CN110684102B - SFTSV detection kit - Google Patents

SFTSV detection kit Download PDF

Info

Publication number
CN110684102B
CN110684102B CN201910885757.6A CN201910885757A CN110684102B CN 110684102 B CN110684102 B CN 110684102B CN 201910885757 A CN201910885757 A CN 201910885757A CN 110684102 B CN110684102 B CN 110684102B
Authority
CN
China
Prior art keywords
artificial sequence
gly
tyr
ser
prt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910885757.6A
Other languages
Chinese (zh)
Other versions
CN110684102A (en
Inventor
吴喜林
吴稚伟
李彦磊
潘逸
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Y Clone Medical Science Co ltd
Original Assignee
Y Clone Medical Science Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Y Clone Medical Science Co ltd filed Critical Y Clone Medical Science Co ltd
Publication of CN110684102A publication Critical patent/CN110684102A/en
Application granted granted Critical
Publication of CN110684102B publication Critical patent/CN110684102B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses

Abstract

The invention relates to an SFTSV detection kit, which comprises a detection antibody, a solid matrix and a coating antibody coated on the solid matrix, wherein the detection antibody and the coating antibody respectively comprise 3 complementarity determining regions CDR1-3, the sequence of CDR1 is or comprises one of the sequences shown in SEQ ID NO. 1-74, the sequence of CDR2 is or comprises one of the sequences shown in SEQ ID NO. 75-151, and the sequence of CDR3 is or comprises one of the sequences shown in SEQ ID NO. 152-232. The invention aims at SFTS with high lethality rate but lacking effective vaccine and specific antiviral drug to develop nano antibody drug development and diagnosis kit, screens the nano antibody VHH specifically combined with GN, identifies the CDR sequence, constructs humanized antibody SNB, provides potential nano antibody new drug for clinical treatment of SFTS, and provides corresponding detection kit for diagnosis of SFTS.

Description

SFTSV detection kit
PRIORITY CLAIM
This application claims priority from PCT application No. PCT/CN2019/097350, filed by the applicant on 23/7/2019, incorporated herein by reference.
Technical Field
The invention relates to the field of biomedicine. More particularly, it relates to a polypeptide capable of binding SFTSV, the application of said polypeptide in the preparation of SFTSV detection agent or SFTSV therapeutic drug, and a SFTSV detection kit.
Background
The fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by a novel bunyavirus (SFTSV) and is mainly transmitted by tick bites or blood and mucous membrane contact of acute patients, and clinically, the fever, thrombocytopenia, leukopenia and multiple organ dysfunction including gastrointestinal tract, liver and kidney are mainly manifested. Since SFTS is reported in Hubei and Henan for the first time in 2009, 16 provinces in China have published case reports, the number of cases diagnosed by 2014 is over 3500, and the incidence rate is increased continuously in recent years; meanwhile, SFTS cases are reported in korea, japan, arabian, and the united states, suggesting that the prevalence area of the disease is expanding. The currently reported mortality rate for SFTS is between 6.3% and 30%; and the mortality rates published in korea and japan in 2016 were as high as 35.4% and 50%, respectively. However, there is no specific therapeutic drug for SFTS in clinical practice, and only broad-spectrum antiviral therapy and symptomatic drug administration are available, which brings great difficulty to prevention and control of SFTS.
The specific neutralizing antibody has good clinical curative effect in antiviral treatment, such as respiratory syncytial virus monoclonal antibody Palivizumab, rabies virus antiserum and the like. Neutralizing antibodies against the SFTSV surface Glycoprotein (Glycoprotein N, GN) have been shown to play an important role in patient survival. Our earlier results also show that the serum anti-GN SFTS patients all recovered well, while the serum non-GN SFTS patients had a mortality rate as high as 66.7%. These studies suggest that neutralizing antibody therapy targeting GN would be an effective approach to combat SFTSV.
In 1993, a novel natural antibody derived from camelidae was found. The antibody naturally lacks a light chain and consists only of a heavy chain comprising two constant regions (CH 2 and CH 3), one hinge region and one heavy chain Variable region (VHH, i.e. antigen binding site) with a relative molecular mass of about 13KDa, which is only 1/10 of that of conventional antibodies, and with a molecular height and diameter at the nanometer level, is the smallest functional antibody fragment currently available, and is therefore also called Nanobody (Nb). Because of the characteristics of high stability (not degraded at 90 ℃), high affinity, homology of more than 80% with a human antibody, low toxicity and immunogenicity and the like, the nano monoclonal antibody is recently widely used for the research and development of immunodiagnosis kits, the research and development of imaging, and the research and development of antibody drugs aiming at the fields of tumors, inflammations, infectious diseases, nervous system diseases and the like.
The SFTS neutralizing antibody against GN is currently known to be Mab4-5 screened by phage display SFTS patient antibody library in 2013, with an IC50 of 2 μ g/ml to 44.2 μ g/ml. The lower the IC50 of the neutralizing antibody, the better, can greatly save the use amount of the antibody so as to save the production cost, and simultaneously can prolong the time for maintaining the effective concentration of the antibody in the body. The antibody titer of the serum of the SFTS patient is detected in the previous period, and the antiserum aiming at GN protein is only about 100 dilution times, which indicates that the establishment of an antibody library by using the serum of the SFTS patient with low antibody titer possibly limits the screening of high-efficiency neutralizing antibodies. Therefore, the new technical means are expected to obtain more efficient and stable SFTSV specific neutralizing antibodies with lower IC 50.
Disclosure of Invention
The invention uses antigen immune camel to obtain camel source nanometer monoclonal antibody and VHH thereof, which is used for diagnosing and treating SFTS patients with acute infection. Based on these studies, the present invention provides a polypeptide that binds to SFTSV comprising 3 complementarity determining regions CDR1-3, the CDR1 sequence being or comprising one of the sequences set forth in SEQ ID NOS 1-74, the CDR2 sequence being or comprising one of the sequences set forth in SEQ ID NOS 75-151, and the CDR3 sequence being or comprising one of the sequences set forth in SEQ ID NOS 152-232.
The invention provides an SFTSV detection kit, which comprises a detection antibody, a solid matrix and a coating antibody coated on the solid matrix, wherein the detection antibody and the coating antibody are respectively one of the polypeptides.
In a specific embodiment, the CDR sequences of the detection antibody are as follows:
CDR1 has the sequence of SEQ ID NO 4, CDR2 has the sequence of SEQ ID NO 84, and CDR3 has the sequence of SEQ ID NO 181; or
CDR1 has the sequence SEQ ID NO 54, CDR2 has the sequence SEQ ID NO 132, and CDR3 has the sequence SEQ ID NO 176; or
The sequence of CDR1 is SEQ ID NO 13, the sequence of CDR2 is SEQ ID NO 99, and the sequence of CDR3 is SEQ ID NO 166.
In a specific embodiment, the CDR sequences of the coated antibody are as follows:
CDR1 has the sequence of SEQ ID NO 4, CDR2 has the sequence of SEQ ID NO 84, and CDR3 has the sequence of SEQ ID NO 181; or
CDR1 has the sequence SEQ ID NO 54, CDR2 has the sequence SEQ ID NO 132, and CDR3 has the sequence SEQ ID NO 176; or
CDR1 has the sequence of SEQ ID NO 6, CDR2 has the sequence of SEQ ID NO 81, and CDR3 has the sequence of SEQ ID NO 158; or
The sequence of CDR1 is SEQ ID NO 13, the sequence of CDR2 is SEQ ID NO 99, and the sequence of CDR3 is SEQ ID NO 166.
In a preferred embodiment of the process according to the invention,
i) The CDR sequences of the detection antibody are as follows: CDR1 has the sequence of SEQ ID NO 4, CDR2 has the sequence of SEQ ID NO 84, and CDR3 has the sequence of SEQ ID NO 181; or the sequence of CDR1 is SEQ ID NO 13, the sequence of CDR2 is SEQ ID NO 99, and the sequence of CDR3 is SEQ ID NO 166;
and the number of the first and second electrodes,
II) the CDR sequences of the coated antibody are as follows: the sequence of CDR1 is SEQ ID NO. 4, the sequence of CDR2 is SEQ ID NO. 84, and the sequence of CDR3 is SEQ ID NO. 181; or CDR1 has the sequence of SEQ ID NO 54, CDR2 has the sequence of SEQ ID NO 132, and CDR3 has the sequence of SEQ ID NO 176; or the sequence of CDR1 is SEQ ID NO. 6, the sequence of CDR2 is SEQ ID NO. 81, and the sequence of CDR3 is SEQ ID NO. 158; or the sequence of CDR1 is SEQ ID NO. 13, the sequence of CDR2 is SEQ ID NO. 99, and the sequence of CDR3 is SEQ ID NO. 166.
In a preferred embodiment of the process according to the invention,
i) The sequence of the detection antibody CDR1 is SEQ ID NO. 4, the sequence of the CDR2 is SEQ ID NO. 84, and the sequence of the CDR3 is SEQ ID NO. 181; and is provided with
II) the CDR sequences of the coated antibody are as follows: CDR1 has the sequence SEQ ID NO 54, CDR2 has the sequence SEQ ID NO 132, and CDR3 has the sequence SEQ ID NO 176; or the sequence of CDR1 is SEQ ID NO 13, the sequence of CDR2 is SEQ ID NO 99, and the sequence of CDR3 is SEQ ID NO 166.
In a further preferred embodiment of the process according to the invention,
i) The sequence of the detection antibody CDR1 is SEQ ID NO 13, the sequence of the CDR2 is SEQ ID NO 99, and the sequence of the CDR3 is SEQ ID NO 166; and is
The CDR sequences of the coated antibody are as follows:
the sequence of CDR1 is SEQ ID NO. 4, the sequence of CDR2 is SEQ ID NO. 84, and the sequence of CDR3 is SEQ ID NO. 181; or CDR1 has the sequence of SEQ ID NO 54, CDR2 has the sequence of SEQ ID NO 132, and CDR3 has the sequence of SEQ ID NO 176; or the sequence of CDR1 is SEQ ID NO. 6, the sequence of CDR2 is SEQ ID NO. 81, and the sequence of CDR3 is SEQ ID NO. 158; or the sequence of CDR1 is SEQ ID NO 13, the sequence of CDR2 is SEQ ID NO 99, and the sequence of CDR3 is SEQ ID NO 166.
The invention aims at SFTS with high lethality rate but lacking effective vaccine and specific antiviral drug to develop nano antibody drug development and diagnosis reagent kit, and selects nano antibody VHH specifically combined with GN by preparing GN protein, immune doublet camel, platform technology for displaying nano monoclonal antibody by using phage library, etc., identifies CDR sequence thereof, and constructs humanized VHH-huFc1 (SNB); at the same time, the efficacy of SNB in treating SFTSV infection was evaluated in vivo using a humanized mouse model. The invention provides a potential nano antibody new drug for the clinical treatment of SFTS, and simultaneously provides a corresponding detection kit for the diagnosis of SFTS.
Drawings
FIG. 1 is the antiserum titer detection curve one week after the 3rd and 4 th immunization of camels with sGN;
FIG. 2 is a graph of the inhibition of SFTSV virus infection of Vero cells in vitro by antiserum one week post the 4 th immunized camel at different dilutions, with pre-immune serum as control;
FIG. 3 is an electrophoretogram of PCR products amplified using a sGN-VHH phage antibody library as a template;
FIG. 4 is the identification of the panning of the sGN-VHH phage antibody library, where A is the ELISA detection statistic after panning of the phage library against sGN protein; b is the second wheel (2) nd ) And a third wheel (3) rd ) 96 clones are selected from the panned phage antibody library respectively to carry out phage ELISA detection statistical chart;
FIG. 5 is a statistical ELISA assay for prokaryotically expressed VHH antibodies, each dot representing a clone, with the ordinate being OD450 for sGN/OD 450 for blank, and a positive value being defined by a ratio greater than 5.0;
FIG. 6 is a statistical chart of experiments on neutralization of SFTSV virus infection by positive VHH antibodies, one dot representing one clone, and the Y-axis being the relative inhibition rate for different viruses;
figure 7 is an ELISA to detect binding of SNB to sGN protein at different purified concentrations, with different colors representing different clone numbers.
FIG. 8 is a photograph of fluorescent staining of SFTSV virus in the presence of different concentrations of antibody, with fluorescent spots representing SFTSV virus, virus-free cells as a No infection control (No infection control), and virus-free cells without antibody as an infection control (infection control);
FIG. 9 is a graph of the inhibition of SFTSV virus by antibodies obtained from the fluorescent spots counted in FIG. 7;
FIG. 10 is a statistical graph of the relative inhibition rates of different mice after challenge, the relative inhibition rates being 1-day 9/day 6 viral loads, with human immunoglobulin as the control (Hu-IgG);
FIG. 11 is a statistical plot of the detection of sGN protein by SNB double antibody sandwich ELISA in which SNB01 (A), SNB02 (B) and SNB37 (C) were used as detection antibodies, respectively; d is coating antibody SNB01, and the double antibody sandwich ELISA detecting antibody SNB37 detects OD450 statistical curves for different concentrations of sGN.
FIG. 12 is a statistical curve of OD450 of SFTSV virus at different concentrations detected by double antibody sandwich ELISA for coating antibody SNB01 and detecting antibody SNB 37.
Detailed Description
1. Preparation of immunogens
Based on GN protein sequence and gene sequence information of HB29SFTSV on NCBI website, we analyzed and designed polypeptide sGN which can effectively induce camel to generate specific antibody aiming at GN protein, and connected His-tag (sGN-His) or rabbit Fc (sGN-rFc) at C terminal for subsequent purification and detection.
2. Camel immunization and antiserum procurement
Priming a doublet camel with an emulsified mixture of 250 mu g of sGN-rFc protein and 250 mu l of Freund's complete adjuvant, boosting the camel with the sGN-rFc protein and 250 mu l of Freund's incomplete adjuvant 3 times on days 14, 28 and 42, and collecting blood to detect the antiserum titer 1 week after 2 and 3 weeks of immunization; after 1 week of the 4 th immunization, 200ml of blood was collected for the construction of phage antibody library.
Antiserum titers were measured by ELISA, assay plates were coated with GN protein at a concentration of 0.5 μ g/ml, and either antiserum diluted in a gradient or purified antibody 100 μ l (control was pre-immune camel serum) was added to each well, incubated at 37 ℃ for 1.5h, washed 2 times, and 1:10000 diluted secondary antibody of horseradish peroxidase labeled Goat anti-Llama IgG (H + L) is incubated for 1H at 37 ℃, after washing for 4-6 times, 100 μ L TMB substrate is added, incubation is carried out for 10min at 37 ℃,50 μ L of 0.2M H 2 SO 4 The reaction was stopped and the OD450nm was measured. ELISA assay serum titers were specified at the highest dilution of OD450 above 2.1 fold over blank and greater than 0.2.
As shown in FIG. 1, the antiserum titers of 3-and 4-immunization were 2.19X 10, respectively 6 And 4.61X 10 6 . It can be seen that this antigen can induce camels to produce high titers of antisera specific for GN protein.
To further verify whether this high titer camelid antiserum was effective in preventing SFTSV virus infection, neutralization experiments for virus infection were performed. Antiserum and preimmune serum at different dilution concentrations were incubated with SFTSV virus for 60min, respectively, then transferred to Vero cells, and infection of SFTSV was judged by immunofluorescent staining of cells for sGN protein 48h later. The results of the neutralization experiments showed that the sGN-induced antiserum inhibition 90% resulted in more than 540-fold dilution of SFTSV infection ID90 (FIG. 2). Taken together, sGN induces high titers of antisera that have the ability to efficiently inhibit SFTSV virus infection.
VHH phage library construction and panning
Collecting 200ml of peripheral blood of the immunized camel, separating by utilizing lymphocyte separation liquid (GE Ficoll-Paque Plus) to obtain PBMC of the camel, extracting RNA according to a TRIzol operation manual, inverting oligo (dT) into cDNA, cloning VHH genes of the camel to phagemid plasmids by utilizing technologies such as primer amplification, molecular cloning and the like, and transforming TG1 bacteria to obtain a VHH phage library. To further identify whether the sGN-VHH phage library was successfully constructed, the VHH target gene of immune sGN camels was amplified by PCR, which revealed a 500bp band with a size that was expected (fig. 3), indicating that the sGN-VHH phage antibody library contained VHH genes. Selecting 50 clones for sequencing, wherein sequencing results show that the sequenced sequences do not have completely consistent repeated sequences; the alignment results show that the most of the different sequences are in the CDR binding region. Through detection, the library capacity of the constructed sGN-VHH phage antibody library is 2.0 multiplied by 10 9 The positive rate is 100%, the sequence Diversity (Diversity) is 100%, and the effective insertion rate (In frame rate) is greater than 95%.
The phage antibody library was recovered from VHH-phagemid transformed bacteria with the help of M13KO7 helper phage and precipitated with PEG/NaCl. The phage antibody library was enriched three times with 50. Mu.g/ml of sGN-His protein coating. And (3) eluting the enriched phage, converting, coating a plate, selecting a monoclonal to perform binding identification of the phage and sGN protein ELISA, sequencing the clone with the binding reading value of more than 1.0, cloning to an expression vector pVAX1, and transfecting 293F cells to express and produce the nano monoclonal antibody.
The panned library was tested for binding to GN protein. The phage ELISA results showed that the binding reading of the sGN-VHH phage library to the sGN protein before enrichment was 0.57, and the reading of the phage library after one, two, and three rounds of enrichment was 0.98, 2.2, and 3.0, respectively (FIG. 4A). To further verify the positive phage rate of sGN-VHH protein binding in the enriched library, 96 clones were selected from each of the enriched libraries from round 2 and round 3 for single phage ELISA detection. The results showed that 24.5% of individual phage clones were positive in the 2nd round library and 67% of phage clones were positive in the 3rd round library, and the mean reading of binding was around 3.0 (FIG. 4B), and that the high binding sGN-VHH phage library was successfully enriched by sGN protein panning.
Construction of VHH prokaryotic expression library and VHH expression
PCR amplification of the enriched 2nd-sGN-VHH and 3rd-sGN-VHH phage antibody libraries from the two and three rounds of panning described above; obtaining and purifying all VHH gene fragments in an antibody library, cloning the VHH gene fragments to a prokaryotic expression vector, converting an SS320 strain, and constructing a prokaryotic expression antibody library of the VHH; coating a plate with the prokaryotic expression antibody library, culturing overnight, randomly picking 1000 monoclonal colonies the next day, using IPTG to induce and express the antibody supernatant, and carrying out ELISA combination detection on the antibody supernatant and the sGN protein.
The results show that there was bacterial supernatant that bound to sGN protein while not binding to the blank, with a reading for sGN binding/reading for the blank of greater than 5.0 (figure 5 and table 1). These sequences were sequenced and aligned to eliminate repeated sequences, and 142 VHH antibody sequences (SEQ ID NOS: 1-142) were finally obtained. Further experiments demonstrated that both the 142 VHH antibodies and CDRs derived from the VHH antibodies specifically bind to SFTSV viruses.
TABLE 1 binding values and sequences of 142 VHH antibodies to sGN protein
Figure BDA0002207249840000081
Figure BDA0002207249840000091
Figure BDA0002207249840000101
Figure BDA0002207249840000111
In order to further verify whether the sequence can well inhibit the SFTSV virus infection, bacterial supernatants of 122 VHH antibodies are treated and incubated with the SFTSV virus, and whether the antibodies can inhibit the SFTSV virus infection is tested. The results of neutralization experiments (fig. 6) show that 23 antibodies can achieve more than 50% of inhibition effects. The antibody numbers G77, G78, G79, G87, G95, G103, G109, G111, G112, G113, G114, G116, G117.1, G121, G124, G125, G126, G131, G133, G145, G170 and G171, respectively.
Eukaryotic expression of VHH-huFc (SNB)
Through a molecular cloning technology, the 23 nanometer monoclonal antibody VHH genes are fused with human Fc genes and inserted into a pVAX1 eukaryotic expression vector to construct and form a Nb-huFc-pVAX1 expression plasmid. The constructed Nb-huFc-pVAX1 is transfected into 293F cells, expressed to produce Nb-huFc (SNB), and purified by Protein G. The purified VHH-huFc1 (SNB) antibodies were collected and tested by ELISA, and some of the antibodies had good binding capacity (FIG. 7). The respective sequence numbers of the constructed humanized antibodies are shown in Table 2.
TABLE 2 original antibody numbering and CDR sequences corresponding to humanized antibodies SNB
Figure BDA0002207249840000112
Coupling 4000RU of sGN protein to CM5 chips by BIAcore x100 instrument according to the amino coupling kit instructions; ethanolamine blocks the coupled chip. The antibody was diluted in a gradient to different concentrations. The Bioevaluation version 4.0 software sets the detection program: detecting the concentration of the antibody from low concentration to high concentration, and performing 2 repeated detections on each concentration; the binding time was set to 180 seconds and the flow rate was 30. Mu.l/min; the dissociation time is set to 180 seconds, and the flow rate is 30 mul/min; setting the flow rate of glycine with the pH value of 2.5 mM as 30 mu l/min for 30 seconds, and activating and regenerating the surface of the chip; PBS was equilibrated for 5 seconds and the flow rate was 30. Mu.l/min. The experimental data were analyzed to obtain binding, dissociation and affinity constants. As a result, as shown in Table 3, the affinity of most antibodies was 10 -9 (Nano molGrade e), two of the antibodies SNB02 and SNB07 can reach 10 -10 (Picomole grade). It can be seen that we obtained VHH-huFc1 (SNB) antibodies with high affinity.
Table 3 summary of SNB affinities.
Figure BDA0002207249840000121
Note: ka is the binding constant, KD is the dissociation constant, and KD is the affinity.
SNB-neutralizing SFTSV infection of Vero cells
SNB02 and SNB16 in VHH-huFc1 were selected for in vitro neutralization experiments. Gradient dilution of antibody to different concentrations, together with SFTSV Virus, in 5% CO 2 Incubating at 37 ℃ for 1 hour, adding 1.5x10 4 Vero cells, 5% CO 2 After incubation at 37 ℃ for 48 hours in an incubator, cell supernatants were removed, fixed with 4% paraformaldehyde for 15min, washed and blocked, rabbit polyclonal serum against GN (1 diluted at 1000), diluted overnight at 4 ℃, washed with PBST, and then incubated with 50. Mu.l of a secondary antibody against rabbits (Alexa Fluor 488Anti-Rabbit IgG (H + L), code:111-545-144, jackson ImmunoResearch laboratories,1 diluted at 1000) at room temperature for 40min, followed by DAPI 10min staining, and after washing, neutralization inhibition and neutralization titers were calculated by counting green spots and fluorescence intensity, inhibition = [1- (mean of fluorescence intensity of sample group-mean of fluorescence intensity of control group CC-mean of fluorescence intensity of control group-mean of fluorescence intensity of cell control group CC-mean of fluorescence intensity of CC of cell control group)]X 100%. Neutralizing titer (ID) 50 Or ND 50 ) Expressed as a multiple of the dilution at 50% inhibition.
As a result, as shown in FIGS. 8 and 9, SNB02 had excellent neutralizing activity, and the inhibition rate reached 83.5% at an antibody concentration of 9. Mu.g/ml. The efficacy of SNB02 was evaluated using a humanized mouse model.
SNB treatment of SFTS in vivo infection
Cg-Prkdcscidil2rgtm1Wjl/SzJ (NCG) mice purchased from Nanjing university model animal institute, similar to NSG mice, at SCThe deletion of the IL2 receptor gene in ID mice resulted in the absence of mouse T cells, B cells and minimal NK cells in vivo. 1.0-15x10 7 PBMC were injected intraperitoneally into NCG mice for 4-6 weeks; three weeks later, human T cells were flow-tested by collecting blood and staining human CD45 + 、CD3 + 、CD4 + And CD8 + . The proportion of human CD45 positive cells reaches more than 5%, and the mice are judged to be successfully humanized. Inoculation 2X10 7 TCID50 SFTSV virus was treated with antibody therapy by intraperitoneal injection of 400. Mu.g SNB02 antibody/mouse (the amount of antibody was approximately 20mg/kg mouse) on days 3 and 6 after infection, respectively. Blood was collected before and on day 9 of each antibody treatment, and the viral load in the blood of mice was examined to determine whether the mice were successfully infected with SFTS virus and whether the SNB antibody could control SFTSV infection of the mice, using human IgG as a control. As shown in fig. 11, after three days of treatment (day 9), the viral load in mice was measured, and the viral load in 9 mice and 7 mice treated with SNB02 was well inhibited, whereas in the control group treated with Hu-IgG, SFTS virus proliferated in vivo, and the inhibition rate of two groups of viruses was compared, so that SNB02 significantly controlled SFTS virus infection (fig. 10).
8. In vivo experiments using SNB02 loaded with AAV viral vectors
Adeno-associated virus (AAV) is derived from non-pathogenic wild adeno-associated virus, and is considered one of the most promising gene transfer vectors due to its high safety, wide host cell range (dividing and non-dividing cells), low immunogenicity, and long time for expressing foreign genes in vivo, and is widely used in gene therapy and vaccine research worldwide.
AAV Helper-Free viral packaging system was purchased from Cell Biolabs, san Diego USA. Inserting the DNA coding sequence of SNB02 into pAAV-MCS plasmid by molecular cloning technology; after the successful construction is proved by sequencing, the constructed plasmid pAAV-Ab and pHelper and pAAV-DJ plasmids are used for co-transfecting AAV-293T cells by using a PEI transfection reagent according to the mass ratio of 1. Supernatants were collected at 48, 72, 96 and 120 hours post transfection and concentrated with 5xPEG8000 (sigma) and finally purified with 1.37g/ml cesium chloride. Purified AAV was dissolved in PBS, identified and aliquoted and stored at-80 ℃.
The humanized mice were inoculated with 2x10 7 TCID50 SFTSV Virus, 3 days post infection blood draw and AAV-SNB02 (1X 10) 11 gc/100 μ l) were injected intramuscularly and blood was collected on days 6, 9 and 12 and viral load was measured with AAV-GFP as control. The results show that the SFTSV viral load in mice injected with AAV-SNB02 was low, while the SFTSV virus in control mice proliferated in large amounts.
9. Double antibody sandwich ELISA
Coating the detection plate with SNB antibodies with different concentrations, incubating at 37 ℃ for 2h and washing for 2-4 times, blocking with 10% bovine serum, incubating at 37 ℃ for 1h and washing for 2-4 times, adding protein and virus or sample diluted in gradient to each well for 100. Mu.l, incubating at 37 ℃ for 1.5h and washing for 2 times, adding 1:200-1:10000 ul of horseradish peroxidase-labeled SNB antibody, incubated at 37 ℃ for 1H, washed 4-6 times, then 100 ul of TMB substrate, incubated at 37 ℃ for 10min,50 ul of 0.2M H 2 SO 4 The reaction was stopped and the OD450nm was measured. The positive samples for the ELISA test were specified to be 2.1 times more than the blank (i.e. no coated assay antigen, labeled Neg in the figure) at OD450 and the highest dilution with optical density values greater than 0.2.
The results show that when the coating antibody is SNB02 or SNB07 and the detection antibody is SNB01, the combination is able to recognize the sGN protein and the negative control protein (fig. 11A); when the detection antibody was SNB02, the ELISA detection background was high (fig. 11B); when the detection antibody was SNB37 and the coating antibodies were SNB01, SNB02 and SNB07, the ELISA was able to recognize the sGN protein and the negative control protein (fig. 11C). The SNB antibody can be applied to the development of a double-antibody sandwich ELISA sGN detection kit, wherein the detection sensitivity of the double-antibody combination of SNB01 and SNB37 is 3.9ng/ml (FIG. 11D). The sensitivity of the combination for detecting SFTSV true virus is measured to be 3.75x10 6 gc/ml (FIG. 12), indicating that the kit can detect not only SFTSV euviruses but also SFTSV euviruses with copy numbers as low as 10^ 6. Therefore, the nano antibody double-antibody sandwich detection kit can be applied to SFTSV virus infection detection.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Sequence listing
<110> Source daolung (Suzhou) medical science and technology, inc
<120> SFTSV detection kit
<150> PCT/CN2019/097350
<151> 2019-07-23
<160> 237
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Gly Phe Thr Phe Asp Asp Ser Asn
1 5
<210> 2
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Gly His Thr Leu Ser Ser Asn Cys
1 5
<210> 3
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Gly Pro Phe Tyr Asn Thr His Cys
1 5
<210> 4
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Gly Asp Thr Ser Thr Ala Tyr Tyr
1 5
<210> 5
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Gly Asp Thr Tyr Ser Ser Ser Cys
1 5
<210> 6
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Gly Phe Thr Ser Cys
1 5
<210> 7
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Gly Val Thr Leu Asp Asp Phe Leu
1 5
<210> 8
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Gly Val Thr Leu Asp Asp Phe Leu
1 5
<210> 9
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Gly Tyr Thr Phe Ser Asn Thr Cys
1 5
<210> 10
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Gly Arg Thr Tyr Arg Thr Tyr Cys
1 5
<210> 11
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Glu Asp Ala His Ser Thr Thr Glu Tyr Ile Val Ser Thr Thr Cys
1 5 10 15
<210> 12
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Gly Val Thr Tyr Gly Ser Tyr Cys
1 5
<210> 13
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Gly Phe Thr Leu Ser Met Tyr Asp
1 5
<210> 14
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Glu Tyr Thr Gly Ser Arg Asn Cys
1 5
<210> 15
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Gly Tyr Thr Tyr Asn Asn Tyr Arg
1 5
<210> 16
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Gly Leu Tyr Tyr Pro Pro Leu Cys
1 5
<210> 17
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Arg Tyr Asp Tyr Ser Arg Thr Cys
1 5
<210> 18
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Gly Phe Thr Val Ser Arg Tyr Asp
1 5
<210> 19
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Gly Ser Ala Tyr Ser Thr Asn Cys
1 5
<210> 20
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Gly Tyr Thr Tyr Ser Ser Asn Cys
1 5
<210> 21
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Gly Phe Thr Phe Asn Ala Tyr Asp
1 5
<210> 22
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Val Tyr Thr Tyr Arg Gly Asn Asn
1 5
<210> 23
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Arg Tyr Thr Pro Thr Ile Thr Arg
1 5
<210> 24
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Val Tyr Thr Ser Ser Thr Met Trp
1 5
<210> 25
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Gly Tyr Thr Ser Thr Ala Tyr Tyr
1 5
<210> 26
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Gly Gly Thr Ser Thr Ala Tyr Tyr
1 5
<210> 27
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
Gly Phe Thr Ser Ser Asn Tyr Asp
1 5
<210> 28
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
Gly Phe Thr Ser Ser Ser Cys Gly
1 5
<210> 29
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
Gly Tyr Thr Tyr Ser Ser Val Cys
1 5
<210> 30
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Gly Tyr Thr Tyr Asn Thr Ala Tyr
1 5
<210> 31
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
Gly Tyr Ala Tyr Ser Thr Tyr Cys
1 5
<210> 32
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 32
Gly Tyr Thr Tyr Asn Glu Tyr Ser
1 5
<210> 33
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 33
Gly Tyr Asn Phe Asn Asn Val Cys
1 5
<210> 34
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 34
Glu Tyr Thr Arg Ser Ser Arg Cys
1 5
<210> 35
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 35
Gly Ser Thr Asp Ser Arg Arg Cys
1 5
<210> 36
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 36
Gly Tyr Pro Tyr Ser Ser Lys Thr Tyr Thr Asn Asn Cys
1 5 10
<210> 37
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 37
Arg Tyr Ser Ser Ser Arg Arg Cys
1 5
<210> 38
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 38
Gly Tyr Ile Tyr Asn Asp Tyr Phe
1 5
<210> 39
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 39
Thr Tyr Asn Tyr Glu Ser Ser Gln
1 5
<210> 40
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 40
Thr Thr Thr Asn Tyr
1 5
<210> 41
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 41
Gly Tyr Thr Tyr Asn Tyr Tyr Cys
1 5
<210> 42
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 42
Gly Tyr Thr Tyr Tyr Asn Ser Asn Cys
1 5
<210> 43
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 43
Gly Gly Ser Val Thr Thr Gly Asn Tyr Tyr
1 5 10
<210> 44
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 44
Gly Tyr Thr Phe Asn Thr Arg Cys
1 5
<210> 45
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 45
Leu Tyr Thr Tyr Ser Tyr Asn Cys
1 5
<210> 46
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 46
Gly Phe Thr Ser Asn Ser Cys Gly
1 5
<210> 47
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 47
Gly Met Met Ser Asn Ala Cys
1 5
<210> 48
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 48
Gly Tyr Thr His Ser Ser Asp Ser
1 5
<210> 49
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 49
Arg Ser Val Asn Arg Asp Thr Cys
1 5
<210> 50
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 50
Ala Phe Ile Ser Asn Asn His Cys
1 5
<210> 51
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 51
Gly Phe Thr Phe Ser Ser Tyr Asp
1 5
<210> 52
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 52
Gly Phe Thr Phe Asp Asp Ser Asp
1 5
<210> 53
<211> 4
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 53
Gly Tyr Arg Cys
1
<210> 54
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 54
Ala Tyr Thr Tyr Arg Gly Asn Asn
1 5
<210> 55
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 55
Gly Ala Thr Tyr Asn Ile Asn Phe
1 5
<210> 56
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 56
Gly Tyr Thr Tyr Ser Asn Tyr
1 5
<210> 57
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 57
Gly Phe Thr Phe Ser Ser Tyr Tyr
1 5
<210> 58
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 58
Gly Ser Ile Tyr Ser Ser Asn Ala
1 5
<210> 59
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 59
Gly Tyr Thr Tyr Ser Ser Ala Cys
1 5
<210> 60
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 60
Gly Met Tyr Ser Asn Thr Cys
1 5
<210> 61
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 61
Gly Gly Ser Ile Thr Thr Asn Tyr Tyr Gly
1 5 10
<210> 62
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 62
Gly Asp Ser Ser Thr Ala Tyr Tyr
1 5
<210> 63
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 63
Gly Asn Thr Tyr Thr Ser Ser Cys
1 5
<210> 64
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 64
Gly Gly Ser Ile Thr Thr Ala Gly Tyr Gly
1 5 10
<210> 65
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 65
Gly Leu Tyr Tyr Leu Pro Leu Cys
1 5
<210> 66
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 66
Gly Leu Trp His Pro Pro Leu Cys
1 5
<210> 67
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 67
Gly Tyr Thr Tyr Gly Ser Tyr Cys
1 5
<210> 68
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 68
Gly Leu Tyr Tyr Ser Pro Leu Cys
1 5
<210> 69
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 69
Arg Tyr Thr Ser Ser
1 5
<210> 70
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 70
Gly Tyr Arg Tyr Asn Ala Cys Ser
1 5
<210> 71
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 71
Gly Asn Pro Ser Gly Arg Lys Phe
1 5
<210> 72
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 72
Gly Ser Ser Gly Leu Ile Phe Ser Gly Ser Ala
1 5 10
<210> 73
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 73
Glu Asp Thr Ser Thr Ala Tyr Tyr
1 5
<210> 74
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 74
Gly Tyr Thr Tyr Ser Ser His Cys
1 5
<210> 75
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 75
Ile Lys Ser Asp Gly Ser Thr Ser
1 5
<210> 76
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 76
Ile Tyr Thr Gly Gly Gly His Thr Tyr
1 5
<210> 77
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 77
Val Tyr Pro His Leu Thr Tyr
1 5
<210> 78
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 78
Ile Tyr Arg Gly Gly Arg Ala Thr Val
1 5
<210> 79
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 79
Ile Cys Ser Asp Gly Ser Ala Ala
1 5
<210> 80
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 80
Ile Tyr Arg Gly Gly His Ser Thr Val
1 5
<210> 81
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 81
Ile Tyr Thr Arg Asp Gly Arg Pro Tyr
1 5
<210> 82
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 82
Ile Asp Ser Glu Gly Arg Ile Asp
1 5
<210> 83
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 83
Ile Asp Ser Gly Gly Ser Ser Ser
1 5
<210> 84
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 84
Ile Tyr Arg Gly Gly Arg Ser Thr Val
1 5
<210> 85
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 85
Ile Asp Arg Ser Gly Ser Ala Ser
1 5
<210> 86
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 86
Ile Thr Ala Leu Ser Ala Thr Ser
1 5
<210> 87
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 87
Thr Tyr Arg His Gly Gly Thr Thr Val
1 5
<210> 88
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 88
Leu Glu Ser Asp Gly Ser Thr Ser
1 5
<210> 89
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 89
Ile Tyr Thr Ser Gly Arg Arg Pro Trp
1 5
<210> 90
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 90
Ile Phe Arg Gly Gly Arg Ser Thr Val
1 5
<210> 91
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 91
Ile Ser Ile Leu Tyr Ser Gly Ile Thr Val Ser Tyr
1 5 10
<210> 92
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 92
Ile Ser Ala Gly Gly Asp His Thr Tyr
1 5
<210> 93
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 93
Ile Asp Arg Asp Gly Ser Thr Ser
1 5
<210> 94
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 94
Ile Cys Ser Asp Gly Ser Thr Ser
1 5
<210> 95
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 95
Ile Phe Val Ser Gly Arg Ser Pro Trp
1 5
<210> 96
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 96
Ile Tyr Arg Asp Asp Gly Thr Thr Tyr
1 5
<210> 97
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 97
Ile Tyr Thr Gly Gly Gly Ser Thr Tyr
1 5
<210> 98
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 98
Ile Phe Arg Ser Gly Arg Thr Ser Trp
1 5
<210> 99
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 99
Ile Phe Thr Ser Gly Arg Arg Pro Trp
1 5
<210> 100
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 100
Ile Thr Ser Thr Gly Thr Arg Gln Tyr
1 5
<210> 101
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 101
Ile Tyr Thr Arg Gly Asp Arg Thr Phe
1 5
<210> 102
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 102
Ile Tyr Arg Gly Asn Gly Ala Thr Gly
1 5
<210> 103
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 103
Ile Trp Arg Gly Gly His Ser Thr Leu
1 5
<210> 104
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 104
Ile Phe Ser Ser Gly Arg Arg Pro Trp
1 5
<210> 105
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 105
Leu His Thr Asp Gly Leu Thr Ser
1 5
<210> 106
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 106
Ile Phe Thr Ser Gly Arg Arg Ser Trp
1 5
<210> 107
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 107
Ile Tyr Thr Ala Thr Gly Arg Thr Tyr
1 5
<210> 108
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 108
Ile Asp Ser Asp Gly Ser Thr Ser
1 5
<210> 109
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 109
Ile Asp Ser Asp Gly Val Thr Asp
1 5
<210> 110
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 110
Ile Asn Ser Val Gly Arg Thr Arg
1 5
<210> 111
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 111
Ile Tyr Thr Ser Ile Gly Arg Thr Tyr
1 5
<210> 112
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 112
Ile Glu His Asp Gly Lys Ile Ile
1 5
<210> 113
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 113
Ile Tyr Arg Gly Ser Gly Thr Thr His
1 5
<210> 114
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 114
Ile Tyr Leu Ala Asn Gly Ala Thr Tyr
1 5
<210> 115
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 115
Ile Tyr His Gly Asp Gly Thr Thr Tyr
1 5
<210> 116
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 116
Ile Gly Ser Asp Gly Thr Thr Lys
1 5
<210> 117
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 117
Val Ser Pro Arg Gly Glu Ser Ile Tyr
1 5
<210> 118
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 118
Ile Phe Ser Ser Arg Asp Tyr Thr Asp
1 5
<210> 119
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 119
Ile Asp Thr Gly Gly Ser Thr Tyr
1 5
<210> 120
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 120
Ile Tyr Thr Arg Asp Ser Arg Thr Tyr
1 5
<210> 121
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 121
Ile Thr Ala Ser Gly Ser Thr Tyr
1 5
<210> 122
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 122
Ile Ser Ala Gly Gly Ile Ser Ile Asp
1 5
<210> 123
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 123
Trp Gly Ser Val Gly Ser Ser Thr Thr Tyr
1 5 10
<210> 124
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 124
Ile Phe Ser Asp Gly Glu Thr Ala
1 5
<210> 125
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 125
Ile Tyr Thr Gly Ile Gly Thr Thr Tyr
1 5
<210> 126
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 126
Ile Tyr Thr Gly Asp Gly Ser Thr His
1 5
<210> 127
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 127
Ile Ser Ala Gln Gly Val Ile Pro Gly
1 5
<210> 128
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 128
Ile Asp Ser Ala Gly Ser Thr Arg
1 5
<210> 129
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 129
Ile Arg Ser Gly Gly Gly Asn Thr Tyr
1 5
<210> 130
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 130
Ile Ser Arg Ile Asp Asn Ser Thr Tyr
1 5
<210> 131
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 131
Phe Val Thr Gly Ala Gly Ser Thr Tyr
1 5
<210> 132
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 132
Ile Thr Val Thr Gly Thr Arg Gln Tyr
1 5
<210> 133
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 133
Ile Asp Asn Asn Gly Trp Ser Thr
1 5
<210> 134
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 134
Ile Asp Thr Asn Gly Ser Thr Ser
1 5
<210> 135
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 135
Ile Tyr Arg Asp Gly Ser Ala Pro Tyr
1 5
<210> 136
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 136
Ile Tyr Thr Gly Gly Ser Ala Thr Ser
1 5
<210> 137
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 137
Ile Cys Ser Asp Gly Ser Ser Ala
1 5
<210> 138
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 138
Ile Tyr Thr Gly Ile Gly Ser Thr Tyr
1 5
<210> 139
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 139
Ile Gly Tyr Ser Gly Ser Thr Tyr
1 5
<210> 140
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 140
Ile Cys Ser Asp Gly Ser Thr Ala
1 5
<210> 141
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 141
Ile Thr Phe Thr Gly Arg Thr Leu
1 5
<210> 142
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 142
Ile Tyr Thr Asn Val Gly Thr Thr Tyr
1 5
<210> 143
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 143
Ile Asp Arg Asp Gly Arg Thr Ser
1 5
<210> 144
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 144
Ile His Thr Asp Gly Ser Thr Ser
1 5
<210> 145
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 145
Ser Asn Thr Asn Gly Gly Ser Thr Tyr
1 5
<210> 146
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 146
Thr Asn Arg Asp Gly Met Ser Tyr
1 5
<210> 147
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 147
Val Tyr Asn Asp Gly Gly His Thr Tyr
1 5
<210> 148
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 148
Ile Phe Thr Arg Gly Thr Thr Lys
1 5
<210> 149
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 149
Leu Tyr Leu Gly Gly Ser Ile Thr Tyr
1 5
<210> 150
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 150
Ile Asp Thr Ile Gly Glu Ile Asp
1 5
<210> 151
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 151
Leu Asp Gly Asp Gly Arg Val Arg
1 5
<210> 152
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 152
Ala Ala Ala Trp Arg Pro Pro Cys Val Pro
1 5 10
<210> 153
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 153
Ala Ala Asp Leu Ser Pro Tyr Asp Cys Tyr Thr Gly Ser Leu Asp Met
1 5 10 15
Ala Ser Arg Phe Thr
20
<210> 154
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 154
Ala Ala Val Leu Cys Thr Asp Asp Tyr Lys Met Ala Pro Ala Asn Tyr
1 5 10 15
<210> 155
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 155
Ala Ala Gly Leu Ala Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Ala
1 5 10 15
Gly Phe Arg Tyr
20
<210> 156
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 156
Ala Ala Arg Arg Thr Trp His Ala Gly Phe Pro Tyr
1 5 10
<210> 157
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 157
Ala Ala Gly Leu Asp Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Ala
1 5 10 15
Gly Phe Arg Tyr
20
<210> 158
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 158
Ala Ala Asn Arg Arg Ala Tyr Pro Tyr Gly Gly Asp Cys Arg Leu Arg
1 5 10 15
His Ala Glu Phe Asp Tyr
20
<210> 159
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 159
Ala Ala Asp Val Pro Gly Arg Arg Glu Val Arg Gly Leu Gly Pro Cys
1 5 10 15
Asp Arg Met Tyr Asp Tyr
20
<210> 160
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 160
Ala Ala Lys Val Pro Phe Gly Arg Gly Ser Cys Ala Tyr Ser Thr Ala
1 5 10 15
His Trp Phe Pro Tyr
20
<210> 161
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 161
Ala Ala Gly Leu Asp Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Asp
1 5 10 15
Gly Phe Arg Tyr
20
<210> 162
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 162
Val Ala Asp Leu Ser Ala Trp Cys Arg Ala Val Arg Pro Gly Val Ile
1 5 10 15
Thr Tyr Asn Tyr
20
<210> 163
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 163
Ala Ala Asp Pro Arg Asp Pro Asn Gly Ser Arg Thr Asp Cys Thr Val
1 5 10 15
Leu Thr Ser Lys Asp Leu Tyr Asn Ser
20 25
<210> 164
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 164
Ala Ala Gly Pro Gly Cys Ser Trp Ser Ser Phe Ala Tyr
1 5 10
<210> 165
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 165
Val Ala Met Thr Trp Asp Gly Thr Cys His Ile Thr Ser Glu Phe Tyr
1 5 10 15
<210> 166
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 166
Ala Ala Val Ile Gly Val Asp Ile Arg
1 5
<210> 167
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 167
Ala Ile Arg Trp Gly Asp Cys Asp Ser Ala Ser Trp Ser Arg Arg Thr
1 5 10 15
Trp Tyr Ala Val
20
<210> 168
<211> 23
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 168
Ala Arg Ser Arg Ala Ser Leu Trp Ser Gly Asn Trp Tyr Arg Ser Leu
1 5 10 15
Ser Glu Asp Glu Tyr Asn Ser
20
<210> 169
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 169
Ala Ala Gly His Asp Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Asp
1 5 10 15
Gly Phe Arg Tyr
20
<210> 170
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 170
Ala Ala Ala Asp Ala Gln Arg Gly Arg Thr Cys Phe Phe Gly Ala
1 5 10 15
<210> 171
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 171
Ala Val Arg Trp Tyr Trp Asp Ala Gly Phe Lys Tyr
1 5 10
<210> 172
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 172
Ala Ala Gly His Asp Gly Arg Ser Gly Tyr Leu Pro Leu Asn Trp Ala
1 5 10 15
<210> 173
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 173
Ala Ala Val Ile Gly Tyr Asp Ile Arg
1 5
<210> 174
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 174
Ala Ala Asp Ser Arg Thr Pro Ser Asp Cys Tyr Ser Gly Ser Trp Leu
1 5 10 15
Glu Lys Tyr Pro Ser Glu Tyr Ser Tyr
20 25
<210> 175
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 175
Ala Ala Asp Val Val Ser Tyr Tyr Ser Asp Tyr Val Cys Thr Asp Ala
1 5 10 15
Ala Asp Phe Gly Tyr
20
<210> 176
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 176
Ala Ala Gly Thr Thr Arg Leu Gly Ser Leu Leu Ala Pro Thr Tyr Arg
1 5 10 15
Tyr
<210> 177
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 177
Ala Ala Gly Phe Met Tyr Gly Glu Thr Arg Ser Pro Asn Trp Val Asn
1 5 10 15
Tyr
<210> 178
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 178
Ala Lys Lys Arg Thr Tyr Asp Cys Tyr Ser Gly Thr Cys Ser
1 5 10
<210> 179
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 179
Ala Ala Gly Leu Tyr Gly Gly Ser Pro Tyr Phe Pro Leu Asn Trp Thr
1 5 10 15
Gly Phe Arg Tyr
20
<210> 180
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 180
Ala Ala Gly Leu Arg Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Ala
1 5 10 15
Gly Phe Arg Tyr
20
<210> 181
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 181
Ala Ala Gly His Asp Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Ala
1 5 10 15
Gly Phe Arg Tyr
20
<210> 182
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 182
Lys Thr Val Lys Asp Pro Thr Ser Pro Pro Gly Cys Ser Arg Gly Tyr
1 5 10 15
<210> 183
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 183
Ala Ala Asp Leu Leu Ile Gly Ala Cys Ser Gln Met Arg Arg Thr Asn
1 5 10 15
Phe Asp Tyr
<210> 184
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 184
Ala Ala Asn Pro Tyr Ser Pro Gly Ala Gly Arg Glu Leu Leu Ser Tyr
1 5 10 15
Pro Tyr Thr Asp
20
<210> 185
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 185
Ala Ala Asn Gln Gly Ser Gly Asp Tyr Cys Tyr Met Ala Met Leu Ile
1 5 10 15
Tyr Gly Met Phe Tyr
20
<210> 186
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 186
Ala Ala Arg Asp Gly Ser Trp Phe Leu Ser Leu Val Pro Ala Thr Tyr
1 5 10 15
Gly Tyr
<210> 187
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 187
Ala Ala Asp Leu Gln Ile Gly Ser Cys Ser Gln Met Arg Arg Tyr Asn
1 5 10 15
Tyr Ala Tyr
<210> 188
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 188
Gly Ala Asp Pro Gly Glu Gly Ser Tyr Cys Ala Tyr Glu Ala Pro Glu
1 5 10 15
Val Gly Ala Leu Asp Ile
20
<210> 189
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 189
Ala Ala Gly Arg Thr Tyr Asp Cys Tyr Pro Gly Thr Cys Ser
1 5 10
<210> 190
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 190
Ala Ala Ser Leu Arg Ala Arg Trp Val Gln Arg Gly Ala Pro Leu Leu
1 5 10 15
Pro Ser Phe Tyr Gly Tyr
20
<210> 191
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 191
Ala Ala Gly Pro Trp Val Ala Thr Pro Glu Ile Ala Asn Glu Tyr Asn
1 5 10 15
Tyr
<210> 192
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 192
Val Ala Gly Ile Trp Thr Cys Gly Arg Ser Ala Leu Thr Asp Pro Asn
1 5 10 15
Asn Tyr
<210> 193
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 193
Ala Ala Thr Phe Gly Leu Phe Trp Glu Ser Trp Glu Trp Tyr Lys Asn
1 5 10 15
Trp His Tyr
<210> 194
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 194
Ala Ala Asp Pro Gly Val Leu Cys Gly Arg Ser Trp Val Gly Arg Phe
1 5 10 15
Pro Tyr
<210> 195
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 195
Ala Ala His Gly Ala Phe Ala Ala Arg Asn Asp Pro Arg Gln Trp Arg
1 5 10 15
Tyr
<210> 196
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 196
Ala Ala Gln Tyr Gly Thr Cys Gln Gly Leu Leu Ser Arg Tyr Phe Ala
1 5 10 15
Tyr
<210> 197
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 197
Ala Ala Ala His Glu Pro Gly Ser Trp Thr Asp Ile Glu Ala Arg Gly
1 5 10 15
Lys Ile Ser Asp Asp Pro Phe Gly Tyr
20 25
<210> 198
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 198
Ala Arg Ala Ser Phe Arg Gly Ser Trp Phe Phe Glu Gly
1 5 10
<210> 199
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 199
Ala Ala Ala Arg Arg Trp Tyr Tyr Glu Asn Ser Cys Leu Lys Val Leu
1 5 10 15
Arg Ser Pro Gly Asp Tyr
20
<210> 200
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 200
Ala Ala Gly Pro Pro Ser Gly Pro Leu Arg Ala Cys His Glu Ser Val
1 5 10 15
Phe Gly Tyr
<210> 201
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 201
Lys Thr Val Arg Asp Pro Ala Ser Pro Pro Ser Cys Ser Gly Gly Tyr
1 5 10 15
<210> 202
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 202
Ala Ala Asp Arg Val Leu Gly Arg Cys Ser Arg Arg Leu Leu Ser Asp
1 5 10 15
Phe Gly Tyr
<210> 203
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 203
Ala Ala Gly Thr Tyr Tyr Ser Asp Tyr Asp Pro Pro Arg Tyr Glu Tyr
1 5 10 15
Lys Tyr
<210> 204
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 204
Ala Ala Lys Glu Arg Pro Leu Cys Gly Ser Phe Trp Glu Arg Gly Asp
1 5 10 15
Glu Tyr Ala Ser
20
<210> 205
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 205
Ala Ala Val Ser Trp Ala Cys Trp Arg Leu Ser Gly Thr Gly Phe Asn
1 5 10 15
Tyr
<210> 206
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 206
Ala Ala Asp Leu Gly Val Asp Asp Tyr Ser Asp Tyr Leu Asp His Pro
1 5 10 15
Phe Gly Tyr
<210> 207
<211> 29
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 207
Ala Ala Asp Ile Gly Gly Ser Trp Pro Arg Lys Pro His Pro Asp Pro
1 5 10 15
Asn Phe Gly Gly Glu Cys Gly Gly Tyr Gly Met Ala Phe
20 25
<210> 208
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 208
Ala Ala Asp Ser Ser Ser Leu Pro Cys Tyr Pro Arg Ala Ala Gln Phe
1 5 10 15
Pro Arg Leu Arg Tyr
20
<210> 209
<211> 26
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 209
Ala Ala Arg Pro Ala Pro Val Ser Gly Ile Thr Arg Phe Arg Leu Asn
1 5 10 15
Arg Ser Leu Leu Pro Asn Glu Tyr Asn Ser
20 25
<210> 210
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 210
Ala Ala Gly Leu Asp Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Ala
1 5 10 15
Asn Phe Arg Tyr
20
<210> 211
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 211
Ala Ala Glu Gly Gly Trp Arg Asp Tyr Val Arg Ser Trp Gly Arg Asn
1 5 10 15
Phe Gly Tyr
<210> 212
<211> 23
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 212
Ala Ala Asp Leu Trp Arg Gly Pro Pro Phe Gly Gly Tyr Trp Ser Pro
1 5 10 15
Thr Lys Ser Glu Phe Ala Tyr
20
<210> 213
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 213
Ala Ala Asn Arg Arg Ala Tyr Pro Tyr Gly Gly Asp Cys Arg Val Arg
1 5 10 15
His Ala Glu Phe Asp Tyr
20
<210> 214
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 214
Ala Ala Arg Phe Arg Ala His Asp Gly Tyr Trp Asn Trp Gln Asn Ala
1 5 10 15
Gly Asn Tyr Asn Tyr
20
<210> 215
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 215
Ala Ala Arg His Tyr Trp Ser Ala Gly Phe Pro Tyr
1 5 10
<210> 216
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 216
Ala Ala Asp Arg Val Leu Gly Arg Cys Ser Arg Arg Ile Leu Ser Asp
1 5 10 15
Phe Gly Tyr
<210> 217
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 217
Ala Arg Ser Ser Pro Arg Thr Val Val Ala Gly Phe Gly Asp
1 5 10
<210> 218
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 218
Ala Ala Gly Leu Asp Gly Gly Asn Gly Tyr Leu Pro Leu Asn Trp Ala
1 5 10 15
Gly Phe Arg Tyr
20
<210> 219
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 219
Ala Arg Gly Pro Phe Gly Cys Tyr Ser Val Ser Gly Cys Tyr Arg Lys
1 5 10 15
Gly Gly Val Asp Asn Tyr
20
<210> 220
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 220
Lys Thr Val Arg Asp Pro Thr Ser Pro Arg Ser Cys Ser Gly Gly Tyr
1 5 10 15
<210> 221
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 221
Ala Ala Tyr His Ser Gly Ser Trp Cys Tyr Leu Pro His Leu Gly Ser
1 5 10 15
Tyr Gly Tyr
<210> 222
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 222
Ala Ala Gly Trp Arg Leu Ser Leu Arg Val Ser Asp Phe Asn Tyr
1 5 10 15
<210> 223
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 223
Ala Ala Ser Phe Arg Pro Thr Trp Phe Cys Arg Gly Leu Ala Pro Tyr
1 5 10 15
Lys Tyr Asn Leu
20
<210> 224
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 224
Thr Ser Ala Ser Leu Thr Trp Asp Gly Gly Asn Trp Tyr Cys Pro Thr
1 5 10 15
His Gly Tyr
<210> 225
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 225
Ala Ala Gly Arg Thr Tyr Asp Cys Tyr Ser Gly Thr Cys Ser
1 5 10
<210> 226
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 226
Ala Ala Gly Ala Tyr Arg Ala Ser Phe Thr Tyr
1 5 10
<210> 227
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 227
Asn Thr Met Trp Gly Ala Arg Gln Asn Lys Asp
1 5 10
<210> 228
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 228
Ala Ala Asn Arg Arg Pro Tyr Pro Tyr Gly Gly Asp Cys Arg Leu Arg
1 5 10 15
His Ala Glu Phe Asp Tyr
20
<210> 229
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 229
Ala Ala Gly Leu Asp Gly Gly Ser Gly Tyr Leu Pro Leu Ala Trp Ala
1 5 10 15
Gly Phe Arg Tyr
20
<210> 230
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 230
Ala Ala Gly Leu Asp Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Ala
1 5 10 15
Arg Phe Arg Tyr
20
<210> 231
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 231
Ala Ala Gly Val Ala Gly Gly Ser Gly Tyr Leu Pro Leu Asn Trp Ala
1 5 10 15
Gly Phe Arg Tyr
20
<210> 232
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 232
Ala Ala Gly Phe Ala Gly Cys Tyr Gly Ser Ser Trp Tyr Gly Ser Ala
1 5 10 15
Asp Phe Gly Tyr
20
<210> 233
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 233
Xaa Xaa Xaa Ser Thr Ala Tyr Tyr
1 5
<210> 234
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 234
Gln Val Arg Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu
1 5 10 15
Thr Leu Arg Leu Ser Cys Thr Ala Ser
20 25
<210> 235
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 235
Met Gly Trp Tyr Arg Gln Gly Pro Gly Asn Glu Cys Glu Met Val Ala
1 5 10 15
Tyr
<210> 236
<211> 36
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 236
Ala Asp Ser Thr Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys
1 5 10 15
His Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly
20 25 30
Val Tyr Tyr Cys
35
<210> 237
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 237
Gly Gln Gly Thr Arg Val Thr Val Ser Ser
1 5 10

Claims (2)

1. An SFTSV detection kit, comprising a detection antibody, a solid substrate and a coating antibody coated on the solid substrate, wherein the detection antibody and the coating antibody respectively comprise 3 complementarity determining regions CDR1-3,
i) The CDR sequences of the detection antibody are as follows: CDR1 has the sequence of SEQ ID NO 4, CDR2 has the sequence of SEQ ID NO 84, and CDR3 has the sequence of SEQ ID NO 181; and is
II) the CDR sequences of the coated antibody are as follows: CDR1 has the sequence SEQ ID NO 54, CDR2 has the sequence SEQ ID NO 132, and CDR3 has the sequence SEQ ID NO 176; or the sequence of CDR1 is SEQ ID NO. 13, the sequence of CDR2 is SEQ ID NO. 99, and the sequence of CDR3 is SEQ ID NO. 166.
2. An SFTSV detection kit, comprising a detection antibody, a solid matrix and a coating antibody coated on the solid matrix, wherein the detection antibody and the coating antibody respectively comprise 3 complementarity determining regions CDR1-3,
the CDR sequences of the detection antibody are as follows: CDR1 has the sequence of SEQ ID NO 13, CDR2 has the sequence of SEQ ID NO 99, and CDR3 has the sequence of SEQ ID NO 166; and is provided with
The CDR sequences of the coated antibody are as follows: CDR1 has the sequence SEQ ID NO 54, CDR2 has the sequence SEQ ID NO 132, and CDR3 has the sequence SEQ ID NO 176.
CN201910885757.6A 2019-07-23 2019-09-19 SFTSV detection kit Active CN110684102B (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/CN2019/097350 WO2021012195A1 (en) 2019-07-23 2019-07-23 Nano-antibody capable of binding to sftsv and application thereof
CNPCT/CN2019/097350 2019-07-23

Publications (2)

Publication Number Publication Date
CN110684102A CN110684102A (en) 2020-01-14
CN110684102B true CN110684102B (en) 2022-10-21

Family

ID=69109550

Family Applications (3)

Application Number Title Priority Date Filing Date
CN201980001738.0A Active CN112105637B (en) 2019-07-23 2019-07-23 Nano antibody capable of combining SFTSV and application thereof
CN201910887917.0A Active CN110713536B (en) 2019-07-23 2019-09-19 Polypeptide capable of combining SFTSV, nucleic acid coding sequence and application thereof
CN201910885757.6A Active CN110684102B (en) 2019-07-23 2019-09-19 SFTSV detection kit

Family Applications Before (2)

Application Number Title Priority Date Filing Date
CN201980001738.0A Active CN112105637B (en) 2019-07-23 2019-07-23 Nano antibody capable of combining SFTSV and application thereof
CN201910887917.0A Active CN110713536B (en) 2019-07-23 2019-09-19 Polypeptide capable of combining SFTSV, nucleic acid coding sequence and application thereof

Country Status (4)

Country Link
JP (1) JP7171737B2 (en)
KR (1) KR102504884B1 (en)
CN (3) CN112105637B (en)
WO (1) WO2021012195A1 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112105637B (en) * 2019-07-23 2022-12-06 源道隆（苏州）医学科技有限公司 Nano antibody capable of combining SFTSV and application thereof
CN112724248A (en) * 2021-01-28 2021-04-30 南京拓峰生物科技有限公司 Nano antibody capable of combining SARS-CoV-2 and application thereof
CN113980125B (en) * 2021-10-15 2024-03-26 中国科学院武汉病毒研究所 Neutralizing monoclonal antibody for resisting SFTSV and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102942629A (en) * 2012-11-21 2013-02-27 江苏省疾病预防控制中心 Humanized antibody for resisting severe fever with thrombocytopenia syndrome bunyavirus (SFTSV)
CN108178796A (en) * 2017-12-28 2018-06-19 江苏省疾病预防控制中心 Fever is with thrombocytopenic syndromes viral glycoprotein immue quantitative detection reagent box

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE374248T1 (en) * 1996-06-27 2007-10-15 Vlaams Interuniv Inst Biotech ANTIBODY MOLECULES THAT INTERACT SPECIFICALLY WITH THE ACTIVE CENTER OR ACTIVE Cleft of a TARGET MOLECULE
AR062123A1 (en) * 2007-07-27 2008-10-15 Inst Nac De Tecnologia Agropec VHH MONOMERIC DOMAIN DERIVED FROM ANTI-VP6 CAMELID ANTIBODIES, DIMERIC DOMAIN, ROTAVIRUS IMMUNODETECTION METHOD, COMPOSITIONS, ROTAVIRUS INFECTION PREVENTION AND TREATMENT METHODS
EP2566894A1 (en) * 2010-05-06 2013-03-13 Novartis AG Compositions and methods of use for therapeutic low density lipoprotein - related protein 6 (lrp6) multivalent antibodies
CN104062443A (en) * 2014-07-14 2014-09-24 江苏省疾病预防控制中心 Quantitative determination kit for neutralizing antibodies of virus and application thereof
KR101785290B1 (en) * 2014-11-05 2017-10-18 대한민국 Production and application of a monoclonal antibody and the development of a competitive enzyme-linked immunosorbent assay(c-ELISA) for detection of severe fever with thrombocytopenia syndrome virus(SFTSV)
JP7328479B2 (en) * 2016-03-23 2023-08-17 ソウル大学校産学協力団 Antibody that binds to outer membrane glycoprotein of severe fever with thrombocytopenic syndrome virus and use thereof
CN107501396B (en) * 2017-09-27 2019-12-13 源道隆（苏州）医学科技有限公司 Protein for diagnosing SFTS patient prognosis condition and application thereof
CN112105637B (en) * 2019-07-23 2022-12-06 源道隆（苏州）医学科技有限公司 Nano antibody capable of combining SFTSV and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102942629A (en) * 2012-11-21 2013-02-27 江苏省疾病预防控制中心 Humanized antibody for resisting severe fever with thrombocytopenia syndrome bunyavirus (SFTSV)
CN108178796A (en) * 2017-12-28 2018-06-19 江苏省疾病预防控制中心 Fever is with thrombocytopenic syndromes viral glycoprotein immue quantitative detection reagent box

Also Published As

Publication number Publication date
JP2021534727A (en) 2021-12-16
JP7171737B2 (en) 2022-11-15
KR102504884B1 (en) 2023-02-28
CN110713536A (en) 2020-01-21
CN112105637B (en) 2022-12-06
CN110684102A (en) 2020-01-14
WO2021012195A1 (en) 2021-01-28
CN110713536B (en) 2021-08-31
KR20210013000A (en) 2021-02-03
CN112105637A (en) 2020-12-18

Similar Documents

Publication Publication Date Title
CN112094342B (en) Alpaca source nano antibody combined with SARS-CoV-2RBD
CN112094343B (en) Alpaca source nano antibody combined with SARS-CoV-2 RBD
CN110903394B (en) Polypeptide capable of binding CD4 and application thereof
CN110684102B (en) SFTSV detection kit
CN111848798B (en) Nanometer antibody capable of combining BCMA and application thereof
CN112724248A (en) Nano antibody capable of combining SARS-CoV-2 and application thereof
WO2021110181A2 (en) Polypeptide able to bind with cd47 and use therefor
CN110922482B (en) Polypeptide capable of binding CD19 and application thereof
CN111808193B (en) Nanobody capable of binding human CD38 and application thereof
CN114276452A (en) Nano antibody capable of being combined with BCMA (brain cell activating antigen) and application thereof
CN113461810A (en) Fully human monoclonal antibody for resisting novel coronavirus spike protein and application thereof
CN115850462A (en) Polypeptide NbM14 capable of recognizing and neutralizing MERS-CoV and application thereof
CN110950957B (en) Polypeptide capable of binding CTLA4 and application thereof
CN110950956B (en) Polypeptide capable of binding PD1 and application thereof
CN110903393B (en) Polypeptide capable of binding IL6R alpha and application thereof
CN111393527B (en) Polypeptide capable of binding PD-L1 and application thereof
CN114213539B (en) Nanobody 4NB357 capable of binding CD4 and application thereof
CN114292329B (en) anti-CD 19 antibodies and uses thereof
CN114249822B (en) Alpaca-derived nanobody combined with SARS-CoV-2RBD
CN114276453A (en) Nanobody 4NB334 capable of binding CD4 and application thereof
CN115850463A (en) Polypeptide NbM9 capable of recognizing and neutralizing MERS-CoV and application thereof
Zhao et al. A high potent synthetic nanobody with broad-spectrum activity neutralizes SARS-Cov-2 virus and Omicron variant through a unique binding mode
CN114249820A (en) Alpaca source nano antibody combined with SARS-CoV-2RBD
CN114249821A (en) Alpaca source nano antibody combined with SARS-CoV-2RBD
CN116023482A (en) Neutralizing antibodies targeting coronaviruses, antigen binding fragments thereof and uses thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant