CN113004390B

CN113004390B - Application of ADAM17 as receptor of hog cholera virus

Info

Publication number: CN113004390B
Application number: CN201911327967.XA
Authority: CN
Inventors: 郑爱华; 袁菲; 李丹丹
Original assignee: Institute of Zoology of CAS
Current assignee: Institute of Zoology of CAS
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2022-08-26
Anticipated expiration: 2039-12-20
Also published as: CN113004390A

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 receptor of hog cholera virus

Technical Field

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

Background

Classical Swine Fever (CSF), previously known as Hog Cholera (Hog Cholera, HC), is a highly contagious disease, one of the major infectious diseases causing great economic losses in the Swine industry, and is listed in the world list of animal health organizations. Hog cholera was first identified in 1810 in tennessee, and then rapidly rolled around the world, widely distributed in asia, eastern europe, russia, and south america; hog cholera was discovered in japan in 1888; the exact time of first finding in china was not documented, but in 1925 chinese scientists have used antiserum to treat swine fever. At present, the swine fever mainly has two prevention policies of killing (non-vaccine) and systemic prevention (vaccine), and the rabbit weakened live vaccine developed based on the C-strain successfully controls the swine fever epidemic situation, so that no wide-range epidemic situation is developed in the past decades, but the effectiveness of the C-strain vaccine is gradually reduced along with the change of virus subtypes, and the swine fever epidemic situation continuously rises in recent years.

Classical Swine Fever Virus (CSFV) is a Swine fever causing pathogen, belonging to the Flaviviridae family (Flaviviridae), the Pestivirus genus (Pestivirus), which also includes Bovine Viral Diarrhea Virus (BVDV), Border Disease Virus (BDV). CSFV enters the host through the oral and nasal mucosa, is first infected with tonsil cells, and then spreads throughout the body via the blood and lymphatic circulation. CSFV has significant tissue tropism for the immune system, such as thymus, spleen, lymph nodes, bone marrow, etc., and causes severe leukopenia.

CSFV has 3 genotypes (I, II, III), each of which has 3 to 4 subtypes, and Shimen strain (Shimen) and SXCDK strain, which are prevalent in China, belong to type I and type II, respectively.

CSFV is a positive-stranded RNA virus with an envelope, the virion diameter is approximately 50nm, and the CSFV genome 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 of 3398 amino acids, which is cleaved into 4 structural proteins (C, Erns, E1, and E2) and 8 nonstructural proteins (Npro, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS 5B).

The structural proteins of CSFV include a capsid protein (C), three envelope glycoproteins (Erns, E1, E2) which are translated as multimeric proteins on the host ribosome and then split into Erns (also known as E0) by the host signal peptidase. Of these, Erns are necessary for the production of infectious virions, and are also target proteins for neutralizing antibodies. Erns mediate the adhesion of virions to hosts through the interaction of positively charged amino acids with cell surface glycosaminoglycans (e.g., heparan sulfate, laminin receptors). However, in 2004, Erns were found to be non-essential for viral invasion of host cells, and only retroviral particles expressing E1, E2 could infect the host.

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

E2 also belongs to a type I transmembrane protein, has an N-terminal ectodomain and a C-terminal hydrophobic helical domain, and anchors E2 to the surface of the viral envelope via the C-terminus. E2 has two dimeric forms: E2-E2, E2-E1; the E2-E2 homodimers were formed first during viral assembly, and the E2-E1 heterodimers were formed after E1 was released from the endoplasmic reticulum. E2 is the most important immunogen in CSFV glycoprotein, comprises epitope, CSFV antibody and vaccine can be developed according to E2, and the mutant E2 glycosylation site can obviously reduce the immunogenicity of E2.

CSFV invasion into the host process is mediated by glycoproteins Erns and E2, of which E2 is indispensable. At the same time, Erns and E2 are also the main targets of neutralizing antibodies, but antibodies against E2 completely neutralize CSFV infection, whereas antibodies against Erns neutralize only a fraction of infections.

Binding of the virus to the host factor is the most critical step in viral infection of the host cell. In 2004, CD46 was identified as a host factor of BVDV, however, the research on CSFV host factor has not been broken through. Research has reported that heparan sulfate and laminin receptor are adhesion factors in the host process of CSFV infection, and the fact is that heparan sulfate is the adhesion factor of many enveloped viruses; furthermore, although laminin receptors can interact with CSFV Erns proteins, antibodies directed against laminin receptors only partially inhibit CSFV infection of the host. This is because although the CSFV entry process into the host is mediated by Erns and E2 proteins, Erns are unnecessary and E2 is indispensable, while the host factor that binds to E2 protein is still a mystery. Therefore, the screening of the swine fever drugs and vaccines and the directional breeding of antiviral pigs lack specific targets.

Disclosure of Invention

In view of the above, the present invention provides the use of ADAM17 as a receptor for swine fever virus. The invention discovers a critical receptor ADAM17 of CSFV (classical swine fever virus) infected pigs, which can be used for designing and screening antiviral vaccines and medicaments and developing targets of antiviral pigs. CSFV E2 cyst membrane glycoprotein specific binding protein ADAM17 is screened by a co-immunoprecipitation technology, and the CSFV infection can be completely blocked by knocking out ADAM17 by a CRISPR-Cas9 technology. The purified recombinant E2 protein and ADAM17 can be specifically combined. The results above demonstrate that ADAM17 is a specific receptor for swine fever virus infected pigs.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of ADAM17 serving as a receptor of E2 protein or classical swine fever virus; the ADAM17 has:

(I) an amino acid sequence shown as SEQ ID No. 1; or

(II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in the (I), and the amino acid sequence has the same or similar functions with the amino acid sequence shown in the (I); or

(III) an amino acid sequence which is at least 80% identical to the sequence of (I) or (II).

In some embodiments of the invention, the nucleotide encoding the ADAM17 has

(IV) a nucleotide sequence shown as SEQ ID No. 2; or

(V) a complementary nucleotide sequence of the nucleotide sequence shown as SEQ ID 2; or

(VI) a nucleotide sequence which encodes the same protein as the nucleotide sequence of (IV) or (V) but which differs from the nucleotide sequence of (IV) or (V) due to the degeneracy of the genetic code; or

(VII) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown In (IV), (V) or (VI), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown In (IV), (V) or (VI); or

(VIII) and a nucleotide sequence having at least 80% identity to the nucleotide sequence of (IV), (V), (VI) or (VII).

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

(I) an amino acid sequence shown as SEQ ID No. 1; or

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

the inhibiting of ADAM17 expression knocks down ADAM17 expression;

the activity of inhibiting ADAM17 is to chelate and/or remove zinc ions at the active center of the metalloprotease domain of ADAM 17.

In some embodiments of the invention, the ADAM17 inhibitor comprises one or more of siRNA (shown in SEQ ID Nos. 5-8), 1,10-phenanthroline and erbasib of ADAM 17.

Wherein, the sequence of the siRNA is shown as follows:

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

(I) an amino acid sequence shown as SEQ ID No. 1; or

On the basis of the above studies, the present invention also provides an agent for preventing and/or treating swine fever, a nucleic acid, protein, compound or salt, composition, complex or mixture thereof, capable of inhibiting the expression of ADAM17 or inhibiting the activity of ADAM 17; the ADAM17 has:

(I) an amino acid sequence shown as SEQ ID No. 1; or

In some embodiments of the present invention, the inhibiting of the expression of ADAM17 in the agent provided by the present invention is knocking down the expression of ADAM 17;

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

(I) an amino acid sequence shown as SEQ ID No. 1; or

The present invention also provides a vaccine for preventing swine fever, a nucleic acid, protein, compound or salt, composition, complex or mixture thereof capable of inhibiting the expression of ADAM17 or inhibiting the activity of ADAM 17; the ADAM17 has:

(I) an amino acid sequence shown as SEQ ID No. 1; or

In some specific embodiments of the present invention, said inhibiting the expression of ADAM17 in the vaccine provided by the present invention is the knocking down of the expression of ADAM 17;

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, ADAM17 was demonstrated to be 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.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

Fig. 1 shows sgRNA recognition sites; wherein, fig. 1(a) shows sgRNA recognition sites in the ADAM17 gene sequence; fig. 1(B) shows sgRNA recognition sites in the ADAM10 gene sequence;

FIG. 2 shows the position of the knockout detection primer;

FIG. 3 shows that ADAM17 is required for CSFV invasion of PK 15; among them, fig. 3(a) shows that the binding of sE2 to PK15 is dependent on ADAM 17; FACS analysis of sE2 binding to PK15, Hela, PK15 ADAM17 knock-out cell lines (PK15 ADAM17-KO), PK15 ADAM10 knock-out cell lines (PK15 ADAM10-KO) and PK15 ADAM17-KO porcine ADAM17 overexpressing cell lines (PK15 ADAM17-KO-pADAM 17); FIG. 3(B) shows that cell lines without ADAM17 were not infected with CSFVpp; the above cell lines were infected with SXCDK and Shimen strains with GFP reporter gene, respectively, VSVPp was used as positive control, and HIV-1, which does not encode the envelope protein, was used as negative control; error bars represent standard deviation; FIG. 3(C) shows the infectivity of CSFVcc in cell lines with and without ADAM17 expression; the above cells were infected with recombinant VSV (rVSV-GFP) having GFP reporter gene and CSFV phoma strain, respectively, and further stained with anti-CSFV E2 (n ═ 3), and error bars represent standard deviations;

FIG. 4 shows that ADAM17 is essential for infection of porcine embryonic fibroblasts with CSFV; PEFs were transfected with two siRNAs to ADAM17 (siADAM17-1, siADAM17-3) and a negative control (siCon.); among them, fig. 4(a) shows that the ADAM17 mRNA expression level was detected using quantitative PCR and calculated as a percentage of negative control (n-3); error bars represent standard deviation; FIG. 4(B) shows the detection of viral RNA levels of the CSFVcc Shimen strain using a quantitative PCR method to determine viral infectivity and calculate the percentage of negative controls; error bars represent standard deviations, and the above data represent three independent experiments;

FIG. 5 shows that ADAM17 binds to CSFV E2 through a metalloprotease domain; wherein, the left panel of figure 5 shows that chelation of the zinc ion of the metalloprotease domain reduces binding of sE2 to PK15 cells; PK15 was treated with different concentrations of 1,10-phenanthroline and binding to sE2 was detected using FACS; figure 5 right panel shows that adrbesib blocks binding of sE2 to PK15 cells by occupying the active center of the metalloprotease domain; various concentrations of aderbasib treated PK15 cells, and then detected binding to sE2 using FACS; the above data represent three independent experiments;

FIG. 6 shows direct binding of ADAM17 to sE 2; wherein, FIG. 6(A) shows a surface plasmon resonance map of binding of purified soluble ADAM17 to sE2 in the absence of zinc ions; fig. 6(B) shows a surface plasmon resonance map of the binding of soluble ADAM17 to sE2 in the presence of zinc ions.

Detailed Description

The invention discloses application of ADAM17 as a receptor of classical swine fever virus, and a person skilled in the art can refer to the content and appropriately modify process parameters to realize the application. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The raw materials and reagents related to the application of ADAM17 provided by the invention in serving as a hog cholera virus receptor are all commercially available.

Interpretation of terms:

hog cholera is a severe swine infectious disease threatening the swine industry, and the pathogen of hog cholera is hog cholera virus, which is a virus of the genus pestivirus of the family flaviviridae, and the host is limited to domestic pigs and wild pigs.

Viral receptors refer to host factors that specifically bind to viruses and mediate viral entry into cells, and most are transmembrane proteins. The receptor is a key host factor for virus infection of a host, and the blocking of the binding of the virus and the receptor can effectively inhibit the virus infection. Therefore, the receptor is an important target of antiviral strategies such as medicaments, vaccines and the like, and is also an important target for screening and selecting antiviral animals in animal breeding.

ADAM17 is a member of the ADAM family of disintegrin matrix metalloproteases 17(a disintegrin and metalloprotease 17, ADAM17) also known as tumor necrosis factor convertase (TACE) which is zinc ion dependent.

CRISPR Cas (Clustered regulated Short Palindromic Repeats, CRISPR) is an RNA-mediated adaptive defense system evolved from bacteria and archaea, and can protect organisms from invasion of viruses and plasmids. The type II CRISPR system from Streptococcus pyogenes (Streptococcus pyogenes) can direct Cas9 protein to perform sequence specific cleavage of targets paired with crRNA, and the 2013 CRISPR/Cas system is transformed into a genome editing tool.

SPR physically refers to Surface Plasmon Resonance, collectively known as Surface Plasmon Resonance, which is an optical technique used to characterize changes in the refractive index of a Surface (typically the interface between a solid phase and a liquid phase) and can be used to track interactions between biomolecules in real time in the native state.

TABLE 1 Main Experimental instruments

TABLE 2 Main commercial reagents and consumables

The invention is further illustrated by the following examples:

EXAMPLE 1 purification and characterization of CSFV E2-Fc protein (sE2)

1. Purification of sE2 protein

The plasmid expressing the sE2 protein was pCAGGS-CSFV-E2-Fc and was stored in the laboratory.

(1) Transferring 293T to 10cm cell culture dish, 30 dishes, and culturing in DMEM containing 8% FBS, 1% penicillin-streptomycin and 1% L-glutamine at 37 deg.C and 5% CO ₂ The cell culture box;

(2) the next day, transfection was started when 293T cells grew to about 90%; the transfection method is calcium phosphate transfection, and the reagent comprises 2.5M CaCl ₂ 、2′HBS(pH7.0)、ddH ₂ O, and preheating in a water bath kettle at 37 ℃ in advance;

(3) transfection system one:

TABLE 3

The first system is arranged in a flow tube and is thoroughly mixed on an oscillator for 1 minute;

(4) dripping 500 mul of 2' HBS one drop by one drop in the system I, and thoroughly mixing on an oscillator for 30 seconds;

(5) adding the mixed system into 293T cells one by one, and carefully mixing up, down, left and right;

(6) 293T cells were placed at 37 ℃ in 5% CO ₂ Continuously culturing the cells in the cell culture box for 12 hours;

(7) preheating a DMEM culture medium containing 2% FBS, 1% penicillin-streptomycin and 1% L-glutamine in a 37 ℃ water bath kettle;

(8) the medium was replaced with the above medium, 5% CO at 37 ℃ ₂ Continuing to culture for 36 hours;

(9) collecting the culture medium, adding 1.0M Tris (pH8.0) in one tenth volume, mixing, centrifuging at 4 deg.C at 5000rpm for 10 min for the first time, and centrifuging at 12000rpm for 15min for the second time to completely remove cell debris;

(10) the following low temperature laboratory operations at 4 ℃;

(11) 500 μ l of ProteinAbeads were taken in the column and 5mL of 10mM Tris (pH8.0) was equilibrated with ProteinAbeads;

(12) passing the medium through ProteinAbeads at a flow rate <1 mL/min;

(13) washing protein A beads with 5mL of 100mM Tris (pH8.0);

(14) washing protein A beads with 5mL of 10mM Tris (pH8.0);

(15) eluting the protein with 50mM glycine (pH3.0), 5mL each time;

(16) taking a plurality of 1.5mL centrifuge tubes, adding 50 μ l of 1M Tris (pH8.0) into the centrifuge tubes, connecting each tube with about 250 μ l of glycine eluent, and rapidly mixing the mixture uniformly;

(17) mu.l of 1' Bradford was taken in a PCR tube and 5. mu.l glycine was added as a control; taking 5 mu l of the eluted solution into 20 mu l of 1' Bradford, uniformly mixing, and placing on white paper to check whether the color turns blue or not, wherein if the color turns blue, protein exists;

(18) elution was until 1' Bradford did not change color;

(19) numbering the eluted solutions according to the sequence of numbers 1-n;

SDS-PAGE analysis of sE2 protein

(1) Preparing SDS-PAGE gel and 10% separating gel;

(2) boiling samples: 5 μ l of 5' SDS-PAGE loading buffer +20 μ l of protein eluent, metal bath at 100 ℃ for 10 min;

(3) electrophoresis

(4) Staining with Coomassie brilliant blue, and decolorizing.

Example 2 establishment of CRISPR Cas9 knockout cell line

sgRNA Synthesis

(1) Utilizing http:// www.rgenome.net/cas-designer/online design website to design sgRNA respectively aiming at the upstream and downstream of the second exon of porcine ADAM17(NC _010445.4, NM _001099926.1) so as to knock out the second exon of ADAM 17; similarly, sgrnas upstream of the second exon and downstream of the third exon of ADAM10(NC _010443.5) were designed by using a website to knock out the second and third exons of ADAM10 (fig. 1), and CACC was added to the 5 'end of the forward primer and AAAC was added to the 5' end of the reverse primer to form sticky ends with the bsi-digested pX330 plasmid (table 4). The strain is handed in Shanghai Czeri Bio-company for synthesis.

TABLE 4 sgRNA sequences

(2) Synthesizing sgRNA double strands:

a. dissolving the sgRNA primer to 100 mu M by using water of RNase/DNase free;

b. prepare the following system in a 600 μ l centrifuge tube and mix well:

TABLE 5

c. Heating the beaker filled with ultrapure water by using a magnetic stirrer until the beaker is boiled;

d. fixing a 600 mu l centrifugal tube on a foam floating plate, putting the centrifugal tube into boiling water, and continuously heating and keeping boiling for 10 minutes;

e. transferring the beaker to a sealed foam box, and slowly cooling the water to room temperature;

f. double strand synthesis, stored at-20 ℃.

pX330 linearization

pX330, collectively designated 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) and has a GFP reporter gene.

(1) Enzyme digestion system:

TABLE 6

The enzyme was digested in a water bath at 37 ℃ for 3 hours.

(2) Glue recovery

Preparing 1% agarose gel, and after the enzyme digestion reaction is finished, performing agarose gel electrophoresis on an enzyme digestion system at the voltage of 130V for 30 min; after the electrophoresis is finished, the specific band with the right size is cut off by a clean scalpel, and the target DNA fragment is recovered by using an agarose gel DNA recovery kit (Promega) by the following operation steps:

a. placing the cut gel containing the target band into a 1.5mL centrifuge tube; weighing gel, and adding 10 μ l of membrane dissolution per 10 mg;

b. incubating in a metal bath at 65 ℃ until the gel is completely dissolved;

c. placing the SV microcolumn in a collection tube;

d. transferring the gel mixed solution into a micro column, and incubating for 1min at room temperature;

e. centrifuging at 16000' g for 1min, and removing effluent;

f. adding 700 μ l membrane wash solution into the micro column, centrifuging at 16000' g for 1min, and discarding the effluent;

g. adding 500 μ l membrane wash solution into the micro column, centrifuging at 16000' g for 1min, and discarding the effluent;

h.16000' g, centrifuging for 5 min;

i. drying at room temperature for 5min to evaporate the rest ethanol;

j. transfer the mini column into a clean 1.5mL centrifuge tube;

k. adding 50 μ l nuclease-free water into the micro column, and incubating at room temperature for 2 min;

l.16000' g, centrifuging for 1min to obtain the target DNA;

measuring DNA concentration using noddrop;

n. store at-20 ℃.

T4 DNA ligase ligation

The linearized pX330 and the synthesized double-stranded sgRNA were ligated in the following ligation system, mixed well, and ligated in a 16 ℃ PCR instrument overnight:

TABLE 7

4. Transformation of

(1) Removing stbl3 competent cells from a-80 ℃ refrigerator, and placing on ice;

(2) after the stbl3 competent cells are thawed, adding 10 mul of the ligation product into 100 mul of the competent cells, gently flicking the fingers for a few times, uniformly mixing, and incubating on ice for 30 minutes;

(3) heat shock at 42 ℃ for 90 seconds, immediately transferring to ice, and incubating for 2 minutes;

(4) adding 1mL of LB (peptone, yeast extract, sodium chloride) culture medium without antibiotics into a clean bench, transferring to a shaking bed, and incubating for 50 minutes at 37 ℃;

(5) centrifuging at room temperature at 4000rpm for 3 min, and sucking most of supernatant in a clean bench while keeping about 100 μ l of supernatant;

(6) the cells and the remaining supernatant were mixed by a pipette, resuspended, uniformly spread on an ampicillin plate using a coating rod, and subjected to inverted culture at 37 ℃ for about 12 hours in an incubator.

5. Bacteria detection

Because the connection efficiency is high and the insert is very short and only 28bp, we adopt the method of direct bacterium selection, small shaking and company sequencing for identification.

(1) Selecting a single colony in 10mL LB culture medium containing ampicillin, shaking the colony at 37 ℃, and culturing the colony overnight;

(2) small upgraded grains:

a. centrifuging to obtain a precipitate: taking 5mL of overnight cultured bacterial liquid in a super clean bench, centrifuging at 12000rpm at room temperature for 3 minutes, sucking away supernatant, and leaving bacterial precipitate;

b. resuspending: resuspend the bacterial pellet with 250. mu.l of BufferP1 and transfer to a 1.5mL centrifuge tube;

c. cracking: adding 250 μ l of BufferP2, reversing the upper part and the lower part, and uniformly mixing for 4-6 times, wherein the cracking time does not exceed 5 minutes;

d. neutralizing: adding 350 μ l of BufferN3, immediately reversing the upside down, and mixing for 4-6 times;

e. centrifuging: centrifuging for 10 minutes in a desktop centrifuge at the rotating speed of 13000 rpm;

f. column passing: adding 800 μ l of the supernatant obtained in step e into a centrifugal column, centrifuging at 13000rpm for 1 minute, and discarding the effluent;

g. washing a centrifugal column: adding 750 ul of BufferPE into a centrifugal column, centrifuging at 13000rpm for 1 minute, and discarding the effluent;

h. removing residual Buffer PE: centrifuging at 13000rpm for 2min, discarding the effluent, and cooling at room temperature for 5 min;

i. and (3) elution: the column was placed in a new 1.5mL centrifuge tube, 50. mu.l of BufferEB (10mM TrisCl, pH 8.5) was added to the column and incubated at room temperature for 2 minutes, 13000rpm, 1 minute;

j. measuring the DNA concentration by using NanoDrop, and sending to Beijing Ongskaceae New industry bio-corporation for sequencing;

k. analyzing the sequencing result;

(3) selecting single colony bacteria liquid with correct sequencing and no mutation, and carrying out mass culture: 1mL of the bacterial solution was cultured overnight in 200mL of LB medium containing ampicillin, shaking at 37 ℃.

6. Large upgrading particle

(1) Centrifuging to obtain a precipitate: collecting overnight cultured bacteria liquid, centrifuging at 4 deg.C with high speed centrifuge 6000' g for 15min, and discarding supernatant;

(2) resuspending: resuspend the cells with 10mL Buffer P1, transfer to a 50mL high speed centrifuge tube;

(3) cracking: adding 10mL Buffer P2, reversing the upper part and the lower part for 4-6 times, thoroughly mixing, and incubating for 5 minutes at room temperature;

(4) neutralizing: adding 10mL of precooled Buffer P3, reversing the upper part and the lower part for 4 to 6 times, thoroughly mixing the mixture, and incubating the mixture on ice for 20 minutes;

(5) centrifuging: centrifuging at 22000 'g for 30min at 4 ℃ to obtain supernatant, and centrifuging again at 22000' g at 4 ℃ for 15 min;

(6) balancing: adding 10mL Buffer QBT into an adsorption column, and naturally dripping by gravity;

(7) column-passing-adsorption: adding the supernatant obtained in the step 5 into the column, and naturally dripping the supernatant by gravity;

(8) and (3) washing the column: adding 30mL Buffer QC into the column, naturally dripping by gravity, and repeating for 2 times;

(9) and (3) elution: putting the column into a new 50mL high-speed centrifugal tube, adding 15mL Buffer QF into the column, and naturally dripping by gravity;

(10) and (3) plasmid precipitation: adding 10.5mL of isopropanol into the eluted DNA, and uniformly mixing; 15000' g at 4 ℃ for 30 minutes; white DNA precipitate was visible and the supernatant was discarded;

(11) washing the DNA: 5mL of 70% ethanol is added at 4 ℃, 15000' g and 10 minutes, the supernatant is discarded, and DNA precipitate is washed;

(12) DNA lysis: drying the DNA precipitate, and adding a proper amount of Buffer EB to dissolve the DNA;

(13) DNA concentration was measured using a NanoDrop and stored at-20 ℃ until use. The constructed plasmids were designated as: pX330-pig ADAM17 KO-up, pX330-pig ADAM17 KO-down, pX330-pig ADAM10 KO-up, pX330-pig ADAM10 KO-down.

7. Transfection of PK15

(1) Transferring PK15 to 6cm cell culture dish, culturing DMEM containing 8% FBS, 1% penicillin-streptomycin and 1% L-glutamine in a 5% CO2 cell culture box at 37 deg.C;

(2) the next day, when the cells were about 90% full, transfection was started with Fugene 6(Promega E2691);

(3) taking 300 ul of opti-MEM culture medium, adding 60 ul of Fugene6 into a 1.5mL centrifugal tube, uniformly mixing by a shaker, centrifuging for a short time, and incubating for 5 minutes at room temperature;

(4) taking 6 micrograms of each of pX330-up and pX330-down in the system, uniformly mixing the mixture by using an oscillator, centrifuging the mixture for a short time, and incubating the mixture for 15 minutes at room temperature;

(5) dropping the transfection system into the cells one by one, and carefully mixing the transfection system up, down, left and right;

(6) continuing to culture the 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 96-well plates, 1 cell per well, to obtain a monoclonal cell line.

(1) Digesting transfected PK15 cells to single cells using pancreatin;

(2) counting by a cell counter, and diluting until the cell concentration is 2' 106 cells/mL;

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

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

(5) sorting green fluorescent positive cells in a 96-well cell culture plate by a Beckman MoFlo XDP multicolor fluorescent flow cytometer, one cell per well;

(6) placing the sorted cells at 37 deg.C with 5% CO ₂ Culturing;

(7) after about one week, the wells in which the monoclonal cells were visible were digested, transferred to 12-well plates and cultured; then transferring to a 6-hole plate for continuous culture;

(8) after the plate is full of 6-hole plates, 2' 10 parts are taken ⁵ Extracting genome DNA from each cell, and transferring the rest cells to a 6cm culture dish for continuous culture;

(9) taking non-operational normal PK 152' 10 at the same time ⁵ Genomic DNA was extracted from each cell as a control.

9. Extraction of genomic DNA

(1) Placing Spin Column in a collecting tube, adding 250 μ l Buffer BL, and centrifuging at 12000' g for 1min to activate silica gel membrane;

(2) taking 20 mu l of proteinase K to the bottom of a 1.5mL centrifuge tube, then adding 200 mu l of high-purity water suspension cell sediment, and carrying out vortex oscillation for 10 seconds; then 200. mu.l of Buffer gA1 was added, vortexed and shaken for 10 seconds, and incubated at 56 ℃ for 1 hour until the cells were completely digested;

(3) after the incubation is finished, adding 200 mu l of absolute ethyl alcohol, and uniformly mixing by vortex oscillation;

(4) transferring all the solution obtained in the step 3 into Spin Column, centrifuging at 12000' g for 1 minute, and discarding the waste liquid;

(5) adding 500 mu l of Buffer PW into Spin Column, centrifuging for 1 minute at 12000' g, and discarding the waste liquid;

(6) repeating the step 5 once;

(7) adding 500 mu l of Wash Buffer into Spin Column, centrifuging for 1 minute at 12000' g, and discarding the waste liquid;

(8) putting the Spin Column back into the collecting pipe, centrifuging at 12000' g for 2 minutes, opening the cover and airing for 1 minute;

(9) the Spin Column was removed and placed into a clean 1.5mL centrifuge tube, centered on the adsorption membrane

Adding 50 μ l TE Buffer, standing at room temperature for 2min, and centrifuging at 12000' g for 2 min;

(10) the DNA solution solubility was determined by NanoDrop and stored at-20 ℃ until use.

PCR detection

(1) The detection primer design positions are shown in FIG. 2.

(2) The following PCR system was configured:

TABLE 8

(3) PCR procedure:

(4) preparing 1% agarose gel, and after the enzyme digestion reaction is finished, performing agarose gel electrophoresis on an enzyme digestion system at the voltage of 130V for 30 min;

(5) analysis of strip size: the bands for successful knockdown were smaller than those of normal cell lines.

Example 3 Flow cytometry (FACS)

(1) Digesting each cell line to a single cell using pancreatin;

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

(3) Counting with a cell counter, two 2' 10 cells per cell line ⁵ The cells were defined as Assay (A) tube and control (C) tube in two 1.5mL centrifuge tubes, respectively;

(4) preparing a working solution: PBS of 1% FBS, placed on ice;

(5) centrifuging at 300g for 2 minutes, and removing supernatant; resuspend 200. mu.l of the working solution and place on ice;

(6) add 2. mu.g sE2 (final concentration 10. mu.g/ml) to tube A of each cell line and mix well; adding PBS with corresponding volume into the tube C, and uniformly mixing; incubating on ice for 50 min, and mixing uniformly for 5 times;

(7) washing cells: centrifuging at 300g and 4 ℃ for 2 minutes by using 1mL of working solution every time, and washing for three times;

(8) preparing a secondary antibody solution: Alexa-Fluor 488 coat anti-human IgG (Thermo Fisher Scientific) is diluted by working solution 1:200, 200 mu l of each tube is used for resuspending cells, the cells are incubated for 45 minutes on ice, and the cells are mixed uniformly for 5 times in the middle;

(9) washing cells: centrifuging for 2 minutes at 300g and 4 ℃ by using 1mL of working solution every time, and washing for three times;

(10) resuspending the cells in 500. mu.l of working solution per tube, transferring into a flow tube, and placing on ice;

(11) detection was analyzed using a BD FACSCalibur flow cytometer.

Example 4 establishment of an overexpression cell line

1. Lentiviral plasmid construction

(1) Cloning ADAM17 homologous genes into a lentiviral vector by using a homologous recombination method;

(2) the porcine ADAM17 is obtained by amplifying cDNA of PK 15;

(3) human ADAM17 was expanded from Hela cells;

(4) mouse ADAM17, jungle fowl ADAM17, and zebrafish ADAM17b were synthesized by Germing Biotech Co., Ltd, Shengyue, Beijing;

(5) the construction process comprises the following steps: PCR amplification of target fragments, linearization of lentiviral vectors, homologous recombination and connection, transformation, bacteria detection, sequencing and mass extraction;

2. establishment of an overexpressing cell line

(1) Transferring 293T to a 10cm culture dish, wherein the culture medium contains 8%FBS, 1% penicillin-streptomycin, 1% L-glutamine DMEM, 37 deg.C, 5% CO ₂ Culturing;

(2) the next day, transfection was started until 293T cells grew to about 90%; the transfection method is calcium phosphate transfection, and the reagent comprises 2.5M CaCl ₂ 、2′HBS(pH7.0)、ddH ₂ O, preheating in a water bath kettle at 37 ℃ in advance;

(3) transfection system one:

TABLE 9

(7) preheating a DMEM culture medium containing 8% FBS, 1% penicillin-streptomycin and 1% L-glutamine in a 37 ℃ water bath kettle;

(9) the day after transfection, PK15 ADAM17-KO cell line was plated on 6cm cell culture dishes;

(10) 293T supernatant is collected 48 hours after transfection, 8 mu g/mL polybrene is added and mixed evenly; centrifuging at 4 deg.C and 12000g for 10 min to remove cell debris;

(11) removing PK15 ADAM17-KO supernatant, adding lentivirus-containing supernatant at 37 deg.C and 5% CO ₂ Continuing to culture for 3 hours;

(12) replacement of PK15 ADAM17-KO with fresh medium at 37 ℃ with 5% CO ₂ Continuously culturing for 2 days;

(13) adding puromycin with the concentration of 3 mu g/mL for screening for 5 days;

(14) replacing with fresh culture medium, continuing culturing, then subculturing and freezing.

3. Detection of overexpressing cell lines

After the cell line was established, Western-Blot (anti-Flag Tag) or immunofluorescence (anti-human ADAM17) was used to detect gene overexpression.

Example 5 pseudovirus packaging

1. Plasmid construction

CSFV is known to contain three subtypes (1, 2, 3), the invention entrusts Beijing Shengyuan scientific union biotechnology Limited to synthesize genetic codon optimized Shimen strain (type 1) (GenBank: AF333000.1), SXCDK strain (type 2) (Genbank: GQ923951.1) envelope protein Ernse1E2 gene fragment, and constructs into XbaI and EcoRI linearized pCAGGS vector by homologous recombination, and the plasmids are named as pCAGGS-CSFV/Shimen-E012 and pCAGGS-CSFV/CDK-E012 respectively.

2. Pseudovirus packaging

(1) Transferring 293T to 10cm culture dish, and culturing in DMEM containing 8% FBS, 1% penicillin-streptomycin and 1% L-glutamine at 37 deg.C and 5% CO ₂ Culturing;

(3) transfection system one:

watch 10

The first system is arranged in a flow tube and is thoroughly mixed on an oscillator for 1 minute; wherein VSV-G expresses VSV glycoprotein for use as a positive control pseudovirus; or glycoprotein expression plasmid without any virus as a negative control (Non-envelope);

(4) adding 500 μ l of 2' HBS drop by drop into the system I, and thoroughly mixing on a shaker for 30 seconds;

(6) 293T cells were placed at 37 ℃ in 5% CO ₂ The cell culture box is continuously cultured for 12 hours;

(9) 293T supernatant is collected 48 hours after transfection, 8 mu g/mL polybrene is added and mixed evenly; centrifuging at 4 deg.C and 12000g for 10 min, collecting supernatant, and freezing at-80 deg.C for use.

3. Pseudovirus titer determination

(1) Transfer PK15 to 96-well plates, 8000 cells per well;

(2) after 24 hours, pseudovirus was diluted in 10-fold gradient, 100. mu.l/well, infected cells, 37 ℃ with 5% CO ₂ Incubating for 3 hours;

(3) the medium was replaced with fresh medium, 5% CO at 37 ℃ ₂ Further culturing for 45 hours

(4) The virus titers were counted under a fluorescent microscope and calculated as GFP-positivity per mL (FFU/mL).

Example 6cell culture Strain (CSFVcc) Virus titre assay

(1) PK15 in 96-well plates, 8000 cells per well;

(2) after 24 hours, the virus was diluted in 10-fold gradient, 100. mu.l/well, infected cells, 37 ℃ C., 5% CO ₂ Incubating for 3 hours;

(3) the medium was changed to 20mM NH4Cl in 5% CO at 37 deg.C ₂ Continuing to culture for 45 hours;

(4) fixing the cells with methanol pre-cooled at-20 ℃;

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

(6) blocking the cells with 1% BSA in PBS for 2 hours at room temperature;

(7)1:500 dilution of murine monoclonal antibody WH303 (anti-CSFV E2 protein), 50. mu.l per well;

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

(9)1, 500 dilution of Alexa-Fluor 488 coat anti-mouse IgG (Thermo Fisher Scientific), 50. mu.l per well;

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

(11) counting under a fluorescence microscope.

Example 7 RNA interference

RNA interference

(1) The non-sense siRNA of the species and the non-sense siRNA of the http:// rnaidesigner. thermofisher. com/rnainexpress/design gene specific siRNAs 3 are submitted to Shanghai Biotech company for synthesis;

the sequence is shown as follows:

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

(3) when the cells grow to about 95% full, the transfection is started;

(4) configuring a first transfection system: adding 18 μ l Lipofectamine RNAimax into 300 μ l Opti-MEM Medium in a 1.5mL centrifuge tube, and mixing;

(5) configuring a transfection system and two: taking 300 mu l of Opti-MEM Medium in a 1.5mL centrifuge tube, adding siRNA to enable the final concentration to be 50nM, and uniformly mixing;

(6) mixing the first system and the second system, uniformly mixing, centrifuging for a short time, and incubating for 5 minutes at room temperature;

(7) adding one drop of the solution obtained in the step 6 into cells, carefully mixing the solution up and down, left and right, and culturing the mixture at 37 ℃ for 36 hours;

2. extraction of cellular RNA

After 36 hours of siRNA transfection, 2' 10 cells were individually selected ⁵ The specific operation steps of each cell, 500 mul Trizol cell lysis and RNA extraction are as follows:

(1)500 μ l Trizol lysed cells for 5 minutes at room temperature;

(2) adding 100 μ l chloroform, mixing by turning upside down, and incubating at room temperature for 2 min;

(3) centrifuging: 12000g at 4 ℃ for 15 minutes;

(4) layering was observed, and the supernatant was carefully removed in a new 1.5mL centrifuge tube;

(5) adding 250 mul of isopropanol, turning upside down and mixing evenly, and incubating for 10 minutes at room temperature;

(6) centrifuging: 12000g at 4 ℃ for 10 minutes;

(7) a white precipitate was visible, the supernatant was carefully aspirated;

(8) adding 1mL of 75% ethanol, and turning upside down for several times;

(9) centrifuging: 7500g at 4 ℃ for 5 minutes;

(10) removing supernatant, and air drying the precipitate at room temperature;

(11) the pellet was dissolved with RNase-free water and the RNA concentration was measured with NanoDrop and used immediately or stored at-80 ℃ until use.

3. Reverse transcription PCR

Reverse transcription PCR was performed using TAKARA PrimeScriptTM RT reagent Kit with gDNA Eraser Kit, as follows:

(1) removing genome DNA reaction: preparing a reaction mixed solution on ice according to the following components, and then incubating for 2 minutes at 42 ℃;

TABLE 11

(2) Reverse transcription reaction: the reaction solution was prepared on ice, and the reaction components were as follows:

TABLE 12

Then, the cDNA is obtained by a procedure on a PCR instrument and is stored at-20 ℃ for later use.

Watch 13

37℃	15min
		85℃	5s
4℃

4. Quantitative PCR

Takara one step SYBR PrimeScript Reverse Transcription (RT) -PCR kit was used as an apparatus Applied Biosystems QuantStudio, beta-actin gene as an internal reference.

The RT-PCR program was:

examples of effects

1. To screen for the identification of classical swine fever virus specific host factors, we cloned the ectodomain sequence (GenBank accession AFF333000.1) encoding glycoprotein E2 of CSFV 1 subtype Shimen strain into a pPUR-TPA-Fc vector and transfected 293T cells, collected the supernatant and purified classical swine fever virus E2 soluble protein sE 2. Porcine ADAM17 protein was obtained by co-immunoprecipitation experiments with sE2 protein in combination with mass spectrometry.

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

cggctagcctgcaaggaagattacaggtacgcaatatcatcaaccaatgagatagggctactcggggccggaggtctcactaccacctggaaagaatacagccacgatttgcaactgaatgacgggaccgttaaggccatttgcgtggcaggttcctttaaaatcacagcacttaatgtggtcagtaggaggtatttggcatcattgcataagggggctttactcacttccgtgacattcgagctcctgttcgacgggaccaacccatcaaccgaagaaatgggagatgacttcgggttcgggctgtgcccgtttgatacgagtcctgttgtcaagggaaagtacaatacaaccttgttgaacggtagtgctttctatcttgtctgcccaatagggtggacgggtgttatagagtgcacagcagtgagcccaacaactctgagaacagaagtggtaaagaccttcaggagagagaagcctttcccacacagaatggattgtgtgaccaccacagtggaaaatgaagatctattctactgtaagttggggggcaactggacatgtgtgaaaggtgaaccagtggtctacacaggggggcaagtaaaacaatgcaaatggtgtggcttcgacttcaacgagcctgacggactcccacactaccccataggtaagtgcattttggcaaatgagacaggttacagaatagtagattcaacggactgtaacagagatggcgttgtaatcagcgcaaaggggagccatgagtgcttgatcggcaacacaactgtcaaggtgcatgcatcagatgaaagactgggccctatgccatgcagacctaaagagattgtctctagtgcaggacctgtaaggaaaacttcctgtacattcaactacgcaaaaactttgaagaacaagtactatgagcccagggacagctacttccagcaatatatgctcaagggcgagtatcagtactggtttgacctggacgtg

protein sequence (shown as SEQ ID No. 3):

RLACKEDYRYAISSTNEIGLLGAGGLTTTWKEYSHDLQLNDGTVKAICVAGSFKITALNVVSRRYLASLHKGALLTSVTFELLFDGTNPSTEEMGDDFGFGLCPFDTSPVVKGKYNTTLLNGSAFYLVCPIGWTGVIECTAVSPTTLRTEVVKTFRREKPFPHRMDCVTTTVENEDLFYCKLGGNWTCVKGEPVVYTGGQVKQCKWCGFDFNEPDGLPHYPIGKCILANETGYRIVDSTDCNRDGVVISAKGSHECLIGNTTVKVHASDERLGPMPCRPKEIVSSAGPVRKTSCTFNYAKTLKNKYYEPRDSYFQQYMLKGEYQYWFDLDV

a DNA sequence of a pig ADAM17 coding region (shown as SEQ ID No. 2):

atgaggcagtgtgcgctcttcctgaccagcttggttcctatcgtgctggcgccgcgaccgccggacgagccgggcttcggctcccctcagcgactcgaaaagcttgattctctgctctcagactacgacatcctctctttatccagcattcgccagcactccgtaaggaaaagggatctgcaggcctcaacacacctagagacactactaactttttcagccttgaacaggcattttaaattatacctgacatcaagtactgaacgcttctcccagaatttcaaagtcgtggtggtcgatggggaagatgaaagtgagtaccccgtcaagtggcaggacttcttcagtggacacgtggttggtgaacctgactctagggttctcgcccacataggagatgatgatattacagtaagaatcaacacagatggggcagaatataatatagagccactttggagactaattaatgatactaaagacaaaagagtgttagtttataagtctgaagatatcaagaatgtttcacgtttgcagtctccaaaagtgtgtggttatataaaggcggataatgaagagttgcttcctaaagggctagtagacagagagccgcctgatgagcttgttcaccgggtgaagagaagagccgaccccaatcccctgaggaacacgtgtaaattattggtggtggcagatcatcgcttttataagtacatgggcagaggggaagagagcacgaccacaaactacctgatagagctaattgacagagttgatgacatctatcggaacacttcatgggacaatgcaggttttaaaggttatggaatacagatagagcagattcgcattctcaagtctccacaagaggtaaaacctggtgaaaggcactacaatatggcaaaaagttacccaaatgaagaaaaggatgcttgggatgtgaagatgttgctagagcaatttagctttgatatagctgaagaagcatctaaagtctgcctggcacatcttttcacctaccaagattttgatatgggaactcttggattagcttatgttggttctcccagagcaaacagtcatggaggtgtttgtccaaaggcttattatagtccaattggaaagaaaaatatctatttaaatagtggtttgaccagcacaaaaaattatggtaaaaccatccttacaaaggaagctgaccttgtgacaactcatgaattggggcacaattttggagcagaacacgatccagatggtttagcagaatgtgccccaaacgaggaccagggaggaaaatacgtcatgtatcccatagccgtgagtggtgatcatgagaacaacaagatgttttcaaactgcagtaaacagtccatctataagaccattgaaagtaaggcccaggagtgttttcaagagcgcagcaacaaagtgtgtggcaactccagggtggatgagggggaggagtgcgaccccggcatcatgtacctgaacaacgacacctgctgcaacagcgactgcaccctgaggccgggcgtccagtgcagtgataggaacagtccttgctgtaaaaactgtcagttcgagacggcccagaagaagtgccaggaggctattaatgccacttgcaaaggcgtgtcttactgcacaggtaacagcagtgagtgcccccctccgggaaacgccgaggacgacacggtgtgcctggacctgggcaggtgcaaggacggcaagtgcgtgcccttctgcgagcgggagcagcggctggagtcctgcgcgtgtaacgaaaccgaccactcgtgcaaggtgtgctgccgggccccctcgggccgttgcctgccctacgtggacgccgaacagaagaacttgtttttgaggaaggggaagccctgtacagtaggattttgtgacatgaatggcaagtgtgagaagcgagtgcaggacgtcatcgagcggttctgggagttcattgacaagctgagcatcaatactttcgggaagttcctggcagacaacatcgtgggctccgtcctggtgttctccctgatgctctggatccccgtcagcatcctcgtccactgcgtggataagaagctggataagcagtacgaatccctgtctctgctgcaccccagcaacgtggagatgctaagcagcatggattcagcatccgttcgcatcatcaagccctttcctgcgccccagaccccaggccgcctgcagcccctgcagcccctgcagcccggccccgtgctgccctctgcgccttcggtgcccgtggctccaaaactggaccaccagcggatggacaccatccaggaggaccccagcacggactcgcacgtggacgaggacggcttcgagaaggaccctttccccaacagcagtgccgctgccaagtcatttgaggatctcacggaccatccggtcacgagaagtgaaaaggcctcgtcctttaagctgcagcgccagagtcgcgttgacagcaaggaaacggagtgc

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

MRQCALFLTSLVPIVLAPRPPDEPGFGSPQRLEKLDSLLSDYDILSLSSIRQHSVRKRDLQASTHLETLLTFSALNRHFKLYLTSSTERFSQNFKVVVVDGEDESEYPVKWQDFFSGHVVGEPDSRVLAHIGDDDITVRINTDGAEYNIEPLWRLINDTKDKRVLVYKSEDIKNVSRLQSPKVCGYIKADNEELLPKGLVDREPPDELVHRVKRRADPNPLRNTCKLLVVADHRFYKYMGRGEESTTTNYLIELIDRVDDIYRNTSWDNAGFKGYGIQIEQIRILKSPQEVKPGERHYNMAKSYPNEEKDAWDVKMLLEQFSFDIAEEASKVCLAHLFTYQDFDMGTLGLAYVGSPRANSHGGVCPKAYYSPIGKKNIYLNSGLTSTKNYGKTILTKEADLVTTHELGHNFGAEHDPDGLAECAPNEDQGGKYVMYPIAVSGDHENNKMFSNCSKQSIYKTIESKAQECFQERSNKVCGNSRVDEGEECDPGIMYLNNDTCCNSDCTLRPGVQCSDRNSPCCKNCQFETAQKKCQEAINATCKGVSYCTGNSSECPPPGNAEDDTVCLDLGRCKDGKCVPFCEREQRLESCACNETDHSCKVCCRAPSGRCLPYVDAEQKNLFLRKGKPCTVGFCDMNGKCEKRVQDVIERFWEFIDKLSINTFGKFLADNIVGSVLVFSLMLWIPVSILVHCVDKKLDKQYESLSLLHPSNVEMLSSMDSASVRIIKPFPAPQTPGRLQPLQPLQPGPVLPSAPSVPVAPKLDHQRMDTIQEDPSTDSHVDEDGFEKDPFPNSSAAAKSFEDLTDHPVTRSEKASSFKLQRQSRVDSKETEC

2. to verify the function of ADAM17, we knocked out the expression of ADAM17 on a susceptible cell PK15 of CSFV using CRISPR-Cas9 technology. The results are shown in FIG. 3: the knockout cell line was unable to bind to sE2 and was unable to infect pseudoviruses and euviruses that do not cause CSFV. Whereas sE2 binding and infection with CSFV virus were all restored after re-overexpression of ADAM 17.

Specifically, fig. 3 (a):

the instrument comprises the following steps: flow cytometer BD FACSCalibur

Software: FlowJo 7.6

The experimental method comprises the following steps: FACS (flow cytometry)

The analysis process comprises the following steps: blue shading is the experimental group plus sE2 and red is the control group. Wild type PK15 (hog cholera virus-susceptible cell line, porcine kidney cell) is combined with sE2, a strong fluorescence signal can be detected, a control group basically has no fluorescence signal, the two groups are analyzed by FlowJo 7.6 software, histograms of the two groups are overlapped, and complete displacement can be seen. While the control cell, Hela (classical swine fever virus-non-susceptible cell line), was unable to bind to sE2, no fluorescence signal was detected, and no fluorescence signal was detected in the control group, and the two histograms were superimposed and no shift was observed as analyzed by FlowJo 7.6 software. The above shows that sE2 specifically binds to classical swine fever virus susceptible cell line PK15 but not to non-susceptible cell line Hela, demonstrating specificity.

Through FACS analysis, after the CRISPR Cas9 knocks down the PK15 cell line ADAM17 gene, the combination change of the CRISPR Cas9 and sE2 is found, and after the ADAM17 knockdown, PK15 cannot be combined with sE2, a fluorescent signal cannot be detected, a control group does not have the fluorescent signal, and the CRISPR Cas9 knockout, the sE2 gene, the fluorescent signal and the fluorescent signal are analyzed by FlowJo 7.6 software, histograms of the two groups are overlapped, and no displacement occurs. Porcine ADAM17cDNA is reintroduced into an ADAM17 knockout cell line, ADAM17 expression is reshaped, FACS analysis can restore the combination with sE2, a strong fluorescence signal can be detected, a control group basically has no fluorescence signal, the two groups are analyzed by FlowJo 7.6 software, histograms of the two groups are overlapped, and complete displacement can be seen. The ADAM family has forty genes, ADAM10 is the most similar gene to ADAM17, the amino acid similarity is 22.6%, but after FACS analysis, after CRISPR Cas9 knocks out the PK15 cell line ADAM10 gene, the combination of the CRISPR Cas9 and sE2 is changed, PK15 can still combine with sE2 after ADAM10 knockout, a strong fluorescence signal can be detected, a control group basically has no fluorescence signal, and the two groups are analyzed by FlowJo 7.6 software, histograms of the two groups are overlapped, and complete displacement can be seen. It is proved that ADAM17 is indispensable for the combination of PK15 and CSFV E2 protein.

TABLE 14 data of FIG. 3(A)

Specifically, fig. 3 (B):

the instrument comprises the following steps: nikon fluorescence microscope

Software: excel and GraphPad Prism6

The experimental method comprises the following steps: pseudovirus titer determination

The analysis process comprises the following steps: pseudoviruses are recombinant viral particles whose core/backbone and envelope proteins are derived from different viruses; in addition, genes within pseudoviruses are often 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 pseudoviruses. Pseudoviruses are able to infect susceptible cell lines, but they can replicate only one round in an infected host cell. Pseudoviruses can be safely manipulated in laboratories with a biological safety level of 2(BSL-2) and are generally easier to manipulate than wild-type viruses. More importantly, the protein conformation of the pseudovirus envelope surface protein is consistent with that of the wild-type virus, and the pseudovirus envelope surface protein can effectively mediate the virus to enter a host cell, so that the pseudovirus is widely applied to the cell tendency, receptor recognition, virus inhibition research, and the development and evaluation of antibodies and vaccines of the virus. In addition, the in vitro and in vivo experiments of the pseudovirus are proved to have good correlation with the experimental results generated by the wild virus.

After 48 hours of infection with pseudovirus, different cell lines were counted and virus titers (FFU/ml) were converted. And converting the data into a base-10 logarithmic function by using Excel, and continuously processing the obtained data by using GraphPad Prism6 to present the final result in a form of a histogram. The result shows that the PK15 of the knockout ADAM17 is not infected with the swine fever viruses of two genotypes at all, the pig ADAM17cDNA is reintroduced to restore the infection to the wild PK15 level, and the knockout ADAM10 is not changed. Meanwhile, VSV-G (vesicular stomatitis virus) is often used as a control virus because it infects almost all cell types, and the results show that various experimental operations do not substantially affect VSV-G infection. The ADAM17 is proved to be indispensable for the invasion of the classical swine fever virus PK 15.

TABLE 15 FIG. 3(B) data

Specifically, fig. 3 (C):

the instrument comprises the following steps: nikon fluorescence microscope

Software: excel and GraphPad Prism6

The experimental method comprises the following steps: cell culture strain (CSFVcc) Virus titer determination

And (3) analysis process: immunofluorescence experiments were performed 48 hours after infection of different cell lines with virus, counted and converted to virus titers (FFU/ml). The data is converted into a logarithmic function with the base 10 by using Excel, and the obtained data is continuously processed by GraphPad Prism6 to present the final result in a form of a histogram. The result shows that PK15 of the knockout ADAM17 is not infected with CSFVcc at all, and the pig ADAM17cDNA is reintroduced to restore the infection to the wild PK15 level, while the knockout ADAM10 is not changed. Meanwhile, rVSV-eGFP-G (recombinant vesicular stomatitis virus) is often used as a control virus because it infects almost all cell types, and the results show that various experimental operations do not affect the infection of the rVSV-eGFP-G basically. It is proved that ADAM17 is indispensable for the infection of PK15 by hog cholera virus.

TABLE 16 FIG. 3(C) data

3. To further investigate the role of ADAM17 in CSFV-infected pigs, siRNA was used to interfere with the expression of ADAM17 in primary Porcine Embryonic Fibroblasts (PEFs) and the effect on CSFV-infected PEFs was examined. Two RNAi primers were designed to transfect PEFs separately.

The instrument comprises the following steps: thermo fisher quantitative PCR instrument

Software: excel and GraphPad Prism6

The experimental method comprises the following steps: RNA interference, cell RNA extraction, reverse transcription PCR and quantitative PCR

And (3) analysis process: the primary porcine embryonic fibroblast cells were analyzed by quantitative PCR for transfection of ADAM17 specific primer (si-ADAM17), irrelevant primer (siCtrl) ADAM17 and reference gene beta-actin, and the number of PCR cycles (Ct value) required for reaching the threshold. The expression (%) of the ADAM17 specific primer (si-ADAM17) relative to the ADAM17 independent primer (siCtrl) was transfected by using Excel preliminary treatment, and the obtained data were further treated with GraphPad Prism6 to present the final results in the form of a histogram. The results showed that the RNA level of ADAM17 was reduced by 70.9% and 78.9%, respectively.

After RNA interference 36, primary pig fibroblasts were infected with CSFVcc, and after 2 days, the virus infection level was measured by quantitative PCR, and the number of PCR cycles (Ct value) required to reach a threshold was determined. Initial treatment with Excel transfected ADAM17 specific primers (si-ADAM17) relative to the CSFVcc content (%) of the transfection-independent primers (siCtrl), the data obtained were further treated with GraphPad Prism6 to present the final results in the form of a histogram. The results show that knocking down ADAM17 expression by two RNAi primers significantly reduced the level of viral infection by 89.2% and 92.4%, respectively.

The results are shown in FIG. 4: after 36 hours, the RNA level of ADAM17 was detected by fluorescent quantitative PCR, and the RNA level of ADAM17 was reduced by 70.9% and 78.9%, respectively. Meanwhile, PEFs cells after 36 hours of RNA interference are infected with CSFVcc, and the virus infection level is detected by quantitative PCR after 2 days. Knocking down ADAM17 expression by two RNAi primers resulted in a significant 89.2% and 92.4% reduction in the level of viral infection, respectively. This result demonstrates that ADAM17 is crucial for CSFV infection of primary porcine cells.

TABLE 17 FIG. 4(A)

Watch 18 fig. 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.

The instrument comprises the following steps: flow cytometer BD FACSCalibur

Software: FlowJo 7.6

The experimental method comprises the following steps: FACS (flow cytometry)

The results are shown in FIG. 5: different concentrations of 1,10-phenanthroline were incubated with pancreatin-treated PK15 on ice for 30 minutes, followed by detection of PK15 binding to sE2 by FACS, and it was found that sE2 binding decreased with increasing concentration and that binding decreased by 74.3% at a1, 10-phenanthroline concentration of 25 mM. The CSFV E2 protein is proved to be combined with the active site of ADAM 17.

A class of ADAM17 inhibitors, including adrvasib, also known as INCB007839, which is a selective inhibitor of ADAM10 and ADAM17, inhibit the activity of metallopeptidases by binding to the active site of the metalloprotease domain, and are currently undergoing clinical trials for cancer treatment. To examine whether or not adarbasib can inhibit binding between ADAM17 and sE2, varying concentrations of adarbasib were incubated with pancreatin-treated PK15 on ice for 30 minutes, and PK15 was similarly examined for binding to sE2 by FACS, and it was found that sE2 binding decreased with increasing concentration and that binding was barely detectable at an adarbasib concentration of 100 μm (right panel of fig. 5). This further illustrates that CSFV E2 protein binds to ADAM17 via a metalloprotease domain.

TABLE 19 left image data of FIG. 5

Table 20 right figure data of fig. 5

5. In order that ADAM17 could be directly bound to CSFV E2, the present invention purified soluble porcine ADAM17 protein (sADAM17), and further verified the interaction between sADAM17 and sE2 using Surface Plasmon Resonance (SPR). Since ADAM17 in an organism must exert biological effects in the presence of zinc ions, the affinity between sADAM17 and sE2 was examined in two environments, the presence and absence of zinc ions, respectively.

The instrument comprises the following steps: surface plasma resonance instrument

Software: excel (Excel)

The experimental method comprises the following steps: surface plasma analysis

The analysis process comprises the following steps: binding sE2 on the sensor surface, injecting different concentrations of sADAM17 solution for expression purification and flowing through the sensor surface (in an environment with zinc ions and an environment without zinc ions respectively) and collecting data. The data were analyzed by Excel to obtain a line graph.

The results are shown in FIG. 6: shows that the affinity of sADAM17 and sE2 is 3.250X 10 under the condition of lacking zinc ions ^- ⁶ mol, 1.122X 10 of affinity in the presence of zinc ions ^-7 mol。

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> institute of animal research of Chinese academy of sciences

<120> use of ADAM17 as receptor for hog cholera 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 (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 (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 (Artificial Sequence)

<400> 5

gcaacaaagu guguggcaat t 21

<210> 6

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

uugccacaca cuuuguugct t 21

<210> 7

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gguguuuguc caaaggcuut t 21

<210> 8

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aagccuuugg acaaacacct t 21

<210> 9

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

caccgagttc ccgccagaca ctac 24

<210> 10

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aaacgtagtg tctggcggga actc 24

<210> 11

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

caccagtgct atcaacctta tacg 24

<210> 12

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aaaccgtata aggttgatag cact 24

<210> 13

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

caccacaggg ctgggtgata gatt 24

<210> 14

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

aaacaatcta tcacccagcc ctgt 24

<210> 15

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cacctgcgtt gaccaaactc tcac 24

<210> 16

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

aaacgtgaga gtttggtcaa cgca 24

<210> 17

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

uucuccgaac gugucacgut t 21

<210> 18

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

acgugacacg uucggagaat t 21

Claims

The application of an ADAM17 inhibitor in preparing a medicament for preventing and/or treating swine fever; the amino acid sequence of the ADAM17 is shown as SEQ ID No. 1;

the inhibitor of ADAM17 is an inhibitor that inhibits ADAM17 expression or inhibits ADAM17 activity;

the inhibiting of ADAM17 expression is knocking down the expression of ADAM 17;

the activity of inhibiting ADAM17 is to chelate and/or remove zinc ions at the active center of the metalloprotease domain of ADAM 17.