CN113004390B - Application of ADAM17 as receptor of hog cholera virus - Google Patents

Abstract

The invention relates to the field of viruses, in particular to application of ADAM17 serving as a receptor of Classical Swine Fever Virus (CSFV). The CSFV E2 cyst membrane glycoprotein specific binding protein ADAM17 is screened by using a co-immunoprecipitation technology, and the CSFV infection can be completely blocked by knocking ADAM17 out by using a CRISPR-Cas9 technology. The purified recombinant E2 protein and ADAM17 can be specifically combined. Above, it was demonstrated that ADAM17 is a specific receptor for swine fever virus. ADAM17 will be an important target for anti-CSFV vaccine and drug design and screening, and also an important target for anti-CSFV pig breeding.

Description

Application of ADAM17 as a receptor for swine fever virus

technical field

The present invention relates to the field of viruses, in particular to the application of ADAM17 as a swine fever virus receptor.

Background technique

Swine fever (Classical Swine Fever, CSF), formerly known as hog cholera (Hog Cholera, HC), is a highly contagious, one of the major infectious diseases that cause huge economic losses to the pig industry, and is listed in the world animal Directory of Health Organizations. Swine fever was first confirmed in Tennessee, United States in 1810, and then quickly swept the world, widely distributed in Asia, Eastern Europe, Russia and South America; swine fever was discovered in Japan in 1888; the exact time of the first discovery in China is not recorded, but In 1925, Chinese scientists had used antiserum to treat swine fever. At present, there are two main prevention policies for swine fever: culling (non-vaccine) and systemic prevention (vaccine). The live attenuated rabbit vaccine developed based on the C-strain has successfully controlled the swine fever epidemic, so there has been no major outbreak in the past few decades. However, with the change of virus subtypes, the effectiveness of the C-strain vaccine has gradually decreased, and the swine fever epidemic has continued to rise in recent years.

Swine fever virus (Classical Swine FeverVirus, CSFV) is the pathogen that causes swine fever, belongs to Flaviviridae (Flaviviridae), Pestivirus, Pestivirus also includes Bovine Viral Diarrhea Virus (BVDV) , Border Disease Virus (BDV). CSFV enters the host through the oral and nasal mucosa, first infecting the tonsil cells, and then spreading throughout the body through the blood and lymphatic circulation. CSFV has obvious tissue tropism to the immune system, such as thymus, spleen, lymph nodes, bone marrow, etc., and causes severe leukopenia.

CSFV has 3 genotypes (I, II, III), and each genotype has 3 to 4 subtypes. The popular Shimen and SXCDK strains in China belong to type I and type II, respectively.

CSFV is an enveloped, positive-strand RNA virus with a virion diameter of approximately 50 nm. The genome of CSFV consists of a 5' non-coding region, an open reading frame and a 3' non-coding region. The open reading frame is translated into a precursor polyprotein consisting of 3398 amino acids, which is cleaved into 4 structural proteins (C, Erns, E1, and E2) and 8 non-structural proteins ( Npro, P7, NS2, NS3, NS4A, NS4B, NS5A and NS5B).

The structural proteins of CSFV include a capsid protein (C), three envelope glycoproteins (Erns, E1, E2). It splits into Erns (also known as E0) under the action of . Among them, Erns is necessary for the production of infectious virus particles, and Erns is also the target protein of neutralizing antibodies. Erns interacts with cell surface mucopolysaccharides (eg, heparan sulfate, laminin receptors) through positively charged amino acids, thereby mediating the adhesion of virions to the host. However, a 2004 study found that Erns is not necessary for viruses to invade host cells, and only retropseudovirions expressing E1 and E2 can infect the host.

E1 is a type I transmembrane protein with an N-terminal ectodomain and a C-terminal hydrophobic helical domain, and anchors E1 to the surface of the viral envelope through the C-terminal. E1 and E2 can form heterodimers through disulfide bonds between cysteine residues, and E2-E1 heterodimers regulate the process of CSFV invasion into the host.

E2 is also a type I transmembrane protein with an N-terminal ectodomain and a C-terminal hydrophobic helical domain, and anchors E2 to the surface of the viral envelope through the C-terminal. E2 has two dimer forms: E2-E2, E2-E1; the E2-E2 homodimer is first formed during virus assembly, and the E2-E1 heterodimer is released after E1 is released from the endoplasmic reticulum form again. E2 is the most important immunogen in the CSFV glycoprotein, including antigenic epitopes. CSFV antibodies and vaccines can be developed based on E2. Mutating the E2 glycosylation site can significantly reduce the immunogenicity of E2.

The process of CSFV invasion into the host is mediated by the glycoprotein Erns and E2, of which E2 is indispensable. At the same time, Erns and E2 are also the main targets of neutralizing antibodies, but anti-E2 antibodies can completely neutralize CSFV infection, while anti-Erns antibodies can only neutralize part of the infection.

The combination of virus and host factor is the most critical step in virus infection of host cells. In 2004, CD46 was identified as a host factor of BVDV, but there has been no breakthrough in the study of CSFV host factor. It has been reported that heparan sulfate and laminin receptor are adhesion factors in the process of CSFV infection of the host. The fact is that heparan sulfate is the adhesion factor of many enveloped viruses; and although laminin receptor can interact with CSFV Erns protein. interaction, but antibodies against the laminin receptor were only able to partially inhibit CSFV infection of the host. This is because although the entry of CSFV into the host is mediated by Erns and E2 proteins, Erns is dispensable and E2 is indispensable, yet the host factor that binds to E2 proteins remains a mystery. Therefore, the screening of swine fever drugs and vaccines and the targeted breeding of anti-virus pigs lack specific targets.

SUMMARY OF THE INVENTION

In view of this, the present invention provides the application of ADAM17 as a receptor for swine fever virus. The present invention discovers ADAM17, a key receptor of swine fever virus (classical swine fevervirus, CSFV) infecting pigs, which can be used for the design and screening of antiviral vaccines and drugs, and a target for the development of antiviral pigs. The CSFV E2 envelope glycoprotein-specific binding protein ADAM17 was screened by co-immunoprecipitation technology. Knockout of ADAM17 by CRISPR-Cas9 technology can completely block CSFV infection. The purified recombinant E2 protein can specifically bind to ADAM17. The above results demonstrate that ADAM17 is a specific receptor for swine fever virus infection.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides the application of ADAM17 as the receptor of E2 protein or swine fever virus; the ADAM17 has:

(I), the amino acid sequence shown in SEQ ID No. 1; or

(II), an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids to the amino acid sequence described in (I), and an amino acid sequence with the same or similar functions as the amino acid sequence shown in (I); or

(III), an amino acid sequence having at least 80% identity to the sequence described in (I) or (II).

In some specific embodiments of the invention, the nucleotides encoding the ADAM17 have

(IV), the nucleotide sequence shown in SEQ ID No.2; or

(V), the complementary nucleotide sequence of the nucleotide sequence shown in SEQ ID 2; or

(VI), a nucleotide sequence encoding the same protein as the nucleotide sequence of (IV) or (V), but different from the nucleotide sequence of (IV) or (V) due to the degeneracy of the genetic code; or

(VII), a nucleotide sequence obtained by substitution, deletion or addition of one or more nucleotide sequences with the nucleotide sequence shown in (IV), (V) or (VI), and with (IV), A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown in (V) or (VI); or

(VIII), a nucleotide sequence that is at least 80% identical to the nucleotide sequence described in (IV), (V), (VI) or (VII).

On the basis of the above research, the present invention also provides the application of an inhibitor of ADAM17 in the preparation of a medicament for preventing and/or treating swine fever; the ADAM17 has:

(I), the amino acid sequence shown in SEQ ID No. 1; or

(II), an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids to the amino acid sequence described in (I), and an amino acid sequence with the same or similar functions as the amino acid sequence shown in (I); or

(III), an amino acid sequence having at least 80% identity to the sequence described in (I) or (II).

In some specific embodiments of the present invention, the inhibitor of ADAM17 is inhibition of ADAM17 expression or inhibition of ADAM17 activity;

The inhibiting ADAM17 expression is knocking down the expression of ADAM17;

The ADAM17-inhibiting activity is the chelation and/or removal of zinc ions from the active center of the metalloprotease domain of ADAM17.

In some specific embodiments of the present invention, the inhibitor of ADAM17 includes one or more of ADAM17 siRNA (as shown in SEQ ID Nos. 5-8), 1,10-phenanthroline, and aderbasib.

Among them, the sequence of siRNA is as follows:

In addition, the present invention also provides the application of knockout of ADAM17 in the preparation or screening of drugs for preventing and/or treating swine fever; the ADAM17 has:

(I), the amino acid sequence shown in SEQ ID No. 1; or

(II), an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids to the amino acid sequence described in (I), and an amino acid sequence with the same or similar functions as the amino acid sequence shown in (I); or

(III), an amino acid sequence having at least 80% identity to the sequence described in (I) or (II).

On the basis of the above research, the present invention also provides drugs for preventing and/or treating swine fever, nucleic acids, proteins, compounds or their salts, compositions, complexes or mixtures that can inhibit the expression of ADAM17 or inhibit the activity of ADAM17; The ADAM17 has:

(I), the amino acid sequence shown in SEQ ID No. 1; or

(II), an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids to the amino acid sequence described in (I), and an amino acid sequence with the same or similar functions as the amino acid sequence shown in (I); or

(III), an amino acid sequence having at least 80% identity to the sequence described in (I) or (II).

In some specific embodiments of the present invention, the inhibition of ADAM17 expression in the medicine provided by the present invention is to knock down the expression of ADAM17;

The ADAM17-inhibiting activity is the chelation and/or removal of zinc ions from the active center of the metalloprotease domain of ADAM17.

In addition, the present invention also provides the application of ADAM17 in the preparation or screening of vaccines for preventing swine fever; the ADAM17 has:

(I), the amino acid sequence shown in SEQ ID No. 1; or

(II), an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids to the amino acid sequence described in (I), and an amino acid sequence with the same or similar functions as the amino acid sequence shown in (I); or

(III), an amino acid sequence having at least 80% identity to the sequence described in (I) or (II).

The present invention also provides a vaccine for preventing swine fever, a nucleic acid, protein, compound or its salt, composition, complex or mixture that can inhibit the expression of ADAM17 or inhibit the activity of ADAM17; the ADAM17 has:

(I), the amino acid sequence shown in SEQ ID No. 1; or

(II), an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids to the amino acid sequence described in (I), and an amino acid sequence with the same or similar functions as the amino acid sequence shown in (I); or

(III), an amino acid sequence having at least 80% identity to the sequence described in (I) or (II).

In some specific embodiments of the present invention, the inhibition of ADAM17 expression in the vaccine provided by the present invention is to knock down the expression of ADAM17;

The ADAM17-inhibiting activity is the chelation and/or removal of zinc ions from the active center of the metalloprotease domain of ADAM17.

In the present invention, the CSFV E2 envelope glycoprotein specific binding protein ADAM17 is screened by co-immunoprecipitation technology, and the knockout of ADAM17 by CRISPR-Cas9 technology can completely block CSFV infection. The purified recombinant E2 protein can specifically bind to ADAM17. All of the above prove that ADAM17 is a specific receptor for swine fever virus. ADAM17 will be an important target for anti-swine fever virus vaccine and drug design and screening, as well as an important target for pig breeding against swine fever virus.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required in the description of the embodiments or the prior art.

Figure 1 shows the sgRNA recognition site; wherein, Figure 1(A) shows the sgRNA recognition site in the ADAM17 gene sequence; Figure 1(B) shows the sgRNA recognition site in the ADAM10 gene sequence;

Figure 2 shows the location of the knockout detection primers;

Figure 3 shows that ADAM17 is necessary for CSFV to invade PK15; among them, Figure 3(A) shows that the binding of sE2 to PK15 is dependent on ADAM17; FACS analysis of sE2 and PK15, Hela, PK15 ADAM17 knockout cell lines (PK15 ADAM17-KO), PK15ADAM10 Binding of knockout cell line (PK15 ADAM10-KO) and PK15 ADAM17-KO porcine ADAM17-overexpressing cell line (PK15ADAM17-KO-pADAM17); Figure 3(B) shows that cell lines without ADAM17 are not infected with CSFVpp; Infected with SXCDK and Shimen strains with GFP reporter gene, VSVpp was used as a positive control, and HIV-1 without encoding envelope protein was a negative control; error bars represent standard deviation; Figure 3(C) shows that CSFVcc is expressed in cells with or without ADAM17 expression Infectivity in lines; the above cells were infected with recombinant VSV (rVSV-GFP) and CSFV Shimen strains with GFP reporter gene, respectively, and further stained with anti-CSFV E2 (n=3), error bars represent standard deviation;

Figure 4 shows that ADAM17 is necessary for CSFV infection of porcine embryonic fibroblasts; PEFs were transfected with two siRNAs against ADAM17 (siADAM17-1, siADAM17-3) and a negative control (siCon.); Quantitative PCR was used to detect ADAM17 mRNA expression level, and the percentage of negative control was calculated (n=3); error bars represent standard deviation; Figure 4(B) shows the use of quantitative PCR to detect viral RNA levels of CSFVcc Shimen strains to determine viral infection and the percentage of negative control was calculated; the error bars represent the standard deviation, and the above data represent three independent experiments;

Figure 5 shows that ADAM17 binds to CSFV E2 through the metalloprotease domain; wherein, the chelation of zinc ions of the metalloprotease domain in the left panel of Figure 5 reduces the binding of sE2 to PK15 cells; different concentrations of 1,10-phenanthroline treat PK15, and Binding to sE2 was detected by FACS; the right panel of Figure 5 shows that aderbasib blocks the binding of sE2 to PK15 cells by occupying the active center of the metalloproteinase domain; PK15 cells were treated with different concentrations of aderbasib, and then the binding to sE2 was detected by FACS; above Data are representative of three independent experiments;

Figure 6 shows the direct binding of ADAM17 to sE2; wherein, Figure 6(A) shows the surface plasmon resonance image of purified soluble ADAM17 binding to sE2 in the absence of zinc ions; Figure 6(B) shows that in the presence of zinc ions Surface plasmon resonance image of soluble ADAM17 bound to sE2.

Detailed ways

The present invention discloses the application of ADAM17 in serving as the receptor of swine fever virus, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

The raw materials and reagents involved in the application of ADAM17 provided by the present invention as a swine fever virus receptor can be purchased from the market.

Terminology Explanation:

Swine fever is a severe swine infectious disease that seriously threatens the swine industry. The pathogen of swine fever is swine fever virus, a virus of the genus Pestivirus of the family Flaviviridae, and its hosts are limited to domestic pigs and wild boars.

Viral receptors refer to host factors that can specifically bind to viruses and mediate virus invasion into cells, most of which are transmembrane proteins. Receptor is a key host factor for virus infection of host, blocking the combination of virus and receptor can effectively inhibit virus infection. Therefore, receptors are important targets for antiviral strategies such as drugs and vaccines, and are also important targets for screening and selecting antiviral animals in animal breeding.

ADAM17 is a disintegrin matrix metalloproteinase 17 (A disintegrin and metalloprotease17, ADAM17) also called tumor necrosis factor-converting enzyme (tumor necrosis factor-converting enzyme, TACE) is a zinc ion-dependent disintegrin metalloprotease ADAM family member.

CRISPR Cas (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR; CRISPR-associated, Cas) is an RNA-mediated adaptive defense system evolved by bacteria and archaea to protect organisms from viruses and plasmids. The type II CRISPR system from Streptococcus pyogenes can direct the Cas9 protein to sequence-specifically cut targets paired with crRNA, and the CRISPR/Cas system was transformed into a genome editing tool in 2013.

SPR refers to Surface Plasmon Resonance in physics, the full name is Surface Plasmon Resonance. Surface (usually the interface between the solid phase and the liquid phase) plasmon resonance is an optical professional technique used to characterize the change of the refractive index of the surface, which can be used in real time. Track interactions between biomolecules in their native state.

Table 1 Main experimental instruments

Table 2 Main commodity reagents and consumables

Below in conjunction with embodiment, the present invention is further elaborated:

Example 1 Purification and identification of CSFV E2-Fc protein (sE2)

1. Purification of sE2 protein

The plasmid expressing sE2 protein is pCAGGS-CSFV-E2-Fc, which is kept in our laboratory.

(1) Transfer 293T to a 10cm cell culture dish, a total of 30 dishes, the medium is DMEM containing 8% FBS, 1% penicillin-streptomycin, 1% L-glutamine, and culture at 37 °C, 5% _CO2 in a cell incubator;

(2) The next day, when the 293T cells grow to about 90%, start transfection; the transfection method is calcium phosphate transfection, and the reagents include 2.5M CaCl ₂ , 2'HBS (pH 7.0), ddH ₂ O, and the Preheat in a 37°C water bath;

(3) Transfection system one:

table 3

Place system 1 in a flow tube and mix thoroughly on a shaker for 1 minute;

(4) Add 500 μl 2'HBS drop by drop to "System 1", and mix thoroughly on the shaker for 30 seconds;

(5) Add the above mixing system drop by drop into the 293T cells, and mix carefully up and down;

(6) Place the 293T cells in a cell culture incubator at 37°C and 5% CO ₂ for 12 hours;

(7) In a 37°C water bath, preheat DMEM medium containing 2% FBS, 1% penicillin-streptomycin, and 1% L-glutamine;

(8) Replaced with the above-mentioned medium, and continued to culture for 36 hours at 37° C., 5% CO ₂ ;

(9) Collect the culture medium, add one-tenth volume of 1.0M Tris (pH8.0) and mix well, the first centrifugation at 4°C, 5000rpm, 10 minutes, and the second centrifugation at 4°C, 12000rpm, 15 minutes , to completely remove cell debris;

(10) The following operation is performed in a low temperature laboratory at 4°C;

(11) Take 500μl of ProteinAbeads into the column, and 5mL of 10mM Tris (pH8.0) to equilibrate the ProteinAbeads;

(12) Pass the culture medium through ProteinAbeads, the flow rate is less than 1 mL/min;

(13) Wash protein A beads with 5 mL of 100 mM Tris (pH 8.0);

(14) Wash protein A beads with 5 mL of 10 mM Tris (pH 8.0);

(15) Elute the protein with 50 mM glycine (pH 3.0), 5 mL each time;

(16) Take several 1.5mL centrifuge tubes, add 50μl of 1M Tris (pH 8.0) to each tube, connect about 250μl of glycine eluate to each tube, and mix quickly;

(17) Take 20μl of 1'Bradford in a PCR tube, add 5μl of glycine as a control; take 5μl of the eluted solution into 20μl of 1'Bradford, mix well, put it on white paper to check whether the color turns blue, if it turns blue the presence of protein;

(18) eluted to 1'Bradford without discoloration;

(19) The eluted solutions are numbered 1 to n in sequence;

2. SDS-PAGE analysis of sE2 protein

(1) Configure SDS-PAGE gel, 10% separation gel;

(2) Cooking sample: 5μl 5′SDS-PAGE loading buffer+20μl protein eluent, 100℃ metal bath, 10 minutes;

(3) Electrophoresis

(4) Coomassie brilliant blue staining, depigmentation.

Example 2 Establishment of CRISPR Cas9 knockout cell line

1. sgRNA synthesis

(1) Using the http://www.rgenome.net/cas-designer/ online design website to design sgRNAs targeting the upstream and downstream of the second exon of porcine ADAM17 (NC_010445.4, NM_001099926.1) to knock out ADAM17 The second exon; in the same way, sgRNAs targeting the upstream and downstream of the second exon of ADAM10 (NC_010443.5) were designed to knock out the second and third exons of ADAM10. (Figure 1), and added CACC to the 5' end of the forward primer and AAAC to the 5' end of the reverse primer to form cohesive ends with the BbsI-digested pX330 plasmid (Table 4). It was handed over to Shanghai Jierui Biological Company for synthesis.

Table 4 sgRNA sequences

(2) Synthesize sgRNA double-stranded:

a. Dissolve sgRNA primers to 100 μM with RNase/DNase free water;

b. Prepare the system in a 600μl centrifuge tube and mix well:

table 5

c. Heat the beaker with ultrapure water with a magnetic stirrer until it boils;

d. Fix the 600μl centrifuge tube on the foam floating plate, put it into boiling water, and continue to heat to keep boiling for 10 minutes;

e. Transfer the beaker to a sealed foam box and let the water temperature slowly drop to room temperature;

f. Double-strand synthesis, stored at -20°C.

2.pX330 linearization

pX330, whose full name is pX330-GFP-U6-chimeric_BB+85-CBh-NLS-hSpCsn1-NLS_HW, is a human codon-optimized SpCas9 and chimeric sgRNA expression plasmid (Cong et al. 2013) with a GFP reporter gene.

(1) Enzyme digestion system:

Table 6

37°C water bath for 3 hours of enzymatic digestion.

(2) Glue recovery

The concentration is 1% agarose gel. After the digestion reaction is completed, the digestion system is subjected to agarose gel electrophoresis, the voltage is 130V, and the electrophoresis time is 30min; after the electrophoresis, the specific band with the correct size is used Cut with a clean scalpel, and use the agarose gel DNA recovery kit (Promega) to recover the target DNA fragments. The operation steps are:

a. Place the excised gel containing the target band in a 1.5mL centrifuge tube; weigh the gel and add 10μl membranebinding solution per 10mg;

b. Heat and incubate in a metal bath at 65°C until the gel is completely dissolved;

c. Place the SV micro-column in the collection tube;

d. Transfer the gel mixture into the micro-column and incubate at room temperature for 1 min;

e. After centrifugation at 16000'g for 1 min, discard the effluent;

f. Add 700 μl membrane wash solution to the micro-column, centrifuge at 16000′g for 1 min, and discard the effluent;

g. Add 500 μl membrane wash solution to the micro-column, centrifuge at 16000′g for 1 min, and discard the effluent;

h.16000′g, centrifuged for 5min;

i. Airing at room temperature for 5 minutes to evaporate the remaining ethanol;

j. Transfer the microcolumn to a clean 1.5mL centrifuge tube;

k. Add 50 μl of nuclease-free water to the mini-column and incubate at room temperature for 2 min;

1. After centrifugation at 16000′g for 1 min, the target DNA was obtained;

m. Use NonoDrop to measure DNA concentration;

n. Store at -20°C.

3. T4 DNA ligase ligation

Connect the linearized pX330 to the synthetic double-stranded sgRNA. The ligation system is as follows, mix well, and connect overnight in a 16°C PCR machine:

Table 7

4. Conversion

(1) Take out the stbl3 competent cells from the -80°C refrigerator and place them on ice;

(2) After the stbl3 competent cells are thawed, add 10 μl of the ligation product to 100 μl of the competent cells, flick the fingers a few times, mix well, and incubate on ice for 30 minutes;

(3) Heat shock at 42°C for 90 seconds, immediately transfer to ice, and incubate for 2 minutes;

(4) In the ultra-clean workbench, add 1 mL of antibiotic-free LB (peptone, yeast extract, sodium chloride) medium, transfer to a shaker, and incubate at 37°C for 50 minutes;

(5) Centrifuge for 3 minutes at room temperature and 4000 rpm, and remove most of the supernatant in the ultra-clean workbench, leaving only about 100 μl of the supernatant;

(6) Use a pipette to mix the cells and the remaining supernatant, resuspend the cells, spread them evenly on the ampicillin plate with a coating rod, and invert them in a 37°C incubator for about 12 hours.

5. Bacterial inspection

Due to the high ligation efficiency and the short insertion fragment of only 28bp, we used the method of direct picking, shaking, and sending the company to sequence for identification.

(1) Pick a single colony in 10 mL of LB medium containing ampicillin, shake at 37°C, and cultivate overnight;

(2) Mini plasmid:

a. Precipitate by centrifugation: Take 5mL of bacterial liquid cultured overnight in the ultra-clean bench, centrifuge at 12,000 rpm for 3 minutes at room temperature, suck off the supernatant, and leave the bacterial sediment;

b. Resuspension: Resuspend the bacterial pellet with 250μl BufferP1 and transfer it to a 1.5mL centrifuge tube;

c. Lysis: Add 250μl BufferP2, invert and mix 4-6 times, and the lysis time should not exceed 5 minutes;

d. Neutralization: Add 350μl BufferN3, and immediately mix up and down 4-6 times;

e. Centrifugation: Centrifuge in a desktop centrifuge for 10 minutes at 13000rpm;

f. Passing the column: Take 800μl of the supernatant obtained in "step e" and add it to the spin column, centrifuge at 13,000 rpm for 1 minute, and discard the effluent;

g. Wash the spin column: add 750 μl BufferPE to the spin column, centrifuge at 13,000 rpm for 1 minute, and discard the effluent;

h. Remove the residual Buffer PE: centrifuge at 13,000 rpm for 2 minutes, discard the effluent, and dry at room temperature for 5 minutes;

i. Elution: put the spin column into a new 1.5mL centrifuge tube, add 50μl BufferEB (10mM TrisCl, pH8.5) to the spin column, incubate at room temperature for 2 minutes, 13000rpm, 1 minute;

j. Use NanoDrop to measure the DNA concentration and send it to Beijing Qingke Xinye Biological Company for sequencing;

k. Analysis of sequencing results;

(3) Select a single colony solution with correct sequencing and no mutation, and carry out large-scale culture: take 1 mL of the bacterial solution in 200 mL of LB medium containing ampicillin, shake at 37°C, and cultivate overnight.

6. Large plasmids

(1) Precipitation by centrifugation: collect the bacterial liquid cultured overnight, centrifuge at 6000′g in a high-speed centrifuge at 4°C for 15 minutes, and discard the supernatant;

(2) Resuspension: Resuspend the cells with 10mL Buffer P1 and transfer to a 50mL high-speed centrifuge tube;

(3) Lysis: add 10mL Buffer P2, invert up and down 4-6 times to mix thoroughly, and incubate at room temperature for 5 minutes;

(4) Neutralization: add 10 mL of pre-cooled Buffer P3, invert up and down 4-6 times to mix thoroughly, and incubate on ice for 20 minutes;

(5) Centrifugation: Centrifuge at 22000'g for 30min at 4°C to obtain the supernatant, and centrifuge again at 22000'g for 15min at 4°C;

(6) Equilibrium: add 10mL Buffer QBT to the adsorption column, and drop it naturally by gravity;

(7) Passing the column-adsorption: adding the supernatant obtained in "step 5" to the column, so that the supernatant drips naturally by gravity;

(8) Washing the column: add 30mL Buffer QC to the column, drop it naturally by gravity, repeat twice;

(9) Elution: put the column into a new 50mL high-speed centrifuge tube, add 15mL Buffer QF to the column, and drop it naturally by gravity;

(10) Precipitate plasmid: add 10.5 mL of isopropanol to the eluted DNA, mix well; 4°C, 15000'g, 30 minutes; white DNA precipitation can be seen, discard the supernatant;

(11) Wash DNA: add 5 mL of 70% ethanol at 4°C, 15000′g for 10 minutes, discard the supernatant, and wash the DNA precipitate;

(12) Dissolve DNA: air dry the DNA precipitate, add an appropriate amount of Buffer EB to dissolve the DNA;

(13) Use NanoDrop to measure DNA concentration, and store at -20°C for later use. The constructed plasmids were named as: pX330-pig ADAM17 KO-up, pX330-pig ADAM17 KO-down, pX330-pig ADAM10 KO-up, pX330-pigADAM10 KO-down.

7. Transfection of PK15

(1) Transfer PK15 to a 6cm cell culture dish, and the medium is DMEM containing 8% FBS, 1% penicillin-streptomycin, and 1% L-glutamine, and culture it in a 37°C, 5% CO2 cell incubator;

(2) The next day, when the cells grow to about 90% full, start transfection, and the transfection reagent is Fugene 6 (PromegaE2691);

(3) Take 300 μl opti-MEM culture medium into a 1.5 mL centrifuge tube, then add 60 μl Fugene6, mix well with a shaker, centrifuge briefly, and incubate at room temperature for 5 minutes;

(4) Take 6 μg of each of pX330-up and pX330-down into the above system, mix with a shaker, centrifuge briefly, and incubate at room temperature for 15 minutes;

(5) Add the transfection system drop by drop to the cells, and mix carefully up and down;

(6) continue culturing cells for 48 hours;

8. Cell Sorting

Since the pX330 plasmid has a GFP reporter gene, cells with green fluorescence can be sorted by flow cytometry in a 96-well plate, 1 cell per well, to obtain a monoclonal cell line.

(1) Use trypsin to digest the transfected PK15 cells to single cells;

(2) Count with a cell counter, and dilute to a cell concentration of 2'106 cells/mL;

(3) Cell sieve filters cells in the flow tube;

(4) Add DMEM medium containing 8% FBS, 1% penicillin-streptomycin, and 1% L-glutamine in a 96-well cell culture plate in advance;

(5) The Beckman MoFlo XDP multicolor fluorescence flow cytometer was used to sort green fluorescence positive cells in a 96-well cell culture plate, one cell per well;

(6) The sorted cells were placed at 37°C and cultured in 5% CO ₂ ;

(7) After about a week, the wells with visible monoclonal cells were digested and transferred to a 12-well plate for continued culture; then transferred to a 6-well plate for continued culture;

(8) After filling the 6-well plate, take 2′10 ⁵ cells to extract genomic DNA, and transfer the rest to a 6cm culture dish to continue culturing;

(9) At the same time, the normal ^PK152'105 cells without manipulation were taken to extract genomic DNA as a control.

9. Extracting Genomic DNA

(1) Put the Spin Column in a collection tube, add 250 μl Buffer BL, centrifuge at 12000′g for 1 minute, and activate the silica gel membrane;

(2) Take 20 μl of proteinase K to the bottom of a 1.5 mL centrifuge tube, then add 200 μl of high-purity water to suspend the cell pellet, vortex for 10 seconds; add 200 μl of Buffer gA1, vortex for 10 seconds, and incubate at 56°C for 1 hour until the cells are completely digested ;

(3) After the incubation, add 200 μl of absolute ethanol, and vortex to mix;

(4) all the solutions obtained in step 3 are transferred into the Spin Column, centrifuged at 12000′g for 1 minute, and the waste liquid is discarded;

(5) Add 500 μl Buffer PW to the Spin Column, centrifuge at 12000′g for 1 minute, and discard the waste liquid;

(6) Repeat step 5 once;

(7) Add 500 μl Wash Buffer to the Spin Column, centrifuge at 12000′g for 1 minute, and discard the waste liquid;

(8) Put the Spin Column back into the collection tube, centrifuge at 12000′g for 2 minutes, open the lid and air dry for 1 minute;

(9) Take out the Spin Column and put it into a clean 1.5mL centrifuge tube, in the center of the adsorption membrane

Add 50 μl TE Buffer to the place, place at room temperature for 2 min, and centrifuge at 12000′g for 2 min;

(10) NanoDrop was used to measure the solubility of DNA solution and stored at -20°C for later use.

10. PCR detection

(1) The design positions of the detection primers are shown in Figure 2.

(2) Configure the following PCR system:

Table 8

(3) PCR program:

(4) The configuration concentration is 1% agarose gel, after the enzyme digestion reaction is completed, the enzyme digestion system is subjected to agarose gel electrophoresis, the voltage is 130V, and the electrophoresis time is 30min;

(5) Analyze the size of the band: the successful knockout band is smaller than that of the normal cell line.

Example 3 Flow cytometry (Flow cytometry, FACS)

(1) Use trypsin to digest each cell line to single cells;

(2) The cell sieve filters the cells in the flow tube;

(3) Counting with a cell counter, take two 2′10 ⁵ cells from each cell line and place them in two 1.5mL centrifuge tubes and define them as Assay (A) tube and Control (C) tube;

(4) Configure working solution: PBS with 1% FBS, put on ice;

(5) Centrifuge at 300g for 2 minutes, remove the supernatant; resuspend in 200μl of working solution and place on ice;

(6) Add 2μg sE2 (final concentration of 10μg/ml) to tube A of each cell line and mix well; add corresponding volume of PBS to tube C and mix well; incubate on ice for 50 minutes, mixing 5 times in between;

(7) Wash cells: 1 mL of working solution each time, centrifuge at 300g for 2 minutes at 4°C, and wash three times;

(8) Prepare secondary antibody solution: dilute Alexa-Fluor 488 goat anti-human IgG (Thermo Fisher Scientific) at 1:200 with working solution, 200 μl per tube, resuspend cells, incubate on ice for 45 minutes, and mix 5 times in between;

(9) Wash cells: 1 mL of working solution each time, centrifuge at 300g for 2 minutes at 4°C, and wash three times;

(10) Resuspend cells in 500 μl working solution per tube, transfer to flow tube, and place on ice;

(11) Analysis and detection using BD FACSCalibur flow cytometer.

Example 4 Establishment of overexpression cell lines

1. Lentiviral Plasmid Construction

(1) using the method of homologous recombination to clone ADAM17 homologous gene into lentiviral vector;

(2) Porcine ADAM17 is amplified from PK15 cDNA;

(3) Human ADAM17 is amplified from Hela cells;

(4) Mouse ADAM17, Jungle ADAM17 and Zebrafish ADAM17b were synthesized by Beijing Shengyuan Kemeng Biotechnology Co., Ltd.;

(5) Construction process: PCR amplification of the target fragment, linearization of the lentiviral vector, homologous recombination ligation, transformation, bacterial detection, sequencing, and mass extraction;

2. Establishment of Overexpression Cell Lines

(1) Transfer 293T to a 10cm petri dish, and the medium is DMEM containing 8% FBS, 1% penicillin-streptomycin, and 1% L-glutamine, and culture at 37°C and 5% CO ₂ ;

(2) The next day, when the 293T cells grow to about 90%, start transfection; the transfection method is calcium phosphate transfection, and the reagents include 2.5M CaCl ₂ , 2'HBS (pH 7.0), ddH ₂ O, and the Preheat in a 37°C water bath;

(3) Transfection system one:

Table 9

Place system 1 in a flow tube and mix thoroughly on a shaker for 1 minute;

(4) Add 500 μl 2'HBS drop by drop to "System 1", and mix thoroughly on the shaker for 30 seconds;

(5) Add the above mixing system drop by drop into the 293T cells, and mix carefully up and down;

(6) Place the 293T cells in a cell culture incubator at 37°C and 5% CO ₂ for 12 hours;

(7) In a 37°C water bath, preheat DMEM medium containing 8% FBS, 1% penicillin-streptomycin, and 1% L-glutamine;

(8) Replaced with the above-mentioned medium, and continued to culture for 36 hours at 37° C., 5% CO ₂ ;

(9) On the second day after transfection, the PK15 ADAM17-KO cell line was transferred to a 6cm cell culture dish;

(10) Collect 293T supernatant 48 hours after transfection, add 8 μg/mL polybrene, and mix well; centrifuge at 4°C, 12000 g, 10 minutes to remove cell debris;

(11) Remove the supernatant of PK15 ADAM17-KO, add the supernatant containing lentiviral particles, and continue to culture for 3 hours at 37°C, 5% CO ₂ ;

(12) Replace the fresh medium for PK15 ADAM17-KO, and continue to culture for 2 days at 37° C., 5% CO ₂ ;

(13) Add 3 μg/mL puromycin to screen for 5 days;

(14) Change to fresh medium, continue culturing, and then passage and cryopreserve.

3. Detection of Overexpressing Cell Lines

After establishing the cell line, use Western-Blot (anti-Flag Tag) or immunofluorescence (anti-humanADAM17) to detect gene overexpression.

Example 5 Pseudovirus packaging

1. Plasmid Construction

It is known that CSFV contains three subtypes (1, 2, 3). The present invention entrusts Beijing Shengyuan Kemeng Biotechnology Co., Ltd. to synthesize Shimen strain (type 1) (GenBank: AF333000.1) and SXCDK strain optimized by human relations codons. (Type 2) (Genbank: GQ923951.1) The gene fragment of the envelope protein ErnsE1E2 was constructed by homologous recombination into the pCAGGS vector linearized by XbaI and EcoRI. The plasmids were named pCAGGS-CSFV/Shimen-E012 and pCAGGS-CSFV, respectively. /SXCDK-E012.

2. Fake virus packaging

(1) Transfer 293T to a 10cm petri dish, and the medium is DMEM containing 8% FBS, 1% penicillin-streptomycin, and 1% L-glutamine, and culture at 37°C and 5% CO ₂ ;

(2) The next day, when the 293T cells grow to about 90%, start transfection; the transfection method is calcium phosphate transfection, and the reagents include 2.5M CaCl ₂ , 2'HBS (pH 7.0), ddH ₂ O, and the Preheat in a 37°C water bath;

(3) Transfection system one:

Table 10

The system 1 was placed in a flow tube and mixed thoroughly on a shaker for 1 minute; VSV-G expressed VSV glycoprotein, which was used as a positive control pseudovirus; or a glycoprotein expression plasmid without any virus was used as a negative. Control (Non-envelope);

(4) Add 500 μl 2'HBS drop by drop to "System 1", and mix thoroughly on the shaker for 30 seconds;

(5) Add the above mixing system drop by drop into the 293T cells, and mix carefully up and down;

(6) Place the 293T cells in a cell culture incubator at 37°C and 5% CO ₂ for 12 hours;

(7) In a 37°C water bath, preheat DMEM medium containing 8% FBS, 1% penicillin-streptomycin, and 1% L-glutamine;

(8) Replaced with the above-mentioned medium, and continued to culture for 36 hours at 37° C., 5% CO ₂ ;

(9) Collect the 293T supernatant 48 hours after transfection, add 8 μg/mL polybrene, and mix well; centrifuge at 4°C, 12000g for 10 minutes, take the supernatant, and freeze it at -80°C for use.

3. Pseudovirus Titer Assay

(1) PK15 was transferred to a 96-well plate with 8000 cells per well;

(2) 24 hours later, 10-fold serial dilution of pseudovirus, 100 μl/well, infect cells, incubate at 37°C, 5% CO ₂ for 3 hours;

(3) Change to fresh medium and continue to culture for 45 hours at 37°C, 5% _CO2

(4) Counted under a fluorescence microscope, the titer of the virus was calculated as the number of GFP-positives per milliliter (FFU/mL).

Example 6 Determination of virus titer of cell culture strain (CSFVcc)

(1) PK15 was transferred to 96-well plate, 8000 cells per well;

(2) After 24 hours, 10-fold serial dilution of virus, 100 μl/well, infect cells, and incubate at 37°C, 5% CO ₂ for 3 hours;

(3) Change to medium containing 20mM NH4Cl, and continue to culture for 45 hours at 37°C, 5% _CO2 ;

(4) Fix the cells with methanol pre-cooled at -20°C;

(5) Wash the cells twice with PBS containing 1% Gelatin;

(6) Block cells with PBS containing 1% BSA at room temperature for 2 hours;

(7) 1:500 dilution of mouse monoclonal antibody WH303 (anti-CSFV E2 protein), 50 μl per well;

(8) Wash the cells three times with PBS containing 1% Gelatin;

(9) 1:500 dilution of Alexa-Fluor 488 goat anti-mouse IgG (Thermo Fisher Scientific), 50 μl per well;

(10) Wash the cells three times with PBS containing 1% Gelatin;

(11) Counting under a fluorescence microscope.

Example 7 RNA interference

1. RNA interference

(1) Use http://rnaidesigner.thermofisher.com/rnaiexpress/ to design 3 pairs of gene-specific siRNAs and species nonsense siRNA, and submit to Shanghai Sangon Biotechnology Co., Ltd. for synthesis;

The sequence looks like this:

(2) Pass the cells to a 6cm cell culture dish;

(3) When the cells grow to about 95% full, start transfection;

(4) Configure transfection system 1: Take 300 μl Opti-MEM Medium in a 1.5 mL centrifuge tube, add 18 μl Lipofectamine RNAimax to it, and mix well;

(5) Configure the transfection system and the second: take 300μl Opti-MEM Medium in a 1.5mL centrifuge tube, add siRNA to make the final concentration 50nM, and mix well;

(6) Mixing system 1 and 2, mix well, centrifuge briefly, and incubate at room temperature for 5 minutes;

(7) Add the obtained in step 6 into the cells drop by drop, carefully mix up and down, left and right, and culture at 37°C for 36 hours;

2. Extracting Cellular RNA

After 36 hours of siRNA transfection, take 2′10 ⁵ cells respectively, lyse the cells with 500 μl Trizol, and extract RNA. The specific operation steps are as follows:

(1) 500 μl Trizol to lyse cells at room temperature for 5 minutes;

(2) Add 100 μl of chloroform, mix upside down and incubate at room temperature for 2 minutes;

(3) Centrifugation: 4°C, 12000g, 15 minutes;

(4) Layers can be seen, carefully take the supernatant into a new 1.5mL centrifuge tube;

(5) Add 250 μl of isopropanol, invert up and down to mix, and incubate at room temperature for 10 minutes;

(6) Centrifugation: 4°C, 12000g, 10 minutes;

(7) White precipitate can be seen, and the supernatant is carefully sucked away;

(8) Add 1 mL of 75% ethanol and turn it upside down several times;

(9) Centrifugation: 4°C, 7500g, 5 minutes;

(10) remove the supernatant, and dry the precipitate at room temperature;

(11) Dissolve the precipitate with RNase-free water, measure the concentration of RNA solution with NanoDrop, use immediately or store at -80°C for later use.

3. Reverse Transcription PCR

Use TAKARA PrimeScriptTM RT reagent Kit with gDNA Eraser kit for reverse transcription PCR, the operation process is as follows:

(1) Removal of genomic DNA reaction: Prepare the reaction mixture on ice according to the following components, and then incubate at 42°C for 2 minutes;

Table 11

(2) Reverse transcription reaction: The reaction solution is prepared on ice, and the reaction components are as follows:

Table 12

Then perform the following procedure on a PCR machine to obtain cDNA, which is stored at -20°C for future use.

Table 13

37℃ 15min 85℃ 5s 4℃

4. Quantitative PCR

Takara one step SYBR PrimerScript reverse transcription (RT)-PCR kit was used, the instrument was Applied Biosystems QuantStudio, and the β-actin gene was used as an internal reference.

The RT-PCR procedure is:

Example of effect

1. In order to screen and identify swine fever virus-specific host factors, we cloned the ectodomain sequence (GenBank accession number AFF333000.1) encoding the glycoprotein E2 of CSFV 1 subtype Shimen strain into the pPUR-TPA-Fc vector, and 293T cells were transfected, the supernatant was collected and the swine fever virus E2 soluble protein sE2 was purified. Porcine ADAM17 protein was obtained by co-immunoprecipitation experiment with sE2 protein combined with mass spectrometry.

DNA sequence of E2 extracellular domain (as shown in SEQ ID No. 4):

cggctagcctgcaaggaagattacaggtacgcaatatcatcaaccaatgagatagggctactcggggccggaggtctcactaccacctggaaagaatacagccacgatttgcaactgaatgacgggaccgttaaggccatttgcgtggcaggttcctttaaaatcacagcacttaatgtggtcagtaggaggtatttggcatcattgcataagggggctttactcacttccgtgacattcgagctcctgttcgacgggaccaacccatcaaccgaagaaatgggagatgacttcgggttcgggctgtgcccgtttgatacgagtcctgttgtcaagggaaagtacaatacaaccttgttgaacggtagtgctttctatcttgtctgcccaatagggtggacgggtgttatagagtgcacagcagtgagcccaacaactctgagaacagaagtggtaaagaccttcaggagagagaagcctttcccacacagaatggattgtgtgaccaccacagtggaaaatgaagatctattctactgtaagttggggggcaactggacatgtgtgaaaggtgaaccagtggtctacacaggggggcaagtaaaacaatgcaaatggtgtggcttcgacttcaacgagcctgacggactcccacactaccccataggtaagtgcattttggcaaatgagacaggttacagaatagtagattcaacggactgtaacagagatggcgttgtaatcagcgcaaaggggagccatgagtgcttgatcggcaacacaactgtcaaggtgcatgcatcagatgaaagactgggccctatgccatgcagacctaaagagattgtctctagtgcaggacctgtaaggaaaacttcctgtacattcaactacgcaaaaactttgaagaacaagtactatgagcccagggacagctacttccagcaatatatgctcaagggcgagtatcagtactggtttgacctggacgtg

Protein sequence (as shown in SEQ ID No. 3):

RLACKEDYRYAISSTNEIGLLGAGGLTTTWKEYSHDLQLNDGTVKAICVAGSFKITALNVVSRRYLASLHKGALLTSVTFELLFDGTNPSTEEMGDDFGFGLCPFDTSPVVKGKYNTTLLNGSAFYLVCPIGWTGVIECTAVSPTTLRTEVVKTFRREKPFPHRMDCVTTTVENEDLFYCKLGGNWTCVKGEPVVYTGGQVKQCKWCGFDFNEPDGLPHYPIGKCILANETGYRIVDSTDCNRDGVVISAKGSHECLIGNTTVKVHASDERLGPMPCRPKEIVSSAGPVRKTSCTFNYAKTLKNKYYEPRDSYFQQYMLKGEYQYWFDLDV

Porcine ADAM17 coding region DNA sequence (as shown in SEQ ID No. 2):

atgaggcagtgtgcgctcttcctgaccagcttggttcctatcgtgctggcgccgcgaccgccggacgagccgggcttcggctcccctcagcgactcgaaaagcttgattctctgctctcagactacgacatcctctctttatccagcattcgccagcactccgtaaggaaaagggatctgcaggcctcaacacacctagagacactactaactttttcagccttgaacaggcattttaaattatacctgacatcaagtactgaacgcttctcccagaatttcaaagtcgtggtggtcgatggggaagatgaaagtgagtaccccgtcaagtggcaggacttcttcagtggacacgtggttggtgaacctgactctagggttctcgcccacataggagatgatgatattacagtaagaatcaacacagatggggcagaatataatatagagccactttggagactaattaatgatactaaagacaaaagagtgttagtttataagtctgaagatatcaagaatgtttcacgtttgcagtctccaaaagtgtgtggttatataaaggcggataatgaagagttgcttcctaaagggctagtagacagagagccgcctgatgagcttgttcaccgggtgaagagaagagccgaccccaatcccctgaggaacacgtgtaaattattggtggtggcagatcatcgcttttataagtacatgggcagaggggaagagagcacgaccacaaactacctgatagagctaattgacagagttgatgacatctatcggaacacttcatgggacaatgcaggttttaaaggttatggaatacagatagagcagattcgcattctcaagtctccacaagaggtaaaacctggtgaaaggcactacaatatggcaaaaagttacccaaatgaagaaaaggatgcttgggatgtgaagatgttgctagagcaatttagctttgatatagctgaagaagcatctaaagtctgcc tggcacatcttttcacctaccaagattttgatatgggaactcttggattagcttatgttggttctcccagagcaaacagtcatggaggtgtttgtccaaaggcttattatagtccaattggaaagaaaaatatctatttaaatagtggtttgaccagcacaaaaaattatggtaaaaccatccttacaaaggaagctgaccttgtgacaactcatgaattggggcacaattttggagcagaacacgatccagatggtttagcagaatgtgccccaaacgaggaccagggaggaaaatacgtcatgtatcccatagccgtgagtggtgatcatgagaacaacaagatgttttcaaactgcagtaaacagtccatctataagaccattgaaagtaaggcccaggagtgttttcaagagcgcagcaacaaagtgtgtggcaactccagggtggatgagggggaggagtgcgaccccggcatcatgtacctgaacaacgacacctgctgcaacagcgactgcaccctgaggccgggcgtccagtgcagtgataggaacagtccttgctgtaaaaactgtcagttcgagacggcccagaagaagtgccaggaggctattaatgccacttgcaaaggcgtgtcttactgcacaggtaacagcagtgagtgcccccctccgggaaacgccgaggacgacacggtgtgcctggacctgggcaggtgcaaggacggcaagtgcgtgcccttctgcgagcgggagcagcggctggagtcctgcgcgtgtaacgaaaccgaccactcgtgcaaggtgtgctgccgggccccctcgggccgttgcctgccctacgtggacgccgaacagaagaacttgtttttgaggaaggggaagccctgtacagtaggattttgtgacatgaatggcaagtgtgagaagcgagtgcaggacgtcatcgagcggttctgggagttcattgacaagctgagcatcaatactttcgggaagtt cctggcagacaacatcgtgggctccgtcctggtgttctccctgatgctctggatccccgtcagcatcctcgtccactgcgtggataagaagctggataagcagtacgaatccctgtctctgctgcaccccagcaacgtggagatgctaagcagcatggattcagcatccgttcgcatcatcaagccctttcctgcgccccagaccccaggccgcctgcagcccctgcagcccctgcagcccggccccgtgctgccctctgcgccttcggtgcccgtggctccaaaactggaccaccagcggatggacaccatccaggaggaccccagcacggactcgcacgtggacgaggacggcttcgagaaggaccctttccccaacagcagtgccgctgccaagtcatttgaggatctcacggaccatccggtcacgagaagtgaaaaggcctcgtcctttaagctgcagcgccagagtcgcgttgacagcaaggaaacggagtgc

Porcine ADAM17 protein sequence (as shown in SEQ ID No. 1):

MRQCALFLTSLVPIVLAPRPPDEPGFGSPQRLEKLDSLLSDYDILSLSSIRQHSVRKRDLQASTHLETLLTFSALNRHFKLYLTSSTERFSQNFKVVVVDGEDESEYPVKWQDFFSGHVVGEPDSRVLAHIGDDDITVRINTDGAEYNIEPLWRLINDTKDKRVLVYKSEDIKNVSRLQSPKVCGYIKADNEELLPKGLVDREPPDELVHRVKRRADPNPLRNTCKLLVVADHRFYKYMGRGEESTTTNYLIELIDRVDDIYRNTSWDNAGFKGYGIQIEQIRILKSPQEVKPGERHYNMAKSYPNEEKDAWDVKMLLEQFSFDIAEEASKVCLAHLFTYQDFDMGTLGLAYVGSPRANSHGGVCPKAYYSPIGKKNIYLNSGLTSTKNYGKTILTKEADLVTTHELGHNFGAEHDPDGLAECAPNEDQGGKYVMYPIAVSGDHENNKMFSNCSKQSIYKTIESKAQECFQERSNKVCGNSRVDEGEECDPGIMYLNNDTCCNSDCTLRPGVQCSDRNSPCCKNCQFETAQKKCQEAINATCKGVSYCTGNSSECPPPGNAEDDTVCLDLGRCKDGKCVPFCEREQRLESCACNETDHSCKVCCRAPSGRCLPYVDAEQKNLFLRKGKPCTVGFCDMNGKCEKRVQDVIERFWEFIDKLSINTFGKFLADNIVGSVLVFSLMLWIPVSILVHCVDKKLDKQYESLSLLHPSNVEMLSSMDSASVRIIKPFPAPQTPGRLQPLQPLQPGPVLPSAPSVPVAPKLDHQRMDTIQEDPSTDSHVDEDGFEKDPFPNSSAAAKSFEDLTDHPVTRSEKASSFKLQRQSRVDSKETEC

2. To verify the function of ADAM17, we knocked down the expression of ADAM17 using CRISPR-Cas9 technology in CSFV-susceptible cell PK15. The results are shown in Figure 3: the knockout cell lines could not bind to sE2, nor could they not be infected with pseudovirus and true virus of CSFV. However, both sE2 binding and CSFV virus infection were completely restored after ADAM17 re-overexpression.

Specifically, Figure 3(A):

Instrument: Flow Cytometer BD FACSCalibur

Software: FlowJo 7.6

Experimental method: FACS (flow cytometry)

Analysis process: the blue shade is the experimental group with sE2, and the red is the control group. Wild-type PK15 (a swine fever virus-susceptible cell line, porcine kidney cells) binds to sE2, and a strong fluorescent signal can be detected, while the control group has almost no fluorescent signal. The two are analyzed by FlowJo 7.6 software, and their histograms are overlapped, showing that complete displacement. The control cells, namely Hela (a swine fever virus insensitive cell line), cannot bind to sE2, and no fluorescent signal can be detected. The control group also has no fluorescent signal. The two are analyzed by FlowJo7.6 software, and the histograms of the two are overlapped, and there is no displacement. . The above shows that sE2 can specifically bind to the swine fever virus-susceptible cell line PK15, but not the non-susceptible cell line Hela, which proves the specificity.

After FACS analysis of CRISPR Cas9 knockout ADAM17 gene in PK15 cell line, it was found that PK15 could not bind to sE2 after ADAM17 knockout, and no fluorescence signal could be detected, and there was no fluorescence signal in the control group. Both were analyzed by FlowJo 7.6 software. Overlap the two histograms, no displacement occurs. After re-introducing porcine ADAM17 cDNA into ADAM17 knockout cell line, after remodeling ADAM17 expression, FACS analysis can restore the binding to sE2, and a strong fluorescent signal can be detected, and the control group has basically no fluorescent signal. Analysis, overlaying the two histograms, shows the complete displacement. ADAM family has more than 40 genes, ADAM10 is the most similar to ADAM17, the amino acid similarity rate is 22.6%, but FACS analysis of CRISPR Cas9 knockout PK15 cell line ADAM10 gene changes in binding to sE2, it is found that PK15 after ADAM10 knockout It can still be combined with sE2, and strong fluorescent signal can be detected, and the control group has basically no fluorescent signal. The two are analyzed by FlowJo 7.6 software, and their histograms are overlapped, showing a complete displacement. It was demonstrated that ADAM17 is indispensable for the binding of PK15 to the CSFV E2 protein.

Table 14 Figure 3(A) Data

Specifically, Figure 3(B):

Instrument: Nikon fluorescence microscope

Software: Excel and GraphPad Prism6

Experimental method: Pseudovirus titer determination

Analysis process: Pseudoviruses are recombinant virus particles whose core/backbone and envelope proteins are derived from different viruses; in addition, the genes inside pseudoviruses are usually altered or modified so that they cannot produce viral surface proteins alone. Therefore, an additional plasmid or stable cell line expressing surface proteins is required to package the pseudovirus. Pseudoviruses are able to infect susceptible cell lines, but they can only replicate once in infected host cells. Pseudoviruses can be safely handled in laboratories at Biosafety Level 2 (BSL-2) and are generally easier to handle than wild-type viruses. More importantly, the protein conformation of pseudovirus envelope surface proteins is consistent with that of wild-type viruses. These surface proteins can effectively mediate virus entry into host cells. Therefore, pseudoviruses are widely used in virus cell tropism and receptor recognition. , viral inhibition studies, and development and evaluation of antibodies and vaccines. Moreover, it has been confirmed that the pseudovirus in vitro and in vivo experiments have a good correlation with the experimental results produced by the wild-type virus.

Virus titers (FFU/ml) were counted and converted 48 hours after infection of the different cell lines with pseudovirus. Use Excel to convert the data into a logarithmic function with base 10, and continue processing the obtained data with GraphPad Prism6 to present the final result in the form of a histogram. The results showed that PK15 knocking out ADAM17 did not infect the two genotypes of swine fever virus at all. After re-introduction of porcine ADAM17 cDNA, the infection could be restored to the level of wild PK15, while knocking out ADAM10 did not change. At the same time, VSV-G (vesicular stomatitis virus) is often used as a control virus because it infects almost all cell types. The results show that various experimental operations basically do not affect VSV-G infection. It was proved that ADAM17 is indispensable for the invasion of PK15 by swine fever virus.

Table 15 Figure 3(B) Data

Specifically, Figure 3(C):

Instrument: Nikon fluorescence microscope

Software: Excel and GraphPad Prism6

Experimental method: Determination of virus titer of cell culture strain (CSFVcc)

Analysis process: After 48 hours of virus infection in different cell lines, immunofluorescence experiments were performed, and the virus titers (FFU/ml) were counted and converted. Use Excel to convert the data into a logarithmic function with base 10, and continue processing the obtained data with GraphPad Prism6 to present the final result in the form of a histogram. The results showed that PK15 knocking out ADAM17 did not infect CSFVcc at all, and after reintroduction of porcine ADAM17 cDNA, the infection could be restored to the level of wild PK15, while knocking out ADAM10 did not change. At the same time, rVSV-eGFP-G (recombinant vesicular stomatitis virus) is often used as a control virus because it infects almost all cell types. The results show that various experimental operations basically do not affect rVSV-eGFP-G infection. It was demonstrated that ADAM17 is indispensable for CSFV infection with PK15.

Table 16 Figure 3(C) Data

3. To further study the role of ADAM17 in CSFV-infected pigs, siRNA was used to interfere the expression of ADAM17 in pig primary embryonic fibroblasts (PEFs), and the effect on CSFV-infected PEFs was detected. Two RNAi primers were designed and synthesized to transfect PEFs respectively.

Instrument: Thermo fisher quantitative PCR instrument

Software: Excel and GraphPad Prism6

Experimental methods: RNA interference, extraction of cellular RNA, reverse transcription PCR, quantitative PCR

Analysis process: The number of PCR cycles (Ct value) required to reach the threshold by transfecting primary porcine embryonic fibroblasts with ADAM17-specific primer (si-ADAM17), irrelevant primer (siCtrl) ADAM17 and internal reference gene β-actin was analyzed by quantitative PCR. . The expression level (%) of ADAM17 transfected with ADAM17-specific primer (si-ADAM17) relative to transfection-independent primer (siCtrl) was preliminarily processed by Excel, and the obtained data was further processed with GraphPad Prism6 to present the final result in the form of a histogram. The results showed that the RNA levels of ADAM17 were reduced by 70.9% and 78.9%, respectively.

The primary porcine fibroblasts after RNA interference 36 were infected with CSFVcc, and the virus infection level was detected by quantitative PCR 2 days later, and the number of PCR cycles (Ct value) required to reach the threshold was determined. The CSFVcc content (%) of the transfected ADAM17-specific primer (si-ADAM17) relative to the transfection-independent primer (siCtrl) was preliminarily processed using Excel, and the data obtained were further processed with GraphPad Prism6 to present the final result in the form of a histogram. The results showed that knockdown of ADAM17 expression by two RNAi primers significantly reduced viral infection levels by 89.2% and 92.4%, respectively.

The results are shown in Figure 4: 36 hours later, the level of ADAM17 RNA was detected by real-time PCR, and the RNA level of ADAM17 was decreased by 70.9% and 78.9%, respectively. At the same time, the PEFs cells after 36 hours of RNA interference were infected with CSFVcc, and the level of virus infection was detected by quantitative PCR 2 days later. Knockdown of ADAM17 expression with two RNAi primers significantly reduced viral infection levels by 89.2% and 92.4%, respectively. This result indicates that ADAM17 is essential for CSFV infection of primary porcine cells.

Table 17 Figure 4(A)

Table 18 Figure 4(B)

4. ADAM17 is a zinc ion-dependent metalloprotease with a zinc ion in the active center of its metalloprotease domain. 1,10-phenanthroline can inhibit its activity by chelating and removing zinc ions.

Instrument: Flow Cytometer BD FACSCalibur

Software: FlowJo 7.6

Experimental method: FACS (flow cytometry)

The results are shown in Figure 5: different concentrations of 1,10-phenanthroline and trypsin-treated PK15 were incubated on ice for 30 minutes, and then the binding of PK15 to sE2 was detected by FACS. It was found that the binding of sE2 decreased as the concentration increased, and Binding was reduced by 74.3% at a 1,10-phenanthroline concentration of 25 mM. It is illustrated that CSFV E2 protein binds to the active site of ADAM17.

A class of ADAM17 inhibitors including aderbasib, also known as INCB007839, is a selective inhibitor of ADAM10 and ADAM17 that inhibits the activity of metallopeptidases by binding to the active site of the metalloprotease domain and is currently undergoing cancer therapy. Clinical Trials. In order to test whether aderbasib can inhibit the binding between ADAM17 and sE2, different concentrations of aderbasib were incubated with trypsin-treated PK15 on ice for 30 minutes, and the binding of PK15 to sE2 was also detected by FACS, and it was found that the binding of sE2 decreased with the increase of concentration , and almost no binding was detected at aderbasib concentration of 100 μM (right panel of FIG. 5 ). This further indicates that CSFV E2 protein binds to ADAM17 through the metalloprotease domain.

Table 19 Figure 5 Left data

Table 20 Figure 5 Right data

5. In order that ADAM17 can directly bind to CSFV E2, the present invention purifies soluble porcine ADAM17 protein (sADAM17), and uses surface plasmon resonance (SPR) to further verify the interaction between sADAM17 and sE2. Because ADAM17 in the living body can play its biological role in the presence of zinc ions, the affinity between sADAM17 and sE2 was detected in the presence and absence of zinc ions, respectively.

Instrument: Surface Plasmon Resonator

Software: Excel

Experimental Method: Surface Plasma Analysis

Analysis process: sE2 was bonded to the sensor surface, and then the expressed and purified sADAM17 solutions of different concentrations were injected into and flowed through the sensor surface (in an environment with zinc ions and in an environment without zinc ions) to collect data. The data was analyzed by Excel to obtain a line graph.

The results are shown in Figure 6: it shows that the affinity between sADAM17 and sE2 is 3.250×10 ^-6 mol in the absence of zinc ions, ^and 1.122×10 ^-7 mol in the presence of zinc ions.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

sequence listing

<110> Institute of Zoology, Chinese Academy of Sciences

<120> ADAM17 as a receptor for swine fever virus

<130> MP1929873

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 833

<212> PRT

<213> Pig ADAM17 (Pig ADAM17)

<400> 1

Met Arg Gln Cys Ala Leu Phe Leu Thr Ser Leu Val Pro Ile Val Leu

1 5 10 15

Ala Pro Arg Pro Pro Asp Glu Pro Gly Phe Gly Ser Pro Gln Arg Leu

20 25 30

Glu Lys Leu Asp Ser Leu Leu Ser Asp Tyr Asp Ile Leu Ser Leu Ser

35 40 45

Ser Ile Arg Gln His Ser Val Arg Lys Arg Asp Leu Gln Ala Ser Thr

50 55 60

His Leu Glu Thr Leu Leu Thr Phe Ser Ala Leu Asn Arg His Phe Lys

65 70 75 80

Leu Tyr Leu Thr Ser Ser Thr Glu Arg Phe Ser Gln Asn Phe Lys Val

85 90 95

Val Val Val Asp Gly Glu Asp Glu Ser Glu Tyr Pro Val Lys Trp Gln

100 105 110

Asp Phe Phe Ser Gly His Val Val Gly Glu Pro Asp Ser Arg Val Leu

115 120 125

Ala His Ile Gly Asp Asp Asp Ile Thr Val Arg Ile Asn Thr Asp Gly

130 135 140

Ala Glu Tyr Asn Ile Glu Pro Leu Trp Arg Leu Ile Asn Asp Thr Lys

145 150 155 160

Asp Lys Arg Val Leu Val Tyr Lys Ser Glu Asp Ile Lys Asn Val Ser

165 170 175

Arg Leu Gln Ser Pro Lys Val Cys Gly Tyr Ile Lys Ala Asp Asn Glu

180 185 190

Glu Leu Leu Pro Lys Gly Leu Val Asp Arg Glu Pro Pro Asp Glu Leu

195 200 205

Val His Arg Val Lys Arg Arg Ala Asp Pro Asn Pro Leu Arg Asn Thr

210 215 220

Cys Lys Leu Leu Val Val Ala Asp His Arg Phe Tyr Lys Tyr Met Gly

225 230 235 240

Arg Gly Glu Glu Ser Thr Thr Thr Asn Tyr Leu Ile Glu Leu Ile Asp

245 250 255

Arg Val Asp Asp Ile Tyr Arg Asn Thr Ser Trp Asp Asn Ala Gly Phe

260 265 270

Lys Gly Tyr Gly Ile Gln Ile Glu Gln Ile Arg Ile Leu Lys Ser Pro

275 280 285

Gln Glu Val Lys Pro Gly Glu Arg His Tyr Asn Met Ala Lys Ser Tyr

290 295 300

Pro Asn Glu Glu Lys Asp Ala Trp Asp Val Lys Met Leu Leu Glu Gln

305 310 315 320

Phe Ser Phe Asp Ile Ala Glu Glu Ala Ser Lys Val Cys Leu Ala His

325 330 335

Leu Phe Thr Tyr Gln Asp Phe Asp Met Gly Thr Leu Gly Leu Ala Tyr

340 345 350

Val Gly Ser Pro Arg Ala Asn Ser His Gly Gly Val Cys Pro Lys Ala

355 360 365

Tyr Tyr Ser Pro Ile Gly Lys Lys Asn Ile Tyr Leu Asn Ser Gly Leu

370 375 380

Thr Ser Thr Lys Asn Tyr Gly Lys Thr Ile Leu Thr Lys Glu Ala Asp

385 390 395 400

Leu Val Thr Thr His Glu Leu Gly His Asn Phe Gly Ala Glu His Asp

405 410 415

Pro Asp Gly Leu Ala Glu Cys Ala Pro Asn Glu Asp Gln Gly Gly Lys

420 425 430

Tyr Val Met Tyr Pro Ile Ala Val Ser Gly Asp His Glu Asn Asn Lys

435 440 445

Met Phe Ser Asn Cys Ser Lys Gln Ser Ile Tyr Lys Thr Ile Glu Ser

450 455 460

Lys Ala Gln Glu Cys Phe Gln Glu Arg Ser Asn Lys Val Cys Gly Asn

465 470 475 480

Ser Arg Val Asp Glu Gly Glu Glu Cys Asp Pro Gly Ile Met Tyr Leu

485 490 495

Asn Asn Asp Thr Cys Cys Asn Ser Asp Cys Thr Leu Arg Pro Gly Val

500 505 510

Gln Cys Ser Asp Arg Asn Ser Pro Cys Cys Lys Asn Cys Gln Phe Glu

515 520 525

Thr Ala Gln Lys Lys Cys Gln Glu Ala Ile Asn Ala Thr Cys Lys Gly

530 535 540

Val Ser Tyr Cys Thr Gly Asn Ser Ser Glu Cys Pro Pro Pro Gly Asn

545 550 555 560

Ala Glu Asp Asp Thr Val Cys Leu Asp Leu Gly Arg Cys Lys Asp Gly

565 570 575

Lys Cys Val Pro Phe Cys Glu Arg Glu Gln Arg Leu Glu Ser Cys Ala

580 585 590

Cys Asn Glu Thr Asp His Ser Cys Lys Val Cys Cys Arg Ala Pro Ser

595 600 605

Gly Arg Cys Leu Pro Tyr Val Asp Ala Glu Gln Lys Asn Leu Phe Leu

610 615 620

Arg Lys Gly Lys Pro Cys Thr Val Gly Phe Cys Asp Met Asn Gly Lys

625 630 635 640

Cys Glu Lys Arg Val Gln Asp Val Ile Glu Arg Phe Trp Glu Phe Ile

645 650 655

Asp Lys Leu Ser Ile Asn Thr Phe Gly Lys Phe Leu Ala Asp Asn Ile

660 665 670

Val Gly Ser Val Leu Val Phe Ser Leu Met Leu Trp Ile Pro Val Ser

675 680 685

Ile Leu Val His Cys Val Asp Lys Lys Leu Asp Lys Gln Tyr Glu Ser

690 695 700

Leu Ser Leu Leu His Pro Ser Asn Val Glu Met Leu Ser Ser Met Asp

705 710 715 720

Ser Ala Ser Val Arg Ile Ile Lys Pro Phe Pro Ala Pro Gln Thr Pro

725 730 735

Gly Arg Leu Gln Pro Leu Gln Pro Leu Gln Pro Gly Pro Val Leu Pro

740 745 750

Ser Ala Pro Ser Val Pro Val Ala Pro Lys Leu Asp His Gln Arg Met

755 760 765

Asp Thr Ile Gln Glu Asp Pro Ser Thr Asp Ser His Val Asp Glu Asp

770 775 780

Gly Phe Glu Lys Asp Pro Phe Pro Asn Ser Ser Ala Ala Ala Lys Ser

785 790 795 800

Phe Glu Asp Leu Thr Asp His Pro Val Thr Arg Ser Glu Lys Ala Ser

805 810 815

Ser Phe Lys Leu Gln Arg Gln Ser Arg Val Asp Ser Lys Glu Thr Glu

820 825 830

Cys

<210> 2

<211> 2499

<212> DNA

<213> Pig ADAM17 (Pig ADAM17)

<400> 2

atgaggcagt gtgcgctctt cctgaccagc ttggttccta tcgtgctggc gccgcgaccg 60

ccggacgagc cgggcttcgg ctcccctcag cgactcgaaa agcttgattc tctgctctca 120

gactacgaca tcctctcttt atccagcatt cgccagcact ccgtaaggaa aagggatctg 180

caggcctcaa cacacctaga gacactacta actttttcag ccttgaacag gcattttaaa 240

ttatacctga catcaagtac tgaacgcttc tcccagaatt tcaaagtcgt ggtggtcgat 300

ggggaagatg aaagtgagta ccccgtcaag tggcaggact tcttcagtgg acacgtggtt 360

ggtgaacctg actctagggt tctcgcccac ataggagatg atgatattac agtaagaatc 420

aacacagatg gggcagaata taatatagag ccactttgga gactaattaa tgatactaaa 480

gacaaaagag tgttagttta taagtctgaa gatatcaaga atgtttcacg tttgcagtct 540

ccaaaagtgt gtggttatat aaaggcggat aatgaagagt tgcttcctaa agggctagta 600

gacagagagc cgcctgatga gcttgttcac cgggtgaaga gaagagccga ccccaatccc 660

ctgaggaaca cgtgtaaatt attggtggtg gcagatcatc gcttttataa gtacatgggc 720

agaggggaag agagcacgac cacaaactac ctgatagagc taattgacag agttgatgac 780

atctatcgga acacttcatg ggacaatgca ggttttaaag gttatggaat acagatagag 840

cagattcgca ttctcaagtc tccacaagag gtaaaacctg gtgaaaggca ctacaatatg 900

gcaaaaagtt acccaaatga agaaaaggat gcttgggatg tgaagatgtt gctagagcaa 960

tttagctttg atatagctga agaagcatct aaagtctgcc tggcacatct tttcacctac 1020

caagattttg atatgggaac tcttggatta gcttatgttg gttctcccag agcaaacagt 1080

catggaggtg tttgtccaaa ggcttattat agtccaattg gaaagaaaaa tatctattta 1140

aatagtggtt tgaccagcac aaaaaattat ggtaaaacca tccttacaaa ggaagctgac 1200

cttgtgacaa ctcatgaatt ggggcacaat tttggagcag aacacgatcc agatggttta 1260

gcagaatgtg ccccaaacga ggaccaggga ggaaaatacg tcatgtatcc catagccgtg 1320

agtggtgatc atgagaacaa caagatgttt tcaaactgca gtaaacagtc catctataag 1380

accattgaaa gtaaggccca ggagtgtttt caagagcgca gcaacaaagt gtgtggcaac 1440

tccagggtgg atgaggggga ggagtgcgac cccggcatca tgtacctgaa caacgacacc 1500

tgctgcaaca gcgactgcac cctgaggccg ggcgtccagt gcagtgatag gaacagtcct 1560

tgctgtaaaa actgtcagtt cgagacggcc cagaagaagt gccaggaggc tattaatgcc 1620

acttgcaaag gcgtgtctta ctgcacaggt aacagcagtg agtgcccccc tccgggaaac 1680

gccgaggacg acacggtgtg cctggacctg ggcaggtgca aggacggcaa gtgcgtgccc 1740

ttctgcgagc gggagcagcg gctggagtcc tgcgcgtgta acgaaaccga ccactcgtgc 1800

aaggtgtgct gccgggcccc ctcgggccgt tgcctgccct acgtggacgc cgaacagaag 1860

aacttgtttt tgaggaaggg gaagccctgt acagtaggat tttgtgacat gaatggcaag 1920

tgtgagaagc gagtgcagga cgtcatcgag cggttctggg agttcattga caagctgagc 1980

atcaatactt tcgggaagtt cctggcagac aacatcgtgg gctccgtcct ggtgttctcc 2040

ctgatgctct ggatccccgt cagcatcctc gtccactgcg tggataagaa gctggataag 2100

cagtacgaat ccctgtctct gctgcacccc agcaacgtgg agatgctaag cagcatggat 2160

tcagcatccg ttcgcatcat caagcccttt cctgcgcccc agaccccagg ccgcctgcag 2220

cccctgcagc ccctgcagcc cggccccgtg ctgccctctg cgccttcggt gcccgtggct 2280

ccaaaactgg accaccagcg gatggacacc atccaggagg accccagcac ggactcgcac 2340

gtggacgagg acggcttcga gaaggaccct ttccccaaca gcagtgccgc tgccaagtca 2400

tttgaggatc tcacggacca tccggtcacg agaagtgaaa aggcctcgtc ctttaagctg 2460

cagcgccaga gtcgcgttga cagcaaggaa acggagtgc 2499

<210> 3

<211> 331

<212> PRT

<213> E2 extracellular domain

<400> 3

Arg Leu Ala Cys Lys Glu Asp Tyr Arg Tyr Ala Ile Ser Ser Thr Asn

1 5 10 15

Glu Ile Gly Leu Leu Gly Ala Gly Gly Leu Thr Thr Thr Trp Lys Glu

20 25 30

Tyr Ser His Asp Leu Gln Leu Asn Asp Gly Thr Val Lys Ala Ile Cys

35 40 45

Val Ala Gly Ser Phe Lys Ile Thr Ala Leu Asn Val Val Ser Arg Arg

50 55 60

Tyr Leu Ala Ser Leu His Lys Gly Ala Leu Leu Thr Ser Val Thr Phe

65 70 75 80

Glu Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr Glu Glu Met Gly Asp

85 90 95

Asp Phe Gly Phe Gly Leu Cys Pro Phe Asp Thr Ser Pro Val Val Lys

100 105 110

Gly Lys Tyr Asn Thr Thr Leu Leu Asn Gly Ser Ala Phe Tyr Leu Val

115 120 125

Cys Pro Ile Gly Trp Thr Gly Val Ile Glu Cys Thr Ala Val Ser Pro

130 135 140

Thr Thr Leu Arg Thr Glu Val Val Lys Thr Phe Arg Arg Glu Lys Pro

145 150 155 160

Phe Pro His Arg Met Asp Cys Val Thr Thr Thr Val Glu Asn Glu Asp

165 170 175

Leu Phe Tyr Cys Lys Leu Gly Gly Asn Trp Thr Cys Val Lys Gly Glu

180 185 190

Pro Val Val Tyr Thr Gly Gly Gln Val Lys Gln Cys Lys Trp Cys Gly

195 200 205

Phe Asp Phe Asn Glu Pro Asp Gly Leu Pro His Tyr Pro Ile Gly Lys

210 215 220

Cys Ile Leu Ala Asn Glu Thr Gly Tyr Arg Ile Val Asp Ser Thr Asp

225 230 235 240

Cys Asn Arg Asp Gly Val Val Ile Ser Ala Lys Gly Ser His Glu Cys

245 250 255

Leu Ile Gly Asn Thr Thr Val Lys Val His Ala Ser Asp Glu Arg Leu

260 265 270

Gly Pro Met Pro Cys Arg Pro Lys Glu Ile Val Ser Ser Ala Gly Pro

275 280 285

Val Arg Lys Thr Ser Cys Thr Phe Asn Tyr Ala Lys Thr Leu Lys Asn

290 295 300

Lys Tyr Tyr Glu Pro Arg Asp Ser Tyr Phe Gln Gln Tyr Met Leu Lys

305 310 315 320

Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Asp Val

325 330

<210> 4

<211> 993

<212> DNA

<213> E2 extracellular domain

<400> 4

cggctagcct gcaaggaaga ttacaggtac gcaatatcat caaccaatga gatagggcta 60

ctcggggccg gaggtctcac taccacctgg aaagaataca gccacgattt gcaactgaat 120

gacgggaccg ttaaggccat ttgcgtggca ggttccttta aaatcacagc acttaatgtg 180

gtcagtagga ggtatttggc atcattgcat aagggggctt tactcacttc cgtgacattc 240

gagctcctgt tcgacgggac caacccatca accgaagaaa tgggagatga cttcgggttc 300

gggctgtgcc cgtttgatac gagtcctgtt gtcaagggaa agtacaatac aaccttgttg 360

aacggtagtg ctttctatct tgtctgccca atagggtgga cgggtgttat agagtgcaca 420

gcagtgagcc caacaactct gagaacagaa gtggtaaaga ccttcaggag agagaagcct 480

ttcccacaca gaatggattg tgtgaccacc acagtggaaa atgaagatct attctactgt 540

aagttggggg gcaactggac atgtgtgaaa ggtgaaccag tggtctacac aggggggcaa 600

gtaaaacaat gcaaatggtg tggcttcgac ttcaacgagc ctgacggact cccacactac 660

cccataggta agtgcatttt ggcaaatgag acaggttaca gaatagtaga ttcaacggac 720

tgtaacagag atggcgttgt aatcagcgca aaggggagcc atgagtgctt gatcggcaac 780

acaactgtca aggtgcatgc atcagatgaa agactgggcc ctatgccatg cagacctaaa 840

gagattgtct ctagtgcagg acctgtaagg aaaacttcct gtacattcaa ctacgcaaaa 900

actttgaaga acaagtacta tgagcccagg gacagctact tccagcaata tatgctcaag 960

ggcgagtatc agtactggtt tgacctggac gtg 993

<210> 5

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 5

gcaacaaagu guguggcaat t 21

<210> 6

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 6

uugccacaca cuuuguugct t 21

<210> 7

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 7

gguguuuguc caaaggcuut t 21

<210> 8

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 8

aagccuuugg acaaacacct t 21

<210> 9

<211> 24

<212> DNA/RNA

<213> Artificial Sequence

<400> 9

caccgagttc ccgccagaca ctac 24

<210> 10

<211> 24

<212> DNA/RNA

<213> Artificial Sequence

<400> 10

aaacgtagtg tctggcggga actc 24

<210> 11

<211> 24

<212> DNA/RNA

<213> Artificial Sequence

<400> 11

caccagtgct atcaacctta tacg 24

<210> 12

<211> 24

<212> DNA/RNA

<213> Artificial Sequence

<400> 12

aaaccgtata aggttgatag cact 24

<210> 13

<211> 24

<212> DNA/RNA

<213> Artificial Sequence

<400> 13

caccacaggg ctgggtgata gatt 24

<210> 14

<211> 24

<212> DNA/RNA

<213> Artificial Sequence

<400> 14

aaacaatcta tcacccagcc ctgt 24

<210> 15

<211> 24

<212> DNA/RNA

<213> Artificial Sequence

<400> 15

cacctgcgtt gaccaaactc tcac 24

<210> 16

<211> 24

<212> DNA/RNA

<213> Artificial Sequence

<400> 16

aaacgtgaga gtttggtcaa cgca 24

<210> 17

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 17

uucuccgaac gugucacgut t 21

<210> 18

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 18

acgugacacg uucggagaat t 21

Claims (1)

1. Application of an inhibitor of ADAM17 in the preparation of a medicine for preventing and/or treating swine fever; the amino acid sequence of ADAM17 is shown in SEQ ID No.1; The inhibitor of ADAM17 is an inhibitor that inhibits the expression of ADAM17 or inhibits the activity of ADAM17; The inhibiting ADAM17 expression is knocking down the expression of ADAM17; The ADAM17-inhibiting activity is the chelation and/or removal of zinc ions from the active center of the metalloprotease domain of ADAM17.

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