CN108178796B - Fever is with thrombocytopenic syndromes viral glycoprotein immue quantitative detection reagent box - Google Patents
Fever is with thrombocytopenic syndromes viral glycoprotein immue quantitative detection reagent box Download PDFInfo
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- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000003014 reinforcing effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000010206 sensitivity analysis Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
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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
-
- 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
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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N2333/175—Bunyaviridae, e.g. California encephalitis virus, Rift valley fever virus, Hantaan virus
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Abstract
The invention discloses it is a kind of can quantitative detection fever with thrombocytopenic syndromes viral glycoprotein kit, the kit includes two monoclonal antibodies for fever with thrombocytopenic syndromes viral glycoprotein Gc, realizes detection fever with the purpose of thrombocytopenic syndromes viral glycoprotein by double antibody sandwich ELISA.Detection kit detectable concentration range of the invention is big, and high sensitivity, high specificity, testing cost is low, easy to use, suitable for promoting on production of vaccine field or clinical diagnosis.
Description
Technical field
The invention belongs to field of biomedicine, it is related to a kind of fever and is quantitatively examined with thrombocytopenic syndromes viral glycoprotein
Test agent box.
Background technique
Fever is with thrombocytopenic syndromes virus (severe fever with thrombocytopenia
Syndrome virus, SFTSV) it is the novel hemorrhagic fever viruse of one kind popular in China Middle Eastern in recent years.The virus
People and animal are infected by the mode that tick worm (haemaphysalis longicornis, H.longicornis) bites, also can cause people by close contact
Border is propagated, and causes huge life, property loss to area is suffered from.China has 17 provinces (city, autonomous region) and confirmed at present
The prevalence of the virus, also someone infects the report of the virus for Chinese Japan, neighbouring country, South Korea etc..
SFTSV belongs to bunyaviridae, Phlebovirus on taxology, genome by big (L), in (M), small
(S) three sub-thread strand RNA segment compositions, viral genome 3 ' end and 5 ' ends similar to other viruses of bunyaviridae
Terminal sequence is complementary.The RNA polymerase that L fragment coding RNA is relied on；M fragment coding glycoprotein precursor, through in host cell after translation
The modification of protease forms two glycoprotein of Gn and Gc；S segment category ambisense RNA is separately encoded nucleocapsid protein NP and non-structural
Albumen NSs.
Prevention for disease of natural focus caused by the virus, in addition to implementing tick eradication, reinforcing the strategies such as Case monitoring
Outside, carrying out effective vaccine inoculation to the Susceptible population for living in epidemic-stricken area is means the most basic.SFTSV inactivated vaccine is current
Just in R&D process, and on effective component-virion envelope in the vaccine glycoprotein (Gn and Gc) it is real-time dynamic,
Quantitative Monitoring is that an important technical is crucial in the vaccine R&D process.
The invention patent describes a kind of sugared come Gc in quantitative detection Virus culture supernatant based on double-antibody sandwich elisa
The content of albumen.Since the mole ratio of two kinds of glycoprotein of the Gn on virus envelope and Gc is 1:1, and the molecular weight of the two is also big
Body is suitable, therefore can be obtained the content of entire glycoprotein by the detection to Gc glycoprotein.
Summary of the invention
One of the objects of the present invention is to provide a kind of fevers with thrombocytopenic syndromes viral glycoprotein quantitative detection
Kit.
The second object of the present invention is to provide above-mentioned fever with thrombocytopenic syndromes viral glycoprotein quantitative detection
Coated antibody and detection antibody in kit.
The third object of the present invention is that providing a kind of detected using mentioned reagent box is generated heat with thrombocytopenic syndromes
The method of glycoprotein in virus.
To achieve the goals above, present invention employs following technical solutions:
The present invention provides a kind of for fever with the monoclonal antibody of thrombocytopenic syndromes viral glycoprotein Gc, institute
Stating monoclonal antibody includes:
Light chain variable region has the ammonia of at least 80% homology with amino acid sequence shown in SEQ ID NO:2 or with it
Base acid sequence；
Heavy chain variable region has the ammonia of at least 80% homology with amino acid sequence shown in SEQ ID NO:1 or with it
Base acid sequence；The monoclonal antibody is named as 1C5-F5.
Preferably, the light chain variable region of 1C5-F5 antibody has amino acid sequence shown in SEQ ID NO:2, weight chain variable
Area has amino acid sequence shown in SEQ ID NO:1.
The present invention provides a kind of for fever with the monoclonal antibody of thrombocytopenic syndromes viral glycoprotein Gc, institute
Stating monoclonal antibody includes:
Light chain variable region has the ammonia of at least 80% homology with amino acid sequence shown in SEQ ID NO:4 or with it
Base acid sequence；
Heavy chain variable region has the ammonia of at least 80% homology with amino acid sequence shown in SEQ ID NO:3 or with it
Base acid sequence；The monoclonal antibody is named as 1H6-B10.
Preferably, the light chain variable region of 1H6-B10 antibody has amino acid sequence shown in SEQ ID NO:4, and heavy chain can
Becoming area has amino acid sequence shown in SEQ ID NO:3.
The antibody of conservative sequence variant including preferred antibody amino acid sequence is also included within the scope of the present invention.
Conserved amino acid sequence variant includes the modification for not significantly changing the amino acid sequence of monoclonal antibody binding property of the invention,
Such as variant caused by the variant of Similar amino acids well known in the art substitution, the missing of amino acid, increase.
Monoclonal antibody of the invention further includes source of people and non-human source antibodies, and is had identical as said monoclonal antibody
Function or all antibody of transformation and optimization.
The monoclonal antibody of disclosure of the invention can include one or more glycosylation positions in heavy chain and light chain variable region
Point, as known in the art, the one or more glycosylation site present in variable region can cause the antibody of enhancing to be exempted from
Epidemic focus, or change the pharmacokinetics of antibody due to changing antigen binding.
Monoclonal antibody of the invention can be designed as comprising modification in the region Fc, usually change 1 of antibody
Or multiple functional characteristics, such as serum half-life, the cytotoxicity that complement combines, Fc receptor combines, and/or antigen relies on.In addition,
Antibody of the invention can be modified by sulphation and (e.g., one or more chemical groups can be connected to antibody), or be modified with
Change its glycosylation, to change the one or more functions characteristic of antibody again.
Another modification that monoclonal antibody of the invention can be designed is Pegylation.Antibody can be by polyethylene glycol
Change, thus, for example, increasing biology (such as serum) half-life period of antibody.In order to make antibody Pegylation, the antibody or its segment
It is anti-with PEG usually under conditions of being suitable for one or more polyethylene glycol (PEG) group and being connected to the antibody or antibody fragment
It answers, such as the active ester or aldehyde derivatives of polyethylene glycol.Preferably, the Pegylation be by with active PEG molecule (or class
As reactive water-soluble polymer) carry out acylation reaction or alkylated reaction and realize.
The present invention also provides the nucleic acid molecules for encoding mentioned-above monoclonal antibody, the nucleic acid molecules have SEQ
Nucleotide sequence shown in ID NO:5-8 or the nucleotide sequence with it at least 80% homology.
Preferably, the nucleic acid molecules have nucleotide sequence shown in SEQ ID NO:5-8.
The sequence of nucleic acid molecules of 1C5-F5 antibody heavy chain variable region is encoded as shown in SEQ ID NO:5, coding 1C5-F5 is anti-
The sequence of nucleic acid molecules of body light chain variable region is as shown in SEQ ID NO:6.
The sequence of nucleic acid molecules of 1H6-B10 heavy chain variable region is encoded as shown in SEQ ID NO:7, encodes 1H6-B10 light chain
The sequence of nucleic acid molecules of variable region is as shown in SEQ ID NO:8.
The nucleic acid molecules of the mentioned-above monoclonal antibody of coding of the invention include having above-mentioned preferred nucleotides sequence
The nucleic acid molecules of the conserved nucleotide sequence variant of column.So-called conserved nucleotide sequence variant derived from genetic code degeneration and
The variant of silencing, substitution, missing and the increase of nucleotide are also included.
The present invention also provides a kind of expression vector, the expression vector includes mentioned-above nucleic acid molecules, in addition, also
Including the expression regulation sequence being operatively connected with the sequence of nucleic acid molecules.
" carrier " word, which refers to, in the present invention can be inserted the polynucleotide for encoding certain albumen and obtain albumen
A kind of nucleic acid delivery vehicle that must be expressed.Carrier can make the inhereditary material of its carrying by conversion, transduction or transfection host cell
Element is expressed in host cell.For example, carrier includes: plasmid；Phasmid；Coemid；Artificial chromosome is such as
The artificial chromosome (PAC) of yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or the source P1；Bacteriophage such as λ bites
Thallus or M13 bacteriophage and animal virus etc..Animal virus type as carrier has retrovirus (including slow virus), gland
Virus, adeno-associated virus, herpesviral (such as herpes simplex virus), poxvirus, baculoviral, papillomavirus, nipple polyoma
Vacuolating virus (such as SV40).A kind of element that carrier may be expressed containing various control, including promoter sequence, transcription initiation sequence
Column, enhancer sequence, selection element and reporter gene.In addition, carrier can also contain replication origin.Carrier is it is also possible to packet
The ingredient for assisting its to enter cell is included, such as virion, liposome or protein coat, but not only there was only these substances.
The present invention also provides a kind of host cell, mentioned-above nucleic acid molecules or front are contained in the host cell
The expression vector.
" host cell " word refers to importing the cell of nucleic acid molecules or carrier, including following many cells in the present invention
Type, such as Escherichia coli or withered grass bacterium prokaryotic cell, such as yeast cells or Aspergillus fungal cell, such as S2 drosophila cell or
The insect cells such as Sf9, or such as fibroblast, Chinese hamster ovary celI, COS cell, NSO cell, HeLa cell, bhk cell, HEK
The zooblast of 293 cells or people's cell.
In specific embodiments of the present invention, the host cell is Chinese hamster ovary celI.
The present invention also provides it is a kind of using mentioned-above host cell generate antibody method, the method includes
Mentioned-above host cell is cultivated under the conditions of suitable and recycles the antibody.
The present invention also provides a kind of antibody generated by the above method.
The present invention also provides a kind of for fever with the monoclonal antibody of thrombocytopenic syndromes viral glycoprotein Gc
Composition, which is characterized in that the monoclonal antibody combination includes 1C5-F5 and 1H6-B10.
A kind of immue quantitative detection reagent box the present invention also provides fever with thrombocytopenic syndromes viral glycoprotein, institute
Stating detection kit includes 1C5-F5 and/or 1H6-B10.
Detection kit of the invention can only include a monoclonal antibody in 1C5-F5 or 1H6-B10, this feelings
Under condition, which detects fever using the association reaction of single antibody and antigen with thrombocytopenic syndromes virus
The presence of glycoprotein.
Detection kit of the invention can include two monoclonal antibodies of 1C5-F5 and 1H6-B10 simultaneously.Such case
Under, which detects fever by double antibody sandwich ELISA with thrombocytopenic syndromes viral glycoprotein
In the presence of.
In specific embodiments of the present invention, 1C5-F5 is applied in combination and two kinds of monoclonal antibodies utilizations of 1H6-B10 are double
Presence of the antibody sandwich ELISA method detection fever with thrombocytopenic syndromes viral glycoprotein.
As a kind of specific embodiment, when detection kit of the invention utilizes 1C5-F5 and 1H6-B10 two simultaneously
When kind monoclonal antibody, coated antibody is 1C5-F5；Detecting antibody is 1H6-B10.
As the embodiment that one kind can substitute, when detection kit of the invention utilizes 1C5-F5 and 1H6- simultaneously
When two kinds of monoclonal antibodies of B10, coated antibody is 1H6-B10；Detecting antibody is 1C5-F5.
Further, the mentioned-above detection kit of the present invention further includes ELISA Plate, confining liquid, cleaning solution, substrate colour developing
Liquid, terminate liquid.
In specific embodiments of the present invention, the confining liquid is 5% milk powder.
In specific embodiments of the present invention, substrate developing solution is TMB and H2O2。
In specific embodiments of the present invention, cleaning solution is phosphate buffer
In specific embodiments of the present invention, terminate liquid is the sulfuric acid of 0.5M.
The present invention also provides a kind of detection fevers of non-diagnostic purpose with thrombocytopenic syndromes viral glycoprotein
Method, the method detect the sugared egg of the virus using 1C5-F5 and/or 1H6-B10 by the immune response of antigen-antibody
It is white.
Preferably, the method is detected using 1C5-F5 and 1H6-B10 by double antibody sandwich ELISA.
ELISA method is routine techniques well known to those skilled in the art, is repeated no more.
The present invention also provides 1C5-F5 and/or 1H6-B10 to generate heat in preparation with the sugared egg of thrombocytopenic syndromes virus
Application in white immue quantitative detection reagent box.
Monoclonal antibody 1C5-F5 and 1H6-B10 of the invention can chemically or by genetic engineering and its
He is conjugated the factor.These factors provide the effect in antibody target required function site or improve or provide for antibody other property
Energy.
Monoclonal antibody according to the present invention can mark chemically or by genetic engineering, detectable to provide
Antibody.Detectable part includes but is not limited to enzyme, prothetic group, fluorescent material, luminescent material, bioluminescent material, radioactivity
Material, positron emitting metal and on-radiation paramagnetic metal ion.
In order to detect and/or analyze and/or diagnostic purpose label is dependent on particular detection/analysis/diagnostic techniques for using
And/or method such as immunohistochemical staining (tissue) sample, flow cytometry, laser scanning Cytometry inspection
Survey, fluorescence immunoassay, enzyme linked immunosorbent assay (ELISA) (ELISA), radiommunoassay (RIA), bioassay (such as phagocytosis make
With measurement), Western blotting application etc..Detection/analysis/diagnostic techniques known in the art and/or method are suitably marked
It is denoted as well known to those skilled in the art.
The term as used herein " monoclonal antibody " refers to the antibody obtained from a kind of substantially uniform group, except a small number of possible
Outside the existing mutation naturally occurred, the single antibody for including in the group is identical.Modifier " monoclonal " only indicates anti-
The characteristic of body is obtained from substantially uniform antibody population, this cannot be construed to need to be produced with any specific process anti-
Body.
" variable " the certain parts for indicating variable region in antibody of the term as used herein are different in sequence, it is formed
Combinations and specificity of the various specific antibodies to its specific antigen.Changeability, which concentrates in light chain and heavy chain variable region, to be known as mutually
It mends in three segments determined in area (CDR) or hypervariable region.Four areas FR are respectively contained in the variable region of native heavy and light chain
(more conservative part in variable region), they are generally in beta sheet configuration, are connected by three CDR of formation connection ring, can shape
At part β-pleated sheet structure.CDR in every chain is by the area FR firmly against together forming together and with the CDR of another chain
The antigen-binding site of antibody.Constant region does not participate in the combination of antibody and antigen directly, but they show different effects
Function such as participates in the cytotoxicity dependent on antibody of antibody.
The advantages of the present invention:
Detection kit of the invention can quantitative detection fever with thrombocytopenic syndromes viral glycoprotein: (1) detect
Concentration range is big；(2) high sensitivity, high specificity are reproducible；(3) it can be used for detection while a small amount of, multiple samples；(4)
Testing cost is low；(5) detection operating procedure is few, easy to use, short the time required to detection.
Skilled person will appreciate that the fever of external preparation is with glycoprotein in thrombocytopenic syndromes viral vaccine
Content be it is unstable, need to be monitored the glycoprotein Content in viral vaccine, detection kit of the invention can pass through
Quantitative Monitoring fever judges that fever is comprehensive with decrease of platelet with glycoprotein Content in thrombocytopenic syndromes viral vaccine
Levy the quality of viral vaccine.
Detailed description of the invention
Fig. 1 shows the linear of the OD450mm value for utilizing double antibody sandwich ELISA detection various concentration viral glycoprotein Gc
Result figure.
Specific embodiment
The present invention is further illustrated below by embodiment.It should be understood that the embodiment of the present invention is for illustrating
The present invention is rather than limiting the invention.The simple modifications that essence according to the present invention carries out the present invention belong to the present invention
Claimed range.
The preparation of the grand antibody of embodiment 1 SFTSV glycoprotein (Gn and Gc) monoclonal antibody
1, Balb/c mouse immune
SFTSV strain (JS-2010-003) is inactivated through beta-propiolactone, low-speed centrifugal removes cell fragment, is surpassed from, molecular sieve layer
Analysis technology and etc. purified.Inactivation of viruses is immunized 5 week old female Balb/c mouse (100 μ g/ only), 100 μ when first immunisation
G antigen is mixed with equal volume of freund's complete adjuvant intraperitoneal injection；3rd Zhou Houyong equivalent amount of antigen is mixed with freund 's incomplete adjuvant
Peritoneal immunity afterwards；5th week the 3rd time immune, and adjuvant is not added.
2, splenocyte fusion with myeloma cells
It merges the last week, recovery murine myeloma cell sp2/0 to OPTI-MEM culture medium (fetal calf serum containing 10%),
37 DEG C are placed in, 5%CO2It cultivates, merges first 3 days in incubator, cell is passed on primary.On the fusion same day, myeloma cell is harvested,
It counts, 5.0 × 107Myeloma cell with serum free medium wash 2 times it is spare.Mouse is plucked 3-5 days after the 3rd time immune
Except eyeball bloodletting, put to death.Mouse spleen is taken out in sterile working, sets in sterilizing plates, separating Morr. cell, counts, spare.
The splenocyte for being equal to 1/2 spleen of mouse is mixed with myeloma cells, 1300rpm is centrifuged 5min, goes as far as possible
Except supernatant.50% PEG of 1.5ml is added in 1.5 minutes, shakes well while adding；Then it is added 20ml's in 8.5 minutes
Serum free medium shakes well while adding.
The cell 1000rpm merged through PEG is centrifuged 5 minutes, supernatant is removed, adds the HAT Selective agar medium weight of 150ml
It is outstanding, fused cell is seeded to sterile 96 orifice plate, 150 hole μ l/, is placed in 37 DEG C, 5%CO2It is cultivated 4 days in incubator, every hole is added
100 μ l Selective agar mediums.
3, the screening of hybridoma and clone
10 days after fusion, 50 μ l supernatants are inhaled from every hole, are added to and are coated with recombination SFTSV Gn (or Gc) 96 hole ELISA
Plate (is closed) with 1% BSA, is incubated at room temperature 1.0 hours；It washes 5 times.Dilute horseradish peroxidase (Horseradish
Peroxidase, HRP) label goat anti-mouse 1:4000, every hole adds 50 μ l, is incubated at room temperature 0.5 hour；Washing 5 times.Every hole
100 μ l HRP substrate (H are added2O2+ TMB), it is incubated at room temperature 10 minutes, 50 μ l 0.5M H are added in every hole2SO4, survey A450nm value.
Positive hole cell is collected, is resuspended in HT Selective agar medium, using limiting dilution assay diluting cells, and is planted in 96
It in porocyte culture plates, observes after 5 days, to the hole for determining the growth of only one cell clone, is identified as ELISA.To sun
Property hole cell carry out limiting dilution, by 3-4 time it is unicellular be separately cultured, until the stable hybridoma cell clone of acquisition.Greatly
Amount culture hybridoma, collects culture supernatant antibody-containing, is purified with proteinG affinity column, and carry out dialysis
In PBS, survey concentration, -20 DEG C freeze it is spare.
The application double-antibody sandwich elisa selection optimum antibody pairing of embodiment 2
Obtain 7 plants of monoclonal antibodies for being directed to glycoprotein altogether by hybridoma technology, the monoclonal antibody molecule that target is Gn is
4E11-C7,3E9-B10,5D7-A6,6C5-E6, the monoclonal antibody molecule that target is Gc is 2D5-E7,1C5-F5,1H6-B10.Using
Double antibody sandwich ELISA makes above-mentioned 7 strain antibody paired with each other, selects optimal combination in favor of glycoprotein detection, narration is such as
Under:
1, the coating of monoclonal antibody
With the NaHCO of 0.1M3/Na2CO3Buffer (pH 9.6) dilutes antibody concentration to 10 μ g/ml, is added to 96 hole enzymes
Target, 100 holes μ l/, 4 DEG C of coatings overnight, are washed 1 time with PBST, and 4 DEG C of milk of 5% closings are added overnight, with PBST wash 1 time it is standby
With.
2, the coupling of monoclonal antibody and HRP
Monoclonal antibody is dialysed into PBS, adjusts concentration to 1mg/ml.Using the work of Beijing Taitianhe biological technology Co., Ltd.
Change HRP to be coupled, concrete operations are referring to its company's specification.In labelled antibody be added 50% glycerol, -20 DEG C freeze it is standby
With.
3, ELISA is operated
5% milk powder of 50 μ l and the inactivation of viruses culture supernatant of 50 μ l is added in every hole, and 37 DEG C of incubation 1hr are washed 5 times,
Then the enzyme labelled antibody (1:1000) of 100 μ l is added in every hole, and 37 DEG C of incubation 0.5hr are washed 5 times, and 100 μ l TMB are added in every hole
+H2O2Substrate is incubated at room temperature 10 minutes, and the sulfuric acid that 100 μ l 0.5M are added in every hole terminates reaction, surveys A450nm value, concrete outcome
As shown in table 1.
1 ELISA of table measures A450nm
As can be seen from the results in the table that only 1H6-B10/1C5-F5 is combined, and only anti-as coating as 1H6-B10
Body, and when 1C5-F5 is as detection antibody, the A450nm value highest of toxin is detected, reaches 0.823, and other combinations
A450nm value is lower.Above-mentioned 2 monoclonal antibodies are Gc glycoprotein specificity, therefore are combined based on 1H6-B10/1C5-F5 monoclonal antibody
Detection is Gc glycoprotein Content in virion.
Double-antibody sandwich elisa sensitivity analysis of the embodiment 3 based on Gc glycoprotein
Using 1H6-B10 as coated antibody, 1C5-F5 and HRP conjugate as detection antibody, recombination Gc glycoprotein is target
Molecule is marked, measures the susceptibility of the ELISA system, concrete operations are same as above.
Using the detection architecture, can be detected from Virus culture supernatant down to about 60ng/ml's (being shown in Table 2 and Fig. 1)
Gc antigen can satisfy SFTSV inactivated vaccine production needs.
Table 2 is directed to the elisa assay of various concentration Gc glycoprotein
The clone of 4 monoclonal antibody 1H6-B10/1C5-F5 of embodiment weight, light-chain variable region gene
The hybridoma of logarithmic growth phase uses oligo using the Trizol extracted total RNA of Invitrogen company
(dT) 20 be primer, and reverse transcription generates cDNA.Then its heavy, light-chain variable region gene is expanded respectively using specific primer PCR.
PCR product clones insertion pMD-18T carrier after purification by electrophoresis, by TA, and sequencing carries out sequence analysis.
The primer of amplified hybridization tumor monoclonal antibody heavy chain variable region VH is as shown in table 3.
The primer sequence of the amplification antibody heavy chain variable region of table 3
The primer sequence of amplified hybridization tumor monoclonal antibody light chain variable region VL is as shown in table 4.
The primer sequence of the amplification antibody's light chain variable region of table 4
As a result:
For 1H6-B10 heavy chain, only primer MHV2 (26-mer)/MHCG2b (21-mer) combination can be amplified
PCR product, identified, the nucleotide sequence of 1H6-B10 heavy chain variable region is as shown in SEQ ID NO.7, amino acid sequence such as SEQ
Shown in ID NO.3.
For 1H6-B10 light chain, only primer MKV2 (30-mer)/MKC (20-mer) combination can amplify PCR
Product, identified, the nucleotide sequence of 1H6-B10 light chain variable region is as shown in SEQ ID NO.8, amino acid sequence such as SEQ ID
Shown in NO.4.
For 1C5-F5 heavy chain, only primer MHV2 (26-mer)/MHCG2b (21-mer) combination can be amplified
PCR product, identified, the nucleotide sequence of 1C5-F5 heavy chain variable region is as shown in SEQ ID NO.5, amino acid sequence such as SEQ
Shown in ID NO.1.
For 1C5-F5 light chain, only primer MKV4 (33-mer)/MKC (20-mer) combination can amplify PCR
Product, identified, the nucleotide sequence of 1C5-F5 light chain variable region is as shown in SEQ ID NO.6, amino acid sequence such as SEQ ID
Shown in NO.2.
Although those skilled in the art should manage above only describes a specific embodiment of the invention example
Solution, these are merely examples, and protection scope of the present invention is defined by the appended claims.Those skilled in the art
Without departing from the principle and essence of the present invention, many changes and modifications may be made, but this
A little changes or modification each fall within protection scope of the present invention.
Sequence table
<110>Jiangsu Prov. Disease Preventing and Controlling Center
<120>fever is with thrombocytopenic syndromes viral glycoprotein immue quantitative detection reagent box
<160> 36
<170> SIPOSequenceListing 1.0
<210> 1
<211> 124
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Glu Val Leu Leu Glu Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Pro Cys Lys Thr Ser Gly Asn Gln Ile Thr Asp Tyr
20 25 30
Ser Met Asp Trp Val Lys Gln Ser His Gly Lys Thr Leu Glu Trp Ile
35 40 45
Gly Asp Ile Asn Pro Asn Asn Gly Gly Ser Ile Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Gly Ile His Tyr Phe Gly Ser Ser Tyr Gln Pro Phe Ala
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Ile Val Ser Ala
115 120
<210> 2
<211> 109
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Asn Ser
20 25 30
Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile
35 40 45
Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser
65 70 75 80
Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Tyr Ser Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala
100 105
<210> 3
<211> 118
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr
1 5 10 15
Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asp Ile Ser Pro Asn Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Tyr Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Gly Asn Tyr Met Arg Thr Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val Ser
115
<210> 4
<211> 113
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser
20 25 30
Ser Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp
85 90 95
Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Ala
<210> 5
<211> 373
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gaggtcctgc tggaacagtc tggacctgag ctggtgaagc ctggggcttc agtgaagatt 60
ccctgcaaga cttctggaaa ccaaatcact gactacagca tggactgggt gaaacagagc 120
catggaaaga cccttgagtg gattggagat attaatccca acaatggtgg tagtatctac 180
aaccagaagt tcaagggcaa ggccacattg actgtagaca agtcctccag cacagcctac 240
atggagctcc gcagtctgac atctgaggac actgcagtct attactgtgc aagatacggc 300
attcattact tcggtagtag ctaccaacct tttgcttact ggggccaagg gactctggtc 360
attgtctctg cag 373
<210> 6
<211> 327
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60
ctcacttgtc gggcaagtca ggacattggt aatagcttaa actggcttca gcaggaacca 120
gatggaacta ttaaacgcct gatctacgcc acatccagtt tagattctgg tgtccccaaa 180
aggttcagtg gcagtaggtc tgggtcagac tattctctca ccatcagcag ccttgagtct 240
gaagattttg tagactatta ctgtctacaa tatgcttatt ctccgtacac gttcggaggg 300
gggaccaaac tggaaataaa acgggct 327
<210> 7
<211> 354
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gaggtcctgc tgcaacagtc tggacctgag ctggtgaagc ctgggacttc agtgaagata 60
ccctgcaagg cttctggata cacattcact gactacaaca tggactgggt gaagcagagc 120
catggaaaga gccttgagtg gattggagat attagtccta acaatggtgg tactatctac 180
aaccagaagt tcaagggcaa ggccacattg actgtagaca agtcctccag cacagcctac 240
atggagctcc gcagcctgac atatgaggac tctgcagtct attactgtgc aagatatggt 300
aattacatga ggactctgga ctactggggt caaggaacct cagtcaccgt ctcc 354
<210> 8
<211> 339
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60
atctcatgca gggccagcca aagtgtcagt acatctagct atagttatat gcactggtac 120
caacagaaac caggacagcc gcccaaactc ctcatcaagt atgcatccaa cctagaatct 180
ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240
cctgtggagg aggaggatac tgcaacatat tactgtcagc acagttggga gattccgtac 300
acgttcggag gggggaccaa gctggaaata aaacgggct 339
<210> 9
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
atgaaatgca gctggggcat sttcttc 27
<210> 10
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
atgggatgga gctrtatcat sytctt 26
<210> 11
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
atgaagwtgt ggttaaactg ggttttt 27
<210> 12
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
atgractttg ggytcagctt grttt 25
<210> 13
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
atggactcca ggctcaattt agttttcctt 30
<210> 14
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
atggcttgtc ytrgsgctrc tcttctgc 28
<210> 15
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
atggratgga gckggrtctt tmtctt 26
<210> 16
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
atgagagtgc tgattctttt gtg 23
<210> 17
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
atggmttggg tgtggamctt gctattcctg 30
<210> 18
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
atgggcagac ttacattctc attcctg 27
<210> 19
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
atggattttg ggctgatttt ttttattg 28
<210> 20
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
atgatggtgt taagtcttct gtacctg 27
<210> 21
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
cagtggatag acagatgggg g 21
<210> 22
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
cagtggatag accgatgggg c 21
<210> 23
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
cagtggatag actgatgggg g 21
<210> 24
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
caagggatag acagatgggg c 21
<210> 25
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
atgaagttgc ctgttaggct gttggtgctg 30
<210> 26
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
atggagwcag acacactcct gytatgggtg 30
<210> 27
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
atgagtgtgc tcactcaggt cctggsgttg 30
<210> 28
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
atgaggrccc ctgctcagwt tyttggmwtc ttg 33
<210> 29
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
atggatttwc aggtgcagat twtcagcttc 30
<210> 30
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
atgaggtkcy ytgytsagyt yctgrgg 27
<210> 31
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
atgggcwtca agatggagtc acakwyycwg g 31
<210> 32
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
atgtggggay ctktttycmm tttttcaatt g 31
<210> 33
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
atggtrtccw casctcagtt ccttg 25
<210> 34
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
atgtatatat gtttgttgtc tatttct 27
<210> 35
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
atggaagccc cagctcagct tctcttcc 28
<210> 36
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
actggatggt gggaagatgg 20
Claims (11)
1. a kind of monoclonal antibody for fever companion thrombocytopenic syndromes viral glycoprotein Gc, which is characterized in that described
Monoclonal antibody includes:
Light chain variable region, amino acid sequence is as shown in SEQ ID NO:2；
Heavy chain variable region, for amino acid sequence as shown in SEQ ID NO:1, which is named as 1C5-F5.
2. a kind of monoclonal antibody for fever companion thrombocytopenic syndromes viral glycoprotein Gc, which is characterized in that described
Monoclonal antibody includes:
Light chain variable region, amino acid sequence is as shown in SEQ ID NO:4；
Heavy chain variable region, for amino acid sequence as shown in SEQ ID NO:3, which is named as 1H6-B10.
3. encoding the nucleic acid molecules of monoclonal antibody of any of claims 1 or 2, which is characterized in that the sequence of nucleic acid molecules
As shown in SEQ ID NO:5-8.
4. a kind of monoclonal antibody combination for fever companion thrombocytopenic syndromes viral glycoprotein Gc, feature exist
In the monoclonal antibody combination includes that monoclonal antibody described in claim 1 and monoclonal as claimed in claim 2 are anti-
Body.
5. a kind of fever is with the detection kit of thrombocytopenic syndromes viral glycoprotein, which is characterized in that the detection examination
Agent box includes monoclonal antibody described in claim 1 and/or monoclonal antibody as claimed in claim 2.
6. detection kit according to claim 5, which is characterized in that the detection kit include coated antibody and/
Or detection antibody, the coated antibody is monoclonal antibody described in claim 1；The detection antibody is claim 2 institute
The monoclonal antibody stated.
7. detection kit according to claim 5, which is characterized in that the detection kit include coated antibody and/
Or detection antibody, the coated antibody is monoclonal antibody as claimed in claim 2；The detection antibody is claim 1 institute
The monoclonal antibody stated.
8. according to the described in any item detection kits of claim 5-7, which is characterized in that the detection kit includes enzyme mark
Plate, confining liquid, cleaning solution, substrate developing solution, terminate liquid.
9. a kind of detection fever of non-diagnostic purpose is with the method for thrombocytopenic syndromes viral glycoprotein, which is characterized in that
It is anti-the method includes being passed through using monoclonal antibody described in claim 1 and/or monoclonal antibody as claimed in claim 2
Original antibody is immunoreacted to detect.
10. according to the method described in claim 9, it is characterized in that, utilizing monoclonal antibody and right described in claim 1
It is required that monoclonal antibody described in 2 is detected by double antibody sandwich ELISA.
It is detected 11. monoclonal antibody of any of claims 1 or 2 is generated heat in preparation with thrombocytopenic syndromes viral glycoprotein
Application in kit.
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| CN108956588B (en) * | 2018-07-09 | 2021-01-05 | 东南大学 | Application of electrochemical luminescence immunosensor in preparation of SFTSV detection kit |
| KR102504884B1 (en) * | 2019-07-23 | 2023-02-28 | 와이-클론 메디컬 사이언시스 컴퍼니 리미티드 | Nanoantibodies capable of binding to SFTSV and applications thereof |
| CN110437332B (en) * | 2019-08-20 | 2020-03-06 | 江苏省疾病预防控制中心(江苏省公共卫生研究院) | SFTSV protein binding molecule for resisting virus infection |
| CN112342316A (en) * | 2020-10-28 | 2021-02-09 | 中国疾病预防控制中心病毒病预防控制所 | Primer probe set and detection method for detecting fever with thrombocytopenia syndrome virus by real-time fluorescent RNA isothermal rapid amplification |
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| CN102809653A (en) * | 2012-04-24 | 2012-12-05 | 万里明 | Preparation and application of ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting novel bunyavirus antigen |
| 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) |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102809653A (en) * | 2012-04-24 | 2012-12-05 | 万里明 | Preparation and application of ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting novel bunyavirus antigen |
| 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) |
Non-Patent Citations (1)
| Title |
|---|
| 《抗发热伴血小板减少综合征病毒结构蛋白单克隆抗体的制备和功能分析》;李阿茜等;《病毒学报》;20150131;第31卷(第1期);第18-23页 |
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