CN104946598B - A kind of monoclonal antibody of anti-XA21 albumen and its application - Google Patents
A kind of monoclonal antibody of anti-XA21 albumen and its application Download PDFInfo
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Abstract
The invention discloses a kind of monoclonal antibody of anti-XA21 albumen and its application.The monoclonal antibody of anti-XA21 albumen provided by the present invention, it is that hybridoma cell line 60,632 18 is secreted, hybridoma cell line 60,632 18 is CGMCC No.10416 in the numbering of registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.Specific good, the potency height of the monoclonal antibody of anti-XA21 albumen provided by the present invention.The monoclonal antibody for the anti-XA21 albumen that the present invention develops can be used for the immunological diagnostic reagent for developing XA21 albumen, such as enzyme-linked diagnostic reagent, colloid gold immune test paper bar, antibody chip, biology sensor etc..
Description
Technical field
The invention belongs to biological technical field, is related to monoclonal antibody and its application of a kind of anti-XA21 albumen.
Background technology
Work about Gene For Resistance To Rice Bacterial Blight Xa21,1977 can be traced back to earliest, at that time India's thremmatology
Family S.Devadath et al. carries out inoculation test to the long medicine wild rice (Oryza longistaminata) of African Mali, finds
The wild rice resists leaf spot bacteria (Xoo) microspecies of all tests.Then, International Rice Research Institute Khush et al. is wild by this
Rice hybridizes with susceptible long-grained nonglutinous rice cultivar IR24, by transformation in 12 years, is obtained in nineteen ninety using IR24 as genetic background
Long-grained nonglutinous rice NIL, IRBB21 is named as, Xoo inoculated identifications show, Xoo physiology all to India and Philippine IRBB21
Microspecies are all resistant.Genetic analysis shows that resistance is determined by single genetic locus, and Khush is wild from long medicine by this
The resistant gene of raw rice is named as Xa21 (Khush G S, Bacalangco E, Ogawa T.A new gene for
resistance to bacterial blight from O.longistaminate.Rice Genet News lett,
1990,7:121-122.)。
Because Xa21 has the characteristics of broad-spectrum disease resistance, so having attracted the sight of molecular genetic scholar.As breeding scholar
Khush by this part of important rice material be supplied to Cornell Univ USA Tanksley teach.Existed at that time
The post-doctor Pamela Ronald in Tanksley laboratories etc. have started Xa21 positioning work.Nineteen ninety-five, Song Wenyuan etc. are in U.S.
University of California Davis of state Pamela Ronald laboratories and with the Chinese Academy of Sciences heredity institute etc. unit cooperation, cloned Xa21
Gene, sequence analysis find that the gene code receptoroid kinases, its ectodomain predicted is the zip-tie rich in leucine
Structure (Lesine-rich repeat, LRR), its intracellular region are protein serine/threonine (Song WY, Wang GL, Chen
LL,Kim HS,Pi LY,Holsten T,Gardner J,Wang B,Zhai WX,Zhu LH,Fauquet C,Ronald
P.A receptor kinase-like protein encoded by the rice disease resistance gene,
Xa21.Science.1995 270(5243):1804-6.).Because Xa21 is to the resistance of wide spectrum and design feature of bacterial leaf-blight,
The gene has important application value in rice breeding, and its study mechanism also has important theory significance.By traditional
Backcross transformation strategy, Xa21 have been introduced into (Zhai Wenxue, Li Xiaobing, Tian Wenzhong, Zhou Yongli, Pan in multiple conventional rice kinds
Bacterial leaf spot resistance gene Xa21 is transferred to me by young tiger, Cao Shouyun, Zhao Xianfeng, Zhao Bin, Zhang Qi, the bright of Zhu Li by agriculture bacillus mediated
5 rice varieties Chinese sciences (C volumes) of state, 2000,43 (4):361-368. Wu's family financial situations, Yang Jianbo, Xu Chuanwan, Li Li, to
Taihe county, Ni great Hu, Wang Xiufeng, Jia Shirong, Tang Yixiong, Zhang Shiping, Fauquet C M. Gene For Resistance To Rice Bacterial Blights Xa21 turn
Trans-genetic hybrid rice and its hybrid paddy rice research Acta Agronomica Sinicas, 2001,27 (1):Zhao 29-34. shows peak, Zhai Wenxue, Li Ping, Wang Chunlian, Pan
Young tiger, Qian Qian, Li Shigui, the field experiment that the bright differences Xa21 Transgenic Hybrid Rices of Zhu Li combine and analysis Acta Agronomica Sinicas are learned,
2002,28(4):521-527.), so, the Xa21 in Testing and appraisal rice material has important application value.Due to Xa21
Sequence, it is known that can easily detect Xa21 presence or absence with the PCR methods expanded, but this amplification based on DNA is simultaneously
Do not know that XA21 protein expressions whether and expression space-time characterisation, so prepare the antibody of anti-XA21 protein, establish
Had important practical significance based on immunologic method and application value.
The content of the invention
It is an object of the invention to provide a kind of monoclonal antibody of anti-XA21 albumen and its application.
The monoclonal antibody of anti-XA21 albumen provided by the present invention is specifically secreted by hybridoma cell line 60632-18 produces
It is raw.Hybridoma cell line 60632-18, enter in the registration of China Committee for Culture Collection of Microorganisms's common micro-organisms center
Volume numbering is CGMCC No.10416.
The hybridoma cell line 60632-18 of the monoclonal antibody of the above-mentioned anti-XA21 albumen of secretion falls within the guarantor of the present invention
Protect scope.
The labelled antibody formed after the monoclonal antibody is marked with label falls within the protection model of the present invention
Enclose.
Wherein, the label can be horseradish peroxidase, it is alkaline phosphatase, biotin, fluorescein isothiocynate, glimmering
Photoinitiator dye Cy3, fluorescent dye Cy5, magnetic bead or agarose.
The hybridoma cell line 60632-18CGMCC No.10416 or described monoclonal antibodies or the labelled antibody
Application in following (a1)-(a4) is any falls within protection scope of the present invention：
(a1) detect or aid in whether to contain rice XA21 albumen in detection testing sample；
(a2) enrichment or separating rice XA21 albumen from the sample containing rice XA21 albumen；
(a3) by forming compound for the structure and/or function to the XA21 albumen with rice XA21 protein bindings
Analyzed；
(a4) by forming compound with rice XA21 protein bindings.
In (a1), the method for the detection including but not limited to utilizes the monoclonal antibody directly and antigen knot
The MBP enzyme linked immuno-adsorbent assay (ELISA) of conjunction, Western blotting (Western Blot), flow cytometry (FACS), immuning tissue
Chemical (IHC) detection or immuno-PCR detection method.In the detection, the monoclonal antibody can individually or with passing through
Key coupling, Electrostatic Absorption or hydrophilic and hydrophobic absorption are learned, and connecting conjugate includes horseradish peroxidase (HRP), alkaline phosphorus
The conjugates such as sour enzyme (AP), biotin (Biotin), fluorescein isothiocynate (FITC), Cy3, Cy5, magnetic bead and agarose connect
Connect.
In (a2), the method for the separation include but is not limited to using agarose is immune, immunomagnetic beads absorption or
Flow cytometry completes Bio- separation method or positive cell sorting.
It is by by the monoclonal antibody or the heavy chain and light-chain variable sequence by the antibody in (a3)
The engineered antibody prepared after recombination expression forms the crystal structure of stable compound with the rice XA21 albumen, and then realizes
The kinases plot structure of rice XA21 albumen is studied.
It is a further object to provide a kind of kit for detecting or aiding in detection rice XA21 albumen.
Containing described in independent packaging in the kit of detection provided by the present invention or auxiliary detection rice XA21 albumen
Monoclonal antibody or the labelled antibody.
Rice XA21 protein standard substances in the kit also containing independent packaging.
The amino acid sequence of the rice XA21 albumen is as shown in sequence 7 in sequence table.
The present invention also protects a kind of antibody of larger range of water resistant rice XA21 albumen.
The antibody of the water resistant rice XA21 albumen, is made up of, the amino acid sequence of the weight chain variable district is such as heavy chain and light chain
In sequence table shown in sequence 3；The amino acid sequence of the light chain variable district is as shown in sequence 4 in sequence table.
Further, the hypotype of the heavy chain constant region of the antibody is IgG1a；The hypotype of the constant region of light chain of institute's antibody is κ
Light chain.
The antibody is secreted by hybridoma cell line 60632-18CGMCC No.10416 to be produced.
The gene of encoding said antibody falls within protection scope of the present invention.
In the encoding gene of the antibody, the encoding gene of the weight chain variable district is specific such as the institute of sequence 5 in sequence table
Show；The encoding gene of the light chain variable district is as shown in sequence 6 in sequence table.
Recombinant vector, recombinant bacterium, recombinant cell lines, recombinant virus or expression cassette containing the gene fall within the present invention
Protection domain.
Advantages of the present invention and beneficial effect:
(1) the Xa21 monoclonal antibodies that the present invention obtains, restructuring XA21 albumen can be identified, has and detect a variety of transgenosis plants
The characteristic of the XA21 albumen of thing, there is wide purposes.
(2) the Xa21 monoclonal antibodies that the present invention obtains are a kind of IgG1a antibody-likes, extremely strong with being combined with for XA21 albumen
Specificity and sensitiveness, the lowest detection of ELISA method are limited to 4ng, when using the Western blot to detect its detection sensitivity for
30ng)。
(3) quantitative analysis can be carried out to the XA21 protein contents in routine and transgenic paddy rice.
(4) the Xa21 monoclonal antibodies that obtain of the present invention can independently or with biotin, horseradish peroxidase, alkaline phosphatase
Enzyme etc. is coupled, to transgenosis the methods of for WB, double-antibody sandwich elisa, indirect ELISA, immunohistochemistry, immuno-PCR
Biology is detected and examination, specificity and high sensitivity, has very high use value.
(5) Xa21 monoclonal antibodies of the invention can carry out the affine of biological specimen by being combined with agarose, magnetic bead
Enrichment and separation.
(6) by the way that the fluorescence molecules such as the Xa21 monoclonal antibodies of the present invention and FITC, Cy3, Cy5 are coupled, for turning base
Flow cytometry and sorting because of cell, and then complete qPCR, ELISpot or single cell analysis.
Preservation explanation
Join the biomaterial (strain) of evidence：60632-18
Scientific description：Hybridoma cell line
Preservation mechanism：China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is referred to as：CGMCC
Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date：On March 31st, 2015
Collection is registered on the books numbering：CGMCC No.10416
Brief description of the drawings
Fig. 1 is expression of the XA21 kinases area protein (XA21K) in Escherichia coli.M：Molecular weight marker；0h：It is induction
Preceding full bacterium ultrasound protein example；S：The Supernatant samples of induced expression bacterium ultrasonication centrifugation.P：Induced expression bacterium surpasses
The deposit sample of the broken centrifugation of sound.
Fig. 2 is the monoclonal antibody detection restructuring of hybridoma cell line 60632-18CGMCC No.10416 of the present invention secretions
XA21K protein.M：Molecular weight marker, applied sample amount：Swimming lane 1-4 applied sample amounts are respectively 150,30,6 and 1.2ng.
Fig. 3 is to be resisted with the monoclonal of hybridoma cell line 60632-18CGMCC No.10416 of the present invention secretions after purification
Rice leaf sample is surveyed in physical examination.M：Molecular weight marker；4021 and CX6221b is the rice material with XA21, TP309 and MH86
For without XA21 rice material；XA21：The XA21 protein of total length；HSP：Applied sample amount is used as by the use of anti-HSP antibody tests
Internal reference.
Fig. 4 is the canonical plotting that double-antibodies sandwich ELISA detects XA21 albumen.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
PGTK carriers：The carrier is provided by Inst. of Genetics and Development Biology, CAS, referring to " Liu, G.Z.,
Pi,L.-Y.,Walker,J.C.,Ronald,P.C.,and Song,W.-Y.Biochemical characterization
of the kinase domain of the rice disease resistance receptor-like kinase
XA21.J.Biol.Chem.2002.277,20264–20269.”。
Rice varieties 4021：There is provided by Inst. of Genetics and Development Biology, CAS, referring to " Shi Jianan, Li Li
Cloud, Xu Wen wait expression study Plant Pathology of the eight WRKY transcription factors of in rice leaf growth and resistance mechanism quietly
Report .2014,44 (1):54-64.”.
Rice varieties CX6221b (or D62B-Xa21)：Carried by Inst. of Genetics and Development Biology, CAS
For, referring to " Gao L, Xia Z, Jiang G, Peng H, Zhao X, Zhai W.Generation of marker-free,
bacterial blight-resistant transgenic sterile line and hybrid rice with
Xa21.Plant Breeding,2011,130:438-443.”。
Rice varieties TP309：It can be obtained from commercial channel." Yan Li Agrobacterium anti insect gene construction of recombinant plasmid and its right
It is also on the books in rice TP309 transfection research Hunan Normal University, Master's thesis in a 2008 " text.
Rice varieties MH86：It can be obtained from commercial channel." such as the Soviet Army, Song Yana, Yao Yuxian turns CryIAb rice not
With the grade of fit Molecular Plant Breedings under growth conditions, 04 phase in 2013 " it is also on the books in a text.
Each solution formula used in ELISA detections is specific as follows in following embodiments：
It is coated with buffer solution：By Na2CO31.59g NaHCO32.93g is dissolved in water, is settled to 1L with water, regulation pH value is 9.6.
Confining liquid：The phosphate buffer of bovine serum albumin(BSA) containing 20g/L (BSA).Wherein, the formula of phosphate buffer
For：By NaCl 8.0g, KH2PO40.2g, Na2HPO41.15g, KCl 0.2g, it is soluble in water, it is settled to 1L with water；PH value is
7.4。
Lavation buffer solution：The phosphate buffer of polysorbas20 containing volume fraction 0.05%.Wherein, phosphate buffer
Formula be：By NaCl 8.0g, KH2PO40.2g, Na2HPO41.15g, KCl 0.2g, it is soluble in water, 1L is settled to water, is adjusted
Whole pH value is 7.4.
Substrate buffer solution：PH5.0 phosphoric acid-citrate buffer solution, compound method are：
A liquid：0.1mol/L citric acids (C6H8O7·H2O), that is, citric acid 19.2g is weighed, is settled to 1000mL；B liquid：
0.2mol/L disodium hydrogen phosphates (Na2HPO4), that is, disodium hydrogen phosphate 71.7g is weighed, is settled to 1000mL.In use, take A liquid
24.3mL, B liquid 25.7mL, add water to 100mL, as substrate buffer solution.
Colorbuffer：Take the μ L of 50 μ L TMB (10mgTMB is dissolved in 1mL DMSO) solution+10mL substrate buffer solutions+10
30% (v/w) hydrogen peroxide, mix.
Terminate liquid：2M H2SO4Buffer solution.
The acquisition of embodiment 1, mouse hybridoma cell system 60632-18
First, the preparation of XA21 kinases area protein (restructuring XA21K albumen) is recombinated
The amino acid sequence of XA21K albumen is as shown in sequence 1 in sequence table, the institute of sequence 2 in its encoding gene such as sequence table
Show.
1st, the structure of recombinant expression carrier
XA21K is cloned into construction expression in expression vector pGTK and clones pGST-XA21K, recombinant expression carrier pGST-
XA21K and its detailed process are referring to document Liu, G.Z., Pi, L.-Y., Walker, J.C., Ronald, P.C., and Song,
W.-Y.Biochemical characterization of the kinase domain of the rice disease
resistance receptor-like kinase XA21.J.Biol.Chem.2002.277,20264–20269.
Recombinant expression carrier pGST-XA21K carries the XA21K genes shown in sequence 2, expressible nucleotide sequence table in ordered list
XA21K albumen shown in middle sequence 1.
2nd, Protein expression and purification
The recombinant expression carrier pGST-XA21K that step 1 is obtained converts e. coli bl21 (DE3) competence, is weighed
Group bacterium.It is incubated overnight, then by 1:The overnight bacteria suspension of single bacterium colony culture is forwarded to 100ml LB by the ratio of 100 (volume ratios)
Culture medium, adds final concentration of 50 μ g/ml kanamycins, and 37 DEG C of shaken cultivations to OD600 are 0.6~0.8.Add
0.1mol/L IPTG, 25 DEG C of concussion and cultivate 8h, ultrasonication after bacterium is received, 12000rpm is centrifuged 20 minutes, collects precipitation respectively
(inclusion body) and supernatant, carry out SDS-PAGE, the expression of detection restructuring XA21K albumen.
As a result it is as shown in Figure 1.As seen from the figure, it is higher to recombinate expression quantity of the XA21K albumen in bacterial supernatant, what is be expected
Molecular weight (about 60kDa) has obvious protein band.
The recombinant protein carries GST labels, and the affinity purification of protein, restructuring after purification are carried out using GST Resin
For the purity of XA21K albumen more than 90%, concentration is about 1.5-2mg/mL, can meet immune animal and antibody screening and identification
Requirement.
2nd, animal immune
It is immune for the first time：The restructuring XA21K albumen that step 1 obtains is emulsified with Freund's complete adjuvant (Sigma companies),
Wherein the volume proportion of XA21 albumen and Freund's complete adjuvant is 1：1, immune 4-6 week old female Balb/c mouse after emulsifying completely
(being purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.), 6 points of every mouse of abdominal part hypodermic, dosage are 60 μ g/
(to recombinate XA21K protein gauge).
Second immune：It is after immune 14 days for the first time, the restructuring XA21K albumen that step 1 obtains is incomplete using Freund
Adjuvant (Sigma companies) emulsify, wherein restructuring XA21K albumen and Freund non-fully adjuvant volume proportion be 1：1), emulsified
3 points of every mouse of abdominal part hypodermic is carried out after complete, dosage is 30 μ g/ (to recombinate XA21K protein gauge).
Third time is immune：After second immune 14 days, carry out belly with the recombinant protein booster immunization without adjuvant once
Every 6 points of mouse is subcutaneously injected, dosage is 50 μ g/ (to recombinate XA21K protein gauge).
4th time immune：Mice serum moderate resistance was detected with indirect ELISA (wavelength 450nm) in 7 days after third time booster immunization
The how anti-potency of immunogene, potency highest mouse is immune with tail vein injection impact, the restructuring XA21K eggs that step 1 is obtained
Mixed in vain with physiological saline, dosage is 50 μ g/ (to recombinate XA21K protein gauge).
The step of above-mentioned indirect elisa method measure serum titer, is specific as follows：
1) it is coated with：The μ g/ml of 100 μ L concentration 5 restructuring XA21K albumen is added in 96 hole elisa Plates, and (solvent is slow for coating
Fliud flushing), 37 DEG C are incubated 2 hours, are washed 3 times with lavation buffer solution.
2) close：With confining liquid shrouding, 37 DEG C of incubation 30min, washed 3 times with lavation buffer solution.
3) sample to be tested is added：The immunized mice serum sample to be measured that 100 μ L are diluted by a certain percentage adds to enzyme mark
In plate, put in wet box 2 hours under the conditions of 37 DEG C, board-washing 4 times.Set simultaneously using non-immunized mice serum and be used as testing sample
Control wells.
4) enzyme-added labeling antibody：Sheep anti-mouse antibody (Beijing Zhong Shan Golden Bridge, the article No. that HRP is marked：PV-6002 1000) are diluted
Times, add 100 μ L per hole, put in wet box 2 hours under the conditions of 37 DEG C, with lavation buffer solution board-washing 3 times.
5) develop the color：100 μ L colorbuffers are added per hole, room temperature lucifuge is developed the color 5 minutes, and it is whole that 50 μ L terminate liquids are added per hole
Only develop the color.
6) reading：With 450nm Single wavelengths measure respectively treat gaging hole OD values, with negative control hole (with non-immunized mouse
Control of the serum as testing sample) ratio (P/N) of OD values is limited more than 2.1, as the critical point for judging serum titer.
ELISA result judgement methods：Potency P/N>The maximum dilution multiple of 2.1 serum represents.
3rd, cell fusion
It is sterile to prepare immune mouse boosting cell suspension up to standard, by mouse boosting cell and murine myeloma cell SP2/0
(ATCC Number CRL-1581) is with 5:1 quantity centrifuges 1500rpm, 5min than mixing.Centrifuge tube is put into 37 after abandoning supernatant
In DEG C water-bath, 1ml PEG1500 (Roche companies) was slowly added in 1 minute, and stir cell.1min is stood in warm water
Afterwards, the IMDM (Sigma companies) of 10ml serum-frees is added, is mixed, centrifuges 1000rpm, 5min.After abandoning supernatant, 10ml blood is added
(PAA companies) is careful clearly gets up cell piping and druming, and adds the thymocyte that 5ml mixes 10 × HAT (Sigma companies), mixes
It is even.Add the Semi-solid cell cultures of IMDM 1640 that 25ml contains 2.1% (2.1g/100ml) methylcellulose (Sigma companies)
Base.Fully mix, then uniformly pour into 20 Tissue Culture Dish.Tissue Culture Dish is put into wet box, is put into 37 DEG C
5%CO2Cultivated in incubator.7 days clone cell group size medium densities after fusion, under anatomical lens, draw round, real, big
Cloning cluster access is ready in 96 well culture plates of culture medium in advance, is put into 37 DEG C of 5%CO2Cultivated in incubator.
4th, ELISA screens positive hybridoma cell
After 3 days, cell concentration constitutes about floor space 2/3, takes 100 μ l supernatants to carry out ELISA screenings.Positive colony changes completely
Liquid, add 200 μ l and contain 1 × 106The IMDM of cell/mL hybridomas feeder cells and 1% (1g/100ml) HT (Sigma companies)
1640 complete mediums.Carry out second of ELISA screening two days later, positive colony be transferred to preprepared culture medium (containing 1 ×
106The IMDM culture mediums of cell/mL hybridomas feeder cells and 1% (1g/100ml) HT) 24 orifice plate cultures.Taken after five days
100 μ l supernatants carry out third time ELISA screenings, and positive colony is gradually transferred to 6 orifice plates and Tissue Culture Flask expands culture and frozen.
The step of above-mentioned ELISA method screening positive cell, is specific as follows：
1) it is coated with：The restructuring XA21K albumen that 100 μ L concentration are 5 μ g/ml is added in 96 hole elisa Plates, and (solvent is coating
Buffer solution), 37 DEG C are incubated 2 hours, are washed 3 times with lavation buffer solution.
2) close：With confining liquid shrouding, 37 DEG C of incubation 30min, washed 3 times with lavation buffer solution.
3) sample to be tested is added：100 μ L Hybridoma Cell Culture liquid to be measured is added in ELISA Plate, puts 37 DEG C of bars in wet box
Lower 2 hours of part, board-washing 4 times.The control wells that testing sample is replaced with sp2/0 cells and supernatants are set simultaneously.
4) enzyme-added labeling antibody：Sheep anti-mouse antibody (Beijing Zhong Shan Golden Bridge, the article No. that HRP is marked：PV-6002 dilutions 1000
Times, add 100 μ L per hole, put in wet box 2 hours under the conditions of 37 DEG C, with lavation buffer solution board-washing 3 times.
5) develop the color：Colorbuffer is added, is added in ELISA Plate hole, per the μ L of hole 100, room temperature lucifuge develops the color 5 minutes, adds
Enter 50 μ L terminate liquid terminating reactions.
6) reading：With 450nm Single wavelengths measure respectively treat gaging hole OD values, with negative control hole (with SP2/0 cell culture
The Qing Dynasty replaces the control of testing sample) ratio (P/N) of OD values is limited more than 2.1, as being judged as positive critical point.ELISA
Result judgement method：If P/N>2.1, then it is determined as positive cell.
Screened through ELISA method, 3 subclonings obtain the monoclonal antibody of 1 plant of energy anti-XA21 albumen of stably excreting
Hybridoma cell line, it is named as 60632-18.The mouse hybridoma cell system has been preserved in the micro- life of China on March 31st, 2015
Thing culture presevation administration committee common micro-organisms center (abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCC No.10416.
Embodiment 2, the preparation of monoclonal antibody and CHARACTERISTICS IDENTIFICATION
First, the preparation of monoclonal antibody
Hybridoma cell line 60632-18CGMCC No.10416 in exponential phase are washed with serum free medium
Wash and hanged, count 5 × 105/1ml.The mouse of paraffin oil sensitization is used in the cell intraperitoneal injection of suspension in advance.Start to receive after 7 days
Collect ascites.The ascites of taking-up centrifuges 4000rpm, 10min in 4 DEG C.Ascites among careful sucking-off is collected in centrifuge tube, 4 DEG C
Or -20 DEG C of preservations.Titration is carried out to ascites using ELISA method, concrete operations are referring to " ELISA method in the step 4 of embodiment 1
Screen positive cell ", wherein step 6) is：With 450nm Single wavelengths measure respectively treat gaging hole OD values, with negative control hole (with
SP2/0 cells and supernatants replace the control of testing sample) ratio (P/N) of OD values is limited more than 2.1, as determination potency
Critical point.ELISA result judgement methods：Potency P/N>The maximum dilution multiple of 2.1 cell culture supernatant represents.Experiment
In triplicate.
As a result show, secrete the hybridoma cell line 60632-18 of the monoclonal antibody of anti-XA21 albumen, its ascites resists
Body potency is 1：106。
2nd, the purifying of monoclonal antibody
With HiTrap rProtein A FF (GE companies) affinity chromatography by specifications antibody purification from ascites.SDS-
PAGE glue identifies purity, Bradford methods measure concentration.The antibody of purifying is stored in -20 DEG C.
3rd, monoclonal antibody subgroup identification
Coating sheep anti-mouse igg (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge) is diluted with 100mM PBS (pH7.4) extremely
0.5 μ g/ml, 100 μ l are added per hole, 4 DEG C, are stayed overnight.Liquid is emptied, with the PBS of the Tween20 containing 0.05% (volume fraction)
(PBS-T) wash 3 times, 200 μ l confining liquids (formula is added per hole：Containing 2% (2g/100ml) BSA and 3% (3g/100ml) sucrose
PBS), 37 DEG C of incubation 1h.Liquid is emptied, is cleaned 3 times with PBS-T.Added per hole on the hybridoma of 0.1ml step 2 after purification
Clearly, 37 DEG C of incubation 1h.Liquid is emptied to be cleaned 3 times with PBS-T.With confining liquid 1：The goat-anti of 1000 (volume ratios) dilution HRP marks
Mouse (κ, λ) antibody (Jackson Products) or 1：2000 (volume ratios) dilution HRP mark sheep anti mouse (IgM, IgG1,
IgG2a, IgG2b, IgG3, IgA) antibody (Southern Biotech companies), 0.1ml is separately added into appropriate hole per hole,
37 DEG C of incubation 1h.Liquid is emptied, is cleaned 3 times with PBS-T.Adding 50 μ l per hole, (Southern Biotech are public containing 0.15%ABTS
Department) and 0.03% (volume fraction) H2O2Citrate buffer solution (pH4.0) carry out chromogenic reaction, measure 405nm in 10-20min
OD values under wavelength.
As a result show, the monoclonal antibody of hybridoma cell line 60632-18CGMCC No.10416 of the invention secretion is
IgG1a type mouse resource monoclonal antibodies, its light chain are κ light chains.
4th, affinity costant determines
Restructuring XA21K albumen prepared by the step 1 of embodiment 1 is coated with, coating concentration is 2 μ g/ml, and 100 μ l/ holes, 4 DEG C are wrapped
Stayed overnight, PBS-T is washed 3 times.37 DEG C of 200 μ l confining liquids are added to close 2h per hole, PBS-T is washed 3 times.The monoclonal purified in step 2
Antibody, from 1：200 start 2 times of gradient dilutions, and last 1 hole blanks control, and 37 DEG C are incubated 1h, and PBS-T is washed 3 times.HRP marks
Sheep anti-mouse antibody (Beijing Zhong Shan Golden Bridge, article No.：PV-6002)1：20000 dilutions, per the μ l of hole 100,37 DEG C are incubated 1h, and PBS-T is washed
3 times.100 μ l are added per hole and contain 0.1% (0.1g/100ml) TMB (Sigma companies) and 0.03% (volume fraction) H2O2Lemon
Acid-phosphate buffer colour developing 10min, adds 50 μ l 0.5M sulfuric acid solution terminating reactions.With ELIASA measure wavelength 450nm suction
Light value.The curve that OD values correspond to antibody extension rate is drawn, finds out extension rate A corresponding to >=1/2 " platform OD values ".Under utilization
Row formula calculates affinity costant.It is repeated three times, results averaged.
Affinity costant ≈ (150000 × A)/antibody original concentration
As a result show, the parent of the monoclonal antibody of hybridoma cell line 60632-18CGMCC No.10416 secretions of the present invention
9 × 10 are up to constant8L/mol。
5th, monoclonal antibody reaction detection
Restructuring XA21K albumen prepared by the step 1 of Example 1, the hybridoma of the present invention is detected with the method for Western blotting
The evident characteristics of the monoclonal antibody of cell line 60632-18CGMCC No.10416 secretions.Immunoblot experiment process is as follows：
The restructuring XA21K albumen of various concentrations is subjected to 12% polyacrylamide gel electrophoresis.According to a conventional method in Bio-Rad electrotransfers
Gel protein band is transferred on pvdf membrane (Millipore companies) in system.Film is placed in containing 5% (5g/100ml) defatted milk
Stayed overnight for 4 DEG C in the TBS-T confining liquids of powder.Add hybridoma cell line 60632-18CGMCC No.10416 secretions after purification
Monoclonal antibody (1：1000 volume ratios dilute, and the concentration of antibody is about 1 μ g/mL after dilution) 4 DEG C be incubated overnight.Use TBS-T
After washing film, 1 is added：The sheep anti mouse secondary antibody (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge) of 5000 volume ratios dilution, room temperature is incubated
Educate 1 hour.TBST washes film again, the super quick nitrite ions (Beijing Puli's lema gene Technology Co., Ltd.) of ECL is added, with ChemiDoc
MP multicolor fluorescences imaging system (Bio-Rad) carries out the collection of chemiluminescence image data.
As a result as shown in Fig. 2 as seen from the figure, using the method for Western blotting, hybridoma cell line 60632- of the present invention
The monoclonal antibody of 18CGMCC No.10416 secretions can identify that (purpose band size is about for about 30ng restructuring XA21 albumen
60kD, consistent with expected results).
6th, the variable region sequences measure of antibody
Fresh hybridoma is cultivated, takes supernatant to carry out antigenic binding property checking, it was demonstrated that the cell line for clone
Really it can secrete the antibody of needs, after results verification, be collected by centrifugation 106Above hybridoma.Trizol methods extract hybridoma
Cell total rna, 9 μ L total serum IgEs are taken, add-the 18primer (10mM) of 2.5 μ L oligo (dT) 12, and 5 μ L dNTPs, mixing is equal
Even, 70 DEG C of insulations 5 minutes are rearmounted 5 minutes on ice, or carry out denaturation operation according to the reverse transcriptase used.Then add 5 μ L RT
Buffer (5X), 2.5 μ L DTT (0.1M) and 1 μ L reverse transcriptases, 42 DEG C are reacted 1 hour.70 DEG C are incubated 15 minutes to terminate instead
Should, the cDNA of acquisition is stored in -20 DEG C.First chain cDNA of acquisition is entered into performing PCR amplification, adds and draws in 50 μ L reaction systems
The sequence of each 25pmol of thing, weight chain variable district and light chain variable district amplification primers puts forth energy chief editor's according to Shen again《Recombinant antibodies》
The design of mouse monoclonal antibody primer sequence and synthesis in (Science Press, publishing for 2005) book.For expanding drawing for weight chain variable district
Thing is as follows, wherein MHV.B1 until MHV.B12 11 primers are sense primer, can respectively with heavy chain anti-sense primer MHC.F groups
Share in amplification heavy chain variable region gene.
MHV.B1：5’-GATGTGAAGCTTCAGGAGTC-3’
MHV.B2：5’-CAGGTGCAGCTGAAGGAGTC-3’
MHV.B3：5’-CAGGTGCAGCTGAAGCAGTC-3’
MHV.B4：5’-AGGTTACTCTGAAAGAGTC-3’
MHV.B5：5’-GAGGTCCAGCTGCAACAATCT-3’
MHV.B6：5’-GAGGTCCAGCTGCAGCAGTC-3’
MHV.B7：5’-CAGGTCCAACTGCAGCAGCCT-3’
MHV.B8：5’-GAGGTGAAGCTGGTGGAGTC-3’
MHV.B9：5’-GAGGTGAAGCTGGTGGAATC-3’
MHV.B10：5’-GATGTGAACTTGGAAGTGTC-3’
MHV.B12：5’-GAGGTGCAGCTGGAGGAGTC-3’
MHC.F：5’-GGCCAGTGGATAGTCAGATGGGGGTGTCGTTTTGGC-3’
Primer for expanding light chain variable district is as follows, and wherein MKV.B1 is until MKV.B10 10 primers draw for upstream
Thing, the variable region gene for expanding Kappa light chains can be combined with light chain anti-sense primer MKC.F respectively.
MKV.B1：5’-GATGTTTTGATGACCCAAACT-3’
MKV.B2：5’-GATATTGTGATGACGCAGGCT-3’
MKV.B3：5’-GATATTGTGATAACCCAG-3’
MKV.B4：5’-GACATTGTGCTGACCCAATCT-3’
MKV.B5：5’-GACATTGTGATGACCCAGTCT-3’
MKV.B6：5’-GATATTGTGCTAACTCAGTCT-3’
MKV.B7：5’-GATATCCAGATGACACAGACT-3’
MKV.B8：5’-GACATCCAGCTGACTCAGTCT-3’
MKV.B9：5’-CAAATTGTTCTCACCCAGTCT-3’
MKV.B10：5’-GACATTCTGATGACCCAGTCT-3’
MKC.F：5’-GGATACAGTTGGTGCAGCATC-3’
Remaining dNTPs and buffer solution are eventually adding the μ L and 1U thermal starting Taq DNA of cDNA templates 1 according to being routinely added to
Polymerase.Set PCR amplification programs be 94 DEG C 40 seconds, 52 DEG C 40 seconds, 72 DEG C 40 seconds, carry out 20 to 25 circulations, last 72 DEG C
Extension 3 minutes, product can be placed in 4 DEG C of standby or direct electrophoresis.20 μ L PCR primers are taken to carry out electrophoretic analysis, in 1.5% agar
Gel extraction is separated on sugared gel, derived heavy chain variable region and light chain variable district are cloned into carrier T sequencing respectively.
As a result show, the monoclonal antibody of hybridoma cell line 60632-18CGMCC No.10416 secretions of the present invention
The coded sequence of weight chain variable district is as shown in sequence 5 in sequence table, the weight chain variable district in polynucleotide shown in sequence 3；Gently
The coded sequence of chain variable region is as shown in sequence 6 in sequence table, the light chain variable district in polynucleotide shown in sequence 4.
The application of monoclonal antibody prepared by embodiment 3, the present invention in immunology detection
The monoclonal antibody for detecting hybridoma cell line 60632-18CGMCC No.10416 secretions of the present invention turns in detection
Whether application effect in XA21 albumen is expressed in trans-genetic hybrid rice.Sequence 7 in the overall amino acid sequence of XA21 albumen such as sequence table
It is shown；Corresponding encoding gene is as shown in sequence 8 in sequence table.
For trying rice：Rice varieties 4021 and CX6221b, it is known that both rice express XA21 albumen.
Rice varieties TP309 and MH86, it is known that both rice do not express XA21 albumen.
1st, pre-treatment obtains testing sample for examination rice
Rice sample (blade) high-throughput tissue grinder (the limited public affairs of Beijing Ding Hao sources science and technology in -70 DEG C will be frozen
Department, TL2010) or fine-powdered is fully ground into by hand, add extracting solution of protein (formula：62.5mmol/L Tris-HCl(pH
7.4)；10% (volume fraction) glycerine；0.1% (v/w) SDS；2mmol/L EDTA；1mmol/L PMSF；5% β-sulfydryl second
Alcohol), 30min is placed in mixing on ice, and 4 DEG C of centrifugation 20min of 12000rpm, it is gross protein to take supernatant.
2nd, Western blot detection testing sample
Carried out with reference to the step 5 of embodiment 2.Setting HSP simultaneously, anti-internal reference HSP primary antibody is Beijing Hua Da egg as internal reference
Co., Ltd of white matter research and development centre Products)；Secondary antibody is sheep anti-mouse antibody (Beijing Zhong Shan Golden Bridge, article No.：PV-6002).
As a result as shown in figure 3, as seen from the figure, rice varieties 4021 and CX6221b detect the mesh that size is about 120kD
Band, it is in the same size with expected XA21 albumen；And rice varieties TP309 and MH86 do not detect purpose band.It can be seen that
Transgenic paddy rice can be detected with the hybridoma cell line 60632-18CGMCC No.10416 of the present invention monoclonal antibodies secreted
In XA21 albumen.
Embodiment 4, double-antibodies sandwich ELISA detection genetically modified plants
First, the composition of kit
The double crush syndrome kit of the present invention includes Xa21 protein standard substances (restructuring XA21K prepared by embodiment 1
Albumen), be coated in ELISA Plate through enzyme (horseradish peroxidase (HRP) or alkaline phosphatase (AP)) mark another plant of Xa21
Monoclonal antibody (the Xa21 monoclonal antibody B71769-3 of BeiJing HuaDa protein Research Center Co., Ltd), by embodiment 2
The monoclonal antibody secreted by mouse hybridoma cell system 60632-18CGMCC No.10416 prepared, other auxiliary reagent bags
Include Sample dilution, confining liquid, nitrite ion and terminate liquid.
The preparation method of Xa21 detection antibody through horseradish peroxidase-labeled：
1st, weigh 6mg HRP (horseradish peroxidase, purchased from Sigma) to be dissolved in 1mL tri-distilled waters, per mL solution dropwise
It is slowly added to the 0.1M NaIO that 0.30mL newly matches somebody with somebody4Solution, 4 DEG C of lucifuges stir 35min, activate HRP, and color is changed into green from brown
Color.Above-mentioned solution is fitted into bag filter, and with 0.01M, pH is 4.4 sodium-acetate buffer, 4 DEG C of dialysed overnights, and color is changed into palm fibre
Green.Precipitation has been seen whether, and has analyzed precipitation character, 10000r/min, 4 DEG C, 10min, centrifugation removes precipitation.Dialysed
HRP afterwards.
2nd, the monoclonal secreted by mouse hybridoma cell system 60632-18CGMCC No.10416 for preparing embodiment 2
Antibody 0.05M, pH are 9.5 4 DEG C of dialysed overnights of carbonic acid buffer.Precipitation has been seen whether, and has analyzed precipitation character,
10000r/min, 4 DEG C, 10min, centrifugation removes precipitation, the antibody after being dialysed.
3rd, the HRP after dialysis is added into 0.16M ethylene glycol (adding 0.1mL per mg enzymes), 4 DEG C of lucifuges stir 1h, then add
Antibody after dialysis；Both use 0.05M, pH after mixing be 9.5 4 DEG C of dialysed overnights of carbonic acid buffer, obtains HRP- antibody and mixes
Close liquid.
4th, 0.1mL 5mg/mL NaBH are added into HRP- antibody mixed liquors4Solution (adds 0.1-0.2mg's per mg enzymes
NaBH4), 4 DEG C of lucifuges stir 3h.Isometric saturation sulfate of ammoniac is added dropwise, 4 DEG C of lucifuges stir 2h.4 DEG C, 10000rpm centrifugations
10min, abandon supernatant.PBS dissolving precipitations, obtain the Xa21 detection antibody through horseradish peroxidase-labeled.
2nd, the detection method of Xa21 protein contents
1st, be coated with buffer solution coating Xa21 capture antibody B71769-3 (the limited public affairs of Beijing Hua Da protein research and development centre
Take charge of product) (2 μ g/mL), as capture antibody, 100 μ L are added per hole in 96 hole elisa Plates, 4 DEG C of coating 12h, make itself and enzyme
Target is combined closely.
2nd, cleaning solution (PBST) board-washing is used after being coated with 6 times, per the μ L of Kong Zaijia 300 2% (v/w) bovine serum albumin as envelope
Close liquid, 37 DEG C of incubation 3h.Closing end is added vegetable protein extract solution to be measured in ELISA Plate hole and (delayed with testing sample and PBS
The volume ratio of fliud flushing is extension rate) and Xa21 protein standard substances (every μ g/mL of hole 5, with 1:5 gradient dilutions are to 10pg/mL, often
Individual sample does three repetitions), 100 μ L/ holes, 37 DEG C of incubation 1h.
3rd, be subsequently added into step 1 preparation horseradish peroxidase-labeled anti-Xa21 detection antibody, 100 μ L/ holes, 37
DEG C be incubated 1h.
4th, developer TMB solution is finally added in the compound of formation, per the μ L of hole 100,3min is incubated at room temperature, in horseradish
Under peroxide enzyme effect, color change occurs for developer, adds 2M, H2SO4, 50 μ L/ holes terminating reactions, according to OD450Value is big
The presence or absence of Xa21 albumen and concentration in small judgement sample.
Result judgement：Taken the logarithm with the concentration of Xa21 protein standard substances and do abscissa, OD450It is worth and makes standard for ordinate
Curve (Fig. 4), obtains calculation formula, according to the OD of testing sample450Value calculates the content of the Xa21 in testing sample.
In Fig. 4, abscissa is that the Xa21 protein standard substance concentration of 5 times of serial dilutions, ordinate are proceeded by from 5 μ g/mL
For OD450 reading value.It is determined that lowest detection is prescribed a time limit, using blank value (hole of the concentration of Xa21 protein standard substances as 0ng/ml
OD450Value) average value add three times standard deviation SD, i.e. blank value average+3SD=0.069+3 × 0.024=0.140, this OD
It is about 4ng/ml that value, which corresponds to Xa21 concentration, and 4ng/mL is limited to the lowest detection of this determination this method.
Claims (11)
1. hybridoma cell line 60632-18, in the registration of China Committee for Culture Collection of Microorganisms's common micro-organisms center
It is CGMCC No.10416 to enter volume numbering.
2. monoclonal antibody, secreted and produced as the hybridoma cell line 60632-18 described in claim 1.
3. the labelled antibody formed after the monoclonal antibody described in claim 2 is marked with label, it is characterised in that：
The label is horseradish peroxidase, alkaline phosphatase, biotin, fluorescein isothiocynate, fluorescent dye Cy3, fluorescence
Dyestuff Cy5, magnetic bead or agarose.
4. the monoclonal antibody described in hybridoma cell line 60632-18 or claim 2 or right described in claim 1 will
Ask application of the labelled antibody in following (a1)-(a4) is any described in 3：
(a1) detect or aid in whether to contain rice XA21 albumen in detection testing sample；
(a2) enrichment or separating rice XA21 albumen from the sample containing rice XA21 albumen；
(a3) by being formed with rice XA21 protein bindings, compound is used for the structure of the XA21 albumen and/or function is divided
Analysis；
(a4) by forming compound with rice XA21 protein bindings.
A kind of 5. kit for detecting or aiding in detection rice XA21 albumen, it is characterised in that：Containing independent in the kit
The labelled antibody described in monoclonal antibody or claim 3 described in the claim 2 of packaging.
6. kit according to claim 5, it is characterised in that：The also rice containing independent packaging in the kit
XA21 protein standard substances.
7. the kit according to claim 5 or 6, it is characterised in that：The sequence of the rice XA21 albumen is sequence table
Shown in middle sequence 7.
8. a kind of antibody of water resistant rice XA21 albumen, is made up of heavy chain and light chain, it is characterised in that：The ammonia of the weight chain variable district
Base acid sequence is as shown in sequence 3 in sequence table；The amino acid sequence of the light chain variable district is as shown in sequence 4 in sequence table.
9. antibody according to claim 8, it is characterised in that：The hypotype of the heavy chain constant region of the antibody is IgG1a；Institute
The hypotype for stating the constant region of light chain of antibody is κ light chains.
10. encode the gene of the antibody of claim 8 or 9, it is characterised in that：The encoding gene of the weight chain variable district such as sequence
In list shown in sequence 5；The encoding gene of the light chain variable district is as shown in sequence 6 in sequence table.
11. recombinant vector, recombinant bacterium, recombinant cell lines, recombinant virus or expression cassette containing gene described in claim 10.
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Non-Patent Citations (3)
| Title |
|---|
| "Q1MX30.1";Xue,Q 等;《GenBank》;20070113;第8页 * |
| "华大蛋白新学期产品促销活动";华大蛋白;《北京华大蛋白质研发中心有限公司（网页）》;20120823;第2页 * |
| "水稻抗白叶枯病基因Xa 21 的研究进展";白辉 等;《遗传》;20061231;第28卷(第6期);第745-753页 * |
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