CN113881742A - Preparation method and application of new crown spike protein in post-fusion state - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of a novel crown spike protein in a post-fusion state. The present invention is based on the previous patent of invention (US11020474B1) and produces spike proteins in a pre-fusion state with an intact furin cleavage site by recombinant means. In the application, in-vitro enzyme digestion experiments show that the spike protein in the pre-fusion state can be digested by furin to generate the spike protein in the post-fusion state with a receptor binding function. The invention proves that the natural spike proteins in the pre-fusion state and the post-fusion state can be used as vaccines or screening sources, so that various functional antibodies capable of neutralizing virus invasion can be obtained.

Description

Preparation method and application of new crown spike protein in post-fusion state

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of a novel crown spike protein in a post-fusion state.

Background

The new coronavirus in 2019 caused a huge disaster all over the world, and new variant viruses are still emerging. The novel coronavirus (SARS-CoV-2 or 2019-nCoV, named after 11/2/2020 by International Committee for viral Classification) is a large family of viruses that can cause more severe diseases such as cold, Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS), and the like. It is known that coronaviruses enter human cells via human angiotensin converting enzyme 2(ACE 2). The common signs of human infection with the novel coronavirus include respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. However, there is no specific treatment for diseases caused by the novel coronavirus, and there are very limited protocols for preventing and treating the novel coronavirus.

The coronavirus spike protein is a class I viral fusion protein. The spike protein exists in the form of a complete Pre-fusion state protein (Pre-fusion) on the cell membrane before entering the host cell, and the protein contains arginine-X- (arginine/lysine) -arginine motif (R-X-K/R-R) and can be recognized by FURIN (FURIN) of the host so as to be cut into two subunits S1 and S2, namely, the Post-fusion state protein (Post-fusion) exists. The Spike protein (Spike Pre-fusion) in the Pre-fusion state can mediate the binding of the virus and the host receptor angiotensin converting enzyme 2(ACE2), is related to the adsorption and membrane fusion of virus particles, and is an important action site of host neutralizing antibodies and a key target point of vaccine design.

It was found that most of the spike proteins on the surface of the new coronavirus particles were in a pre-fused state, i.e., they existed as intact new corona spike proteins. The host cell FURIN (FURIN) can convert the spike protein in the pre-fusion state into the spike protein in the post-fusion state containing two subunits S1 and S2 with a broken sequence by enzyme digestion. This cleavage process is necessary for the invasion and replication of human cells by the new coronavirus. Mutations or deletions in the furin cleavage site can affect viral entry and replication. Therefore, the inhibition of the activity of furin or the blocking of furin cleavage sites by antibodies can inhibit the conversion of the spike protein in the pre-fusion state into the spike protein in the post-fusion state, and can possibly inhibit the invasion of new coronavirus into human cells.

The recombinant Spike protein extracellular domain (S-ECD) is widely applied to the fields of protein structure research, cell function tests, immunodetection reagents, recombinant protein drugs, diagnostic reagent development, vaccines and the like. The conformation and yield of the recombinant spike protein are particularly critical in the above applications.

However, the spike protein of the novel coronavirus contains a furin cleavage site, and is easily incompletely cleaved by furin of a host during recombination of a host cell into a mixture of a pre-fusion protein and a post-fusion protein with two subunits S1 and S2. This creates three problems: the first one cannot obtain a high purity of the protein in the pre-fusion state. Secondly, post-fusion state proteins are not available. The third two yields were not high. To solve this problem, arginine/lysine at the furin recognition site may be mutated to other amino acids. But this produces a protein of non-native structure. Brings new troubles in function and use. Mutated furin recognition sites, fail to produce corresponding neutralizing antibodies. The mutated protein cannot serve as a substrate for furin and cannot be converted into a protein in the post-fusion state. Based on the previous invention, we further provide a method for preparing post-fusion spike protein.

Thus, the pre-fusion state of the spike protein is more important. In addition, the new crown spike protein in the pre-fusion state has a virus natural structure and a furin enzyme cutting site, and is an ideal target and immunogen. The novel crown spike protein, particularly the receptor binding domain RBD thereof, is an important target for current vaccines and neutralizing antibodies. However, the effectiveness of these vaccines and neutralizing antibodies is decreasing as new mutations are continually present in the receptor binding domain RBD.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a preparation method and application of a novel crown spike protein in a post-fusion state, and aims to solve part of problems in the prior art or at least alleviate part of problems in the prior art.

The invention is realized in such a way that a preparation method of a new crown spike protein in a post-fusion state utilizes furin to carry out enzyme digestion on the spike protein in a natural pre-fusion state.

The invention also provides the application of the novel coronary spike protein in post-fusion state in the preparation of a binding agent of angiotensin converting enzyme 2.

Further, the application of the post-fusion state of the new coronary spike protein as a new coronary virus treatment target.

Further, the new crown spike protein in post-fusion state is used as the target of the new crown virus vaccine.

Further, the new crown spike protein in post-fusion state is used as a target of a new crown virus neutralizing antibody.

Further, the use of the novel corona spike protein in post-fusion state as an immunogen for novel corona viruses.

Based on the previous research content, the invention uses CRISPR-Cas9 technology to knock out the furin gene of host expression cells (such as other expression cells like HEK293 and CHO); then, expressing and purifying the spike protein of the extracellular region in the recombinant prefusion state by using a recombinant protein technology; the post-fusion state protein can be generated by the fusion state protein before the in vitro furin enzyme digestion.

The specific method and operation are as follows:

1. the post-fusion protein is obtained by carrying out enzyme digestion on the pre-fusion protein by furin in vitro, and the purification effect is compared.

2. The ACE2 protein is used for identifying and detecting the biological activity of the spike protein in a post-fusion state, and the high activity and the high quality of the spike protein are verified.

In the aspects of scientific research and diagnosis, the recombinant spike protein in the fusion state can be used as an antigen to generate an antibody for producing scientific research antibodies; can be used as binding molecules for various detection experiments, such as blood transfusion, organ transplantation or new crown diagnosis. In the aspect of prevention and treatment, the spike protein in the recombinant fusion state can be used as a medicament for intervening or blocking the binding of a new crown to a human receptor; can be used as a protective group of high-efficiency vaccines.

This method of expression purification of the recombinant fusion state spike protein can also be applied to similar other viruses having furin cleavage sequence without changing the protein sequence of interest, such as CoV-ZXC21(MG772934), SARS-CoV (NC _004718.3), SARS-like BM4821(MG772934), HCoV-OC43(AY391777), HKU9-1(EF065513), HCoV-NL63(KF530114.1), HCoV229E (KF514433.1), MERS-CoV (NC019843.3), and HKU1(NC _ 006577.2). And their natural mutants. Basically, the same construction vector is transfected into a cell with a gene knocked out of the furin enzyme, and the fusion state spike proteins are obtained by expression, purification and enzyme digestion. These viruses are used as a therapeutic service for downstream diagnostic reagents to prevent and treat, interfering with cell binding, antibody antigen binding molecules, diagnostic therapeutic vaccines against the virus.

In summary, the advantages and positive effects of the invention are:

the present invention is based on the previous patent of invention (US11020474B1) and produces spike proteins in a pre-fusion state with an intact furin cleavage site by recombinant means. In the application, in-vitro enzyme digestion experiments show that the spike protein in the pre-fusion state can be digested by furin to generate the spike protein in the post-fusion state with a receptor binding function. While the mutant furin cleavage site can obtain a pre-fusion state protein with a similar structure, a post-fusion state protein cannot be further obtained.

Therefore, the furin enzyme cutting site on the spike protein in the recombinant prefusion state has biological activity, can be used as a target antigen to immunize animals, and can also be used for screening and generating a neutralizing antibody aiming at the site, thereby being capable of blocking the invasion of new coronavirus. The furin cleavage site is only 10 or less amino acids, and the probability of mutation is far less than that of mutation due to hundreds of amino acid combinations in the RBD domain. The antibody has far more advantages than RBD when being used as a vaccine and screening a neutralizing antibody.

Two subunits of the complete post-fusion protein can form trimer with equal proportion, and the structure is stable, so that the protein can be stably expressed in cells without being degraded by other proteases in the cells, and the yield of the protein is much higher than that of wild cells. Therefore, is very valuable for downstream treatment or industrial production. The post-fusion state protein retains the original natural conformation of the virus surface protein, and can better serve the fields of protein structure research, cell function test, immunodetection reagent, recombinant protein medicine, diagnostic reagent development, neutralizing monoclonal antibody, vaccine and the like.

In vitro binding experiments of the invention show that the RBD domain of the spike protein in post-fusion state can also bind to the receptor ACE 2. And this binding is superior to the natural pre-fusion state spike protein or the mutant pre-fusion state spike protein. The spike protein in the fused state can then also be used as a target and immunogen. The s protein before virus invasion is pre-fusion, the space conformation after the invasion of host cells is completely different from that of the pre-fusion protein after enzyme digestion, the two proteins are completely different, otherwise, the virus cannot invade cells. There is no technology in the prior art that is capable of producing the post-fusion recombinant proteins of the present application.

In conclusion, the recombinant natural spike proteins in pre-fusion state and post-fusion state can be used as vaccines or screening antigens, so as to obtain various functional antibodies capable of neutralizing virus invasion.

Drawings

FIG. 1 is an SDS-PAGE pattern of S-ECD-PFS protein;

FIG. 2 is an SDS-PAGE pattern of furin;

FIG. 3 is a SDS-PAGE pattern of the cleavage of S-ECD-PFS by furin;

FIG. 4 is a SDS-PAGE pattern of the cleavage of S-ECD-PFS and S-ECD-MT by furin;

FIG. 5 shows the results of experiments on the binding activity of S-ECD-PFS, S-ECD-MT and S-ECD-AF to ACE 2.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.

For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.

In the following examples of the present invention, the temperature is not particularly limited, and all of the conditions are normal temperature conditions. The normal temperature refers to the natural room temperature condition in four seasons, no additional cooling or heating treatment is carried out, and the normal temperature is generally controlled to be 10-30 ℃, preferably 15-25 ℃.

The genes, proteins or fragments thereof involved in the present invention may be naturally purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant techniques.

The invention discloses a preparation method and application of a novel crown spike protein in a post-fusion state. The amino acid sequence of furin Asp23-Ala574 is shown in SEQ ID NO. 1. The optimized S-ECD (AA Val16-Gln1208, with the nucleotide sequence shown in SEQ ID NO.2 and the amino acid sequence shown in SEQ ID NO.3) was subcloned into pcDNA vector (accession # YP _009724390.1) by 5'XbaI/3' AgeI. S-ECD (AA Met1-Gln1208, nucleotide sequence shown as SEQ ID NO.4, first 45 bases as a signal peptide coding part, and amino acid sequence shown as SEQ ID NO.5) is subcloned into pcDNA vector by 5'XbaI/3' AgeI.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

Examples

Referring to published patent US11020474B1, S-ECD-PFS (Pre-fusion state) was purified by transfecting S-ECD expression vector into HEK293 Furin-/-cells (see FIG. 1, with a single band intact, about 180 kDa). Thus, S-ECD-AF (after fur factor clearance) was produced in vitro using it as a substrate.

The nucleotide sequence of the Furin gene (AA108-574) [ 10 ] is shown in SEQ ID NO6, and the amino acid sequence is shown in SEQ ID NO. 7. The Furin gene with the CD33 signal peptide sequence and C-terminal tag Flag tag was synthesized in vitro into pUC57 vector. 5 'is XbaI cleavage site and 3' is XhoI cleavage site. Then, XhoI/XbaI cleavage was performed on pUC57-Furin and pcDNA (Invitrogen). The former recovers DNA fragment containing Furin, the latter recovers vector skeleton, and then uses T4 DNA to link the product to construct recombinant plasmid pcDNA-Furin-C-Flag (T4 DNA Ligase NEB M0202S).

The linking system is as follows:

the method for transforming and identifying the ligation product comprises the following steps: adding the connection product of the enzyme-cut carrier skeleton and the double-stranded DNA fragment into competent bacteria, supplementing a culture medium, culturing for half an hour, coating on a resistant plate, culturing overnight, selecting a single colony, performing amplification culture, harvesting a bacterial liquid, extracting recombinant plasmids, performing sequencing identification, and storing the recombinant plasmids with correct identification.

The Furin expression vector was transfected into HEK293 Furin-/-cells. And collecting the supernatant after 4-6 days of culture, performing affinity purification by using Flag m2 and eluting by using Flag polypeptide to obtain the recombinant furin. (see FIG. 2, Furin protein was purified using flag m2 affinity gel, and the purified protein was separated by SDS-PAGE, with a size of about 60 kDa).

In vitro reaction of 5ug S-ECD-PFS, 10ng recombinant Furin, 0.2mM CaCl₂20mM HEPES, pH 7.5. The results of the experiment are shown in FIG. 3, and it is expected that the 180KD protein is apparently cut into two bands of S1 with 120KD and S2 with 80 KD. After dialysis, furin was cleared by Flag antibody coupled to protein G or Flag M2 gel (Sigma). The cleaved post-fusion spike protein was then exchanged into 1 XPBS buffer for further analysis.

In contrast, the S-ECD containing the double mutations R683A and R685A (Cat # Abclonal RP01260MT) was resistant to Furin cleavage (FIG. 4, S-ECD-PFS in lane 1, cleavable by Furin; S-ECD-MT in lane 2, resistant to Furin).

The following investigations were carried out in the present application as to whether S-ECD-AF is able to produce an activity similar to or higher than that of S-ECD-PFS on human ACE 2.

SARS-CoV-2 enters human cells primarily through binding of its RBD domain on the spike protein to the receptor ACE 2. Thus, the binding activity to ACE2 allows the quality of the protein to be assessed.

The binding activity of the protein to ACE2 was tested by enzyme-linked immunosorbent assay (ELISA) in this application. The specific operation is as follows: S-ECD was immobilized on a 96-well plate at 2ug/mL (100uL/well) and recombinant ACE2 with Fc domain was added. Recombinant ACE2 with Fc domain was serially diluted 5-fold at 2ug/mL (100/well) and 6 times. Blank was used as negative control. Conjugated recombinant ACE2-Fc (Abclonal RP01275) was detected with anti-human Fc-HRP antibody. OD values were measured with Origin8 software and EC50 values were calculated.

EC50 of S-ECD-AF is 3.82 ng/mL; EC50 of S-ECD-PFS is 4.33 ng/mL; the EC50 for S-ECD-MT was 10.38 ng/mL. The lower the EC50, the better the binding activity. The experimental results show that S-ECD-AF has better ACE2 binding activity (see FIG. 5).

The above results indicate that S-ECD-AF is more suitable than S-ECD mutants for producing antibodies as antigens for use in detection or diagnosis, intervention in SARS-CoV-2 cell binding therapy or as vaccines (producing SARS-CoV-2 antibodies).

The invention relates to parts of materials and equipment

Qiagen Middling kit (Cat.No./ID:12843)

293 expression medium (Gibco # A1435102)

Opti-MEM I reducing serum Medium (Gibco #31985088)

Expifeacylamine 293 transfection kit (GIbco # A14524)

PBS (1X) (Gibco #10010-023 or equivalent)

Ni-NTA agarose (Qiagen #30230 or equivalent)

NuPAGE 4-12% bistis protein gel (Invitrogen Catalog number: NP0329BOX)

HEK293 cell (Gibco)

Ni-NTA Agarose (Qiagen #30230 or other equivalents)

NuPAGE 4-12％Bis-Tris Protein Gels(Invitrogen Catalog number:NP0329BOX)

A2220-ANTI-

Affinity gel-Sigma-Aldrich

Furin mAb antibody(Abclonal A5043)

6XHis antibody(AbclonalAE068 lot#3561557103)

ActinmAb antibody(Abclonal AC026)

Recombinant protein S-ECD-MT Recombinant S-ECD R683A, R685A mutation (Abclonal RP01260MT)

Recombinant protein Recombinant ACE2(AbclonalRP01275)

The invention relates to the following detailed steps:

1. procedure of experiment

1) HEK293 cells were seeded into 12-well plates 12-24 hours before transfection to achieve 70% confluency of cells before transfection.

2) Turning over for 5-10 times before using the transfection reagent to fully mix the mixture. Taking a clean sterile 1.5ml centrifuge tube, adding 150ul Optiman, adding 2ul transfection reagent, blowing and beating the mixture by a gun, and standing the mixture at room temperature for 5min to obtain a solution A;

3) taking a clean sterile 1.5ml centrifuge tube, adding 150ul Optiman, adding 2ug of plasmid, and gently blowing and mixing by using a gun to obtain a solution B;

4) the two dilutions A and B were combined (total volume about 304ul), gently mixed by blowing with a gun, and incubated at room temperature for 20 min.

5) And slowly adding the incubated compound mixed solution into a twelve-well plate by using a 1ml pipette, adding the mixed solution into the whole hole uniformly as much as possible while adding, and then, slightly swinging in a shape of 8 to mix uniformly.

6) And the supernatant was collected 4 days after transfection.

Note that: to avoid operator error, a positive control containing GFP fluorescent protein and a negative control without the plasmid and transfection reagent complex are required for each batch of items during transfection.

2. WB detection

Sample preparation: the supernatant 4 days after HEK293 transfection was sampled by 10ul and 40ul3 loading and heated at 97 ℃ for 5 min. Sample 5ul, WB.

SDS-PAGE electrophoresis: the concentration of the separation gel is selected according to the molecular weight of the target protein. Electrophoresis is carried out in a constant-pressure mode, the gel is concentrated at 80V by 5%, when marker begins to separate for about 25min, the gel is adjusted to 120V, and the bromophenol blue reaches the bottom of the separation gel to terminate electrophoresis.

Film transfer: the assembly sequence is as follows: the transfer film is clamped with a black surface (negative electrode), a spongy cushion, 3 layers of filter paper, glue, a film, 3 layers of filter paper, the spongy cushion and a red surface (positive electrode). Film transfer time: 200mA, 90-180min

And (3) sealing: marking and washing the membrane transfer solution (TBST, 5minX2 times) after the membrane transfer is finished; the cleaned membrane was placed in a container with 3% skimmed milk (made in TBST) and sealed at room temperature for 60-90 min.

6 × His primary antibody incubation: after the sealing is completed, the sealing liquid is poured off. Adding primary antibody solution diluted by 3% skimmed milk (TBST) at a ratio of 1:7000, shaking gently on a shaker, and incubating at room temperature for 2h or overnight at 4 deg.C (after incubation at 4 deg.C, incubating at room temperature for 15-30 min). After the primary antibody incubation is finished, pouring out the primary antibody solution; the membrane was rinsed 4 times 5min each with TBST.

And (3) secondary antibody incubation: before the primary antibody incubation was completed, the enzyme-labeled secondary antibody 1: 5000 dilutions were made to the amount required for the experiment (TBST dilution). And (3) putting the washed membrane into a container containing the secondary antibody solution, slowly shaking on a shaking table, and incubating at room temperature for 60-80 min. After the secondary antibody incubation is finished, pouring out the secondary antibody solution; the membrane was rinsed 4 times 5min each with TBST.

Exposure: the membrane was removed from the TBST with forceps, drained appropriately, and placed on a gel tray. Mixing with equal volume of ECL Solution I and Solution II, adding onto the membrane, and covering completely. The substrate reacts with the membrane for about 30 seconds and is placed in a chemiluminescent imaging system. Setting the exposure time to be 3S; 10S; 30S; 60S; 120S.

WB detection objectives include: whether the protein expression size is correct; comparing with a high expression sample, and acquiring high/low protein expression amount information; for the non-stripe item in WB, the high exposure determines that there is no expression if a stripe can be found, and the non-stripe determines that there is no expression.

3. ELISA experiments:

sterile water-redissolved protein lyophilized powder is diluted with PBS,

1. coating quilt

Taking 833.7 mu L of coating solution, taking 11.5 mu L of each spike protein stock solution, adding the coating solution, diluting the spike protein to 2 mu g/mL, uniformly mixing, taking 100 mu L/hole, adding 8 holes, adding an enzyme label plate, and placing in a refrigerator at 4 ℃ for coating overnight.

2. Sealing of

The coated plate was taken out, the coating solution was discarded, 200. mu.L of the blocking solution was added thereto, and the mixture was left in an incubator at 37 ℃ for 2 hours.

3. Adding reaction protein

Dilution ACE2 was diluted in 7 gradients, 5-fold gradient dilution, with a maximum reaction concentration of 2 μ g/mL, 1 dilution control.

And (3) taking out the ELISA plate, washing the ELISA plate with washing liquid for 1 time, 300 mu L/hole and 1.5 min/time, removing the washing liquid, respectively taking 100 mu L of protein diluent to the hole of the corresponding ELISA plate, and placing the diluted protein diluent in a constant temperature box at 37 ℃ for 2 hours.

4. Adding antibodies

The ELISA plate was removed, washed with washing solution 3 times (300. mu.L/well) for 1.5 min/time, and the antibody was diluted at a ratio of 1:1000 (100. mu.L/well), and placed in a 37 ℃ incubator for 1 hour.

5. Color development

And (3) taking out the ELISA plate, washing the ELISA plate with washing liquid for 4 times, 300 mu L/hole and 1.5 min/time, adding color development liquid TMB, 100 mu L/hole, and developing in dark for about 15min-25min (less than 30min), wherein the color development is 7min in the experiment.

6. Adding stopping liquid

Stop solution was added to the microplate at 50. mu.L/well.

EC50 values were calculated by measuring OD450nm and OD630nm with a microplate reader.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

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ggcttccaac caaccaatgg agtgggctac caaccataca gggtggtggt gctgtccttt 1500

gaactgctcc atgcccctgc cacagtgtgt ggaccaaaga agagcaccaa cctggtgaag 1560

aacaagtgtg tgaacttcaa cttcaatgga ctgacaggca caggagtgct gacagagagc 1620

aacaagaagt tcctgccatt ccaacagttt ggcagggaca ttgctgacac cacagatgct 1680

gtgagggacc cacagacctt ggagattctg gacatcacac catgttcctt tggaggagtg 1740

tctgtgatta cacctggcac caacaccagc aaccaggtgg ctgtgctcta ccaggatgtg 1800

aactgtactg aggtgcctgt ggctatccat gctgaccaac ttacaccaac ctggagggtc 1860

tacagcacag gcagcaatgt gttccagacc agggctggct gtctgattgg agcagagcat 1920

gtgaacaact cctatgagtg tgacatccca attggagcag gcatctgtgc ctcctaccag 1980

acccagacca acagcccaag gagggcaagg tctgtggcaa gccagagcat cattgcctac 2040

acaatgagtc tgggagcaga gaactctgtg gcttacagca acaacagcat tgccatccca 2100

accaacttca ccatctctgt gaccacagag attctgcctg tgagtatgac caagacctct 2160

gtggactgta caatgtatat ctgtggagac agcacagagt gtagcaacct gctgctccaa 2220

tatggctcct tctgtaccca acttaacagg gctctgacag gcattgctgt ggaacaggac 2280

aagaacaccc aggaggtgtt tgcccaggtg aagcagattt acaagacacc tccaatcaag 2340

gactttggag gcttcaactt cagccagatt ctgcctgacc caagcaagcc aagcaagagg 2400

tccttcattg aggacctgct gttcaacaag gtgaccctgg ctgatgctgg cttcatcaag 2460

caatatggag actgtctggg agacattgct gccagggacc tgatttgtgc ccagaagttc 2520

aatggactga cagtgctgcc tccactgctg acagatgaga tgattgccca atacacctct 2580

gccctgctgg ctggcaccat cacctctggc tggacctttg gagcaggagc agccctccaa 2640

atcccatttg ctatgcagat ggcttacagg ttcaatggca ttggagtgac ccagaatgtg 2700

ctctatgaga accagaaact gattgccaac cagttcaact ctgccattgg caagattcag 2760

gactccctgt ccagcacagc ctctgccctg ggcaaactcc aagatgtggt gaaccagaat 2820

gcccaggctc tgaacaccct ggtgaagcaa ctttccagca actttggagc catctcctct 2880

gtgctgaatg acatcctgag cagactggac aaggtggagg ctgaggtcca gattgacaga 2940

ctgattacag gcagactcca atccctccaa acctatgtga cccaacaact tatcagggct 3000

gctgagatta gggcatctgc caacctggct gccaccaaga tgagtgagtg tgtgctggga 3060

caaagcaaga gggtggactt ctgtggcaag ggctaccacc tgatgagttt tccacagtct 3120

gcccctcatg gagtggtgtt cctgcatgtg acctatgtgc ctgcccagga gaagaacttc 3180

accacagccc ctgccatctg ccatgatggc aaggctcact ttccaaggga gggagtgttt 3240

gtgagcaatg gcacccactg gtttgtgacc cagaggaact tctatgaacc acagattatc 3300

accacagaca acacctttgt gtctggcaac tgtgatgtgg tgattggcat tgtgaacaac 3360

acagtctatg acccactcca acctgaactg gactccttca aggaggaact ggacaaatac 3420

ttcaagaacc acaccagccc tgatgtggac ctgggagaca tctctggcat caatgcctct 3480

gtggtgaaca tccagaagga gattgacaga ctgaatgagg tggctaagaa cctgaatgag 3540

tccctgattg acctccaaga actgggcaaa tatgaacaat acatcaagtg gccacatcat 3600

caccaccatc actaa 3615

<210> 3

<211> 1199

<212> PRT

<213> S-ECD

<400> 3

Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser

1 5 10 15

Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val

20 25 30

Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr

35 40 45

Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe

50 55 60

Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr

65 70 75 80

Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp

85 90 95

Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val

100 105 110

Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val

115 120 125

Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val

130 135 140

Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe

145 150 155 160

Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu

165 170 175

Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His

180 185 190

Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu

195 200 205

Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln

210 215 220

Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser

225 230 235 240

Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln

245 250 255

Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp

260 265 270

Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu

275 280 285

Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg

290 295 300

Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu

305 310 315 320

Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr

325 330 335

Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val

340 345 350

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

355 360 365

Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser

370 375 380

Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr

385 390 395 400

Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly

405 410 415

Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly

420 425 430

Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro

435 440 445

Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro

450 455 460

Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr

465 470 475 480

Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val

485 490 495

Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro

500 505 510

Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe

515 520 525

Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe

530 535 540

Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala

545 550 555 560

Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser

565 570 575

Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln

580 585 590

Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala

595 600 605

Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly

610 615 620

Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His

625 630 635 640

Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys

645 650 655

Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val

660 665 670

Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn

675 680 685

Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr

690 695 700

Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser

705 710 715 720

Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn

725 730 735

Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu

740 745 750

Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala

755 760 765

Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly

770 775 780

Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg

785 790 795 800

Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala

805 810 815

Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg

820 825 830

Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro

835 840 845

Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala

850 855 860

Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln

865 870 875 880

Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val

885 890 895

Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe

900 905 910

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

915 920 925

Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu

930 935 940

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

945 950 955 960

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

965 970 975

Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr

980 985 990

Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn

995 1000 1005

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg

1010 1015 1020

Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser

1025 1030 1035 1040

Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln

1045 1050 1055

Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala

1060 1065 1070

His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe

1075 1080 1085

Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn

1090 1095 1100

Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn

1105 1110 1115 1120

Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu

1125 1130 1135

Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly

1140 1145 1150

Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile

1155 1160 1165

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

1170 1175 1180

Leu Gln Glu Leu Gly Lys Tyr Glu Gln His His His His His His

1185 1190 1195

<210> 4

<211> 3660

<212> DNA

<213> S-ECD

<400> 4

atgtttgtgt tcctggtgct gctgccactg gtgtccagcc agtgtgtgaa cctgaccacc 60

aggacccaac ttcctcctgc ctacaccaac tccttcacca ggggagtcta ctaccctgac 120

aaggtgttca ggtcctctgt gctgcacagc acccaggacc tgttcctgcc attcttcagc 180

aatgtgacct ggttccatgc catccatgtg tctggcacca atggcaccaa gaggtttgac 240

aaccctgtgc tgccattcaa tgatggagtc tactttgcca gcacagagaa gagcaacatc 300

atcaggggct ggatttttgg caccaccctg gacagcaaga cccagtccct gctgattgtg 360

aacaatgcca ccaatgtggt gattaaggtg tgtgagttcc agttctgtaa tgacccattc 420

ctgggagtct actaccacaa gaacaacaag tcctggatgg agtctgagtt cagggtctac 480

tcctctgcca acaactgtac ctttgaatat gtgagccaac cattcctgat ggacttggag 540

ggcaagcagg gcaacttcaa gaacctgagg gagtttgtgt tcaagaacat tgatggctac 600

ttcaagattt acagcaaaca cacaccaatc aacctggtga gggacctgcc acagggcttc 660

tctgccttgg aaccactggt ggacctgcca attggcatca acatcaccag gttccagacc 720

ctgctggctc tgcacaggtc ctacctgaca cctggagact cctcctctgg ctggacagca 780

ggagcagcag cctactatgt gggctacctc caaccaagga ccttcctgct gaaatacaat 840

gagaatggca ccatcacaga tgctgtggac tgtgccctgg acccactgtc tgagaccaag 900

tgtaccctga aatccttcac agtggagaag ggcatctacc agaccagcaa cttcagggtc 960

caaccaacag agagcattgt gaggtttcca aacatcacca acctgtgtcc atttggagag 1020

gtgttcaatg ccaccaggtt tgcctctgtc tatgcctgga acaggaagag gattagcaac 1080

tgtgtggctg actactctgt gctctacaac tctgcctcct tcagcacctt caagtgttat 1140

ggagtgagcc caaccaaact gaatgacctg tgtttcacca atgtctatgc tgactccttt 1200

gtgattaggg gagatgaggt gagacagatt gcccctggac aaacaggcaa gattgctgac 1260

tacaactaca aactgcctga tgacttcaca ggctgtgtga ttgcctggaa cagcaacaac 1320

ctggacagca aggtgggagg caactacaac tacctctaca gactgttcag gaagagcaac 1380

ctgaaaccat ttgagaggga catcagcaca gagatttacc aggctggcag cacaccatgt 1440

aatggagtgg agggcttcaa ctgttacttt ccactccaat cctatggctt ccaaccaacc 1500

aatggagtgg gctaccaacc atacagggtg gtggtgctgt cctttgaact gctccatgcc 1560

cctgccacag tgtgtggacc aaagaagagc accaacctgg tgaagaacaa gtgtgtgaac 1620

ttcaacttca atggactgac aggcacagga gtgctgacag agagcaacaa gaagttcctg 1680

ccattccaac agtttggcag ggacattgct gacaccacag atgctgtgag ggacccacag 1740

accttggaga ttctggacat cacaccatgt tcctttggag gagtgtctgt gattacacct 1800

ggcaccaaca ccagcaacca ggtggctgtg ctctaccagg atgtgaactg tactgaggtg 1860

cctgtggcta tccatgctga ccaacttaca ccaacctgga gggtctacag cacaggcagc 1920

aatgtgttcc agaccagggc tggctgtctg attggagcag agcatgtgaa caactcctat 1980

gagtgtgaca tcccaattgg agcaggcatc tgtgcctcct accagaccca gaccaacagc 2040

ccaaggaggg caaggtctgt ggcaagccag agcatcattg cctacacaat gagtctggga 2100

gcagagaact ctgtggctta cagcaacaac agcattgcca tcccaaccaa cttcaccatc 2160

tctgtgacca cagagattct gcctgtgagt atgaccaaga cctctgtgga ctgtacaatg 2220

tatatctgtg gagacagcac agagtgtagc aacctgctgc tccaatatgg ctccttctgt 2280

acccaactta acagggctct gacaggcatt gctgtggaac aggacaagaa cacccaggag 2340

gtgtttgccc aggtgaagca gatttacaag acacctccaa tcaaggactt tggaggcttc 2400

aacttcagcc agattctgcc tgacccaagc aagccaagca agaggtcctt cattgaggac 2460

ctgctgttca acaaggtgac cctggctgat gctggcttca tcaagcaata tggagactgt 2520

ctgggagaca ttgctgccag ggacctgatt tgtgcccaga agttcaatgg actgacagtg 2580

ctgcctccac tgctgacaga tgagatgatt gcccaataca cctctgccct gctggctggc 2640

accatcacct ctggctggac ctttggagca ggagcagccc tccaaatccc atttgctatg 2700

cagatggctt acaggttcaa tggcattgga gtgacccaga atgtgctcta tgagaaccag 2760

aaactgattg ccaaccagtt caactctgcc attggcaaga ttcaggactc cctgtccagc 2820

acagcctctg ccctgggcaa actccaagat gtggtgaacc agaatgccca ggctctgaac 2880

accctggtga agcaactttc cagcaacttt ggagccatct cctctgtgct gaatgacatc 2940

ctgagcagac tggacaaggt ggaggctgag gtccagattg acagactgat tacaggcaga 3000

ctccaatccc tccaaaccta tgtgacccaa caacttatca gggctgctga gattagggca 3060

tctgccaacc tggctgccac caagatgagt gagtgtgtgc tgggacaaag caagagggtg 3120

gacttctgtg gcaagggcta ccacctgatg agttttccac agtctgcccc tcatggagtg 3180

gtgttcctgc atgtgaccta tgtgcctgcc caggagaaga acttcaccac agcccctgcc 3240

atctgccatg atggcaaggc tcactttcca agggagggag tgtttgtgag caatggcacc 3300

cactggtttg tgacccagag gaacttctat gaaccacaga ttatcaccac agacaacacc 3360

tttgtgtctg gcaactgtga tgtggtgatt ggcattgtga acaacacagt ctatgaccca 3420

ctccaacctg aactggactc cttcaaggag gaactggaca aatacttcaa gaaccacacc 3480

agccctgatg tggacctggg agacatctct ggcatcaatg cctctgtggt gaacatccag 3540

aaggagattg acagactgaa tgaggtggct aagaacctga atgagtccct gattgacctc 3600

caagaactgg gcaaatatga acaatacatc aagtggccac atcatcacca ccatcactaa 3660

<210> 5

<211> 1214

<212> PRT

<213> S-ECD

<400> 5

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

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

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

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

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

675 680 685

Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

705 710 715 720

Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

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

965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu

1010 1015 1020

Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val

1025 1030 1035 1040

Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala

1045 1050 1055

Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu

1060 1065 1070

Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His

1075 1080 1085

Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val

1090 1095 1100

Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr

1105 1110 1115 1120

Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr

1125 1130 1135

Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu

1140 1145 1150

Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp

1155 1160 1165

Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp

1170 1175 1180

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

1185 1190 1195 1200

Gln Glu Leu Gly Lys Tyr Glu Gln His His His His His His

1205 1210

<210> 6

<211> 1513

<212> DNA

<213> Furin gene (AA108-574) 【10】

<400> 6

tctagagaag cttgccacca tgccactgct gctactgctg cccctgctgt gggcaggggc 60

cctggctgga tccgacgtgt accaggagcc cacagacccc aagtttcctc agcagtggta 120

cctgtctggt gtcactcagc gggacctgaa tgtgaaggcg gcctgggcgc agggctacac 180

agggcacggc attgtggtct ccattctgga cgatggcatc gagaagaacc acccggactt 240

ggcaggcaat tatgatcctg gggccagttt tgatgtcaat gaccaggacc ctgaccccca 300

gcctcggtac acacagatga atgacaacag gcacggcaca cggtgtgcgg gggaagtggc 360

tgcggtggcc aacaacggtg tctgtggtgt aggtgtggcc tacaacgccc gcattggagg 420

ggtgcgcatg ctggatggcg aggtgacaga tgcagtggag gcacgctcgc tgggcctgaa 480

ccccaaccac atccacatct acagtgccag ctggggcccc gaggatgacg gcaagacagt 540

ggatgggcca gcccgcctcg ccgaggaggc cttcttccgt ggggttagcc agggccgagg 600

ggggctgggc tccatctttg tctgggcctc ggggaacggg ggccgggaac atgacagctg 660

caactgcgac ggctacacca acagtatcta cacgctgtcc atcagcagcg ccacgcagtt 720

tggcaacgtg ccgtggtaca gcgaggcctg ctcgtccaca ctggccacga cctacagcag 780

tggcaaccag aatgagaagc agatcgtgac gactgacttg cggcagaagt gcacggagtc 840

tcacacgggc acctcagcct ctgccccctt agcagccggc atcattgctc tcaccctgga 900

ggccaataag aacctcacat ggcgggacat gcaacacctg gtggtacaga cctcgaagcc 960

agcccacctc aatgccaacg actgggccac caatggtgtg ggccggaaag tgagccactc 1020

atatggctac gggcttttgg acgcaggcgc catggtggcc ctggcccaga attggaccac 1080

agtggccccc cagcggaagt gcatcatcga catcctcacc gagcccaaag acatcgggaa 1140

acggctcgag gtgcggaaga ccgtgaccgc gtgcctgggc gagcccaacc acatcactcg 1200

gctggagcac gctcaggcgc ggctcaccct gtcctataat cgccgtggcg acctggccat 1260

ccacctggtc agccccatgg gcacccgctc caccctgctg gcagccaggc cacatgacta 1320

ctccgcagat gggtttaatg actgggcctt catgacaact cattcctggg atgaggatcc 1380

ctctggcgag tgggtcctag agattgaaaa caccagcgaa gccaacaact atgggacgct 1440

gaccaagttc accctcgtac tctatggcac cgccgctagc gactacaaag acgacgatga 1500

caaataactc gag 1513

<210> 7

<211> 495

<212> PRT

<213> Furin gene (AA108-574) 【10】

<400> 7

Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala

1 5 10 15

Gly Ser Asp Val Tyr Gln Glu Pro Thr Asp Pro Lys Phe Pro Gln Gln

20 25 30

Trp Tyr Leu Ser Gly Val Thr Gln Arg Asp Leu Asn Val Lys Ala Ala

35 40 45

Trp Ala Gln Gly Tyr Thr Gly His Gly Ile Val Val Ser Ile Leu Asp

50 55 60

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

65 70 75 80

Gly Ala Ser Phe Asp Val Asn Asp Gln Asp Pro Asp Pro Gln Pro Arg

85 90 95

Tyr Thr Gln Met Asn Asp Asn Arg His Gly Thr Arg Cys Ala Gly Glu

100 105 110

Val Ala Ala Val Ala Asn Asn Gly Val Cys Gly Val Gly Val Ala Tyr

115 120 125

Asn Ala Arg Ile Gly Gly Val Arg Met Leu Asp Gly Glu Val Thr Asp

130 135 140

Ala Val Glu Ala Arg Ser Leu Gly Leu Asn Pro Asn His Ile His Ile

145 150 155 160

Tyr Ser Ala Ser Trp Gly Pro Glu Asp Asp Gly Lys Thr Val Asp Gly

165 170 175

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

180 185 190

Arg Gly Gly Leu Gly Ser Ile Phe Val Trp Ala Ser Gly Asn Gly Gly

195 200 205

Arg Glu His Asp Ser Cys Asn Cys Asp Gly Tyr Thr Asn Ser Ile Tyr

210 215 220

Thr Leu Ser Ile Ser Ser Ala Thr Gln Phe Gly Asn Val Pro Trp Tyr

225 230 235 240

Ser Glu Ala Cys Ser Ser Thr Leu Ala Thr Thr Tyr Ser Ser Gly Asn

245 250 255

Gln Asn Glu Lys Gln Ile Val Thr Thr Asp Leu Arg Gln Lys Cys Thr

260 265 270

Glu Ser His Thr Gly Thr Ser Ala Ser Ala Pro Leu Ala Ala Gly Ile

275 280 285

Ile Ala Leu Thr Leu Glu Ala Asn Lys Asn Leu Thr Trp Arg Asp Met

290 295 300

Gln His Leu Val Val Gln Thr Ser Lys Pro Ala His Leu Asn Ala Asn

305 310 315 320

Asp Trp Ala Thr Asn Gly Val Gly Arg Lys Val Ser His Ser Tyr Gly

325 330 335

Tyr Gly Leu Leu Asp Ala Gly Ala Met Val Ala Leu Ala Gln Asn Trp

340 345 350

Thr Thr Val Ala Pro Gln Arg Lys Cys Ile Ile Asp Ile Leu Thr Glu

355 360 365

Pro Lys Asp Ile Gly Lys Arg Leu Glu Val Arg Lys Thr Val Thr Ala

370 375 380

Cys Leu Gly Glu Pro Asn His Ile Thr Arg Leu Glu His Ala Gln Ala

385 390 395 400

Arg Leu Thr Leu Ser Tyr Asn Arg Arg Gly Asp Leu Ala Ile His Leu

405 410 415

Val Ser Pro Met Gly Thr Arg Ser Thr Leu Leu Ala Ala Arg Pro His

420 425 430

Asp Tyr Ser Ala Asp Gly Phe Asn Asp Trp Ala Phe Met Thr Thr His

435 440 445

Ser Trp Asp Glu Asp Pro Ser Gly Glu Trp Val Leu Glu Ile Glu Asn

450 455 460

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

465 470 475 480

Leu Tyr Gly Thr Ala Ala Ser Asp Tyr Lys Asp Asp Asp Asp Lys

485 490 495

Claims (7)

1. A method for preparing a novel crown spike protein in a post-fusion state is characterized in that: the spike protein in the native prefusion state is cleaved with furin.

2. Application of the new coronary spike protein in post-fusion state in preparing angiotensin converting enzyme 2 binding agent.

3. Use according to claim 2, characterized in that: the new crown spike protein in post-fusion state is used as the new crown virus treating target.

4. Use according to claim 3, characterized in that: the new coronary spike protein in post-fusion state is used as the target of new coronary virus vaccine.

5. Use according to claim 3, characterized in that: the new crown spike protein in post-fusion state is used as the target of new crown virus neutralizing antibody.

6. Use according to claim 2, characterized in that: the new crown spike protein in post-fusion state is used as immunogen of new crown virus.

7. Use according to any one of claims 2 to 6, characterized in that: the method for preparing the novel crown spike protein in post-fusion state is as described in claim 1.

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