CN108486063A - The monoclonal antibody and application of hybridoma cell strain Anti-CLas McAb2 and its secretion - Google Patents
The monoclonal antibody and application of hybridoma cell strain Anti-CLas McAb2 and its secretion Download PDFInfo
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- CN108486063A CN108486063A CN201810132721.6A CN201810132721A CN108486063A CN 108486063 A CN108486063 A CN 108486063A CN 201810132721 A CN201810132721 A CN 201810132721A CN 108486063 A CN108486063 A CN 108486063A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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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
Abstract
The present invention provides a kind of hybridoma cell strain Anti CLas McAb2, are mouse hybridoma cell (Mus musculus Hybridoma) Anti CLas McAb2, and deposit number is CCTCC NO：C2017123.The monoclonal antibody of the recombination Huanglong germ CLas McAb2 generated the present invention also provides the preparation method of the hybridoma cell strain and by the hybridoma cell strain or the secretion of its passage cell strain.The present invention also provides the applications of the monoclonal antibody of recombination Huanglong germ CLas McAb2, including detection kit and immune colloidal gold detection test paper strip, utilize the specific detection of the monoclonal antibody of recombination Huanglong germ CLas McAb2.Have the advantages that the good, strong interference immunity of specificity, it is at low cost, be widely used, can fast and effeciently detect Huanglong germ present in citrus material.
Description
Technical field
The present invention relates to a kind of hybridoma cell strain and monoclonal antibody more particularly to hybridoma cell strain Anti-CLas
The monoclonal antibody and application of McAb2 and its secretion.
Background technology
Citrus Huanglongbing pathogen is that prevention is most difficult to, endangers a kind of most heavy, the maximum destructive disease of threat on Orange Producing, by people
Be referred to as citrus on " AIDS ", be widely distributed in the almost all of citrus main producing region in China.As a kind of destructive disease,
Citrus infected plant often loses bearing capacity or death in 2~3 years, or even causes to ruin garden.The rampant danger of Citrus Huanglongbing pathogen
Evil and sprawling diffusion, cause serious threat to the stability and development of Citrus Industry, have become the weight of Aspects In The Development of Citrus Industry
Big obstacle.
Effectively preventing medicament and ideal resistant variety are still lacked to Citrus Huanglongbing pathogen at present, therefore integrated control is to work as
The main method of the preceding disease control.Wherein, stringent plant quarantine is to protect the premise of no lesion and new district citrus, establishes nothing
Viral nursery, cultivation and the virus-free nursery stock of plantation are the bases for preventing yellow twig, nip in the bud and pass diseased wood lice and thoroughly excavate disease
Tree is the key measure for preventing plant disease epidemic.However, the development of these work all must be set up in clear citrus material whether
On the basis of Huanglong germ.Therefore, to Huanglong germ, rapidly and accurately detect and diagnose is that current disease synthesis is anti-
The important prerequisite of control.
A variety of methods and techniques have been developed in the existing detect and diagnose to Citrus Huanglongbing pathogen, such as field diagnosis, instruction
Plant identification, micro- sem observation, Serologic detection, nucleic acid hybridization check and PCR detections etc..Although these methods promote people
Intensification to Citrus Huanglongbing pathogen understanding, however different degrees of limitation is but shown in citrus production application.Such as exist
During the diagnosis of field, since Disease symptoms are complicated and changeable, and easily mutually obscure with symptoms such as nutritional deficiency, virus disease and poisoning, because
This often results in diagnostic error；And it grafts the problems such as success rate is not high and stability is poor and affects and identified by indicator plant
Efficiency, and this method takes longer (often to pass through some months or even longer time can just learn qualification result)；By aobvious
The identification of the methods of micro mirror, serology then needs the experimental skill of specific instrument and equipment and profession；Nucleic acid hybridizes and various PCR
Although technology can rapidly and accurately detect yellow twig, with the methods of microscope, serology, expensive instrument is needed to set
Standby, reagent material and skilled experimental skill.Therefore, there is an urgent need to develop the new technologies in a kind of practice applied to Orange Producing
Or new product, it realizes to Huanglong germ quickly and accurately detect and diagnose.
Monoclonal antibody be the height that is generated by single B cell clone it is uniform, only for the anti-of a certain specific antigen epitope
Body, referred to as monoclonal antibody.Prepared by generally use hybridoma technology, hybridoma (hybridoma) antibody technique is in cell
On the basis of integration technology, by the sensitization B cell with secreting specificity antibody ability and the marrow with Immortalization ability
Oncocyte is fused to B cell hybridoma.With the single Hybridoma Cell Culture for having this characteristic at cell mass, it can prepare and be directed to
A kind of specific antibody, that is, monoclonal antibody of epitope.Documents 1：Patent No. " CN102212134A ", it is entitled
The patent of " Citrus Huanglongbing pathogen bacterial outer membrane protein polyclonal antibody and its preparation method and application ", discloses anti-Citrus Huanglongbing pathogen bacterium
The polyclonal antibody of outer membrane protein.Prepared polyclonal antibody can be used for the detection of field infection Citrus Huanglongbing pathogen bacterium sample.
The patent disclosure is polyclonal antibody, because polyclonal antibody itself is there are the poor drawback of unavoidable specificity, and mandarin orange
There are many kinds of the outer membrane proteins of orange dragon germ, which kind of outer membrane protein to make monoclonal antibody detection citrus Huanglong using as antigen
The effect of germ preferably has not been reported, and grinding in terms of the current monoclonal antibody not yet having for Huanglong's germ differential protein
Study carefully report.
Invention content
It is an object of the invention to overcome the defect of the prior art, hybridoma cell strain Anti-CLas McAb2 are provided
And CLas McAb2 monoclonal antibodies, have specificity good, anti-dry for the monoclonal antibody of CLas McAb2 Membrane surface proteins
Immunity is strong, at low cost, the advantages of being widely used, and can fast and effeciently detect Huanglong germ present in citrus material.
The invention is realized in this way：
It is that Mouse Hybridoma Cells are thin one of the objects of the present invention is to provide hybridoma cell strain Anti-CLas McAb2
Born of the same parents (Mus musculus Hybridoma) Anti-CLas McAb2, deposit number are CCTCC NO： C2017123.
The second object of the present invention is to provide the monoclonal antibody of recombination Huanglong germ CLas McAb2, for for weight
The monoclonal antibody specific of group Huanglong germ CLas McAb2 albumen, the Dan Ke of the recombination Huanglong germ CLas McAb2
Hybridoma cell strain Anti-CLas McAb2 or the secretion of its passage cell strain described in grand antibody are generated.
The third object of the present invention is to provide the hybridoma cell strain Anti-CLas preparation methods of McAb2, including such as
Lower step：
Step 1, amplification Huanglong germ CLas McAb2 coding region genes, by containing specific cleavage site (BamH
I/Xho I) specific primer be packed into prokaryotic expression carrier pET-28a, obtain recombinant expression plasmid pET28a-McAb2；
Step 2 converts recombinant expression plasmid pET28a-McAb2 to Escherichia coli Rosetta bacterial strains, and derivant is added
Induce the expression of CLas McAb2 albumen, thalline after being induced；
Step 3 collects and is crushed thalline after induction, then passes through immunization experiment animal, animals following immunization spleen cell and bone
Myeloma are merged, and hybridoma cell strain is obtained, and hybridoma cell strain obtains only secreting by otherness ELISA screening methods is directed to CLas
The positive cell strain of McAb2 protein antibodies.
The fourth object of the present invention is to provide the application of the monoclonal antibody of recombination Huanglong germ CLas McAb2, pass through
Indirect elisa method detects Citrus Huanglongbing pathogen bacterium using the monoclonal antibody of the recombination Huanglong germ CLas McAb2.
The fifth object of the present invention is to provide a kind of ELISA detection kit of Huanglong germ, including any of the above institute
The monoclonal antibody of the recombination Huanglong germ CLas McAb2 stated.
The sixth object of the present invention is to provide a kind of immune colloidal gold detection test paper strip of Huanglong germ, including to take up an official post
The monoclonal antibody of recombination Huanglong germ CLas McAb2 described in one.
The invention has the advantages that：Have the advantages that the good, strong interference immunity of specificity, it is at low cost, be widely used,
Huanglong germ present in citrus material can fast and effeciently be detected.
Wherein, hybridoma cell strain of the invention the deposit date is on October 11st, 2017, deposit number CCTCC
NO：C2017123.It is named as hybridoma cell strain Anti-CLas McAb2, the Latin name Mus of source kind
Musculus, the entitled China typical culture collection center of depositary institution, address are Wuhan University of Wuhan, China city, postcode：
430072。
Description of the drawings
Fig. 1 is the preparation flow of the monoclonal antibody for the recombination Huanglong germ CLas McAb2 that the embodiment of the present invention 1 provides
Figure；
Fig. 2 is the plasmid map for the carrier pET-28a that the embodiment of the present invention 1 provides；
Fig. 3 detects CLas McAb2 albumen after purification for the SDS-PAGE that the embodiment of the present invention 1 provides；
Fig. 4 is that the ascites that the SDS-PAGE that present invention implementation 2 provides is detected after purification recombinates Huanglong germ CLas McAb2
Monoclonal antibody；
Fig. 5 is that the present invention implements the 3 Western-blot experimental verification ascites recombination Huanglong germ CLas McAb2 provided
Monoclonal antibody specificity.
Specific implementation mode
The foundation of 1 hybridoma cell strain Anti-CLas McAb2 of embodiment
One, preparing experiment material and reagent
The various sterilized consumptive materials needed for following table are placed into irradiation-sterilize in super-clean bench before operation.Various specifications pipettor
And mating pipette tips, homogenizer, scissors and tweezers, glass dish, foam pad, syringe needle, cell screen clothes, filter paper, 96 hole cells trainings
Support plate (sterile).
Table 1
Two, experimental method
1, the preparation of recombinant expression plasmid pET28a-McAb2
First design is for Huanglong germ McAb2 (the nucleotide sequence such as SEQ of recombination Huanglong germ CLas McAb2 albumen
ID NO：Shown in 1) specific primer, according to carrier specific structure design upstream and downstream primer, the restriction enzyme site used is BamH
I/Xho I, upper and lower primer are respectively 5 '-CGGGATCCATGATCGATAACTTGGATACG-3 ' (nucleotide sequence such as SEQ ID
NO：Shown in 2) and 5 '-CCCTCGAGAGTATTAGCACGACATC-3 ' (nucleotide sequence such as SEQ ID NO：Shown in 3), it uses
Round pcr expands McAb2 coding region genes (high fidelity enzyme) from Huanglong's germ genome, is packed into after amplified production recovery purifying
Carrier pET-28a obtains recombinant expression plasmid pET28a-McAb2, which is transferred to TOP10 bacterial strains by heat-shock transformed method
Expand it is numerous, after sequence verification is errorless, by recombinant plasmid pET28a-McAb2 be transferred to Rosetta express bacterial strain.
Blank control group：Empty carrier plasmid pET-28a without McAb2 genes.It is illustrated in figure 2 carrier pET-28a's
Plasmid map.
2, the preparation of immunizing antigen
(1) bacterium liquid activation：Recombinant expression plasmid pET28a-McAb2 inoculation fluid nutrient medium shakes bacterium, take 20ul bacterium solutions in
LB culture medium activation (37 DEG C, 16h) of the 2ml containing Kan+；Blank control group：Empty carrier plasmid pET- without McAb2 genes
28a does identical processing.
(2) induced expression is tested：Picking single bacterium is fallen in the LB culture mediums containing Kan+, when shaking bacterium to OD600=0.6,
0.8mM IPTG induced expressions (37 DEG C, 4h).It is compared simultaneously with McAb2/Rosetta of non-induced expression.It is centrifuged after induction
12000rpm, 1min collect bacterial sediment and isometric 2 × SDS-PAGE loading buffer are added and boil after PBS is resuspended
Sample preparation is boiled, 12%SDS-PAGE analyzes expression.Judged according to SDS-PAGE results, McAb2/Rosseta normal expressions.It is empty
White control group：Empty carrier plasmid pET-28a without McAb2 genes does identical induction processing.
(3) it takes the bacterium solution of activation to expand culture, when OD600=0.6,0.2mM IPTG inductions (20 DEG C, 16h) is added, receive
Collect thalline.Ultrasonic wave breaks bacterium 15min, power 390W, and work 2s stops 4s, 9000rpm, centrifuges 10min, collects supernatant, and to become
Property buffer solution precipitation is resuspended, sample preparation respectively, 12%SDS-PAGE tests and analyzes its expression status.Blank control group：It is free of
The empty carrier plasmid pET-28a of McAb2 genes does the identical processing that spreads cultivation.Judged in conjunction with its volume according to SDS-PAGE results,
Main expression is in precipitation [inclusion body].
(4) protein purification：The inclusion body for being dissolved in denaturation buffer is taken, sephadex separation, ion exchange etc. are passed through
Method purifies destination protein, and the albumen after purifying is dissolved in by buffer exchange in PBS solution, as shown in figure 3, SDS-
The high CLas McAb2 albumen of purity is obtained known to PAGE detections.Wherein, in the protein solution of purifying buffer main component
And concentration such as the following table 2.Blank control group：Empty carrier plasmid pET-28a without McAb2 genes does identical purification process.
Table 2
| Ingredient | Na2HPO4 | NaH2PO4 | NaCl | KCl |
| Concentration (mmol/L) | 8 | 1.47 | 137 | 2.7 |
3, mouse is immune
The female mouse of pure lines BALB/C 10 for selecting the Quick off the mark of 4~6 week old, are divided into two groups, i.e. experimental group and control
Each 5 of group takes subcutaneous multi-point injection first immunisation mouse respectively, and the immunizing dose of every mouse of control group is 10ul/, real
Test group carry out 4 times it is immune, each immunization interval time is 7-15 days.
Table 3
| Serial number | Time | Working node | Action |
| 1 | 2016.6.17 | Negative blood is adopted before immune | Negative serum >=10ul/ is only |
| 2 | 2016.6.17 | It is immune for the first time | Immunizing antigen 100ug/ is only |
| 3 | 2016.7.2 | Second immune | Immunizing antigen 100ug/ is only |
| 4 | 2016.7.16 | Third time is immune | Immunizing antigen 100ug/ is only |
| 5 | 2016.7.30 | 4th time immune | Tail vein 100ug/ is only |
4, the fusion of cell
It is as follows：After the 4th immune 3d of tail vein, a small amount of blood is acquired, -20 DEG C of serum of separation freezes, as
Positive control when screening.Immunized mice, 75% alcohol soaking disinfection 5min, sterile extracting spleen cell are put to death by bio-safety method
It is merged under PEG (MW4000) effects with the myeloma cell SP2/0 in exponential phase, it is huge with Balb/c mouse peritoneals
As feeder cells, the cell and feeder cells HAT culture mediums that have merged suspend phagocyte, dispense 96 orifice plates, set 37 DEG C,
It is cultivated in 6%CO2 incubators.Fresh HAT medium is added after 5d, uses HAT complete mediums after 10d instead and is cultivated, periodically
It observes, change liquid and detection.
5, the screening of specific positive cells strain
After cell fusion 10 days, with micro- sem observation, it can be seen that hybrid cell forms microcolony.Replace fresh HAT
Complete medium, 100 holes μ l/.To the hole with hybridoma, indirect enzyme-linked immunosorbent assay is detected using ELISA, is utilized
PET-28a carries out positive-selecting, cell hole to be screened with lysate after pET28a-McAb2 bacteria-inductions as detection antigen
Supernatant reacts with it respectively simultaneously, is only reacted with lysate coating hole after pET28a-McAb2 bacteria-inductions, that is, with
The reaction of CLas McAb2 protein-specifics is set to candidate strain.The OD450 values of supernatant and the OD450 values of blank control wells it
Than being judged as that positive hole, hybridoma therein can be described as positive cell strain more than 2.1.Pass through this principle specificity
Screening can obtain the cell strain just for CLas McAb2 protein antibodies, so as to be obtained in cell culture by this method
Clear liquid or ascites.
The OD values that ELISA detection indirect enzyme-linked immunosorbent assays measure the monoclonal antibody are 2.896.
6, subclone screening
The 3 plants of positive cell strains screened in step 6 are subcloned respectively using limiting dilution assay, it is sub- twice in succession
False positive cell strain is rejected after clone, it is final to obtain the specific monoclonal secreted respectively just for CLas McAb2 albumen
The hybridoma cell strain of antibody, this is capable of the hybridoma cell strain of stably excreting monoclonal antibody, expands liquid nitrogen cryopreservation after culture.
Hybridoma cell strain as provided by the invention, the deposit date is on October 11st, 2017, deposit number was CCTCC NO：
C2017123.It is named as hybridoma cell strain Anti-CLas McAb2, the Latin name Mus of source kind
Musculus, the entitled China typical culture collection center of depositary institution, address are Wuhan University of Wuhan, China city, postcode：
430072。
Embodiment 2：The preparation of monoclonal antibody
1, prepared by ascites
Take the BALB/c mouse of 6 week old or more that freund 's incomplete adjuvant is injected intraperitoneally, 0.5ml/ is only；After three days, abdominal cavity connects
Kind PBS or the diluted hybridoma of incomplete culture medium, every mouse 1-5 × 106/0.5ml；Interval is after 3 days, daily
Mouse ascites production is observed, if abdomen obviously expands, when touching, skin has tension, you can is adopted with 10ml syringe needles
Collect ascites；By ascites centrifugation (12000rpm centrifuges 10min), supernatant is collected, supernatant is frozen in -20 DEG C of refrigerators.
2, ascites antibody purifies
(1) serum pre-processes:0.22 aperture filter filtering serum, mixes with isometric PBS (pH7.4), is adjusted to pH7.8,
Affine hanging column effect is preferable at this time；
(2) affinity column is balanced:Pillar is cleaned with the PBS (pH7.4) of 10 times of volumes, while by pillar lower end and nucleic acid-protein
Detector inlet is connected and fixed；
(3) loading：When PBS balances cylinder, nucleic acid-protein detector numerical stability is adjusted 0 or so, sample is added
Sample column bed, numerical value slowly increases on nucleic acid-protein detector, collects liquid；
(4) after loading, albumen is washed with PBS (pH7.2), when the numerical value of nucleic acid-protein detector drops to balance number
Value when no longer declining, stops collection and flows through liquid；
(5) antibody elution：When the numerical value of nucleic acid-protein detector keeps relatively low numerical value and stablizes, wait for that column bed liquid level flows soon
When dry, eluent (0.1M Gly-HCl, pH2.7) is gently added on column bed, tries not to rush column of getting up；When nucleic acid-protein is examined
Survey numerical value on instrument and be sequentially in charge of the liquid for accepting elution rapidly when increasing rapidly, and use rapidly neutralizer (0.5M Tris-Cl,
PH8.0 it) neutralizes, accepts appropriate to the occasion multitube and sequentially pick up, and record sequence；
(6) after eluting, when numerical value is lower and no longer changes, with the PBS of at least 5 times bed volumes with 2~4ml/
Min flow velocitys cleaning balance pillar；
(7) column bed is preserved with liquid (PBS for containing 20% ethyl alcohol) is preserved, and closes pillar upper and lower ends, 4 DEG C of preservations；
(8) antibody dialyses to PBS (pH7.2) 3 times, each 4h or more；
(9) antibody concentration, antibody concentration 2mg/ml are surveyed, and antibody purity is analyzed by SDS-PAGE.
As shown in figure 4, be for first Marker in wherein Fig. 4, second and third road are the negative control of BSA, the 4th
The monoclonal antibody for the cell strain subclone secretion that road is Anti-CLas McAb2 provided by the invention, that is, be directed to CLas
The monoclonal antibody of McAb2 albumen.
The Characteristics Detection of 3 monoclonal antibody of embodiment
1, antibody titer detects
(1) it is coated with：McAb2 antigens are coated with by 1ug/ml, the holes 100ul/, and 4 DEG C overnight.
(2) it closes：Liquid in ELISA Plate is patted dry, the holes 150ul/ confining liquid (1%BSA in TBST), 37 DEG C of incubations are added
60min。
(3) primary antibody：Pat dry liquid in ELISA Plate, the holes 350ul//time, TBST is washed 3 times, is patted dry, by embodiment 2 after purification
Antibody with 1:3000 and 3 times of doubling dilution (1%BSA dilutions), the holes 100ul/, 37 DEG C of incubation 60min.
(4) secondary antibody：Pat dry liquid in ELISA Plate, the holes 350ul//time, TBST is washed 3 times, is patted dry, and HRP sheep anti-Mouses are added
(1:3000 confining liquids dilute) holes 100ul/, 37 DEG C of incubation 60min.
(5) it develops the color：Pat dry liquid in ELISA Plate, the holes 350ul//time, TBST is washed 3 times, is patted dry, and single substrate TMB is added
The holes 100ul/, develop the color 3-5min.
(6) it terminates：The holes terminate liquid 50ul/, OD450 readings is added.
Experiment display, the potency for recombinating the monoclonal antibody of Huanglong germ CLas McAb2 are 1：7.29×105。
2, the identification of monoclonal antibody specificity
Destination protein is subjected to SDS-PAGE electrophoresis, using negative serum as negative control group.After electrophoresis, carry out
Western-blot is tested, and albumen is transferred on NC films using wet robin, be then incubated by skimmed milk power closing, primary antibody,
Secondary antibody be incubated and etc. after, be exposed colour developing.
As shown in figure 5,1 swimming lane is negative control in figure, it is positive reaction in swimming lane 2, wherein the band of 19KD is the positive
Band (size of CLas McAb2 albumen is 19KD), as a result shows that prepared monoclonal antibody can be with yellow twig positive citrus leaf
Destination protein is specifically bound in piece, without being reacted with to the control of healthy Citrus leaf.
Embodiment 4 recombinates the application of the monoclonal antibody of Huanglong germ CLas McAb2
1, recombination Huanglong germ McAb2 monoclonal antibodies obtained are applied to prepare ELISA kit/immune colloid gold inspection
Test the various forms of products such as paper slip, immunofluorescence, immunoturbidimetry, chemiluminescence.
(1) ELISA kit for recombinating Huanglong germ McAb2, selected from following any：
A, box body, the ELISA Plate being located in box body and the reagent being stored in box body, shown reagent include recombination yellow twig
The monoclonal antibody of bacterium McAb2, standard items are the recombination Huanglong germ McAb2 albumen purified, detection antibody or competition marker；
B, box body, the ELISA Plate for the monoclonal antibody for being coated with recombination Huanglong germ McAb2 being located in box body and storage
Reagent in box body, shown reagent include recombination Huanglong germ McAb2 albumen, the detection antibody or competing that standard items purify
Strive marker.
(2) immune colloidal gold detection test paper strip of Huanglong germ, including sample pad, gold-labelled pad, nitrocellulose filter and suction
Pad is received, the sample pad and the gold-labelled pad are closely coupled, and the gold-labelled pad and the nitrocellulose filter are close
It is connected, the nitrocellulose filter and the water absorption pad are closely coupled, and inspection is provided on the nitrocellulose filter
Survey line, for the detection line close to the gold-labelled pad, the nitric acid of side of the detection line far from the gold-labelled pad is fine
It is provided with nature controlling line on the plain film of dimension, wherein by preserving number be CCTCC NO：The recombination that the hybridoma cell strain of C2017123 generates
The monoclonal antibody and colloidal gold of Huanglong germ CLas McAb2 forms gold labeling antibody after combining, and the gold labeling antibody is sprayed at
Gold-labelled pad is formed on bonding pad, is CCTCC NO by preserving number：The recombination yellow twig that the hybridoma cell strain of C2017123 generates
For the monoclonal antibody of bacterium CLas McAb2 as detection antibody, the detection antibody, which is sprayed on nitrocellulose filter, forms inspection
Survey line.
2, by indirect ELISA method, the citrus in prepared antibody or detection kit detection vegetable material is utilized
Huanglong germ, operating procedure are as follows：
(1) the citrus branch of maturation to be detected, blade middle arteries, petiole or root, with sharp disposable blade to be checked are taken
This progress of test sample is crosscutting, at the same handle health Citrus leaf and infection yellow twig Citrus leaf respectively as negative control and
Positive control is transferred to crosscutting face balance is uniform on nitrocellulose filter, about 8-12 seconds；
(2) nitrocellulose filter after transfer is closed 2 hours at room temperature in SuperBlock confining liquids；
(3) it is added the monoclonal antibody of the described recombination Huanglong germ CLas McAb2 according to appropriate dilution proportion, 37
DEG C 120rpm is incubated 1.5 hours；
(4) PBST buffer solutions wash 3 times, every time 10 minutes；
(5) two antiantibodys marked according to the enzyme of appropriate dilution proportion are added, 37 DEG C of 120rpm are incubated 1.0 hours；
(6) PBST buffer solutions wash 3 times, every time 10 minutes；
(7) substrate is added, is protected from light colour developing 30 minutes；
(8) color reaction is observed and recorded, it is positive reaction (infection Huanglong germ) to have color, and no color is feminine gender
It reacts (being uninfected by Huanglong germ).
This method can quickly detect Citrus Huanglongbing pathogen bacterium, easy, economical；Quickly, sensitive；Without speciallyying permit instrument and equipment；Nothing
Need special training, general orchard worker that can operate.
A, the sensitivity of Anti-CLas McAb2 monoclonal antibodies detection Citrus Huanglongbing pathogen sample
Field acquires the blade of the Citrus Cultivars such as Ponkan, local morning, Man tangerines, sweet orange, and infects citrus Huang by grafting
The blade of the catharanthus roseus of imperial germ and healthy catharanthus roseus, 10 samples of each kind, by above-mentioned indirect ELISA method, respectively
With the recombination Huanglong germ McAb2 monoclonal antibodies obtained of outer membrane protein polyclonal antibody and the present invention in documents 1
It is detected as primary antibody.As a result 4 are see the table below, the recall rate of recombination Huanglong produced by the present invention germ McAb2 monoclonal antibodies
The outer membrane protein polyclonal antibody being much higher than in documents 1.
4 monoclonal antibody of the present invention of table is compared with polyclonal antibody recall rate in documents 1
B, the specificity of Anti-CLas McAb2 monoclonal antibodies detection Citrus Huanglongbing pathogen sample
The symptom of Citrus leaf caused by certain nutritional deficiencys, virosis etc. is similar to Citrus Huanglongbing pathogen Disease symptoms, leads to people
The two is obscured in field indetification, therefore this experiment examines this kind of symptom blade using prepared monoclonal antibody
It surveys, to determine that the monoclonal antibody can only specific detection Citrus Huanglongbing pathogen.Field acquires the symptoms leaves such as zinc-deficiency, manganese deficiency, magnesium deficiency
Piece, and the blade of citrus broken mosaic virus and decline virus is infected, with coated elisa plate after coating buffer solution grinding, utilization is made
Standby antibody carries out indirect ELISA experiment.It the results are shown in Table 5, it can be seen that prepared antibody specific detection citrus Huanglong
Germ, and positive reaction does not occur with samples such as the broken leaf disease of zinc-deficiency, magnesium deficiency, manganese deficiency and citrus, decline diseases.
5 Anti-CLas McAb2 monoclonal antibodies of table detect specificity identification
Note："+" indicates that specific reaction, "-" indicate no specific reaction
C, detection of the Anti-CLas McAb2 monoclonal antibodies to the Citrus Huanglongbing pathogen bacterium isolate in different regions source
The sample for acquiring the ground such as Jiangxi, Fujian, Guangdong, Guangxi, Hunan, Yunnan, Hainan infection Citrus Huanglongbing pathogen, with coating
Coated elisa plate after buffer solution grinding carries out indirect ELISA examination using prepared Anti-CLas McAb2 monoclonal antibodies
It tests, the results showed that Anti-CLas McAb2 monoclonal antibodies can be with the citrus Huanglong of specific recognition China difference geographic origin
Germ in sick sample all has preferable detection result (being shown in Table 6).
Testing result of the 6 Anti-CLas McAb2 monoclonal antibodies of table to different regions Citrus Huanglongbing pathogen bacterium isolate
Note："+" indicates that specific reaction, "-" indicate no specific reaction
Above-described vector plasmid pET-28a full name are pET-28a (+).
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
With within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention god.
Sequence table
<110>Hua Zhong Agriculture University
<120>The monoclonal antibody and its application of hybridoma cell strain Anti-CLas McAb2 and its secretion
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 420
<212> DNA
<213>Huanglong germ (liberibacter)
<400> 1
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caggagcaat tttcttcatc tgttggtgat tctgtgtttt ttgataccag ttcttattcc 120
attcgcccag cagatattca agtgttatct aatttaggat cttggcttga aaaacatgat 180
tgtgattttc ttatagaagg tcatgcagat gaactaggtt cccgcaacag tagtattgct 240
ttgggacttc gtcgtgctta tgcagttttt aattattttg tagctagagg gattagcgca 300
tctcgcatga aggttacttc atatggaaag gagatgccat ctgtatatgg tcatgatgag 360
gatgcttatg cgaaaaatcg tcgtgcgatc gtttttttaa agggatgtcg tgctaatact 420
<210> 2
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
cgggatccat gatcgataac ttggatacg 29
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ccctcgagag tattagcacg acatc 25
Claims (10)
1. hybridoma cell strain Anti-CLas McAb2, which is characterized in that it is mouse hybridoma cell (Mus musculus
Hybridoma) Anti-CLas McAb2, deposit number are CCTCC NO：C2017123.
2. a kind of preparation method of hybridoma cell strain Anti-CLas McAb2 described in claim 1, which is characterized in that packet
Include following steps：
Step 1, amplification Huanglong germ CLas McAb2 coding region genes, pass through the specific primer containing specific cleavage site
It is packed into prokaryotic expression carrier pET-28a, obtains recombinant expression plasmid pET28a-McAb2；
Step 2 converts recombinant expression plasmid pET28a-McAb2 to Escherichia coli Rosetta bacterial strains, and derivant induction is added
The expression of CLas McAb2 albumen, thalline after being induced；
Step 3 collects and is crushed thalline after induction, then passes through immunization experiment animal, animals following immunization spleen cell and myeloma
Fusion obtains hybridoma cell strain, and hybridoma cell strain obtains only secreting by otherness ELISA screening methods is directed to CLas
The positive cell strain of McAb2 protein antibodies.
3. the preparation method of hybridoma cell strain Anti-CLas McAb2 as claimed in claim 2, which is characterized in that described
The primer up and down of amplification Huanglong germ CLas McAb2 coding region genes is respectively in step 1
5′-CGGGATCCATGATCGATAACTTGGATACG-3′、
With 5 '-CCCTCGAGAGTATTAGCACGACATC-3 '.
4. the preparation method of hybridoma cell strain Anti-CLas McAb2 as claimed in claim 2, which is characterized in that described
Otherness ELISA screening methods in step 3 are that will contain the induction of recombinant plasmid pET28a-McAb2 and empty carrier plasmid pET-28a
Cellular lysate object be coated with solid phase carrier, respectively with the antibody response secreted by cell strain of monoclonal antibody to be screened, only with containing
The inducing lysis object of recombinant plasmid pET28a-McAb2 reacts, without being reacted with the inducing lysis object of empty carrier plasmid pET-28a
Cell strain be determined as the positive, to screening obtain only secrete for CLas McAb2 protein antibodies positive cell strain.
5. recombinating the monoclonal antibody of Huanglong germ CLas McAb2, which is characterized in that the recombination Huanglong germ CLas
The monoclonal antibody of McAb2 is by hybridoma cell strain Anti-CLas McAb2 described in claim 1 or its passage cell strain point
Secrete generation.
6. the monoclonal antibody of recombination Huanglong germ CLas McAb2 as claimed in claim 5, which is characterized in that it was prepared
Journey is as follows：It obtains being directed to CLas McAb2 eggs containing specificity in the hybridoma cell strain culture or injection experiments animal body
The cell ascites or supernatant of Bai Kangti further isolates and purifies to obtain the special of recombination Huanglong germ CLas McAb2 albumen
Property monoclonal antibody.
7. recombinating the application of the monoclonal antibody of Huanglong germ CLas McAb2, which is characterized in that utilized by indirect elisa method
The monoclonal antibody of any recombination Huanglong germ CLas McAb2 of claim 5-6 detects Citrus Huanglongbing pathogen bacterium.
8. the application of the monoclonal antibody of recombination Huanglong germ CLas McAb2 as claimed in claim 7, which is characterized in that its
Detecting Citrus Huanglongbing pathogen bacterium, steps are as follows：
(1) the citrus branch of maturation to be detected, blade middle arteries, petiole or root, with sharp disposable blade to sample to be detected are taken
This progress is crosscutting, is transferred to crosscutting face balance is uniform on nitrocellulose filter, about 8-12 seconds；
(2) nitrocellulose filter after transfer is closed 2 hours at room temperature in SuperBlock confining liquids；
(3) list of any recombination Huanglong germ CLas McAb2 of claim 5-6 according to appropriate dilution proportion is added
Clonal antibody, 37 DEG C of 120rpm are incubated 1.5 hours；
(4) PBST buffer solutions wash 3 times, every time 10 minutes；
(5) two antiantibodys marked according to the enzyme of appropriate dilution proportion are added, 37 DEG C of 120rpm are incubated 1.0 hours；
(6) PBST buffer solutions wash 3 times, every time 10 minutes；
(7) substrate is added, is protected from light colour developing 30 minutes；
(8) color reaction is observed and recorded, it is the positive to have color signal, represents and contains Citrus Huanglongbing pathogen bacterium.
9. a kind of ELISA detection kit of Huanglong germ, which is characterized in that including any recombinations of claim 5-6
The monoclonal antibody of Huanglong germ CLas McAb2.
10. a kind of immune colloidal gold detection test paper strip of Huanglong germ, which is characterized in that any described including claim 5-6
Recombination Huanglong germ CLas McAb2 monoclonal antibody.
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| CN109897829A (en) * | 2019-03-11 | 2019-06-18 | 浙江大学 | Secrete anti-Citrus Huanglongbing pathogen bacterium monoclonal antibody hybridoma cell strain and its monoclonal antibody application |
| CN110713985A (en) * | 2019-10-23 | 2020-01-21 | 华南农业大学 | Hybridoma cell strain 5H4 secreting monoclonal antibody against CLas membrane protein Cmp1, monoclonal antibody and application |
| CN110760482A (en) * | 2019-10-23 | 2020-02-07 | 华南农业大学 | Hybridoma cell strain 5A4 secreting monoclonal antibody against CLas membrane protein Cmp1, monoclonal antibody and application |
| CN111117969A (en) * | 2020-01-15 | 2020-05-08 | 华南农业大学 | Hybridoma cell strain 1D4 secreting monoclonal antibody against CLas transporter Ctp2, monoclonal antibody and application |
| CN111154729A (en) * | 2020-01-15 | 2020-05-15 | 华南农业大学 | Hybridoma cell strain 1D6 secreting monoclonal antibody against CLas transporter Ctp2, monoclonal antibody and application |
| CN111424018A (en) * | 2020-01-21 | 2020-07-17 | 华南农业大学 | Hybridoma cell strain 4A12 secreting monoclonal antibody against Ctp4 of C L as transporter and application |
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