CN113429476A - Dual-function fusion protein combined with coronavirus, preparation method and application thereof - Google Patents

Dual-function fusion protein combined with coronavirus, preparation method and application thereof Download PDF

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CN113429476A
CN113429476A CN202010208906.8A CN202010208906A CN113429476A CN 113429476 A CN113429476 A CN 113429476A CN 202010208906 A CN202010208906 A CN 202010208906A CN 113429476 A CN113429476 A CN 113429476A
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coronavirus
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glu
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王皓
钱卫珠
张大鹏
李晶
陶静
李伟
李继阳
张浔敏
李云峰
袁秀珍
焦树阁
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Taizhou Mabtech Pharmaceutical Co ltd
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract

The invention discloses a dual-function fusion protein combined with coronavirus, wherein the combining areas at two ends of the fusion protein are respectively combined with different epitopes of coronavirus S protein (Spike protein), and the fusion protein simultaneously has the functions of resisting SARS-CoV and SARS-CoV-2 and is combined with ACE 2. The difunctional fusion protein combined with coronavirus provided by the invention has the capability of treating virus infected cells, and also has the effects of resisting virus, reducing lung inflammation, reducing cytokine storm and treating lung injury.

Description

Dual-function fusion protein combined with coronavirus, preparation method and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly discloses a bifunctional fusion protein combined with coronavirus, and a preparation method and application thereof.
Background
2019-novel coronavirus (2019 novel coronavirus, 2019-nCoV, SARS-CoV-2) is an enveloped positively charged single-stranded RNA virus, and belongs to the genus of beta coronavirus and the subgenus Sarbecovirus. After a person is infected with SARS-CoV-2 (2019-nCoV, SARS-CoV-2), most patients can have symptoms such as fever, cough, vomiting, diarrhea, mild pneumonia and the like, some patients can have severe pneumonia, and patients with severe illness can have acute respiratory failure, shock and even death.
In the whole process of SARS-CoV-2 (2019-nCoV, SARS-CoV-2) infection and disease, an important metalloprotease-angiotensin converting enzyme 2 (ACE2) in human body plays an important role (Lu R, ZHao X, Li j,et al. Genomic characterization and epidemiology of 2019 novel coronavirus：implications for virus origins and receptor binding [J]. Lancet，2020.[Epub ahead of print]）。
ACE2 is a homologue of angiotensin converting enzyme, is I-type transmembrane protein composed of 805 amino acids, has 2 domains, namely an amino terminal catalytic domain and a carboxyl terminal domain, the catalytic structure contains 1 active site-zinc metalloprotein domain, and ACE2 can be expressed in various tissues of human heart, kidney, lung, liver, testis, intestine and the like.
The role of ACE2 in the course of new coronary pneumonia, in addition to acting as a receptor for viral binding, ACE2 is involved in mediating the immune process. The expression of ACE2 in lung adenocarcinoma tissues is increased, which indicates that the patients possibly have higher susceptibility to new coronavirus, and possibly miR-125b-5p-ACE2-IL6 axis further possibly causes the phenomena of cytokine storm and pneumonia aggravation. Meanwhile, the Expression of ACE2 can be further increased after the infection of new Coronavirus, thereby being more beneficial to the infection of virus (Wang, P.H. (2020). Advance Host Cellular Receptor-Angiotensin-Converting Enzyme 2 (ACE2) Expression by Coronavir major efficacy 2019-nCoV infection. bioRxiv.).
Angiotensin converting enzyme 2 (ACE2) is a double-edged sword: in the process of virus infection, it is a membrane-bound receptor of new coronavirus, and is related to the fusion and invasion process of virus and host cells; under the conditions of inflammation and acute lung injury caused by influenza virus and SARS, ACE2 promotes the conversion of angiotensin II into angiotensin 1-7, and inhibits the NF-kB signal mediated by angiotensin II, thereby avoiding cytokine storm caused by a large amount of inflammation signals and slowing down the course of disease.
Previous studies found that once bound by SARS-CoV, the amount of ACE2, mRNA levels, and enzyme activity at the surface of host cells were significantly reduced. Although this may reduce viral entry, it may also exacerbate the inflammatory response.
Interleukin-6 (Interleukin-6; IL-6) is the major factor responsible for cytokine storm, and after infection with SARS-CoV-2 (2019-nCoV), activated pathogenic T cells produce granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-6. GM-CSF can activate CD14 + CD16 + inflammatory monocytes, further producing more cytokines, including IL-6. IL-6 plays an integral part in this positive feedback cycle, eventually contributing to the loss of control of the immune system.
At this time, a large amount of immune cells and interstitial fluid in the lung may block the exchange of gas between alveoli and capillaries, resulting in acute respiratory distress syndrome. Once a cytokine storm is formed, the immune system can kill many normal cells in the lung while removing viruses, thereby seriously destroying the ventilation function of the lung and further leading to death of the patient
Research shows that the expression of ACE2 of a lung cancer patient is up-regulated, and the susceptibility of new coronary pneumonia is increased; however, the role of ACE2 in different stages of the course of new coronary pneumonia is different, and despite mediating viral invasion, maintaining normal expression of ACE2 plays an important role in avoiding cytokine storm. (the Long Chen group of the chemical company, et al. Lung Adenocarcinoma Patients Own Higher Risk of SARS-CoV-2 Infection. Preprints. 26 Feb, 2020.）
SARS-CoV-2 (2019-nCoV) uses angiotensin converting enzyme 2 (ACE2) as the entry receptor, as does SARS coronavirus, both of which are bound to ACE2 via the S protein (SPIKE protein) on the virion (viral SPIKE glycoprotein), the viral membrane and cell membrane fuse, and then the RNA virus will replicate its gene intracellularly and eventually produce new viral particles that will be secreted to infect other cells. (Zhou P, Yang XL, Wang XG,et al：Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin. BioRxiv. 2020.01.22.914852）。
the small receptor binding domain of the RBD (receptor-binding domain) SARS virus S protein (Spike protein) has been shown to be a key domain for binding of the virus to the ACE2 receptor during cell entry, and the use of this binding domain has been shown to be effective in preventing SARS entry into cells for proliferation. (Wong SK, Li W, Moore MJ,et al：A193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. J Biol Chem. 2004; 279(5):3197-3201）。
in a method of coronavirus treatment, if an antibody that binds to ACE2 protein is used, it binds to normal cells expressing ACE2 in the human body and causes an inflammatory response in normal tissues. The use of ACE2-Fc, which binds to the S protein of coronavirus (Spike protein), prevents binding of coronavirus to ACE2, further preventing entry of coronavirus into the cell.
However, one potential limitation of the ACE2-Fc strategy is that an increase in extracellular ACE2 levels may have an unknown effect on the human body, which has been found to secrete small amounts of extracellular ACE2, and thus entry of this extracellular domain into the blood circulation is not unprecedented. (Shao Z, Shrestha K, Borowski AG,et al：Increasing serum soluble angiotensin-converting enzyme2 activity after intensive medical therapy is associated with better prognosis in acute decompensated heart failure. J Card Fail2013, 19(9) 605-610). After the recombinant human ACE2 (rhACE 2) protein with activity is applied to an acid-damaged ACE2 gene knockout mouse and a wild-type mouse, acute lung injury symptoms such as pulmonary edema and the like are improved (Imai, Y., Kuba, K., Rao, S., Huan, Y., Guo, F., Guan, B.&Crackower, M.A. (2005). Angiotensin-converting enzyme 2 technologies from segment acid failure. Nature, 436(7047), 112-. In animals and in phase ii clinical studies with rhACE2 injection treatment of diagnosed Acute Respiratory Distress Syndrome (ARDS) patients, rapid decreases in Ang ii levels and increases in Ang1-7 levels were found, preliminary showing that supplementation with ACE2 in humans can remodel the ACE2/ACE balance (Khan, a., Benthin, c.& Hardes, K. (2017). A pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome. Critical Care, 21(1), 234. Treml, B., Neu, N., Kleinsasser, A., Gritsch, C., Finsterwalder, T., Geiger, R..&Loeckinger, a. (2010), Recombinant angiotensin-converting enzyme 2 improves pulmonary blood flow and oxidation in lipid polysaccharide-induced lung in peptides, clinical care media, 38(2), 596), ACE2 has the potential to reduce pulmonary inflammation and has tolerable safety.
Antiviral S glycoprotein IgG disturbs the macrophage response during acute SARS-CoV infection resulting in severe acute lung injury. The use of S-IgG, although significantly reducing viral titers, caused lung injury at the early stages of infection, primarily by abrogating the macrophage response and TGF- β production of wound healing, while promoting the production of the inflammatory cytokine IL-8, and MCP1 and the accumulation of inflammatory macrophages. (Anti-spike IgG mice lung acid not in the road by skin with capillary stress reducing acid SARS-CoV infection. [ J ]. JCI inertia, 2019, 4(4): e 123158.).
After the outbreak of SARS (Severe Acute Respiratory Syndrome), various therapeutic approaches have emerged, along with the production of drugs against SARS, including antibody-based drugs. In patent US11667640 a composition of binding molecules capable of neutralizing the SARS coronavirus (SARS-CoV) is disclosed, said composition comprising at least two immunoglobulins capable of binding to the SARS-associated coronavirus (SARS-CoV) and having SARS-CoV neutralizing activity.
In the patent US11667640, an antibody capable of binding to ASRS coronavirus is disclosed, and it is disclosed that the antibody binds to amino acid 318-510 of the S protein (Spike protein) of SARS-CoV. Although the drug described in this patent is a composition, the antibodies constituting the composition are all antibodies directed to the amino acids at position 318-510 of the S protein (Spike protein) of SARS-CoV, and even if the specific binding site of each antibody is different amino acids at position 318-510, there are disadvantages in that they affect each other.
Because the antibody belongs to macromolecular protein and because of the characteristics of its space structure, when the antibody and antigen are combined together, the mutual influence between antibody can be existed, the compositions provided by said patent are all the amino acids combined in 318-th and 510-th positions of S protein (Spike protein) of SARS-CoV, when they are combined with virus, the action effect of antibody neutralization SARS-CoV coronavirus can be influenced.
By 2019-new coronavirus (2019 novel coronavirus, 2019-nCoV, SARS-CoV-2) outbreak, multi-scientists are researching the infection route of SARS-CoV-2, Chinese scientist Chen Shinan and the team thereof, and through research, SARS-CoV-2 can invade host cells through a new route of CD 147: s eggThe white blood (Spike protein) binds to the receptor CD147 on the host cell, mediating viral invasion (zhinan. Chen, ping. zhu,et al：SARS-CoV-2 invades host cells via a novel route: CD147-spike protein，BioRxiv）。
disclosure of Invention
In order to solve the technical problems of the prior art, the present invention provides a bifunctional fusion protein specifically binding to coronavirus.
The dual-function fusion protein specifically combined with coronavirus has the functions of resisting SARS-CoV and SARS-CoV-2, has Fc function effect, and has the capability of treating virus infected cells, and also has the functions of resisting virus, reducing lung inflammation and reducing cell factor storm.
The difunctional fusion protein specifically binding to coronavirus provided by the invention has a wide neutralization function, has the capability of neutralizing SARS-CoV (SARS-like CoV) related to animals, also plays a neutralization function in the treatment of undiscovered viruses similar to SARS-CoV or SARS-CoV-2, can be used for virus treatment which has been discovered to use ACE2 as a receptor at present and treatment of future novel viruses which have not been discovered to use ACE2 as a receptor, and also has a treatment function on lung injury diseases mediated by an ACE2 axis.
The two-end binding region of the bifunctional fusion protein specifically binding to coronavirus provided by the invention is respectively bound to different epitopes of coronavirus S protein (Spike protein), and the bifunctional fusion protein specifically binding to coronavirus has a special structure, so that after being bound to coronavirus S protein (Spike protein), a steric hindrance effect is generated, other binding sites where coronavirus S protein (Spike protein) can be bound to host cells are blocked, and other ways for coronavirus to invade the host cells through the binding of S protein (Spike protein) are blocked, such as: the CD147 receptor binding pathway (binding of the coronavirus S protein to the receptor CD147 on the host cell, thereby invading the host cell).
The difunctional fusion protein specifically binding to the coronavirus can block the binding of the coronavirus to host cells in multiple ways, effectively reduce the invasion and infection of the coronavirus, resist the virus, reduce the pulmonary inflammation, reduce the cytokine storm and effectively treat diseases caused by the coronavirus.
The structure diagram of the dual-function fusion protein specifically binding to coronavirus provided by the invention is shown in figure 1, and the fusion protein has a coronavirus binding end resisting SARS-CoV or SARS-CoV-2 and an end with ACE 2.
The invention provides a difunctional fusion protein specifically binding to coronavirus, wherein the Fab end of an anti-SARS-CoV or SARS-CoV-2 antibody can be combined with S protein (Spike protein) of coronavirus, and the combination site is different from the combination site of ACE2 and cannot competitively bind to RBD of coronavirus with ACE 2. The Fab end of the anti-SARS-CoV or SARS-CoV-2 antibody can synergistically enhance the binding of ACE2 and coronavirus RBD, enhancing antiviral activity while avoiding possible viral enhancement.
The dual-function fusion protein specifically binding to coronavirus provided by the invention has the advantages that one end of ACE2 can be bound to coronavirus, ACE2 on the cell surface can be protected from being bound by coronavirus, the normal action of ACE2 is not influenced, certain active ACE2 is supplemented, cytokine storm can be reduced and cannot be caused, inflammatory factors are prevented from being generated, the effect of reducing inflammation is achieved, the ACE2 binding path of coronavirus and normal cells is blocked, inflammation caused by ACE2 reduction can be relieved, pneumonia caused by the ACE2 path is avoided, the pneumonia symptoms of coronavirus infected persons are relieved, and the cytokine storm effect is reduced.
The above mentioned bifunctional fusion protein specifically binding to coronavirus is bound to coronavirus S protein (Spike protein) via different binding sites, and due to its specific structure, it generates steric hindrance effect, blocks other binding sites where coronavirus S protein can bind to host cell, and blocks other routes of invasion of coronavirus into host cell via S protein binding, for example: the CD147 receptor binding pathway (binding of the coronavirus S protein to the receptor CD147 on the host cell, thereby invading the host cell).
Moreover, the antibody part of the bifunctional fusion protein specifically binding to coronavirus provided by the invention can target a target molecule to the virus, and can reduce the risk of blood pressure reduction caused by using ACE2 alone. ACE2 binds to the S1, S2 interface of SARS coronavirus (Aiping Wu, Peihua Niu, Lulan Wang, et al. bioRxivMarch 02, 2020）。
Currently, SARS-CoV-2 viruses of Wuhan, Shenzhen, hong Kong and France in China are detected, and the phenomenon that RBD (receptor-binding domain) of SARS-CoV-2 has mutation is found, but the mutant virus strains have stronger ACE2 binding capacity (Junxian Ou, Zhonghua Zhou,et al：RBD mutations from circulating SARS-CoV-2 strains enhance the structure stability and infectivity of the spike protein. bioRxiv). As mentioned above, the bifunctional fusion protein specifically binding to coronavirus has the characteristics of blocking the ACE2 pathway of coronavirus and has stronger advantages than the ACE2-Fc alone, so that the bifunctional fusion protein specifically binding to coronavirus provided by the invention can more effectively cope with mutant virus strains of coronavirus.
The difunctional fusion protein specifically binding to coronavirus provided by the invention can be effectively used as a drug stock for treating SARS and SARS-CoV-2 (2019-nCoV) and similar virus outbreak in the future, and can also be used for treating any other novel viruses entering cells through ACE2 receptors in the future. And can be used as passive immunoprotection agent for dangerous people, and can be used for preventing SARS and SARS-CoV-2 related diseases.
The invention discloses a bifunctional fusion protein specifically binding to coronavirus, which is respectively bound to different binding sites of coronavirus.
The bifunctional fusion protein as described above specifically binds to a coronavirus having a binding site for a Spike protein.
A bifunctional fusion protein as described above which specifically binds to a coronavirus having an ACE2 binding site.
The bifunctional fusion protein specifically binding to coronavirus as described above, which binds to ACE2 binding site of coronavirus, and binds to Spike protein of coronavirus at the same time.
A bifunctional fusion protein as described above that specifically binds to coronavirus having the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof.
The bifunctional fusion protein specifically binding to coronavirus as described above, having the amino acid sequence of SEQ ID NO: 6 and the amino acid sequence of the heavy chain shown in SEQ ID NO: 2, or a light chain amino acid sequence set forth in seq id no.
The invention discloses a composition, which comprises the difunctional fusion protein specifically binding to coronavirus and a pharmaceutically acceptable excipient.
The invention discloses a preparation method of a difunctional fusion protein specifically binding to coronavirus, which comprises the following steps: a) synthesizing gene sequence of the dual-function fusion protein specifically combined with coronavirus to prepare an expression vector containing the dual-function fusion protein gene; b) transfecting the expression vector to a host cell, culturing the host cell and expressing the bifunctional fusion protein; c) and (3) separating and purifying the bifunctional fusion protein.
The method for preparing the bifunctional fusion protein capable of specifically binding to coronavirus is characterized in that the host cell is eukaryotic mammalian cell CHO cell.
The method for preparing the bifunctional fusion protein capable of specifically binding to coronavirus is characterized in that the host cell is cultured in a serum-free medium without animal-derived components.
The invention discloses application of a bifunctional fusion protein specifically binding to coronavirus, which comprises application of the bifunctional fusion protein specifically binding to coronavirus in preparation of an anti-coronavirus drug.
Use of a bifunctional fusion protein as described above which specifically binds to a coronavirus having the ACE2 infection pathway.
The application of the dual-function fusion protein specifically binding to coronavirus is characterized in that the coronavirus is SARS-CoV related coronavirus, MERS-CoV related coronavirus or SARS-CoV-2 related virus.
Drawings
FIG. 1 is a schematic structural diagram of a bifunctional fusion protein that specifically binds to coronavirus;
FIG. 2, CoV022 light chain full-length expression vector construction diagram;
FIG. 3, construction diagram of CoV022 heavy chain variable region cloning vector;
FIG. 4, CoV022 heavy chain full-length expression vector construction diagram;
FIG. 5 is a diagram of construction of CoV022 light chain and heavy chain full-length single plasmid expression vectors;
FIG. 6, construction diagram of ACE2-609-Fc fusion protein cloning vector;
FIG. 7 shows construction diagrams of ACE2-609-Fc fusion protein expression vectors;
FIG. 8, construction diagram of ACE2-615-Fc fusion protein cloning vector;
FIG. 9, construction diagram of ACE2-615-Fc fusion protein expression vector;
FIG. 10 shows construction of ACE2-609-Fc fusion protein (Fc in Hole) expression vector;
FIG. 11 is a drawing showing construction of an ACE2-615-Fc fusion protein (Fc in Hole) expression vector;
FIG. 12, construction of cloning vector for the variable region of the CoV022 heavy chain (Knob);
FIG. 13, construction of CoV022 heavy chain full length (Knob) expression vector;
FIG. 14 depicts the construction of individual plasmids of CoV022 light chain, heavy chain full length (Knob) expression vectors;
FIG. 15, SDS-PAGE detection profile of CHO host cells expressing ACE2-609-Fc, CoV022, ACE2-609-Fc/CoV022 bifunctional fusion proteins;
FIG. 16, SDS-PAGE detection profile of CHO host cells expressing ACE2-615-Fc, CoV022, ACE2-615-Fc/CoV022 bifunctional fusion proteins;
FIG. 17, CoV022, ACE2-609-Fc, ACE2-609-Fc/CoV022 bifunctional fusion protein and SARS-CoV-2 Spike protein binding ability ELISA detection map;
FIG. 18, CoV022, ACE2-615-Fc, ACE2-615-Fc/CoV022 bifunctional fusion protein and SARS-CoV-2 Spike protein binding ability ELISA detection map.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
Example 1 Gene Synthesis of bifunctional fusion proteins that specifically bind to coronavirus
(1) Construction of expression vector for antibody binding to coronavirus S protein (Spike protein)
CoV022 is an antibody that binds to the coronavirus S protein (Spike protein).
Construction of full-length expression vector for CoV022 light chain:
the CoV022 light chain full-length cloning vector is subjected to double enzyme digestion by HindIII and EcoR1 through the whole gene synthesis, a pL101 expression vector is subjected to double enzyme digestion, a target fragment of about 750bp and an expression vector fragment of about 9kb are respectively recovered through gel extraction, the target fragment and the expression vector fragment are connected and transformed by T4 DNA ligase, and the plasmid is extracted in a small amount and is subjected to double enzyme digestion identification by HindIII and EcoR 1.
The full length of the CoV022 light chain has SEQ ID NO: 1, SEQ ID NO: 2.
As shown in fig. 2: the agarose concentration was 1%, and the arrow indicates the right 1: DL2000 DNA Marker, right 2: lambda-HindIII digest DNA Marker.
Construction of CoV022 heavy chain full-length cloning vector:
the CoV022 heavy chain variable region cloning vector is subjected to double enzyme digestion by HindIII and NheI through the whole gene synthesis, meanwhile, an IgG1 constant region gene cloning vector is subjected to double enzyme digestion, about 450bp of target fragments and about 4kb of cloning vector fragments are respectively recovered through gel extraction, the target fragments and the cloning vector fragments are connected and transformed by T4 DNA ligase, and plasmids are extracted in small quantity and identified by double enzyme digestion by HindIII and NheI.
As shown in fig. 3: the agarose concentration was 1%, and the arrow indicates the fragment of interest of about 450bp, right 1: DL2000 DNA Marker, left 1: lambda-HindIII digest DNA Marker.
Construction of the CoV022 heavy chain full-length expression vector:
the CoV022 heavy chain full-length cloning vector is subjected to double digestion by HindIII and EcoR1, meanwhile, the pL102 expression vector is subjected to double digestion, about 1500bp of target fragments and about 5kb of expression vector fragments are respectively recovered by glue, the target fragments and the about 5kb expression vector fragments are connected and transformed by T4 DNA ligase, and plasmids are extracted in small quantity and subjected to double digestion identification by HindIII and EcoR 1.
As shown in fig. 4: the agarose concentration was 1%, and the arrow indicates the fragment of interest of about 1500bp, right 1: DL2000 DNA Marker, right 2: lambda-HindIII digest DNA Marker.
The full length of the CoV022 heavy chain has SEQ ID NO: 3, SEQ ID NO: 4.
Construction of CoV022 light chain and heavy chain full-length single plasmid expression vector:
the CoV022 light chain full-length expression vector and the CoV022 heavy chain full-length expression vector are subjected to double enzyme digestion by SalI and NotINotI respectively, about 9kb light chain full-length expression fragments and about 4kb heavy chain full-length expression vector fragments are recovered respectively, the two fragments are connected and transformed by T4 DNA ligase, a small amount of plasmid is extracted and is subjected to double enzyme digestion identification by SalI and NotINotI.
As shown in fig. 5: agarose concentration is 1%, arrows indicate the full-length light chain expression vector fragment of about 9kb and the full-length target fragment of heavy chain around 4kb, right 1: DL2000 DNA Marker, right 2: lambda-HindIII digest DNA Marker.
(2) Construction of fusion protein expression vector binding to ACE2 binding site of coronavirus
a) Construction of expression vector of ACE2-609-Fc fusion protein
Construction of ACE2-609-Fc fusion protein cloning vector:
the ACE2-609 cloning vector is synthesized by whole gene and is double-digested by HindIII and NheI, meanwhile, the Fc/pGEM-T cloning vector is double-digested, about 2.0kb target fragment and about 3.7kb Fc cloning vector fragment are respectively recovered, the target fragment and the Fc cloning vector fragment are connected and transformed by T4 DNA ligase, a small amount of plasmid is extracted and identified by double-digestion of HindIII and NheI.
As shown in fig. 6: the agarose concentration was 1%, and the arrow indicates an about 2.0kb fragment of interest, right 1: DL2000 DNA Marker, right 2: lambda-HindIII digest DNA Marker.
Construction of an ACE2-609-Fc fusion protein expression vector:
the cloning vector of ACE2-609-Fc/pGEM-T is subjected to double digestion by HindIII and EcoR1, the pL101 expression vector is subjected to double digestion, about 2.7kb target fragment and about 9kb expression vector fragment are respectively recovered by gel, the target fragment and the expression vector fragment are connected and transformed by T4 DNA ligase, a small amount of plasmid is extracted, and the cloning vector is subjected to double digestion identification by HindIII and EcoR 1.
ACE2-609-Fc has the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or a pharmaceutically acceptable salt thereof.
As shown in fig. 7: the agarose concentration was 1%, and the arrow indicates an about 2.7kb fragment of interest, right 1: DL2000 DNA Marker, right 2: lambda-HindIII digest DNA Marker.
b) Construction of expression vector of ACE2-615-Fc fusion protein
Construction of an ACE2-615-Fc fusion protein cloning vector:
the ACE2-615 cloning vector is synthesized by whole gene and is double digested by HindIII and NheI, and simultaneously the Fc/pGEM-T cloning vector is double digested, about 2.0kb target fragment and about 3.7kb Fc cloning vector fragment are respectively recovered, the two are connected and transformed by T4 DNA ligase, plasmid is extracted in small quantity and identified by HindIII and NheI double digestion.
As shown in fig. 8: the agarose concentration was 1%, and the arrow indicates an about 2.0kb fragment of interest, right 1: DL2000 DNA Marker, right 2: lambda-HindIII digest DNA Marker.
Construction of an ACE2-615-Fc fusion protein expression vector:
the cloning vector of ACE2-615-Fc/pGEM-T is double-digested by HindIII and EcoR1, the pL101 expression vector is double-digested simultaneously, about 2.7kb target fragment and about 9kb expression vector fragment are recovered respectively, the target fragment and the expression vector fragment are connected and transformed by T4 DNA ligase, the plasmid is extracted in small quantity and identified by double-digestion of HindIII and EcoR 1.
ACE2-615-Fc has the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.
As shown in fig. 9: the agarose concentration was 1%, and the arrow indicates an about 2.7kb fragment of interest, right 1: DL2000 DNA Marker, right 2: lambda-HindIII digest DNA Marker.
(3) Construction of ACE2-609-Fc, ACE2-615-Fc fusion protein and CoV022 bifunctional fusion protein expression vector (Knob in Hole)
Construction of ACE2-609-Fc fusion protein (Fc in Hole) expression vector:
the ACE2-609-Fc cloning vector is subjected to double digestion by HindIII and EcoR1, the pL101 expression vector is subjected to double digestion, about 2.7kb target fragment and about 9kb expression vector fragment are recovered respectively, the target fragment and the expression vector fragment are connected and transformed by T4 DNA ligase, and the plasmid is extracted in a small amount and identified by double digestion by HindIII and EcoR 1.
ACE2-609-fc (fc in hole) has the sequence of SEQ ID NO: 11, SEQ ID NO: 12.
As shown in fig. 10: the agarose concentration was 1%, and the arrow indicates an about 2.7kb fragment of interest, right 1: DL2000 DNA Marker, right 2: lambda-HindIII digest DNA Marker.
Construction of ACE2-615-Fc fusion protein (Fc in Hole) expression vector
The ACE2-615 fragment is synthesized by whole gene and is double-digested by HindIII and EcoR1, the pL101 expression vector is double-digested simultaneously, about 2.0kb target fragment and about 9kb expression vector fragment are recovered respectively, the target fragment and the expression vector fragment are connected and transformed by T4 DNA ligase, the plasmid is extracted in small quantity and is identified by double-digestion of HindIII and EcoR 1.
ACE2-615-fc (fc in hole) has the sequence of SEQ ID NO: 13, SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof.
As shown in fig. 11: the agarose concentration was 1%, and the arrow indicates an about 2.0kb fragment of interest, right 1: DL2000 DNA Marker, right 2: lambda-HindIII digest DNA Marker.
Construction of the CoV022 heavy chain full-length cloning vector (Knob):
the CoV022 heavy chain variable region cloning vector is subjected to double enzyme digestion by HindIII and NheI through a whole gene synthesis method, meanwhile, an IgG1 constant region cloning vector (Knob) is subjected to double enzyme digestion, about 450bp of target fragments and about 4kb of cloning vector fragments are respectively recovered through gel extraction, the target fragments and the cloning vector fragments are connected and transformed by T4 DNA ligase, a small amount of plasmid is extracted, and the cloning vector is subjected to double enzyme digestion identification by HindIII and NheI.
As shown in fig. 12: the agarose concentration was 1%, and the arrow indicates the fragment of interest of about 450bp, right 1: DL2000 DNA Marker, left 1: lambda-HindIII digest DNA Marker.
Construction of full-length expression vector for the CoV022 heavy chain (Knob):
the CoV022 heavy chain full-length (Knob) cloning vector is double-digested by HindIII and EcoR1, meanwhile, a pL102 expression vector is double-digested, about 1500bp of target fragments and about 5kb of expression vector fragments are respectively recovered by glue, the target fragments and the expression vector fragments are connected and transformed by T4 DNA ligase, a small amount of plasmid is extracted and is double-digested by HindIII and EcoR1 for identification.
The full length of the CoV022 heavy chain (Knob) has the sequence of SEQ ID NO: 5, SEQ ID NO: 6.
As shown in fig. 13: the agarose concentration was 1%, and the arrow indicates the fragment of interest of about 1500bp, right 1: DL2000 DNA Marker, right 2: lambda-HindIII digest DNA Marker.
Construction of single plasmid for CoV022 light chain, heavy chain full length (Knob) expression vector:
the CoV022 light chain full-length expression vector and the CoV022 heavy chain full-length (knob) expression vector are subjected to double digestion by SalI and NotI respectively, about 9kb light chain full-length expression fragments and about 4kb heavy chain full-length expression vector fragments are recovered respectively, the light chain full-length expression fragments and the heavy chain full-length expression vector fragments are connected and transformed by T4 DNA ligase, and plasmids are extracted in a small quantity and are subjected to double digestion identification by SalI and NotI.
As shown in fig. 14: agarose concentration is 1%, arrows indicate the full-length light chain expression vector fragment of about 9kb and the full-length target fragment of heavy chain around 4kb, right 1: DL2000 DNA Marker, right 2: lambda-HindIII digest DNA Marker.
Example 2 preparation of bifunctional fusion proteins that specifically bind to coronavirus
(1) Preparation of bifunctional fusion protein specifically binding to coronavirus with ACE2-609-Fc/CoV 022:
purifying an expression vector of the CoV022, ACE2-609-Fc and ACE2-609-Fc/CoV022 bifunctional proteins by using a column, transiently transfecting the obtained plasmid to host cell Expi CHO-S cells, adding a feed medium for continuous culture for 8-12 days, and culturing the host cells and expressing target proteins by using a serum-free culture medium without animal-derived components; the supernatant of the host cell culture solution was collected, and separated and purified by using a Protein A affinity column.
SDS-PAGE confirms the molecular weight of the purified CoV022, ACE2-609-Fc and ACE2-609-Fc/CoV022 bifunctional proteins.
(2) Preparation of bifunctional fusion protein specifically binding to coronavirus with ACE2-615-Fc/CoV 022:
purifying an expression vector of the CoV022, ACE2-615-Fc and ACE2-615-Fc/CoV022 bifunctional proteins by using a column, transiently transfecting the obtained plasmid to host cell Expi CHO-S cells, adding a feed medium, continuously culturing for 8-12 days, collecting the supernatant of the culture solution of the host cells, and separating and purifying by using a Protein A affinity chromatography column.
SDS-PAGE confirms the molecular weight of the purified CoV022, ACE2-615-Fc and ACE2-615-Fc/CoV022 bifunctional proteins.
The SDS-PAGE electrophoretograms of CoV022, ACE2-609-Fc, ACE2-615-Fc, ACE2-609-Fc/CoV022 and ACE2-615-Fc/CoV022 are respectively shown in FIG. 15 and FIG. 16, wherein the sample sequence of FIG. 15 is as follows: 1. the sample in lane 2 is ACE2-609-Fc, the sample in lane 3 and 4 is CoV022, and the sample in lane 5 and 6 is ACE2-609-Fc/CoV 022; the sample sequence of fig. 16 is: 1. the sample in lane 2 is ACE2-615-Fc, the sample in lane 3 and 4 is CoV022, and the sample in lane 5 and 6 is ACE2-615-Fc/CoV 022.
Example 3 detection of the binding Capacity of the bifunctional fusion protein specifically binding to coronavirus
The ELISA detection method detects the binding capacity of the bifunctional fusion protein specifically binding to coronavirus and coronavirus protein:
adding SARS-CoV-2S protein (Spike protein) to the ELISA plate using the coating solution, and coating for 1 hour at 37 ℃; removing the coating solution, adding sealing solution, sealing at 37 deg.C for 1.5 hr at 350 μ l/hole; diluting the CoV022, ACE2-609-Fc, ACE2-609-Fc/CoV022 bifunctional proteins, and ACE2-615-Fc, ACE2-615-Fc/CoV022 bifunctional proteins with a diluent step by step, adding into an enzyme label plate, and carrying out water bath at 37 ℃ for 1 hour; washing for 5-7 times by a plate washing machine; diluting rabbit anti-human-HRP with diluent, adding into an enzyme label plate, and carrying out water bath at 37 ℃ for 1 hour; discarding the supernatant, and washing for 5-7 times by a plate washing machine; adding TMB color development liquid to prevent light for color development, adding equal volume of stop solution to stop color development, and rapidly mixing.
Reading by a microplate reader, and detecting results are shown in figure 17 and figure 18, wherein figure 17 is the binding capacity of the ELISA detection CoV022, ACE2-609-Fc, ACE2-609-Fc/CoV022 bifunctional protein and SARS-CoV-2S protein (Spike protein); FIG. 18 shows ELISA detection of binding ability of CoV022, ACE2-615-Fc, ACE2-615-Fc/CoV022 bifunctional protein and SARS-CoV-2S protein (Spike protein).
According to the experimental results, the bifunctional fusion proteins ACE2-609-Fc/CoV022 and ACE2-615-Fc/CoV022 which specifically bind to coronavirus all bind to ARS-CoV-2S protein (Spike protein), and the bifunctional fusion proteins ACE2-609-Fc/CoV022, ACE2-615-Fc/CoV022 and ARS-CoV-2S protein (Spike protein) all have stronger binding capacity than CoV022, ACE2-609-Fc or ACE2-615-Fc and ARS-CoV-2S protein (Spike protein).
Example 4 detection of the binding Capacity of the bifunctional fusion protein specifically binding to coronavirus
The affinity of CoV022, ACE2-609-Fc, ACE2-609-Fc/CoV022 bifunctional fusion proteins, ACE2-615-Fc, ACE2-615-Fc/CoV022 bifunctional fusion proteins to SARS-CoV-2S Protein was detected using SPR Surface Plasmon Resonance (SPR) technology.
ARS-CoV-2S Protein is coupled on the surface of a CM5 chip, then CoV022, ACE2-609-Fc, ACE2-609-Fc/CoV022 bifunctional fusion proteins, ACE2-615-Fc, ACE2-615-Fc/CoV022 bifunctional fusion proteins with sequentially increased concentrations are detected, and five samples and ARS-CoV-2S Protein are evaluated for affinity parameters. Regeneration treatment is performed between samples. Results were collected using a Biacore instrument and analyzed for data using a Biacore Evaluation software to evaluate affinity parameters (KD values) and the results are shown in table 1.
The results show that the affinity of the bifunctional fusion proteins ACE2-609-Fc/CoV022 and ACE2-615-Fc/CoV022 and ARS-CoV-2S protein (Spike protein) is higher than that of CoV022, ACE2-609-Fc or ACE2-615-Fc and ARS-CoV-2S protein (Spike protein), and the bifunctional fusion proteins ACE2-609-Fc/CoV022 and ACE2-615-Fc/CoV022 have better potential virus neutralization effects.
Table 1 shows the affinity results of the bifunctional fusion Protein for detecting ACE2-609-Fc, ACE2-615-Fc, CoV022, ACE2-609-Fc/CoV022 and ACE2-615-Fc/CoV022 and SARS-CoV-2S Protein by the SPR method.
TABLE 1 SPR measurement results
Sample (I) KD（M）
CoV022 6.198 × 10-9
ACE2-609-Fc 1.013× 10-8
ACE2-609-Fc/CoV022 1.276 × 10-9
ACE2-615-Fc 1.296× 10-8
ACE2-615-Fc/CoV022 1.411 × 10-9
Sequence listing
<110> Taizhou Mibo Taike pharmaceutical Co., Ltd
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Lys
<210> 7
<211> 2478
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 7
cagtccacca ttgaggaaca ggccaagaca tttttggaca agtttaacca cgaagccgaa 60
gacctcttct atcaaagttc acttgcttct tggaattata acaccaacat tactgaagag 120
aatgtccaaa acatgaataa tgctggggac aaatggtctg cctttctgaa ggaacagtcc 180
acacttgccc aaatgtatcc actgcaagaa attcagaatc tcacagtcaa gctccagctg 240
caagctcttc agcaaaatgg gtcttcagtg ctctcagaag acaagagcaa acggttgaac 300
acaattctca atacaatgag caccatctac agtactggaa aagtttgtaa cccagataat 360
ccacaagaat gcctgctcct tgaaccaggt ttgaatgaaa taatggcaaa cagtctggac 420
tacaatgaaa ggctctgggc ttgggaaagc tggagatccg aggtcggcaa gcagctgagg 480
ccactgtatg aagagtatgt ggtcctgaag aatgagatgg caagagcaaa tcattatgag 540
gactatgggg attattggag gggagactat gaagtaaatg gggtagatgg ctatgactac 600
agccgcggcc agttgattga agatgtggaa catacctttg aagagattaa accactctat 660
gaacatcttc atgcctatgt gagggcaaag ttgatgaatg cctatccttc ctatatcagt 720
cctattggat gcctccctgc tcatttgctt ggtgatatgt ggggtagatt ttggacaaat 780
ctgtactctc tgacagttcc ctttggacag aaaccaaaca tagatgttac tgatgcaatg 840
gtggaccagg cctgggatgc acagagaata ttcaaggaag ccgagaagtt ctttgtatct 900
gttggtcttc ctaatatgac acaaggattc tgggaaaatt ccatgctgac agacccagga 960
aatgttcaga aagcagtctg ccatcccaca gcttgggacc tggggaaggg cgacttcagg 1020
attcttatgt gcacaaaggt gacaatggac gacttcctga cagctcatca tgagatgggg 1080
catatccagt atgatatggc atacgctgca caaccttttc tgctgagaaa tggagctaat 1140
gaaggattcc atgaagctgt tggggaaatc atgtcacttt ctgctgccac acctaagcat 1200
ctgaaatcca ttggtcttct gtctcccgat tttcaagaag acaatgaaac agaaatcaac 1260
ttcctgctca aacaagcact caccattgtt gggactctgc catttactta catgctggag 1320
aagtggaggt ggatggtctt taaaggggaa attcccaaag accagtggat gaaaaagtgg 1380
tgggagatga agcgggagat agttggggtg gtggaacctg tgccccatga tgaaacatac 1440
tgtgaccccg catctctgtt ccatgtttct aatgattact cattcattcg atattacaca 1500
aggacccttt accagttcca gtttcaagaa gcactttgtc aagcagctaa acatgaaggc 1560
cctctgcaca aatgtgacat ctcaaactct acagaagctg gacagaaact gttcaatatg 1620
ctgaggcttg gaaaatcaga accctggacc ctcgcattgg aaaatgttgt aggagcaaag 1680
aacatgaatg taaggccact gctcaactac tttgagcccc tgtttacctg gctgaaagac 1740
cagaacaaga actcttttgt gggatggagt accgacgcta gcgagcccaa atcttgtgac 1800
aaaactcaca catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc 1860
ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 1920
gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 1980
gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 2040
gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 2100
aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 2160
cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac 2220
caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 2280
gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 2340
ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 2400
gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 2460
tccctgtccc cgggtaaa 2478
<210> 8
<211> 826
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 8
Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn
1 5 10 15
His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn
20 25 30
Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala
35 40 45
Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln
50 55 60
Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu
65 70 75 80
Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser
85 90 95
Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr
100 105 110
Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu
115 120 125
Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg
130 135 140
Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg
145 150 155 160
Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala
165 170 175
Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val
180 185 190
Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp
195 200 205
Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His
210 215 220
Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser
225 230 235 240
Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg
245 250 255
Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro
260 265 270
Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln
275 280 285
Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro
290 295 300
Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly
305 310 315 320
Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys
325 330 335
Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe
340 345 350
Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr
355 360 365
Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His
370 375 380
Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His
385 390 395 400
Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu
405 410 415
Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr
420 425 430
Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys
435 440 445
Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys
450 455 460
Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr
465 470 475 480
Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile
485 490 495
Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu
500 505 510
Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser
515 520 525
Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly
530 535 540
Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys
545 550 555 560
Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr
565 570 575
Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp
580 585 590
Ala Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
595 600 605
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
610 615 620
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
625 630 635 640
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
645 650 655
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
660 665 670
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
675 680 685
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
690 695 700
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
705 710 715 720
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
725 730 735
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
740 745 750
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
755 760 765
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
770 775 780
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
785 790 795 800
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
805 810 815
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
820 825
<210> 9
<211> 2496
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 9
cagtccacca ttgaggaaca ggccaagaca tttttggaca agtttaacca cgaagccgaa 60
gacctcttct atcaaagttc acttgcttct tggaattata acaccaacat tactgaagag 120
aatgtccaaa acatgaataa tgctggggac aaatggtctg cctttctgaa ggaacagtcc 180
acacttgccc aaatgtatcc actgcaagaa attcagaatc tcacagtcaa gctccagctg 240
caagctcttc agcaaaatgg gtcttcagtg ctctcagaag acaagagcaa acggttgaac 300
acaattctca atacaatgag caccatctac agtactggaa aagtttgtaa cccagataat 360
ccacaagaat gcctgctcct tgaaccaggt ttgaatgaaa taatggcaaa cagtctggac 420
tacaatgaaa ggctctgggc ttgggaaagc tggagatccg aggtcggcaa gcagctgagg 480
ccactgtatg aagagtatgt ggtcctgaag aatgagatgg caagagcaaa tcattatgag 540
gactatgggg attattggag gggagactat gaagtaaatg gggtagatgg ctatgactac 600
agccgcggcc agttgattga agatgtggaa catacctttg aagagattaa accactctat 660
gaacatcttc atgcctatgt gagggcaaag ttgatgaatg cctatccttc ctatatcagt 720
cctattggat gcctccctgc tcatttgctt ggtgatatgt ggggtagatt ttggacaaat 780
ctgtactctc tgacagttcc ctttggacag aaaccaaaca tagatgttac tgatgcaatg 840
gtggaccagg cctgggatgc acagagaata ttcaaggaag ccgagaagtt ctttgtatct 900
gttggtcttc ctaatatgac acaaggattc tgggaaaatt ccatgctgac agacccagga 960
aatgttcaga aagcagtctg ccatcccaca gcttgggacc tggggaaggg cgacttcagg 1020
attcttatgt gcacaaaggt gacaatggac gacttcctga cagctcataa tgagatgggg 1080
aatatccagt atgatatggc atacgctgca caaccttttc tgctgagaaa tggagctaat 1140
gaaggattcc atgaagctgt tggggaaatc atgtcacttt ctgctgccac acctaagcat 1200
ctgaaatcca ttggtcttct gtctcccgat tttcaagaag acaatgaaac agaaatcaac 1260
ttcctgctca aacaagcact caccattgtt gggactctgc catttactta catgctggag 1320
aagtggaggt ggatggtctt taaaggggaa attcccaaag accagtggat gaaaaagtgg 1380
tgggagatga agcgggagat agttggggtg gtggaacctg tgccccatga tgaaacatac 1440
tgtgaccccg catctctgtt ccatgtttct aatgattact cattcattcg atattacaca 1500
aggacccttt accagttcca gtttcaagaa gcactttgtc aagcagctaa acatgaaggc 1560
cctctgcaca aatgtgacat ctcaaactct acagaagctg gacagaaact gttcaatatg 1620
ctgaggcttg gaaaatcaga accctggacc ctcgcattgg aaaatgttgt aggagcaaag 1680
aacatgaatg taaggccact gctcaactac tttgagcccc tgtttacctg gctgaaagac 1740
cagaacaaga actcttttgt gggatggagt accgactgga gtccttatgc agacgctagc 1800
gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 1860
gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 1920
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 1980
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 2040
tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 2100
ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 2160
atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 2220
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 2280
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 2340
cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 2400
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 2460
tacacgcaga agagcctctc cctgtccccg ggtaaa 2496
<210> 10
<211> 832
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 10
Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn
1 5 10 15
His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn
20 25 30
Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala
35 40 45
Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln
50 55 60
Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu
65 70 75 80
Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser
85 90 95
Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr
100 105 110
Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu
115 120 125
Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg
130 135 140
Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg
145 150 155 160
Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala
165 170 175
Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val
180 185 190
Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp
195 200 205
Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His
210 215 220
Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser
225 230 235 240
Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg
245 250 255
Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro
260 265 270
Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln
275 280 285
Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro
290 295 300
Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly
305 310 315 320
Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys
325 330 335
Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe
340 345 350
Leu Thr Ala His Asn Glu Met Gly Asn Ile Gln Tyr Asp Met Ala Tyr
355 360 365
Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His
370 375 380
Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His
385 390 395 400
Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu
405 410 415
Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr
420 425 430
Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys
435 440 445
Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys
450 455 460
Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr
465 470 475 480
Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile
485 490 495
Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu
500 505 510
Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser
515 520 525
Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly
530 535 540
Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys
545 550 555 560
Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr
565 570 575
Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp
580 585 590
Trp Ser Pro Tyr Ala Asp Ala Ser Glu Pro Lys Ser Cys Asp Lys Thr
595 600 605
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
610 615 620
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
625 630 635 640
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
645 650 655
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
660 665 670
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
675 680 685
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
690 695 700
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
705 710 715 720
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
725 730 735
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
740 745 750
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
755 760 765
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
770 775 780
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
785 790 795 800
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
805 810 815
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
820 825 830
<210> 11
<211> 2478
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 11
cagtccacca ttgaggaaca ggccaagaca tttttggaca agtttaacca cgaagccgaa 60
gacctcttct atcaaagttc acttgcttct tggaattata acaccaacat tactgaagag 120
aatgtccaaa acatgaataa tgctggggac aaatggtctg cctttctgaa ggaacagtcc 180
acacttgccc aaatgtatcc actgcaagaa attcagaatc tcacagtcaa gctccagctg 240
caagctcttc agcaaaatgg gtcttcagtg ctctcagaag acaagagcaa acggttgaac 300
acaattctca atacaatgag caccatctac agtactggaa aagtttgtaa cccagataat 360
ccacaagaat gcctgctcct tgaaccaggt ttgaatgaaa taatggcaaa cagtctggac 420
tacaatgaaa ggctctgggc ttgggaaagc tggagatccg aggtcggcaa gcagctgagg 480
ccactgtatg aagagtatgt ggtcctgaag aatgagatgg caagagcaaa tcattatgag 540
gactatgggg attattggag gggagactat gaagtaaatg gggtagatgg ctatgactac 600
agccgcggcc agttgattga agatgtggaa catacctttg aagagattaa accactctat 660
gaacatcttc atgcctatgt gagggcaaag ttgatgaatg cctatccttc ctatatcagt 720
cctattggat gcctccctgc tcatttgctt ggtgatatgt ggggtagatt ttggacaaat 780
ctgtactctc tgacagttcc ctttggacag aaaccaaaca tagatgttac tgatgcaatg 840
gtggaccagg cctgggatgc acagagaata ttcaaggaag ccgagaagtt ctttgtatct 900
gttggtcttc ctaatatgac acaaggattc tgggaaaatt ccatgctgac agacccagga 960
aatgttcaga aagcagtctg ccatcccaca gcttgggacc tggggaaggg cgacttcagg 1020
attcttatgt gcacaaaggt gacaatggac gacttcctga cagctcatca tgagatgggg 1080
catatccagt atgatatggc atacgctgca caaccttttc tgctgagaaa tggagctaat 1140
gaaggattcc atgaagctgt tggggaaatc atgtcacttt ctgctgccac acctaagcat 1200
ctgaaatcca ttggtcttct gtctcccgat tttcaagaag acaatgaaac agaaatcaac 1260
ttcctgctca aacaagcact caccattgtt gggactctgc catttactta catgctggag 1320
aagtggaggt ggatggtctt taaaggggaa attcccaaag accagtggat gaaaaagtgg 1380
tgggagatga agcgggagat agttggggtg gtggaacctg tgccccatga tgaaacatac 1440
tgtgaccccg catctctgtt ccatgtttct aatgattact cattcattcg atattacaca 1500
aggacccttt accagttcca gtttcaagaa gcactttgtc aagcagctaa acatgaaggc 1560
cctctgcaca aatgtgacat ctcaaactct acagaagctg gacagaaact gttcaatatg 1620
ctgaggcttg gaaaatcaga accctggacc ctcgcattgg aaaatgttgt aggagcaaag 1680
aacatgaatg taaggccact gctcaactac tttgagcccc tgtttacctg gctgaaagac 1740
cagaacaaga actcttttgt gggatggagt accgacgcta gcgagcccaa atcttctgac 1800
aaaactcaca catgcccacc ctgcccagca cctgaactcc tggggggacc atccgtcttc 1860
ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 1920
gtggtggtgg acgtgtctca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 1980
gtggaggtgc ataatgccaa gacaaagcca agggaagagc agtacaacag cacatacaga 2040
gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 2100
aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 2160
cagcccagag aaccacaggt gtgcaccctg cccccatcca gggacgagct gaccaagaac 2220
caggtcagcc tgtcctgcgc cgtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 2280
gagagcaatg ggcagcccga gaacaactac aagaccacac ctcccgtgct ggactccgac 2340
ggctccttct tcctcgtgag caagctcacc gtggacaaga gcaggtggca gcaggggaac 2400
gtcttctcct gctccgtgat gcatgaggct ctgcacaacc actacaccca gaagagcctc 2460
tccctgtctc ccggtaaa 2478
<210> 12
<211> 826
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 12
Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn
1 5 10 15
His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn
20 25 30
Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala
35 40 45
Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln
50 55 60
Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu
65 70 75 80
Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser
85 90 95
Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr
100 105 110
Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu
115 120 125
Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg
130 135 140
Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg
145 150 155 160
Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala
165 170 175
Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val
180 185 190
Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp
195 200 205
Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His
210 215 220
Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser
225 230 235 240
Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg
245 250 255
Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro
260 265 270
Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln
275 280 285
Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro
290 295 300
Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly
305 310 315 320
Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys
325 330 335
Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe
340 345 350
Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr
355 360 365
Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His
370 375 380
Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His
385 390 395 400
Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu
405 410 415
Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr
420 425 430
Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys
435 440 445
Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys
450 455 460
Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr
465 470 475 480
Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile
485 490 495
Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu
500 505 510
Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser
515 520 525
Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly
530 535 540
Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys
545 550 555 560
Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr
565 570 575
Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp
580 585 590
Ala Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys
595 600 605
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
610 615 620
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
625 630 635 640
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
645 650 655
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
660 665 670
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
675 680 685
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
690 695 700
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
705 710 715 720
Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu
725 730 735
Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr
740 745 750
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
755 760 765
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
770 775 780
Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
785 790 795 800
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
805 810 815
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
820 825
<210> 13
<211> 2490
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 13
cagtccacca ttgaggaaca ggccaagaca tttttggaca agtttaacca cgaagccgaa 60
gacctcttct atcaaagttc acttgcttct tggaattata acaccaacat tactgaagag 120
aatgtccaaa acatgaataa tgctggggac aaatggtctg cctttctgaa ggaacagtcc 180
acacttgccc aaatgtatcc actgcaagaa attcagaatc tcacagtcaa gctccagctg 240
caagctcttc agcaaaatgg gtcttcagtg ctctcagaag acaagagcaa acggttgaac 300
acaattctca atacaatgag caccatctac agtactggaa aagtttgtaa cccagataat 360
ccacaagaat gcctgctcct tgaaccaggt ttgaatgaaa taatggcaaa cagtctggac 420
tacaatgaaa ggctctgggc ttgggaaagc tggagatccg aggtcggcaa gcagctgagg 480
ccactgtatg aagagtatgt ggtcctgaag aatgagatgg caagagcaaa tcattatgag 540
gactatgggg attattggag gggagactat gaagtaaatg gggtagatgg ctatgactac 600
agccgcggcc agttgattga agatgtggaa catacctttg aagagattaa accactctat 660
gaacatcttc atgcctatgt gagggcaaag ttgatgaatg cctatccttc ctatatcagt 720
cctattggat gcctccctgc tcatttgctt ggtgatatgt ggggtagatt ttggacaaat 780
ctgtactctc tgacagttcc ctttggacag aaaccaaaca tagatgttac tgatgcaatg 840
gtggaccagg cctgggatgc acagagaata ttcaaggaag ccgagaagtt ctttgtatct 900
gttggtcttc ctaatatgac acaaggattc tgggaaaatt ccatgctgac agacccagga 960
aatgttcaga aagcagtctg ccatcccaca gcttgggacc tggggaaggg cgacttcagg 1020
attcttatgt gcacaaaggt gacaatggac gacttcctga cagctcataa tgagatgggg 1080
aatatccagt atgatatggc atacgctgca caaccttttc tgctgagaaa tggagctaat 1140
gaaggattcc atgaagctgt tggggaaatc atgtcacttt ctgctgccac acctaagcat 1200
ctgaaatcca ttggtcttct gtctcccgat tttcaagaag acaatgaaac agaaatcaac 1260
ttcctgctca aacaagcact caccattgtt gggactctgc catttactta catgctggag 1320
aagtggaggt ggatggtctt taaaggggaa attcccaaag accagtggat gaaaaagtgg 1380
tgggagatga agcgggagat agttggggtg gtggaacctg tgccccatga tgaaacatac 1440
tgtgaccccg catctctgtt ccatgtttct aatgattact cattcattcg atattacaca 1500
aggacccttt accagttcca gtttcaagaa gcactttgtc aagcagctaa acatgaaggc 1560
cctctgcaca aatgtgacat ctcaaactct acagaagctg gacagaaact gttcaatatg 1620
ctgaggcttg gaaaatcaga accctggacc ctcgcattgg aaaatgttgt aggagcaaag 1680
aacatgaatg taaggccact gctcaactac tttgagcccc tgtttacctg gctgaaagac 1740
cagaacaaga actcttttgt gggatggagt accgactgga gtccttatgc agacgagccc 1800
aaatcttctg acaaaactca cacatgccca ccctgcccag cacctgaact cctgggggga 1860
ccatccgtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 1920
gaggtcacat gcgtggtggt ggacgtgtct cacgaagacc ctgaggtcaa gttcaactgg 1980
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc caagggaaga gcagtacaac 2040
agcacataca gagtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 2100
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 2160
aaagccaaag ggcagcccag agaaccacag gtgtgcaccc tgcccccatc cagggacgag 2220
ctgaccaaga accaggtcag cctgtcctgc gccgtcaaag gcttctatcc cagcgacatc 2280
gccgtggagt gggagagcaa tgggcagccc gagaacaact acaagaccac acctcccgtg 2340
ctggactccg acggctcctt cttcctcgtg agcaagctca ccgtggacaa gagcaggtgg 2400
cagcagggga acgtcttctc ctgctccgtg atgcatgagg ctctgcacaa ccactacacc 2460
cagaagagcc tctccctgtc tcccggtaaa 2490
<210> 14
<211> 830
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 14
Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn
1 5 10 15
His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn
20 25 30
Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala
35 40 45
Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln
50 55 60
Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln Leu
65 70 75 80
Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser
85 90 95
Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr
100 105 110
Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu
115 120 125
Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg
130 135 140
Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg
145 150 155 160
Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala
165 170 175
Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val
180 185 190
Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp
195 200 205
Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His
210 215 220
Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser
225 230 235 240
Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg
245 250 255
Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro
260 265 270
Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln
275 280 285
Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro
290 295 300
Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly
305 310 315 320
Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys
325 330 335
Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe
340 345 350
Leu Thr Ala His Asn Glu Met Gly Asn Ile Gln Tyr Asp Met Ala Tyr
355 360 365
Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His
370 375 380
Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His
385 390 395 400
Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu
405 410 415
Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr
420 425 430
Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys
435 440 445
Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys
450 455 460
Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr
465 470 475 480
Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile
485 490 495
Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu
500 505 510
Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser
515 520 525
Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly
530 535 540
Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys
545 550 555 560
Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr
565 570 575
Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp
580 585 590
Trp Ser Pro Tyr Ala Asp Glu Pro Lys Ser Ser Asp Lys Thr His Thr
595 600 605
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
610 615 620
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
625 630 635 640
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
645 650 655
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
660 665 670
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
675 680 685
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
690 695 700
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
705 710 715 720
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro
725 730 735
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val
740 745 750
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
755 760 765
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
770 775 780
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
785 790 795 800
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
805 810 815
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
820 825 830

Claims (16)

1. A bifunctional fusion protein that binds to a coronavirus, wherein said bifunctional fusion protein binds to a different epitope of the coronavirus, respectively.
2. The coronavirus-binding bifunctional fusion protein of claim 1, wherein the coronavirus has a Spike protein.
3. The coronavirus-binding bifunctional fusion protein of claim 1, wherein the coronavirus has an ACE2 binding site.
4. The coronavirus-binding bifunctional fusion protein of claim 1, which binds to the ACE2 binding site of coronavirus and simultaneously binds to the Spike protein of coronavirus.
5. The coronavirus-binding bifunctional fusion protein of claim 1, which has the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof.
6. The coronavirus-binding bifunctional fusion protein of claim 1, which has the amino acid sequence of SEQ ID NO: 6 and the amino acid sequence of the heavy chain shown in SEQ ID NO: 2, or a light chain amino acid sequence set forth in seq id no.
7. A composition comprising the coronavirus binding bifunctional fusion protein of claims 1, 4-6 and a pharmaceutically acceptable excipient.
8. A method for preparing a coronavirus-binding bifunctional fusion protein, comprising the steps of: a) gene synthesizing gene sequence of the dual-function fusion protein combined with coronavirus of claim 1, 4-6, preparing expression vector containing the gene of the dual-function fusion protein; b) transfecting the expression vector to a host cell, culturing the host cell and expressing the bifunctional fusion protein; c) and (3) separating and purifying the bifunctional fusion protein.
9. The method of claim 8, wherein the host cell is a eukaryotic mammalian cell, CHO cell.
10. The method of claim 8, wherein the host cell is cultured in a medium free of serum and animal-derived components.
11. Use of a coronavirus-binding bifunctional fusion protein according to claims 1, 4-6, 8-10 for the preparation of an anti-coronavirus drug.
12. Use of a coronavirus-binding bifunctional fusion protein according to claim 7 for the preparation of an anti-coronavirus agent.
13. Use of a bifunctional fusion protein binding to a coronavirus according to claim 11, wherein the coronavirus has the ACE2 infection pathway.
14. Use of a bifunctional fusion protein binding to a coronavirus according to claim 12, wherein the coronavirus has the ACE2 infection pathway.
15. The use of a bifunctional fusion protein that binds to a coronavirus according to claim 11, wherein the coronavirus is a SARS-CoV-associated coronavirus, a MERS-CoV-associated coronavirus, or a SARS-CoV-2-associated virus.
16. The use of a bifunctional fusion protein that binds to a coronavirus according to claim 12, wherein the coronavirus is a SARS-CoV-associated coronavirus, a MERS-CoV-associated coronavirus, or a SARS-CoV-2-associated virus.
CN202010208906.8A 2020-03-23 2020-03-23 Dual-function fusion protein combined with coronavirus, preparation method and application thereof Pending CN113429476A (en)

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CN112707968A (en) * 2020-12-17 2021-04-27 江苏普瑞康生物医药科技有限公司 Recombinant receptor binding protein and recombinant receptor protein for detecting neutralizing antibody of novel coronavirus

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EP4192859A1 (en) * 2020-08-06 2023-06-14 BioNTech SE Binding agents for coronavirus s protein
CN114369141B (en) * 2021-12-06 2023-09-01 浙江大学 ACE2 targeting peptide for targeting binding ACE2 protein and application thereof

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US7728110B2 (en) * 2006-05-19 2010-06-01 Amgen, Inc. Antibodies to SARS coronavirus
CN104403004B (en) * 2014-11-24 2017-10-13 苏州丁孚靶点生物技术有限公司 The preparation and use of antibody interferon heterodimer

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112707968A (en) * 2020-12-17 2021-04-27 江苏普瑞康生物医药科技有限公司 Recombinant receptor binding protein and recombinant receptor protein for detecting neutralizing antibody of novel coronavirus
CN112707968B (en) * 2020-12-17 2023-10-20 苏州科锐迈德生物医药科技有限公司 Recombinant receptor binding proteins, recombinant receptor proteins for detection of novel coronavirus neutralizing antibodies

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