CN102021146A - West nile virus NS1 protein monoclonal antibody, identified B cell epitope thereof and application - Google Patents
West nile virus NS1 protein monoclonal antibody, identified B cell epitope thereof and application Download PDFInfo
- Publication number
- CN102021146A CN102021146A CN2010105416354A CN201010541635A CN102021146A CN 102021146 A CN102021146 A CN 102021146A CN 2010105416354 A CN2010105416354 A CN 2010105416354A CN 201010541635 A CN201010541635 A CN 201010541635A CN 102021146 A CN102021146 A CN 102021146A
- Authority
- CN
- China
- Prior art keywords
- monoclonal antibody
- wnv
- west nile
- protein
- nile virus
- 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.)
- Granted
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a west nile virus NS1 protein monoclonal antibody, an identified B cell epitope thereof and application. A hybridoma cell strain which stably secretes anti-WNV (West Nile Virus)-NS1 protein monoclonal antibody is obtained by screening and has the microorganism preservation number of CGMCC No.4242. The monoclonal antibody WN-3D10 secreted by the hybridoma cell strain carries out specific reaction with WNV-NS1 protein but does not react with JEV (Japanese Encephalitis Virus)-NS1 protein. The monoclonal antibody and the WNV-NS1 protein virus specific B cell epitope identified by the monoclonal antibody can be used to be prepared into a reagent for identifying or diagnosing west nile virus and Japanese encephalitis virus, and lay a foundation for the serology differential diagnosis method for the WNV and Flavivirus virus.
Description
Technical field
The present invention relates to a strain of hybridoma strain and excretory monoclonal antibody thereof, relate in particular to a strain and secrete the hybridoma cell strain of anti-west nile virus NS1 protein monoclonal antibody and secreted monoclonal antibody thereof; The invention still further relates to a B cell epitope, relate in particular to the west nile virus NS1 protein B cell epitope of discerning by said monoclonal antibody; The invention still further relates to the application in preparation diagnosis, prevention or treatment west nile virus medicine of above-mentioned hybridoma cell strain, monoclonal antibody and B cell epitope, belong to the anti-system field of west nile virus disease.
Background technology
(West Nile Fever is a kind of by west nile virus (West Nile virus, the acute infectious diseases common to human beings and animals that WNV) causes and pass through mosquitoes spread WNF) to west Nile fever.WNV is the sub-thread positive chain RNA virus, belong to the flaviviridae Flavivirus, and WNV and japanese encephalitis virus (Japanese encephalitis virus, JEV), St. Louis encephalitis virus (Saint-Louis encephalitis virus, SLEV) and Murray Valley encephalitis virus (Murray Valley encephalitis virus, MVEV) belong to japanese encephalitis virus serogroups (Japanese Encephalitis Virus Serocomplex) together, have serious serological cross reaction between them.December nineteen thirty-seven scientist be separated to first from the heating women's in the ugandan Xi Niluo in Africa area blood should virus, so with its called after " west nile virus ".Birds are its important hosts, and it is its main circulation way that mosquito (mainly being culex) bites.People, animal and birds are susceptible animal.Since scientist after WNV is found first in Africa, the France of the Egypt of the 1950's and Israel, the sixties and the South Africa of the seventies also all had should disease report, but the Rumanian WNF outburst up to 1996, this virus just really causes people's attention.1996-1997 Romania morbidity people more than 500, mortality ratio is near 100%.WNV came across the western hemisphere first in 1999, and the great outburst of WNF takes place in the U.S., this virus rapid spread U.S. whole area, and propagate into Canada and Central American countries.It is reported, heating paresthesia can appear in the infected of about 20% behind the humans and animals infection WNV, and the infected of about 1/150 can develop into encephalitis, meningitis or meningoencephalitis, but in the outburst of WNF several times recently, sickness rate, the ratio that the encephalitis symptom occurs and mortality ratio all increase to some extent, the WNF of U.S.'s outburst in 1999 has caused the morbidity of 20000 people and 25000 dry goods by the end of 2005,800 people and 5000 dry goods death are wherein arranged.
Up to the present, China does not also have the report of WNV cases of infection.Chinese in recent years domestic investigator detects some geographic mosquitoes and birds, and its result also is the WNV feminine gender.But in 1987 and the epidemiology survey carried out in 2006 WNV antibody positive person has appearred all, there has been the WNF outburst in Chinese in addition surrounding countries and area, so the research of reinforcement to this virus is badly in need of in China, carries out the tachnical storage that can carry out prevention and control and monitoring to it.Simultaneously because humans and animals often is in inferior clinical symptom or asymptomatic after infecting WNV, so it is particularly necessary to set up the laboratory diagnostic method of WNV.In addition, have serious cross reaction between WNV and other flavivirus, and China is the popular district of JEV, so should set up the method that to carry out differential diagnosis to the infection of WNV and JEV.
NS1 albumen is the conservative non-structural glycoprotein of of WNV, contains 352 amino acid, and molecular mass is about 42kDa.NS1 albumen is having important utility value aspect the eqpidemic disease diagnosis.NS1 albumen just can be detected behind organism infection in 3-8 days, reached maximum after infection on the 5th day, and the time that this time will occur early than clinical symptom is so NS1 albumen can be used to the early diagnosis of virus infection.This viral viremia time is short, the virus particle level is low in the blood, be difficult for directly detecting cause of disease, and this viral operational requirement carried out in the level of 3 grades of Biosafeties, this has brought inconvenience all for the diagnosis of eqpidemic disease, and, then can when eqpidemic disease is diagnosed, distinguish natural infected animal and weak malicious seedling immune animal, and diagnostic work can be finished in common lab if to detecting at the proteic antibody of NS1 in the serum to be checked.In addition, owing to have serious serological cross reaction between the flavivirus, so the diagnostic method of antibody can not effectively belong to each virus to this and carries out differential diagnosis in the general detection serum, and existing studies show that carries more specificity site in the NS1 albumen, can induce a plurality of in flavivirus the specific antibody of no cross reaction, and if filter out the hybridoma cell strain that a strain can secreting specificity antibody, and identify the WNV-NS1 protein-specific B cell epitope that its secreted monoclonal antibody is discerned, so just can set up the ELISA detection method that can carry out differential diagnosis to WNV and other flavivirus.
Summary of the invention
One of the object of the invention provides the hybridoma cell strain of strain secretion west nile virus NS1 protein monoclonal antibody;
Two of the object of the invention provides a kind of by the secreted monoclonal antibody of above-mentioned hybridoma cell strain, this monoclonal antibody can with WNV-NS1 albumen generation specific reaction, and do not react with JEV-NS1 albumen;
Three of the object of the invention is to identify the proteic WNV specific b cells of a WNV-NS1 epi-position.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The present invention utilizes the pMAL-c2x prokaryotic expression carrier that the WNV-NS1 gene is carried out prokaryotic expression, with behind the expressed solubility WNV-NS1 protein purification that goes out as immunogen, immune BALB/c mouse is got its splenocyte and SP2/0 myeloma cell and is merged.In addition, the present invention also utilizes baculovirus expression system that the WNV-NS1 gene is carried out eukaryotic expression, WNV-NS1 albumen to expressed inclusion body form carries out using antigen as detecting after sex change, purifying and the renaturation, setting up indirect ELISA detection method screens positive hybridoma cell, final screening obtains the hybridoma cell strain of the anti-WNV-NS1 protein monoclonal antibody of a strain stably excreting, and its microbial preservation number is: CGMCC No.4242; The classification name is: the hybridoma cell strain of mouse monoclonal antibody; The preservation time: on October 13rd, 2010; Depositary institution is: Chinese common micro-organisms culture presevation administrative center; The preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
It is a kind of by the secreted monoclonal antibody of above-mentioned hybridoma cell strain that the present invention also provides, its called after WN-3D10, Western b1ot detected result show WN-3D10 can with WNV-NS1 albumen generation specific reaction, and do not react with JEV-NS1 albumen.
The present invention utilizes display technique of bacteriophage and pepscan that the proteic B cell epitope of the WNV-NS1 that WN-3D10 discerned is identified that this epi-position is accurately orientated as the most at last:
27AWMDRYKYYP
36(SEQ ID No.1).Simultaneously, the sequence alignment result shows
27AWMDRYKYYP
36Be the peculiar and conservative B cell epitope of WNV.
In sum, the present invention's preparation has also identified a specific specificity at the proteic monoclonal antibody of WNV-NS1, the conservative B cell epitope of the WNV-NS1 prion specificity that monoclonal antibody of the present invention and this monoclonal antibody are discerned can be used for being prepared into to be differentiated or diagnosis west nile virus (West Nile virus, WNV) or japanese encephalitis virus (Japanese encephalitis virus, JEV) reagent is for the serology differential diagnosis method of setting up WNV and flavivirus is laid a good foundation.
Description of drawings
Fig. 1 uses Western blot test and detects monoclonal antibody WN-3D10 and WNV-NS1 albumen and the proteic reactive result of JEV-NS1.1,3,5:WNV-NS1 albumen; 2,4,6:JEV-NS1 albumen; M: standard protein Marker.
Fig. 2 phage clone sequencing result reaches with the WNV-NS1 protein similar and compares.
The fifteen amino acid small peptide SDS-PAGE of Fig. 3 prokaryotic expression analyzes; 1.MBP-15VP; M: standard protein Marker.
The reactive Western blot of the fifteen amino acid small peptide of Fig. 4 prokaryotic expression and WN-3D10 analyzes M: standard protein Marker; The reaction of 1:MBP-15VP and WN-3D10; The reaction of 2:MBP and WN-3D10.
The reactive indirect ELISA analysis of the fifteen amino acid small peptide of Fig. 5 prokaryotic expression and WN-3D10.
In Fig. 6 table 1 the reactive indirect ELISA of synthetic small peptide and WN-3D10 analyze the reaction of 1:NS1-15VP and WN-3D10; The reaction of 2:NS1-13ET and WN-3D10; The reaction of 3:NS1-11AE and WN-3D10; The reaction of 4:NS1-9WP and WN-3D10; The reaction of 5:NS1-7MY and WN-3D10.
In Fig. 7 table 2 the reactive indirect ELISA of synthetic small peptide and WN-3D10 analyze the reaction of 1:NS1-10AP and WN-3D10; The reaction of 2:NS1-10WE and WN-3D10; The reaction of 3:NS1-8WY and WN-3D10; The reaction of 4:NS1-8MP and WN-3D10.
Fig. 8 identifies the virus-specific analysis of B cell epitope.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
Main experiment material and source
1. albumen, cell, seed culture of viruses and antibody
The WNV-NS1 albumen of WNV-NS1 albumen, eukaryotic expression and the purifying of prokaryotic expression and purifying, SP2/0 cell, Sf9 cell and the recombinant baculovirus BACV-NS1 that carries the WNV-NS1 gene preserve by this laboratory; The JEV-NS1 albumen of prokaryotic expression is so kind as to give (above-mentioned albumen and antibody also can prepare according to the method for relevant document record) with anti-JEV-NS1 protein monoclonal antibody JE-1H6 (having verified that it can only react with JEV-NS1 albumen) by Harbin veterinary institute Hua Ronghong assistant researcher; Flavivirus NS1 protein monoclonal antibody Flavi-4G4 (having verified that it all can react with flavivirus NS1 albumen) is so kind as to give (above-mentioned antibody also can prepare according to the method for relevant document record) by Australian animal health Ros professor s of institute.
2. main agents and medicine
Foetal calf serum (FBS), DMEM dry powder and L-glutaminate are available from GIBCOL company; Freund's adjuvant, 50%PEG, 50 * HAT, 50 * HT and 8-azaguanine (8-AG) and fluorescein isothiocyanate (FITC) mark goat anti-mouse IgG are all available from Sigma company; The anti-mouse IgG of horseradish peroxidase (HRP) labelled goat is Bioisystech Co., Ltd of a Golden Bridge import packing product; SBA ClonotypingTMSystem/HRP antibody subgroup identification test kit is purchased the company in Southern Biotechnology; Storage capacity is 2.7 * 10
9The host bacterium ER2738 of M13 phage random peptide library Ph.D.-12TM, tsiklomitsin (Tet) resistance and prokaryotic expression carrier pMAL-c2X all available from NEB company; Ex Taq archaeal dna polymerase, T
4Dna ligase, restriction enzyme EcoR I and Sal I are available from precious biotechnology (Dalian) company.
3. laboratory animal
6 the week age BALB/c mouse provide by Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center.
1. mouse immune
With the reorganization of the prokaryotic expression behind purifying NS1 protein immunization female BALB/c mouse in age in 56 weeks, immunity is 3 times altogether, each immunity is two weeks at interval, one exempts from reorganization NS1 albumen and isopyknic Freund's complete adjuvant mixing and emulsifying, two exempt from and three exempt from reorganization NS1 albumen and isopyknic Freund's incomplete adjuvant mixing and emulsifying, immunizing dose is 50 μ g/, and immunization route is a peritoneal immunity.
Exempt from and three exempt from one week of back to the mouse blood sampling of docking two respectively, (4 ℃, 10000rpm 20min), detects antibody horizontal with indirect ELISA to separation of serum.In cytogamy preceding 3 days, the BALB/c mouse of good immune effect is carried out booster immunization again, every mouse peritoneal is injected 50 μ g immunizing antigens (not adding adjuvant).
2. cytogamy
Merge and prepared feeder cell in preceding 1 day, it is stand-by to go the BALB/c mouse peritoneal macrophage to be laid in the 96 porocyte culture plates according to ordinary method.Disconnected neck is put to death the mouse of waiting to get spleen, and aseptic spleen and the separating Morr. cell got carries out cytogamy in splenocyte and 4: 1 ratio of SP2/0 myeloma cell with PEG, and the cell after the fusion is laid on the ready feeder cell.
3. the screening of positive hybridoma cell strain and clone
Utilize the eukaryotic expression WNV-NS1 albumen behind the purifying to set up the strain of indirect ELISA detection method screening positive hybridoma cell, hybridoma enlarged culturing to reacting positive, carry out the clone of positive hybridoma cell simultaneously with limiting dilution assay, clone 3 takes turns, and the positive hybridoma cell that the clone is good is in time frozen.The final hybridoma cell strain that obtains a strain can the anti-WNV-NS1 protein monoclonal antibody of stably excreting, its microbial preservation number are: CGMCC No.4242, and with its excretory monoclonal antibody called after WN-3D10.
4. a large amount of preparations of monoclonal antibody
Give the healthy BALB/c mouse abdominal injection Freund's incomplete adjuvant about 10 ages in week, 0.5mL/, 1 all pneumoretroperitoneum injections 1 * 10
6Individual hybridoma extracts ascites when the mouse web portion extreme expansion behind the 7-10d, take out once every 2d, with the centrifugal 10min of ascites 10000r/min that extracts, removes upper strata grease and precipitation, and the supernatant packing is stored in-20 ℃.
The evaluation of embodiment 2 monoclonal antibodies
1. the subgroup identification of monoclonal antibody
According to SBA ClonotypingTM System/HRP antibody subgroup identification test kit process specifications embodiment 1 resulting monoclonal antibody is carried out subgroup identification.
The result shows that the heavy chain of monoclonal antibody WN-3D10 of the present invention is IgG
1, light chain is the κ chain.
2.Western blot test
WNV-NS1 albumen and JEV-NS1 albumen are carried out the SDS-PAGE electrophoresis carry out electric transfer printing then, electric commentaries on classics condition is 18V voltage 30min.Nitrocellulose filter after the transfer printing is put into the 4 ℃ of sealings of PBS confining liquid that contain the 50g/L skim-milk and is spent the night; The nitrocellulose filter that sealing is good is put into positive hybridoma nutrient solution supernatant room temperature effect 1h, with PBST (the PBS damping fluid that contains the pH7.4 of 0.5mL/L tween 20) washing 3 times, again with goat anti-mouse IgG HRP traget antibody room temperature effect 1h through 5000 times of dilutions of PBS, PBST washing 3 times, the colour developing of DAB colouring reagents box, sweep record.
In order to confirm the specificity of prepared monoclonal antibody of the present invention and WNV-NS1 albumen test, this experiment has also been set JE-1H6 and two kinds of monoclonal antibodies of Flavi-4G4 in contrast, existing experimental result show JE-1H6 only with JEV-NS1 albumen generation specific reaction, and do not react with WNV-NS1 albumen, Flavi-4G4 then reacts with two kinds of albumen.
Test-results confirms, the prepared monoclonal antibody WN-3D10 of the present invention can with WNV-NS1 albumen generation specific reaction, and do not react with JEV-NS1 albumen, set contrast JE-1H6 and Flavi-4G4 all set up (Fig. 1) simultaneously, infer that according to this experimental result the WNV-NS1 protein B cell epitope that WN-3D10 discerned is a linear epitope simultaneously.
The evaluation of embodiment 3 B cell epitopes
1. the biological elutriation of phage display random peptide library and phage genome sequencing
With reference to NEB company phage display 12 peptide storehouse operational manuals at random, with the prepared monoclonal antibody WN-3D10 that is purified into random peptide library is carried out 3 and take turns elutriation, thereby elutriation goes out the displayed polypeptide Duan Keyu of institute monoclonal antibody specificity bonded phage.3 take turns elutriation monoclonal antibody package amount is 100 μ g/mL, 150 μ L/ holes, and 3 take turns that tween 20 concentration is respectively 0.1%, 0.3% and 0.5% in the used PBST damping fluid of elutriation.
Take turns from the 3rd and to get 2 μ L the phage that elutes and do 10 times of serial dilutions, each extent of dilution is got 10 μ L and is inoculated 200 μ L logarithmic phase ER2738 host bacterium, behind the effect 5min whole bacterium liquid are mixed with the 3mL nutrient agar and be poured on the solid medium that contains IPTG and X-gal 37 ℃ of overnight incubation.
From the flat board of total amount less than 100 plaques, 10 plaques of picking are inoculated 8 pipe 1mL logarithmic phase ER2738 host bacterium respectively at random, and 37 ℃ of shaking tables are cultivated 4.5h.Afterwards with culture 10000rpm, centrifugal 30s, supernatant move in the new pipe 10000rpm once more, and centrifugal 30s gets 600 μ L supernatants and is amplification phage storage liquid.Can from above-mentioned phage storage liquid, extract phage genome DNA according to the method for introducing in the operational manual afterwards, and transfer to Shanghai Ying Jun Bioisystech Co., Ltd and carry out sequencing.
There are 8 to have some common sequences (Fig. 2) in 10 phage clones that the elutriation of phage sequencing result demonstration WNV-3D10 institute goes out, finally determine a concensus sequence DRYKYY by relatively integrating, and this sequence of existence on the WNV-NS1 albumen, so think that DRYKYY should be the linear epitope that WN-1C10 discerns, or core sequence of this epi-position.
2.B the preliminary evaluation of cell epitope
With the similar section on the above-mentioned NS1 albumen is core, respectively extend 4-5 amino acid to albumen n end and C end respectively, finally choosing length is that 15 amino acid whose small peptides (VEAWMDRYKYYPETP) are research object, carries out the preliminary evaluation of WNV-NS1 protein B cell epitope.Concrete grammar is as follows:
(1) a pair of complementary oligonucleotide chain of synthetic, above-mentioned 15 amino acid short peptides of encoding respectively.
Coding VEAWMDRYKYYPETPWNVNS1-15VP-EcoR I
F:5 '-aattcgaggcttggatggaccggtacaagtattaccctgaaacgccacaataag-3 ' (SEQ ID No.2), the EcoR I viscosity extending end of line part for introducing.
WNVNS1-15VP-Sal?I
R:5 '-tcgacttattgtggcgtttcagggtaatacttgtaccggtceatccaagcctcG-3 ' (SEQ ID No.3), the Sal I viscosity extending end of line part for introducing.
Above primer is synthetic by Nanjing Genscript Biotechnology Co., Ltd.
(2) two couples of direct 54 ℃ of annealing 10min of complementary strand are formed the double-stranded DNA that has sticky end, with EcoR I and Sal I pMAL-c2x is carried out double digestion simultaneously.
(3) double-stranded DNA that two annealing are formed is connected with pMAL-c2x after enzyme is cut
(4) connect product and transform the BL21 competent cell, with upstream chain in two pairs of complementary strands and carrier downstream primer M13-47 recombinant plasmid is carried out PCR and identify, recombinant plasmid difference called after pMAL-NS1-15VP.
(5) the single positive bacterium colony incubated overnight of picking, be inoculated in the 40mL LB liquid nutrient medium with 1: 100 afterwards, 37 ℃ are cultured to bacterium liquid OD600 and are about 0.6, adding inductor IPTG is 0.5mM to final concentration, 37 ℃ of abduction delivering 5h, and it is resuspended with 4mL PBS damping fluid to collect thalline, the ultrasonic disruption thalline, 10000rpm, 4 ℃, centrifugal 20min.Cleer and peaceful precipitation in the collection, and respectively it is carried out the SDS-PAGE electrophoretic analysis, to determine the expression (Fig. 3) of small peptide.
The result shows the small peptide successful expression, and the entrained maltose binding protein (MBP) of itself and pMAL-c2x carrier forms fusion rotein, with this fusion rotein called after MBP-15VP.
(6) after the affirmation small peptide successful expression, utilize WN-3D10 that small peptide is carried out Western blot and analyze, and small peptide 100ng/ hole bag is carried out indirect ELISA by the ELISA Sptting plate detect.
The result show expressed fifteen amino acid small peptide can with WN-3D10 take place specific reaction (Fig. 4, Fig. 5).And then with WNV-NS1 protein B cell epitope Primary Location that WN-3D10 discerned in
25VEAWMDRYKYYPETP
39
3.B the accurate location of cell epitope
Utilize pepscan to synthesize following small peptide (table 1), and it is carried out indirect ELISA detect, package amount is the 100ng/ hole, and then accurately locatees the B cell epitope.
Table 1 is at WN-3D10 institute synthetic small peptide
The result shows that NS1-15VP, NS1-13ET, NS1-11AE and NS1-9WP all can be positive with WN-3D10, the reaction and NS1-7MY and WN-3D10 are negative.But the OD492 value of NS1-9WP and WN-3D10 reaction is low is about 0.2, the joint efficiency that both are described is lower, there is some key amino acid between NS1-11AE and the NS1-9WP thereby further specify, determined the joint efficiency (Fig. 6) of epi-position small peptide and monoclonal antibody.Thereby the epi-position that WN-3D10 discerned is accurate between NS1-11AE and the NS1-7MY.
Further synthesize following 4 small peptides (table 2) according to above-mentioned indirect ELISA detected result, finally finish the accurate location that WN-3D10 discerns WNV-NS1 protein B cell epitope.
Table 2 is at WN-3D10 institute synthetic small peptide
Small peptide in the his-and-hers watches 2 carries out the indirect ELISA detection and shows that NS1-10AP and NS1-10WE all can be positive with WN-3D10, but the OD492 value of NS1-10WE and WN-3D10 reaction is hanged down and is about 0.19, and NS1-8WY and the NS1-8MP reaction (Fig. 7) that all is negative.The A that the The above results explanation is 27 is a key amino acid to the epi-position that WN-3D10 discerned, its disappearance can significantly reduce the joint efficiency of WN-3D10 and corresponding epi-position, 37 E then is a nonessential amino acid concerning the epi-position that WN-3D10 discerned, and its disappearance can not influence the joint efficiency of WN-3D10 and corresponding epi-position.
In sum, the present invention accurately orientates the WNV-NS1 protein B cell epitope that WN-3D10 discerned as
27AWMDRYKYYP
36
4.B the conservative type of cell epitope and virus-specific analysis
The aminoacid sequence Blast program of utilizing European Bioinformatics Institute (EBI) database to provide, the WNV-NS1 protein B cell epitope that is identified is carried out the conservative property analysis, whether simultaneously this epitope sequences and other flavivirus NS1 albumen respective section are compared, analyzing this epi-position is the WNV specificity epitope.
Blast result shows that the WNV-NS1 protein B cell epitope identified guards in each strain of WNV, and and this epi-position of relatively demonstration of other flavivirus NS1 albumen respective section be WNV peculiar (Fig. 8).
Claims (6)
1. the hybridoma cell strain of anti-west nile virus (West Nile virus) NS1 protein monoclonal antibody is secreted in a strain, and its microbial preservation number is: CGMCC No.4242.
2. the described hybridoma cell strain excretory of claim 1 monoclonal antibody.
3. by the linear B cell epitope of west nile virus NS1 albumen of the described monoclonal antibody identification of claim 2, it is characterized by: its amino acid is
27AWMDRYKYYP
36
4. the application of the described hybridoma cell strain of claim 1 in preparation diagnosis, prevention or treatment west nile virus medicine.
5. the application of the described monoclonal antibody of claim 2 in preparation diagnosis, prevention or treatment west nile virus medicine.
6. the application of the linear B cell epitope of the described west nile virus NS1 of claim 3 albumen in preparation diagnosis, prevention or treatment west nile virus medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105416354A CN102021146B (en) | 2010-11-12 | 2010-11-12 | West nile virus NS1 protein monoclonal antibody, identified B cell epitope thereof and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105416354A CN102021146B (en) | 2010-11-12 | 2010-11-12 | West nile virus NS1 protein monoclonal antibody, identified B cell epitope thereof and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102021146A true CN102021146A (en) | 2011-04-20 |
| CN102021146B CN102021146B (en) | 2012-06-27 |
Family
ID=43862931
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010105416354A Expired - Fee Related CN102021146B (en) | 2010-11-12 | 2010-11-12 | West nile virus NS1 protein monoclonal antibody, identified B cell epitope thereof and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102021146B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827261A (en) * | 2012-08-31 | 2012-12-19 | 任瑞文 | Recombinant antigenic protein for detecting west nile virus antibody, kit and application thereof |
| CN104628831A (en) * | 2015-03-20 | 2015-05-20 | 浙江省农业科学院 | Duck tembusu virus (DTMUV) protein E linear B cell antigenic epitope polypeptide and application thereof |
| CN106085964A (en) * | 2016-06-03 | 2016-11-09 | 山东农业大学 | A kind of duck tembusu virus NS1 protein monoclonal antibody and the B cell epi-position of identification thereof and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2871888A1 (en) * | 2004-06-22 | 2005-12-23 | Pasteur Institut | Method for diagnosis and detection of flavivirus, especially West Nile virus, infection, by immunoassay using new antibodies specific for the secreted form of non-structural protein 1 |
| JP2008058194A (en) * | 2006-08-31 | 2008-03-13 | Kobe Univ | Discrimination method of westnile virus infection |
| CN101235085A (en) * | 2008-01-24 | 2008-08-06 | 中国科学院微生物研究所 | Monoclonal antibody of membrane protein E for resisting West Nile virus and application thereof |
-
2010
- 2010-11-12 CN CN2010105416354A patent/CN102021146B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2871888A1 (en) * | 2004-06-22 | 2005-12-23 | Pasteur Institut | Method for diagnosis and detection of flavivirus, especially West Nile virus, infection, by immunoassay using new antibodies specific for the secreted form of non-structural protein 1 |
| JP2008058194A (en) * | 2006-08-31 | 2008-03-13 | Kobe Univ | Discrimination method of westnile virus infection |
| CN101235085A (en) * | 2008-01-24 | 2008-08-06 | 中国科学院微生物研究所 | Monoclonal antibody of membrane protein E for resisting West Nile virus and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 《中国预防兽医学报》 20090831 马健男等 西尼罗病毒NS1基因的原核表达与抗原性鉴定 第592-595页 1-6 第31卷, 第8期 2 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827261A (en) * | 2012-08-31 | 2012-12-19 | 任瑞文 | Recombinant antigenic protein for detecting west nile virus antibody, kit and application thereof |
| CN104628831A (en) * | 2015-03-20 | 2015-05-20 | 浙江省农业科学院 | Duck tembusu virus (DTMUV) protein E linear B cell antigenic epitope polypeptide and application thereof |
| CN106085964A (en) * | 2016-06-03 | 2016-11-09 | 山东农业大学 | A kind of duck tembusu virus NS1 protein monoclonal antibody and the B cell epi-position of identification thereof and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102021146B (en) | 2012-06-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102636644B (en) | Double-antibody sandwich ELISA (enzyme linked immunosorbent assay) kit used for detecting avian leukosis group specific antigen | |
| CN103751774A (en) | Recombinant cell line for stably expressing classical swine fever virus E2 protein, and applications of the same in preparation of subunit vaccines and diagnosis reagents of classical swine fever | |
| CN102766207B (en) | Bactrian camel heavy-chain (HC) variable-domain antibody resisting porcine circovirus 2 as well as preparation method and application thereof | |
| CN104017080B (en) | A kind of dog source single-chain antibody and construction method thereof and application | |
| CN105992594A (en) | Antibodies specific for enteroviruses that infect humans | |
| CN107033250A (en) | Bovine coronavirus recombinant multi-epitope antigens and its application | |
| CN103992988A (en) | Hybridoma cell line and anti-canine distemper virus N protein monoclonal antibody produced through hybridoma cell line | |
| CN102021146B (en) | West nile virus NS1 protein monoclonal antibody, identified B cell epitope thereof and application | |
| CN103740650A (en) | Monoclonal antibody BTV16-3G10 resistant to bluetongue virus serum 16 type VP2 protein, B-cell epitope polypeptide identified by monoclonal antibody BTV16-3G10, and applications of monoclonal antibody BTV16-3G10 | |
| CN107058239A (en) | A kind of swine fever virus resistant E2 protein monoclonal antibodies cell line and its application | |
| CN105906712A (en) | Porcinized single-chain antibody resisting porcine epidemic diarrhea virus and preparation method of porcinized single-chain antibody | |
| CN103740649B (en) | The B cell epi-position of anti-blue tongue virus serum 16 type VP2 protein monoclonal antibody BTV16-2B4 and identification thereof and application | |
| CN103305470A (en) | Monoclonal antibody BTV8-VP2-3E11 resistant to bluetongue virus serum 8 type VP2 protein, B-cell epitope peptide identified thereby and application | |
| CN101492495A (en) | A group of antigen epitope polypeptide and uses thereof | |
| CN104744572A (en) | Avian and porcine hepatitis e virus shared antigen, monoclonal antibody and preparation method and application | |
| CN104710529B (en) | A kind of single-chain antibody of anti-fishes virus haemorrhagic septicaemia virus | |
| CN109406788A (en) | A kind of monoclonal antibody and its application | |
| CN103305471B (en) | Monoclonal antibody BTV8-VP2-4D9 resist ant to bluetongue virus serum 8 type VP2 protein, B-cell epitope peptide identified thereby and application | |
| CN101921337B (en) | Antibody against lactate dehydrogenase of plasmodium vivax, related preparation method, hybridoma cell strain and application | |
| CN103642757A (en) | Bluetongue virus NS2 protein monoclonal antibody BTV-4D4, B cell epitope recognized thereby and applications | |
| CN103725697A (en) | Chemically synthesized staphylococcus aureus surface protein FnBPA gene fragment and expression and application thereof | |
| CN110016466A (en) | The monoclonal antibody and its hybridoma cell strain of specific detection blue tongue virus and application | |
| CN102584952A (en) | Antigen conformation epitope mimic peptide of hepatitis C virus F protein and application thereof | |
| CN106399294A (en) | Preparation of monoclonal antibody 7H8 capable of resisting epitope at N terminal of human procalcitonin protein | |
| CN101186644B (en) | H3 type flu virus hemagglutinin space conformation simulation antigen epitope and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Wu Donglai Inventor after: Yang Tao Inventor after: Liu Nihong Inventor after: Wang Qun Inventor after: Sun Encheng Inventor after: Lin Yan Inventor after: Tian Ye Inventor before: Wu Donglai Inventor before: Yang Tao Inventor before: Liu Nihong Inventor before: Wang Qun Inventor before: Sun En Inventor before: Cheng Linyan Inventor before: Tian Ye |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: WU DONGLAI YANG TAO LIU NIHONG WANG QUN SUN EN CHENG LINYAN TIAN YE TO: WU DONGLAI YANG TAO LIU NIHONG WANG QUN SUN ENCHENG LIN YAN TIAN YE |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120627 Termination date: 20211112 |