequence of the siRNA sense strand is as shown in SEQ ID NO: 3, and the nucleotide sequence of the siRNA antisense strand is as shown in SEQ ID NO: 4.

[0007] 2. The SDC2 gene interfering agent according to item 1, which acts to inhibit the expression or activity of the SDC2 gene and its encoded protein.

[0008] 3. Application of SDC2 gene inhibitors in the preparation of drugs for resistance to African swine fever virus infection.

[0009] 4. The use according to item 3, wherein the inhibitor inhibits the expression or activity of the SDC2 gene or its encoded protein at the transcriptional or translational level;

[0010] The inhibitors include RNA interference agents, antisense oligonucleotides, small molecule compounds, antibodies or gene editing agents.

[0011] 5. The use according to item 4, wherein the RNA interference agent comprises miRNA, siRNA, dsRNA or shRNA; or comprises a plasmid or viral vector containing the miRNA, siRNA, dsRNA or shRNA.

[0012] 6. The use according to item 4, wherein the RNA interference agent is the interference agent described in item 1 or 2.

[0013] 7. A kit for resisting African swine fever virus infection, comprising the interfering agent described in item 1 or 2.

[0014] 8. Use of SDC2 gene inhibitor in the preparation of a kit for resisting African swine fever virus infection.

[0015] 9. A molecular marker for breeding animals resistant to African swine fever virus, wherein the nucleotide sequence is shown in SEQ ID NO: 1, and the animal is a mammal.

[0016] 10. Use of the SDC2 gene inhibitor or the molecular marker described in item 9 or the interfering agent described in item 1 or 2 or the kit described in item 7 in breeding animals resistant to African swine fever virus.

[0017] Beneficial effects:

[0018] The present application provides a new target gene SDC2 related to resistance to African swine fever virus infection, and for the first time uses molecular biology technology combined with cell biological experimental methods to prove that inhibiting the expression of the SDC2 gene can significantly reduce the replication of African swine fever virus, and has broad application prospects in the cultivation or preparation of new animal breeding materials resistant to African swine fever virus, and the research and development of antiviral vaccines and drugs.

[0019] This application discovered for the first time that the SDC2 gene interacts with African swine fever virus endocytosis-related proteins and can serve as a potential receptor for African swine fever virus invasion, providing a basis for subsequent in-depth research on the molecular mechanism of the SDC2 gene in regulating the replication of African swine fever virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The results of RT-qPCR detection of SDC2 gene expression level after adding SDC2 siRNA and NC siRNA to PAM cells.

[0021] Figure 2 This is the result of Western blot detection of SDC2 gene expression level after adding SDC2 siRNA and NC siRNA to PAM cells.

[0022] Figure 3 The results are shown in the figure below: After adding SDC2 siRNA and NC siRNA to PAM cells, the expression level of viral protein p30 in cells was detected by indirect immunofluorescence.

[0023] Figure 4 Statistical analysis results of indirect immunofluorescence quantification based on the relative proportion of ASFV-infected PAM cells (i.e., p30-positive cells (%) = number of infected cells / total number of cells) after adding SDC2 siRNA and NC siRNA to PAM cells.

[0024] Figure 5 The results of indirect immunofluorescence detection of the expression level of viral protein p30 in 3D4 cells after overexpression of SDC2 gene.

[0025] Figure 6 Statistical analysis results of indirect immunofluorescence quantification based on the relative proportion of ASFV-infected PAM cells (i.e., p30-positive cells (%) = number of infected cells / total number of cells) after overexpression of the SDC2 gene in 3D4 cells.

[0026] Figure 7 The results of SDS-PAGE detection of the expression of purified SDC2 protein.

[0027] Figure 8 The results are shown in Figure 2, which are the results of SDS-PAGE detection of the expression of purified p22 protein.

[0028] Fig. 9 The results are shown in Figure 2, which are the results of SDS-PAGE detection of the expression of purified p30 protein.

[0029] Fig.10 The results of SPR detection of the binding affinity between SDC2 protein and p22 protein.

[0030] Fig.11 The results of SPR detection of the binding affinity between SDC2 protein and p30 protein. DETAILED DESCRIPTION

[0031] The present application is further described below in conjunction with examples. It should be understood that the examples are only used to further describe and illustrate the present application and are not used to limit the present application.

[0032] Unless otherwise defined, the technical and scientific terms in this specification have the same meaning as those generally understood by those skilled in the art. Although similar or identical methods and materials as described herein may be used in experiments or practical applications, materials and methods are described herein below. In the event of a conflict, the present specification including the definitions therein shall prevail. In addition, materials, methods and examples are provided for illustration only and are not restrictive. The present application is further described below in conjunction with specific embodiments, but is not intended to limit the scope of the present application.

[0033] definition

[0034] The terms "comprising", "including" or "containing", including their grammatical synonyms used in this document, should generally be understood as open and non-limiting, for example, not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0035] The term "siRNA" used herein refers to ribonucleic acid (RNA) that can inhibit the expression of a target gene and includes a sense RNA fragment region and an antisense RNA fragment region.

[0036] The term "shRNA" as used herein refers to an RNA molecule having a stem-loop structure, which includes a first region and a second region of complementary sequence, the degree of complementarity and the orientation of the regions are sufficient to allow base pairs to appear between the regions, the first region and the second region are connected by the loop region, and the loop is generated by the lack of base pairing between nucleotides (or nucleotide analogs) in the loop region.

[0037] The term "miRNA" as used herein is a short, naturally occurring, non-coding, single-stranded RNA molecule of approximately 16-26 nucleotides in length that is generally involved in regulating gene expression in vivo.

[0038] As used herein, the term "inhibit" may be used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms, and includes any level of inhibition.

[0039] SDC2 gene disruptor

[0040] The present application provides a SDC2 gene interfering agent, the interfering agent includes miRNA, siRNA, dsRNA or shRNA; or includes a plasmid or viral vector containing the miRNA, siRNA, dsRNA or shRNA. In some embodiments, the interfering agent is siRNA or a plasmid or viral vector containing the siRNA, the nucleotide sequence of the siRNA sense strand is shown in SEQ ID NO: 3 (GGAGAAGCACUCAGACAAUTT), and the nucleotide sequence of the siRNA antisense strand is shown in SEQ ID NO: 4 (AUUGUCUGAGUGCUUCUCCTT).

[0041] Among them, the syndecan 2 (SDC2) belongs to the cell surface transmembrane heparan sulfate proteoglycan (HSPG) family. SDC2 can bind to a series of ligands through its heparan sulfate side chain, and is widely involved in biological processes such as tissue differentiation and organ development. It plays a key role in the formation and metastasis of various tumors. SDC2 has also been shown to be a common receptor for some viruses (such as human herpes simplex virus type-1 (HSV-1) and human immunodeficiency virus type 1 (HIV)) to invade host cells.

[0042] The present application is not intended to limit the mode of action of the interfering agent, which can be obtained by conventional methods in the art, such as chemical synthesis, in vitro transcription and expression vector method. The expression vector method is to express the interfering agent in a suitable plasmid or viral vector (such as a lentiviral vector, an adenoviral vector and an adeno-associated viral vector).

[0043] In some embodiments, the interfering agent acts to inhibit the expression or activity of the SDC2 gene and its encoded protein.

[0044] SDC2 gene inhibitor

[0045] The present application provides the use of the SDC2 gene as a target in the preparation of drugs for resisting African swine fever virus infection.

[0046] The present application provides an application of an SDC2 gene inhibitor in the preparation of a drug for resisting African swine fever virus infection.

[0047] In some embodiments, the inhibitor inhibits the expression or activity of the SDC2 gene or its encoded protein at the transcriptional or translational level; the inhibitor includes RNA interference agents, antisense oligonucleotides, small molecule compounds, antibodies or gene editing agents.

[0048] In some embodiments, the inhibitor is an RNA interference agent. In some embodiments, the RNA interference agent includes miRNA, siRNA, dsRNA or shRNA; or includes a plasmid or viral vector containing the miRNA, siRNA, dsRNA or shRNA.

[0049] In some embodiments, the RNA interference agent is siRNA or a plasmid or viral vector containing the siRNA, the nucleotide sequence of the siRNA positive chain is shown in SEQ ID NO: 3 (GGAGAAGCACUCAGACAAUTT), and the nucleotide sequence of the siRNA antisense chain is shown in SEQ ID NO: 4 (AUUGUCUGAGUGCUUCUCCTT).

[0050] In some embodiments, the inhibitor is an antisense oligonucleotide.

[0051] Among them, the term "antisense oligonucleotide (ASO)" refers to a nucleotide fragment expressed in vivo or artificially synthesized, which is complementary to a certain region in the target gene or mRNA, and can bind to the target gene / mRNA through the principle of base complementarity, thereby inhibiting the expression of the target gene.

[0052] In some embodiments, the inhibitor is a small molecule compound.

[0053] The small molecule compound may be natural or chemically synthesized.

[0054] In some embodiments, the inhibitor is an antibody.

[0055] Among them, the term "antibody" is used in the broadest sense and covers various antibody structures, including not only intact (i.e., full-length) antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), variants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies (scFV), VHH antibodies, multispecific antibodies, multispecific single-chain antibodies), and any other modified configurations of immunoglobulin molecules that contain antigen recognition sites of desired specificity, including antibody glycosylation variants, antibody amino acid sequence variants, and covalently modified antibodies.

[0056] In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the antibody is a mouse antibody, a rabbit antibody, a chimeric antibody, a humanized antibody or a fully human antibody. In some embodiments, the antibody can be further labeled.

[0057] In some embodiments, the inhibitor is a gene editing agent.

[0058] Herein, the term "gene editing reagent" refers to the components required for gene editing tools and may include enzymes, riboproteins, solutions, cofactors, etc. For example, gene editing reagents include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and one or more components required for clustered regularly interspaced short palindromic repeat system (CRISPR / Cas) gene editing.

[0059] Kits and applications

[0060] The present application provides a kit for resisting African swine fever virus infection, wherein the kit comprises the above-mentioned interfering agent.

[0061] The present application provides the use of the SDC2 gene or the above-mentioned SDC2 gene inhibitor in the preparation of a kit for resisting African swine fever virus infection.

[0062] Molecular markers for breeding and their application

[0063] The present application provides a molecular marker for breeding animals resistant to African swine fever virus, and the nucleotide sequence of the molecular marker is shown in SEQ ID NO: 1.

[0064] Wherein, the sequence of SEQ ID NO: 1 is as follows:

[0065] .

[0066] The present application provides the use of SEQ ID NO: 1 as a molecular marker for breeding animals resistant to African swine fever virus.

[0067] The present application provides a method for breeding animals resistant to African swine fever virus, the method comprising: detecting the expression level of the SDC2 gene in the animal through the above-mentioned molecular markers, wherein the animal with reduced SDC2 gene expression level is the African swine fever virus resistant animal.

[0068] The present application provides the use of the SDC2 gene or the above-mentioned SDC2 gene inhibitor or the above-mentioned molecular marker or the above-mentioned interfering agent or the above-mentioned kit in the breeding of animals resistant to African swine fever virus.

[0069] In addition, the present application also provides a method for breeding animals resistant to African swine fever virus, the method comprising: knocking out the SDC2 gene in the animal to obtain SDC2-deficient African swine fever virus-resistant animals.

[0070] In some embodiments, the method comprises: introducing the above-mentioned SDC2 gene inhibitor or the above-mentioned interfering agent or the above-mentioned kit into an animal, and breeding and screening to obtain African swine fever virus-resistant animals with reduced SDC2 gene expression.

[0071] In some embodiments, the method comprises: knocking out the above-mentioned SDC2 gene in animal cells, performing somatic cell nuclear transplantation and breeding screening to obtain SDC2 gene knockout animals resistant to African swine fever virus.

[0072] In some embodiments, the cell is a somatic cell or a fertilized egg cell.

[0073] The introduction can be performed by conventional techniques well known to those skilled in the art.

[0074] In some embodiments, the screening is performed by the above-mentioned molecular markers. Specifically, the expression level of the SDC2 gene in the animal is detected by using molecular markers, thereby screening and obtaining SDC2 gene knockout resistant African swine fever virus animals.

[0075] In actual operation, the expression level of the SDC2 gene is based on the general level of expression of the gene in animals in the field. The expression level is relative. The high or low expression level compared to the general level in the field can help determine whether the animal is resistant to African swine fever virus. The lower the expression level of the SDC2 gene, the stronger the resistance, and vice versa.

[0076] Wherein, the above-mentioned animal resistant to African swine fever virus is a mammal, such as a pig.

[0077] This application uses molecular biology technology combined with cell experimental methods to find that in the target cells infected with African swine fever virus - porcine alveolar macrophages (PAMs), inhibiting the expression of the SDC2 gene can significantly reduce the replication of African swine fever virus; at the same time, it was found in non-susceptible 3D4 cells that overexpression of SDC2 can promote the replication of African swine fever virus; and through protein interaction analysis, it was found that SDC2 has a strong binding force with African swine fever virus endocytosis-related proteins p22 and p30. The above results prove that SDC2 can mediate the invasion and replication of African swine fever virus, and is directly related to African swine fever virus infection.

[0078] In summary, the present application provides a new target gene for anti-African swine fever virus, which can inhibit African swine fever virus infection by down-regulating the expression of SDC2 gene or its encoded protein at the transcriptional or translational level using interfering agents, antisense oligonucleotides, small molecule compounds, antibodies or gene editing reagents. In practical applications, the SDC2 gene can be used as a gene marker target for the cultivation or preparation of new animal breeding materials resistant to African swine fever virus, and can be used to prepare genetic markers to provide molecular markers for breeding, and has broad application prospects in the development of vaccines and drugs against African swine fever virus infection.

[0079] Example

[0080] The present application content will be described below in conjunction with specific examples, but the scope of the present application is not limited thereto. Unless otherwise specified, the reagents and instruments used in the following examples are all conventional reagents and instruments in this area and can be obtained commercially. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the scheme and obtain corresponding results according to the embodiments.

[0081] Example 1 SDC2 gene silencing can significantly inhibit ASFV infection

[0082] (1) Design of interfering RNA (siRNA)

[0083] The RNA interference target sequences of the SDC2 gene (Genebank No. 100152754; its CDS sequence is shown in SEQ ID NO: 1, and the encoded amino acid sequence is shown in SEQ ID No: 2) were designed: SDC2 siRNA (SEQ ID NO: 3-4) and control siRNA (NC siRNA) (SEQ ID NO: 5-6); SDC2 siRNA and NC siRNA were synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0084] Wherein, the sequence shown in SEQ ID NO: 1 is as follows:

[0085] ATGCGGCGCGTTTGGATCCTGCTCACCTTGGGCTTGGTGGCCTGCGTGTCGGCGGAGTCGCGGGCAGAGCTGACGTCTGATAAAGACATGTACCTTGACAACAGCTCCATTGAAGAAGCTTCAGGAGTGTATCCTATTGATGATGACGACTATGCTTCTGCTTCAGGCTCAGGGGCCGATGAGGACGTAGAGAGTCCAGAGCTGACCACATCTCGACCCCTTCCAAAGATACCGATCACCAGTGCCGCTCCGAGAGTGGAAACCACGACGCTGGACAAGATGCAGAACAAGATCCCTGCTCAGACAAAGTCACCTGAAGAAATTGATAAGGAGAAAGTTCACCTCTCTGACTCAGAAAGGAAAACGGACCCAGCTGAAGAGGATACAAATGTGTATACGGAGAAGCACTCAGACAATCTGTTTAAAAGAACAGAAGTCCTGGCAGCTGTCATTGCTGGCGGAGTTATCGGCTTTCTCTTTGCCATTTTCCTTATTCTGCTGTTGGTGTATCGCATGAGAAAGAAGGATGAAGGAAGTTATGACCTTGGAGAACGCAAACCATCCAGCGCTGCTTACCAGAAGGCACCGACTAAGGAGTTTTATGCGTGA;

[0086] The sequence shown in SEQ ID NO: 2 is as follows:

[0087] MRRVWILLTLGLVACVSAESRAELTSDKDMYLDNSSIEEASGVYPIDDDDYASASGSGADEDVESPELTTSRPLPKIPITSAAPRVETTTLDKMQNKIPAQTKSPEEIDKEKVHLSDSERKTDPAEEDTNVYTEKHSDNLFKRTEVLAAVIAGGVIGFLFAIFLILLLVYRMRKKDEGSYDLGERKPSSAAYQKAPTKEFYA;

[0088] The sequence shown in SEQ ID NO: 3 is as follows:

[0089] GGAGAAGCACUCAGACAAUTT;

[0090] The sequence shown in SEQ ID NO: 4 is as follows:

[0091] AUUGUCUGAGUGCUUCUCCTTT;

[0092] The sequence shown in SEQ ID NO: 5 is as follows:

[0093] UUCUCCGAACGUGUCACGUTT;

[0094] The sequence shown in SEQ ID NO: 6 is as follows:

[0095] ACGUGACACGUUCGGAGAATT.

[0096] (2) Evaluation of the effect of SDC2 gene on ASFV infection

[0097] Porcine alveolar macrophages (PAMs) were isolated from 1-month-old Large White pigs, resuspended in 1640 medium (containing 10% FBS (Gibco), 100 U / mL penicillin and 100 U / mL streptomycin), and inoculated in 48-well plates (5×10 5 / well), and cultured in a 37°C, 5% CO2 incubator overnight; SDC2 siRNA (110 pM) and NC siRNA (110 pM) were transfected into PAM cells using JetPRIME transfection reagent (PolyPlus, B180306), and cultured in an incubator for 8 hours. The culture medium was replaced and cultured for another 24 hours. The expression level of SDC2 gene was analyzed by reverse transcription real-time fluorescence quantitative PCR (RT-qPCR) and protein immunoblot (Western blot) experiments; GADPH was used as an internal reference in the Western blot experiment.

[0098] The results are as follows Figure 1-Figure 2 As shown, compared with NC siRNA, the expression of SDC2 gene at both transcriptional and protein levels in PAM cells transfected with SDC2 siRNA was significantly decreased, indicating that SDC2 siRNA has an inhibitory effect on the expression of SDC2 gene.

[0099] (3) Evaluation of ASFV infection effects

[0100] ASFV with a multiplicity of infection of 0.1 (MOI=0.1) was inoculated into PAM cells transfected with SDC2 siRNA and NC siRNA. After 24 hours of infection, an indirect immunofluorescence experiment was performed using an ASFV-p30 protein monoclonal antibody, in which DAPI was used to stain the cell nucleus.

[0101] The results are as follows Figure 3-Figure 4 As shown, ASFV replication was significantly reduced in PAM cells transfected with SDC2 siRNA compared with NC siRNA.

[0102] Example 2 SDC2 gene overexpression can promote ASFV replication

[0103] (1) Construction and packaging of lentiviral expression vector

[0104] The CDS sequence of the SDC2 gene (SEQ ID NO: 1) was synthesized into the lentiviral vector pLVX-T2A-mCherry-Puro to construct an SDC2 overexpression plasmid, which was constructed by GenScript Biotech Co., Ltd.

[0105] One day before transfection, human embryonic kidney cells (HEK293T) were inoculated into T75 cell culture flasks. When the cell density reached 80%-90%, transfection was performed. The culture medium was discarded, and 5 mL of fresh DMEM culture medium (containing 2% FBS and no antibiotics) was added. PMD2.G plasmid, psPAX2 plasmid, and 24 μg SDC2 overexpression plasmid (the mass ratio of each plasmid was 1:2:3) were diluted with 500 μL JetPrime buffer using JetPRIME transfection reagent (PolyPlus, B180306). After mixing, 40 μL JetPRIME was added, and the mixture was allowed to stand at room temperature for 10 minutes. The mixture was carefully added to the cell culture medium, and the cells were transfected for 6-8 hours. 5 mL of DMEM culture medium (containing 1% double antibody and 2% FBS) was added, and the cells were cultured for 24 hours. 10 mL of DMEM culture medium (containing 1% double antibody and 2% FBS) was added again, and the culture was continued for 60 hours. The supernatant was harvested by centrifugation and washed with 0.45 The lentivirus was finally obtained by filtering through a μm filter and centrifuging again to discard the supernatant. The lentivirus was resuspended in 200 μL pre-cooled DMEM medium (containing 1% double antibody and 10% FBS).

[0106] (2) Construction of a 3D4 cell line stably expressing SDC2

[0107] Porcine alveolar macrophages (3D4) were inoculated in 6-well plates. When the cell density reached 40%-50%, the obtained lentivirus (1 mL) was used for infection, and polybrene (final concentration of 8 μg / mL) was added. One day after infection, the cells were transferred to T25 cell culture flasks and cultured for 2 days. Puromycin (final concentration of 3 μg / mL) was added to kill the cells not infected with the lentivirus, and the surviving cells were inoculated in 10 cm dishes, culture medium (containing puromycin) was added and cultured for another 7 days. The cells were scraped with a glass needle and transferred to a 24-well plate, and the culture was expanded to finally obtain a 3D4 cell line stably expressing SDC2.

[0108] (3) Evaluation of ASFV infection effects

[0109] Uninfected 3D4 cells (control MOCK group) and 3D4 cells stably expressing SDC2 (SDC2 OE group) were inoculated in 48-well plates and cultured overnight. The cells were infected with ASFV at MOI = 50. After 24 hours of infection, indirect immunofluorescence experiments were performed using ASFV-p30 protein monoclonal antibodies, in which DAPI was used to stain the cell nucleus.

[0110] The results are as follows Figure 5-Figure 6 As shown, compared with the MOCK group, overexpression of the SDC2 gene can promote the replication of ASFV in 3D4 cells.

[0111] Example 3 Interaction analysis between host gene SDC2 and ASFV endocytosis-related proteins

[0112] (1) Construction of a eukaryotic expression vector for the extracellular region of the SDC2 protein

[0113] The DNA sequence encoding its extracellular membrane region (amino acids 1-147 SEQ ID NO: 7) was selected for human codon optimization, and the SDC2 nucleotide sequence containing the HRV3C restriction site and 6×His tag was synthesized by Beijing Qingke Biotechnology Co., Ltd., and the eukaryotic expression vector pCAGGS-SDC2 was obtained through construction.

[0114] Wherein, the sequence shown in SEQ ID NO: 7 is as follows:

[0115] MRRVWILLTLGLVACVSAESRAELTSDKDMYLDNSSIEEASGVYPIDDDDYASASGSGADEDVESPELTTSRPLPKIPITSAAPRVETTTLDKMQNKIPAQTKSPEEIDKEKVHLSDSERKTDPAEEDTNVYTEKHSDNLFKRTEVL.

[0116] (2) Expression and purification of SDC2 protein

[0117] The vector was transfected using the ExpiFectamine™ 293 transfection kit. pCAGGS- SDC2 (30 μg) was diluted in 1.5 mL Opti-MEM™ I reduced serum medium. At the same time, 80 μL of Expi Fectamine™ 293 reagent was diluted in 1.5 mL Opti-MEM™ I reduced serum medium, incubated at room temperature for 5 minutes, added to the diluted pCAGGS-SDC2, and incubated at room temperature for 15 minutes. The incubated solution was added to Expi293F suspension cells (Thermo Fisher Scientific) (7.5×10 5 cells), cultured in a cell suspension incubator at 37°C, 8% CO2, 120rpm for 16-18 hours, added 1.5 mL of the mixture (mixed by Expi Fectamine™ 293 transfection enhancer 1 and 2) and continued to culture until the cell supernatant was harvested on the 6th day.

[0118] The cell supernatant was affinity purified using a nickel column (Ni Sepharose column, GE Healthcare 17526801). The harvested cell supernatant was concentrated and added to the Ni Sepharose column. Gradient elution was performed with an elution buffer containing 20 mM, 40 mM, 80 mM, 250 mM, and 500 mM imidazole, and the eluate was collected for polyacrylamide gel electrophoresis (SDS-PAGE) detection. The elution buffer with higher protein purity was collected and combined, further separated by an ion exchange column, and gradient elution was performed with different concentrations of NaCl solution, and the eluate was collected for SDS-PAGE detection.

[0119] The results are as follows Figure 7 As shown, the size of the expressed SDC2 protein varies depending on its glycosylation degree, ranging from 40 to 150 kD, which is consistent with the theoretical prediction.

[0120] (3) Construction of prokaryotic expression vectors for ASFV endocytosis-related proteins p22 and p30

[0121] The nucleotide sequences encoding p22 and p30 proteins from the China_HLJ_2018 ASFV strain (HLJ / 18) (SEQ ID NOs: 8 and 9) were downloaded from the ASFV database (ASFVdb, http: / / asfvdb.popgenetics.net / ), and the Escherichia coli source codons were optimized. The p22 and p30 nucleotide sequences containing 6×His tags were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the prokaryotic expression vectors pET-sumo-p22 and pET-sumo-p30 were obtained by construction.

[0122] Among them, the sequence shown in SEQ ID NO: 8 is as follows:

[0123] ATGTTTAATATTAAAATGACAATTTCTACATTGCTTATTGCTCTTATTATACTACTTATTATTATTTTAGTAGTGTTTTTATACTATAAGAAACAACAACCACCGAAAAAGGTCTGTAAAGTAGATAAAGATTGTGGTAGTGGAGAGCATTGTGTTCGTGGATCATGTAGCTCATTGAGCTGCTTAGATGCCGTAAAAATGGACAAACGAAATATTAAGATAGATTCTAAGATTTCCTCATGCGAATTCACTCCCAATTTTTACCGTTTTACGGATACTGCTGCTGATGAGCAGCAAGAATTTGGAAAAACACGGCATCCTATAAAAATAACTCCATCTCCAAGTGAATCCCATAGCCCCCAAGAGGTGTGTGAAAAATATTGTTCATGGGGAACCGATGACTGTACAGGTTGGGAATATGTTGGTGATGAAAAGGAGGGAACATGTTATGTATATAATAATCCACATCACCCGGTTCTTAAATATGGTAAGGATCACATCATAGCCTTACCTAGAAATCATAAACATGCATAA;

[0124] The sequence shown in SEQ ID NO: 9 is as follows:

[0125] .

[0126] (4) Expression and purification of p22 and p30 proteins

[0127] pET-sumo-p22 and pET-sumo-p30 were transformed into Escherichia coli DE3 competent cells, respectively, and cultured at 37°C overnight after coating on LB plates. Single clone colonies were picked and inoculated into LB liquid culture medium, and cultured at 37°C until the bacteria grew into the logarithmic phase (OD600=0.4-0.6). IPTG inducer (0.5 mM) was added and induced for expression at 16°C for 16 hours. Ultrasonic disruption was performed, and the supernatant was harvested and concentrated. The concentrated protein was affinity purified and separated by ion exchange using a Ni Sepharose column (same as step (2) in Example 3), followed by SDS-PAGE and Coomassie Brilliant Blue staining.

[0128] The results are as follows Figure 8-Figure 9 As shown, the size of the expressed p22 protein is 40 kD ( Figure 8 ); the expressed p30 protein size is 45 kD ( Fig. 9), which are consistent with the theoretical predictions.

[0129] (5) Evaluation of the interaction between SDC2 protein and p22 and p30 proteins

[0130] Surface plasmon resonance (SRP) technology was used to evaluate the interaction between SDC2 protein and p22 and p30 proteins. The purified SDC2 protein was dissolved in HBS-EP (PH7.4) buffer and coated on Series S Sensor Chip CM5 (GE Healthcare) using the biomolecular interaction analysis system Biacore T200. At the same time, the purified p22 and p30 proteins were gradiently diluted with HBS-EP (PH7.4) buffer to 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM and 200 nM, respectively. 10 mM glycine was used as the regeneration buffer, and the instrument detected the affinity of SDC2 protein binding to p22 and p30 proteins. The binding curves were recorded and curve fitting was performed using evaluation software, and the kinetic parameters were calculated based on the fitting curves.

[0131] The results are as follows Figure 10-11 As shown in Figure 2, the equilibrium dissociation constant (K) of SDC2 protein and p22 protein D ) value is 980 pM ( Fig.10 ); K of SDC2 protein and p30 protein D The value is 78 pM ( Fig.11 ), the results showed that SDC2 protein had a strong binding affinity with viral endocytosis-related proteins p22 and p30.

[0132] The above is only the preferred embodiment of the present application, and does not limit the present application in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. An SDC2 gene interfering agent, comprising miRNA, siRNA, dsRNA or shRNA; or comprising a plasmid or viral vector containing the miRNA, siRNA, dsRNA or shRNA, the nucleotide sequence of the siRNA sense strand is as shown in SEQ ID NO: 3, and the nucleotide sequence of the siRNA antisense strand is as shown in SEQ ID NO:

4.

2. The SDC2 gene interfering agent according to claim 1, wherein the function of the interfering agent is to inhibit the expression or activity of the SDC2 gene and its encoded protein.

3. Application of SDC2 gene interfering agent in the preparation of drugs for resisting African swine fever virus infection.

4. The use according to claim 3, wherein: The SDC2 gene interfering agent is the interfering agent described in claim 1 or 2.

5. A kit for resisting African swine fever virus infection, comprising the interfering agent according to claim 1 or 2.

6. Application of SDC2 gene interfering agent in the preparation of a kit for resisting African swine fever virus infection.

7. The use according to claim 6, wherein: The SDC2 gene interfering agent is the interfering agent described in claim 1 or 2.

8. A molecular marker for breeding animals resistant to African swine fever virus, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the animal is a pig.

9. Use of the molecular marker described in claim 8, the interfering agent described in claim 1 or 2, or the kit described in claim 5 in breeding animals resistant to African swine fever virus.

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