CN106047820A - Preparation and applications of giant panda follicle-stimulating hormone beta subunit monoclonal antibody - Google Patents
Preparation and applications of giant panda follicle-stimulating hormone beta subunit monoclonal antibody Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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Abstract
The present invention discloses preparation and applications of a giant panda follicle-stimulating hormone beta subunit monoclonal antibody, wherein an anti-giant panda FSH[beta] subunit monoclonal antibody hybridoma cell line 09199 is provided, and the preservation number is CCTCC NO: C2016144. According to the present invention, a cell fusion technology is used, the hybridoma cell line secreting the anti-FSH[beat] subunit monoclonal antibody is established, the cell line culture supernatant is collected and purified, the highly-specific FSH[beta] subunit monoclonal antibody is obtained, application of the highly-specific FSH[beta] subunit monoclonal antibody in Western Blot protein detection is established to provide information for FSH protein location and tissue expression, and the deep researches on the ligand, the antagonist and the anti-antagonist of the FSH receptor are performed with the highly-specific FSH[beta] subunit monoclonal antibody so as to create the conditions for the founding of the specific ligand, the antagonist and the anti-antagonist; and the new data and the new idea are provided for the further exploration of the FSH secretion, the further regulation of the genes, and the revealing of the reproductive physiological laws.
Description
Technical field
The present invention relates to the preparation of a species specific monoclonal antibody, especially for the inspection of giant panda follicle stimulating hormone albumen
Survey.
Background technology
Follicle stimulating hormone (FSH) is the glycoprotein hormones of anterior pituitary basophil secretion.In ovary, follicle stimulating hormone
Stimulating the growth of the most immature follicle, follicle can discharge inhibin to block the further of follicule-stimulating hormone (FSH) in growth course
Synthesis.This mechanism ensure that the selectivity of ovulation.At the end in luteinization stage, follicule-stimulating hormone (FSH) level also has and carries by a small margin
Rise, may be relevant with the beginning of next ovulatory cycle.In testis, follicule-stimulating hormone (FSH) improves sertoli's cell synthesis male and swashs
The protein-bonded level of element, combining closely of induction sertoli's cell, secrete inhibin simultaneously, play in becoming Process of Spermatozoa
Vital effect.Follicle stimulating hormone (FSH) activity form is glycosylated heterodimer, is made up of two subunits of α and β, β
Subunit determines specific antigenicity and the exceptional function of hormone, but need to be combined with α subunit and just have biologic activity.Lutropin,
Thyroxin, waits glycoprotein hormones to have employed the structure similar to follicle stimulating hormone.They share same α subunit, and β subunit
Then different with the difference of hormone.Therefore, FSH β subunit is the unique special subunit of FSH, by the albumen of 129 aminoacid codings,
About 14kd, active dimer FSH about 28kd.Due to species variation, currently without special anti-giant panda FSH
Specific antibody.
Summary of the invention
It is an object of the present invention to provide a kind of anti-giant panda FSH β subunit monoclonal antibody hybridoma cell strain, anti-giant panda
FSH β subunit monoclonal antibody and preparation method, solve do not have asking of special anti-giant panda FSH specific antibody in prior art
Topic.
The technical scheme is that anti-giant panda FSH β subunit monoclonal antibody hybridoma cell strain 09199, preservation is compiled
Number: CCTCC NO:C2016144, carry out preservation in China typical culture collection center (CCTCC) on July 20th, 2016.
Anti-giant panda FSH β subunit monoclonal antibody hybridoma cell strain 09199 is used for producing anti-giant panda FSH β subunit list
Clonal antibody.
Anti-giant panda FSH β subunit monoclonal antibody, is produced by hybridoma cell strain 09199, and hypotype is IgG1.
The application in western blot technology of the anti-giant panda FSH β subunit monoclonal antibody.
The preparation method of anti-giant panda FSH β subunit monoclonal antibody, step is as follows:
(1), shown in design giant panda FSH β distinguished sequence SEQ ID NO:1, aminoacid sequence is as follows:
LVYKDPARPNIQKICTFKELAYETVKVPGC, and with KLH coupling, as produce specific antibody immunogen
FSHβ-KLH；
(2) the immunogen FSH β-KLH immune mouse prepared with step (1)；
(3) cell is warm obtains anti-giant panda FSH β subunit monoclonal antibody hybridoma cell strain 09199；
(4) amplification culture obtains anti-giant panda FSH β subunit monoclonal antibody.
The present invention compared with prior art has the advantage that
The present invention utilizes cell-fusion techniques, sets up the hybridoma cell strain secreting anti-FSH β subunit monoclonal antibody, receives
Collection purifying cells strain culture supernatant, it is thus achieved that high specific FSH β subunit monoclonal antibody, and the FSH β setting up high specific is sub-
Base monoclonal antibody is applied to Western Blot Protein Detection, for location and the tissue expression information of FSH albumen, and utilizes
The part of fsh receptor, antagonist, anti-antagonist are furtherd investigate, for spy by the monoclonal antibody of FSH β subunit high specific
Specific ligand, antagonist, the discovery of anti-antagonist create conditions.Adjust further for discussion FSH secretion and gene further simultaneously
Control, discloses reproductive physiology rule, it is provided that new data and thinking.
Preservation information: the anti-giant panda FSH β subunit monoclonal antibody hybridoma cell strain 09199 of the present invention, in 2016
July 20 carried out preservation in China typical culture collection center (CCTCC), deposit number: CCTCC NO:C2016144, ground
Location: Wuhan, China. Wuhan University
Accompanying drawing explanation
Fig. 1 is the canonical plotting of cellular protein concentration in embodiment 2.
Fig. 2 is Western-blot testing result in embodiment 2.
Detailed description of the invention
The preparation method of specificity anti-giant panda FSH β monoclonal antibody, has steps of:
1) design of specific polypeptides fragment and synthesis: according to giant panda FSH β protein subunit sequence (NP_001291794),
Analyze with reference to other species sequence, devise shown in the sequence SEQ ID NO:1 that giant panda FSH β subunit is special:
LVYKDPARPNIQKICTFKELAYETVKVPGC, the polypeptide fragment that synthetic this section is special, and with KLH coupling, enhancing is exempted from
Epidemic focus is as the immunogen (FSH β-KLH) of production specific antibody, and this part entrusts Shanghai Sheng Gong biotech firm to complete.
2) mouse immune: use adjuvant: the mixed liquor of antigen=1:1 (coupled peptide FSH β-KLH), after emulsifying completely, abdominal cavity
Injection.First immunisation adjuvant is dress ornament Freund's complete adjuvant, and follow-up immunization adjuvant is dress ornament Freund's incomplete adjuvant, first immunisation mice it
Before, afterbody takes mouse blood, separates serum, as negative control.Repeatedly immunity, till serum titer is up to standard.
3) cell merge: include conventional method carry out cell fusion (include the preparation of SP2 cell, prepared by feeder layer cells,
The preparation of splenocyte), HAT selectivity cultivate and ELISA method screening, obtain hybridoma.
4) hybridoma obtained above is carried out the screening of positive colony, selects positive cell hole to carry out cell gram
Grand, obtain anti-giant panda FSH β subunit monoclonal antibody hybridoma cell strain 09199 by limiting dilution assay, and carry out hypotype mirror
Fixed.
5) the specificity anti-giant panda FSH β subunit monoclonal antibody hybridoma cell strain mass propgation obtained, collects cell
Culture supernatant, purification is also concentrated to give specificity anti-giant panda FSH β subunit monoclonal antibody.
Anti-giant panda FSH β monoclonal antibody, in the application of western blot technology, has steps of:
(1) extraction of destination protein expression tissue total protein: grow up giant panda testis and the extraction of ovary tissue total protein,
Protein Extraction Reagent kit: C510004-0020, Sangon Biotech (Shanghai) Co., Ltd., all operations step presses reagent
Box description is carried out.
(2) testis and the mensuration of ovary tissue total protein concentration: BCA test kit, C503021-0500, raw work biological engineering
(Shanghai) limited company, all operations step is carried out by test kit description.
(3) Western blot detection application: Western blot technical scheme is carried out routinely, first destination protein electricity
Swimming separates, then transferring film, closes, then carries out anti-giant panda FSH β subunit monoclonal antibody and hatch and two anti-Peroxidase-
Conjugated AffiniPure Goat Anti-Mouse IgG (H+L) is hatched, and finally carries out ECL exposure.
Embodiment 1
The preparation of the most anti-giant panda FSH β monoclonal antibody
The design of 1.1 specific polypeptides fragments and synthesis: according to ncbi database giant panda FSH β yldeneamino acid sequence, step on
Record number: NP_001291794, sequence is:
MKSVQLCFLFCCWRAICCKSCELTNITITVEKEECRFCISINTTWCAGYCYTRDLVYKDPARPNIQKICTFKELAYE
TVKVPGCAHQADSLYTYPVATECHCGKCDSDSTDCTVRGLGPSYCSFNEMKE, and analyze with reference to other species sequence,
Devise giant panda FSH β distinguished sequence
LVYKDPARPNIQKICTFKELAYETVKVPGC (FSH β-1), and by synthetic, and with KLH (spoon sky blood
Azurin) coupling, composition produces the immunogen (FSH β-KLH) of specific antibody.For the synthesis of polypeptide fragment, this technique ratio
More ripe, by sequence disclosed in the present application, Sangon Biotech (Shanghai) Co., Ltd. can produce polypeptide fragment
FSH β-1 and immunogen FSH β-KLH
1.2 antigen mouse immunes: with adjuvant: the mixed liquor inoculation of antigen=1:1, (amount of antigen can mix with sodium chloride not
After conjunction again with adjuvant emulsion).Head exempts from Freund's complete adjuvant, after immunity incomplete Freund's adjuvant.It is ready to before immunity
The syringe of sterilizing, tee T, disposable syringe.First with the good syringe of sterilizing by antigen and NaCl inspiration syringe
(400ul, the amount of four altogether), then adjuvant inhalation syringe (will be total to 400ul, the amount of four) with the syringe that sterilizing is good；?
After the syringe that sterilizing is good be connected in tee T carry out emulsifying (general more than 10 minutes of emulsifying, to Water-In-Oil state),
After proceed to disposable syringe is injected by the mixed liquor merged again.
1st day: lumbar injection, 200ul mono-.(amount of antigen is 100ug/)
14th day: lumbar injection, 200ul mono-.(antigen is all 50ug/ afterwards)
21st day: lumbar injection, 200ul mono-.
27th day: lumbar injection, 200ul mono-.
…………………
(within after immunity 3 to 4 days for the third time, can take a blood sample by mouse tail, 12000rmp, 8min, take its titer of determination of serum, with
FSH β-1, being coated concentration is that 2ug/ml detects serum titer as antigen, ELISA method, is i.e. ready for merging after titer is up to standard,
The not high enough person of titer need to continue immunity until up to standard.)
1.3 cells merge:
1.3.1 the recovery of myeloma cell (SP2): first take out frozen good cell from liquid nitrogen, be quickly put in 37 DEG C of water
Bath melts, makes cell loose；Again cell is put in the centrifuge tube of 15ml, then take the PBS of general 5ml, mixing,
1000rpm, 5min, centrifugal, abandon supernatant, be repeated twice cleaning SP2 cell, cell supported in culture bottle, carry out labelling, finally
Culture bottle is put into 37 DEG C, 5%CO2Incubator is cultivated.
1.3.2SP2 the passing on of cell: at the bottom of the cell in culture bottle covers with bottle about 80%, so that it may carry out passage.
With rifle head, cell is blown and beaten, by 1000r/min in culture fluid sucking-off to 15ml centrifuge tube, centrifugal 5 minutes；Abandon supernatant, add
PBS5ml, piping and druming mixing, recentrifuge is abandoned supernatant, is repeated PBS washing step 2 times.Add 2ml 10% after having washed to cultivate completely
Liquid re-suspended cell, takes appropriate cell in culture bottle, puts in CO2 gas incubator and cultivate.
1.3.3 the preparation (merge and be ready for the previous day) of trophoderm macrophage:
It is lethal that mice takes off cervical vertebra, reduces the compressing to abdominal cavity, it is to avoid damage intraperitoneal blood vessel when noting disconnected cervical vertebra as far as possible, with
Anti-feeder cells are contained within a large amount of hemocyte.Mice is soaked in 75% ethanol 5 minutes, the most portable live mousetail, on
Under rinse for several times in ethanol.It is placed in sterilized petri dishes.Cut off skin by sterile scissors from rear abdomen, with hands, both sides skin is torn
Open, expose abdominal part, be careful not to damage peritoneum.With cotton ball soaked in alcohol wiping peritoneum.With syringe draw 6-8ml containing dual anti-not
Complete medium is injected into abdominal cavity (dual anti-: not exclusively culture fluid=1:100), notes mentioning peritoneum with tweezers during injection, it is to avoid
Syringe needle stings the abdominal visceras such as intestinal tube.With cotton balls abdomen massage one minute gently, the culture fluid that sucking-off is injected, proceed to centrifuge tube
In.1000r/min is centrifuged 5min, supernatant discarded.Wash four times with PBS again.Cell is resuspended in the complete culture solution of 10%.
Above-mentioned cell suspension adds 96 orifice plates, and every hole adds 100ul, finally 96 orifice plates is put into CO2Incubator cultivate.
(macrophage is difficult to too much, can optionally abandon the cell that a part is collected.)
1.3.4 prepared by immune spleen cell:
(1) mouse spleen is taken
Take the mice reaching immunity requirement.It is first noted that sterile working, in case cell contamination, after putting to death mice, by it
The ethanol of 75% soaks about five minutes, is placed in aseptic plate, be placed in the position (super-clean bench of beneficially oneself operation
In), dissect.First with shears, mouse tail cut an osculum, then cut fur layer open with hands, gently wipe with cotton ball soaked in alcohol and cut open
Position, then that layer of translucent thin film of parcel internal organs is provoked with tweezers, cut off, exposed spleen, taken out spleen gently,
And remove fatty tissue above as far as possible, the spleen taken out is washed as in PBS.
(2) prepared by splenocyte suspension
First wash spleen with PBS, rinse about 3 times, spleen is placed in plate, with shears, spleen is cut as far as possible
Broken, add PBS washing and filtering, not histiocyte, collect the splenocyte suspension separated, 1000r/min, centrifugal 5 minutes, abandon
Clearly；Wash with 5mlPBS again, 1000r/min, centrifugal 5 minutes；In triplicate, the incomplete culture fluid of 2ml is added after having washed
(DMEM) re-suspended cell, diluting cells suspension 100 times or 1000 times is used for cell counting, and remaining cell is placed in 37 DEG C of water-baths
Standby.
(3) preparation of SP2 cell suspension: the thin film bottom Cell sap air-breathing piping and druming culture bottle will be drawn with glue head straw
(cell be suspension or slight adherent growth), then Cell sap sample-adding is robbed be transferred to 1000r/min in 15ml centrifuge tube, from
The heart 5 minutes；Abandon supernatant, then add PBS5ml, mixing, 1000r/min, centrifugal 5 minutes, repeated washing twice, add 2ml after having washed
Not exclusively culture fluid (DMEM) re-suspended cell, diluting cells suspension 100 times or 1000 times is used for cell counting, and remaining cell is placed in
In 37 DEG C of water-baths standby.
1.3.5PEG cell fusion is carried out under effect
SP2 and splenocyte are mixed in the ratio of 1:4 (between 1:10 to 1:4), centrifugal, 600rpm, 3min；Abandon
Supernatant.Gently at the bottom of attack centrifuge tube, make cell precipitation slightly loose dynamic.37 DEG C of preheated 0.6ml 50% it are slowly added in 1min
PEG solution, limit edged gentle agitation and knocking.1min is stood after adding.37 DEG C of preheated incomplete culture fluid 10ml are with end
Only PEG effect, drips while knocking rotating centrifugal pipe, at the uniform velocity adds, and stands 2min after adding.Centrifugal, 800rpm, 5min, abandon
Supernatant；Wash 2 times with PBS or not exclusively culture fluid, to remove PEG again.
Abandon supernatant after having washed, select culture fluid re-suspended cell with 10mlHAT.Above-mentioned cell is added to existing feeder layer
96 orifice plates in (be the macrophage plate prepared before, first by the liquid sucking-off in hole not, the more full culture fluid that cannots be used up
Washed once, sucking liquid), every hole adds 100ul；Culture plate is put CO2Incubator is cultivated.After 4 hours again in hole
Adding the complete culture solution (19.6ml10% complete culture solution+0.4mlHAT) containing HAT, every hole adds 100ul；Culture plate is put
CO2Incubator is cultivated.
1.3.6 select to cultivate
(1) merge the 4th day half liquid method cultivated and change HAT culture fluid: with sample loading gun, every for the supernatant in 96 orifice plates hole is inhaled
Take 100ul, discard, add new every hole 100ulHAT culture fluid (HAT culture fluid is formulated as: the complete culture solution of 10%:
HAT=1:50), every piece of plank needs to add 200ul 50 × HAT in 10ml complete culture solution.
(2) merge the 7th day half liquid method cultivated and change HT culture fluid: with sample loading gun, every for the supernatant in 96 orifice plates hole is inhaled
Take 100ul, discard, add new every hole 100ulHT culture fluid (being formulated as of HT culture fluid: the complete culture solution of 10%: HT
=1:50), every piece of plank needs to add 200ul 50 × HT in 10ml complete culture solution.
1.3.7 positive colony screening
Obvious clone cell in cultivating about 7 days visible holes, (probably the 12nd when clone cell is looked abundant
About it), culture fluid can be drawn and detect it with or without secretory antibody.
(1) first by corresponding ELISA (being coated FSH β-1, being coated concentration the is 2ug/ml) plank that is coated before from refrigerator
Taking out and allow it recover room temperature, then add each hole of culture fluid 100ul to be detected in plate, holes does yin and yang attribute comparison, negative
Compare the culture fluid with 10%, the positive control serum (serum of mouse orbit blood sampling before merging) of dilution 10000 times.
(2) hatch: hatch 1 hour with in the rearmounted 37 DEG C of incubators of shrouding film shrouding.
(3) washing: carefully take shrouding film off, washs 4 times, and finally button solid carbon dioxide divides as far as possible.
(4) add dual anti-: dual anti-dilution 10000 times, the every hole of 50ul.
(5) hatch: hatch 30 minutes with rearmounted 37 DEG C of shrouding film shrouding.
(6) washing: carefully take shrouding film off, washs 4 times, and finally button solid carbon dioxide divides as far as possible.
(7) colour developing: every hole adds developer TMB100ul, mixing of vibrating gently, the incubator general 10min of colour developing.
(8) colorimetric determination: every hole adds stop buffer 50ul (concentrated sulphuric acid of stop buffer=21.5ml is settled to 200ml), gently
Vibration mixing, sets microplate reader wavelength as 450nm, measures each hole value.
Choose positive findings height person (at least 4 times of negative control), as positive colony hole.
1.3.7 limiting dilution assay screens anti-giant panda FSH β subunit monoclonal antibody hybridoma cell strain
(1) counting of positive porocyte: limiting dilution can be done in the hole, positive colony hole screening obtained.First thin by hole
Dysuria with lower abdominal colic moves on to (piping and druming limit, limit rotates, and makes cell suspension) in the centrifuge tube of 15ml, then adds the complete culture solution of 10% to 2ml；
Count with counting chamber again, take the Cell sap containing only 1000 cells after counting and carry out next step experiment (owing to 1 hole has only to
One cell, 96 orifice plate one plates are accomplished by about 100 cells, and 10 plates are exactly 1000 cells).
(2) Cell sap is added in 200ml complete culture solution, mixing, 96 orifice plate sample-addings, 200ul/ hole, totally ten piece of 96 hole
Plate.
(3) finally culture plate is put in CO2 incubator and cultivate.
(4) after cultivating 4-5 days, visible little cell clone on inverted microscope, the growing state of observation of cell, note
The hole that record individual cells growth is assembled.
(5) cultivate the 5th day and carry out changing liquid to the hole having record individual cells growth to assemble, add 10% complete culture solution
100ul/ hole.
When (6) the 8-9 days, naked eyes visible cell is cloned, and carries out antibody test in time, by assembled by individual cells growth
Hole and the reasonable hole of growing state carry out the detection (ELSIA detection) of culture fluid, and positive strong hole is anti-giant panda FSH
β subunit monoclonal antibody hybridoma cell strain.The present invention finally screens and obtains a strain anti-giant panda FSH β subunit monoclonal antibody
Hybridoma cell strain 09199.
1.3.8 hypotype is identified
Pierce Rapid ELISA Mouse mAb Isotyping Kit 37503 test kit is used to carry out hypotype mirror
Fixed.
Preparation: TBS in test kit is dissolved in 500ml distilled water, for dilute sample, 870ml distilled water with
30ml30X Wash Buffer mixes, and is used for washing plate, determines how many planks of needs according to the amount of done sample, and remaining puts back to 4
DEG C Refrigerator store, prepares the sample diluting liquid of 450ul, draws the cell culture fluid of 20ul and adds in the TBS of 980ul and mix.
Experimental procedure: plank equilibrates to room temperature, addition testing sample is to every hole, and 50ul/ hole, each sample need to add 8 holes i.e.
Article one, adding the Goat Anti-Mouse IgG+IgA+IgM HRP in 50ul/ hole, soft plank mixing of rocking covers shrouding
Film, incubated at room one hour, wash plate 4 times, button solid carbon dioxide divides, and adds the TMB nitrite ion colour developing in 75ul/ hole, it will be seen that in hole
Liquid becomes blue, and the stop buffer adding 75ul/ hole after colour developing 5-15min terminates reaction, and liquid is become yellow from blueness.Warp
The antibody subtype secreted by anti-giant panda FSH β subunit monoclonal antibody hybridoma cell strain 09199 obtained by evaluation and screening is
IgG1 type.
1.3.9 the amplification culture of monoclonal antibody and purification, concentrates.
(1) Batch Culture: will carry out hypotype qualification, result is that the cell hole of monoclonal antibody transfers to (limit rotation in 24 orifice plates
Limit is blown and beaten, and makes cell suspension, shifts completely) cultivate, and 10% complete culture solution adding 600ul is cultivated.
(2) growing state of observation of cell, carries out titration after comparison of looking many, and the cell that titer is high carries out turning
Move, transfer to little culture bottle is cultivated and (first the cell in 24 orifice plates is blown and beaten and make cell suspension, with sample loading gun, cell is being turned
Move on in culture bottle, add the complete culture solution 7ml of 10%).
(3) observation of cell growing state, transfer to afterwards of looking relatively good is cultivated in big culture bottle.One little training
Foster bottle shifts respectively in two big culture bottles and carries out cultivating (passage).
(4) several culture bottle can be passed cultivate, frozen a part of cell more, then take culture fluid to carry out antibody and cross column purification.Institute
With pillar be filler used be Pierce Protein G Agarose, and with 10000kda super filter tube concentrate and purify anti-big
Panda FSH β monoclonal antibody ,-20 degree save backup.
1.3.10 cell strain of monoclonal antibody is frozen
The anti-giant panda FSH β subunit monoclonal antibody hybridoma cell strain 09199 identified is cultivated after stablizing, will cultivate
Cell piping and druming in Ping makes cell suspension (cell is normally suspended in culture fluid or adherent growth) in culture fluid, by cell
It is transferred in the centrifuge tube of 15ml.Centrifugal, 1000 turns/5 minutes.Wash twice with PBS: first the supernatant in centrifuge tube is inhaled
Go out, discard, add PBS, mixing, centrifugal 1000 turns/5 minutes, finally it is repeated once.Last sample loading gun sucking-off supernatant of using again, then
Appropriate frozen stock solution (frozen stock solution=5ml serum+4mlDMEM+1mlDMSO, reverse mixing are filtered standby) is added in cell, mixed
Even.It is eventually adding in cryopreservation tube, often pipe 1ml Cell sap.Put into freezing storing box, first put-80 DEG C overnight, then cell strain B10 is put into
Liquid nitrogen is medium-term and long-term to be saved backup.
Embodiment 2
The application in western blot technology of the anti-giant panda FSH β subunit monoclonal antibody.
1. the extraction of the tissue total protein of mesh
Application Protein Extraction Reagent kit: C510004-0020, Sangon Biotech (Shanghai) Co., Ltd. provides, carries
Take adult giant panda testis and ovary tissue total protein, and by BCA method, total protein concentration is measured.
2 glues: preparation polyacrylamide gel, concentrate glue 5%, separation gel 12%.
4. sample preparation: albumen applied sample amount is 80ug
5. electrophoresis: concentrate glue 80V, 30min；Separation gel 120V, 90min.
6. transferring film: 250mA, 40min.
7. close: 5% skimmed milk, 37 DEG C of slow concussion 2h.
8. hatching one to resist: hybridoma cell strain 09199 (2), the equal 1:500 of C12 (3) antibody dilute, 4 DEG C slow vibrated
Night, next day 37 DEG C of rewarming 1h.
9. hatch two to resist: 1:8000 dilutes, and hatches 1h for 37 DEG C.
10.ECL exposes.
Application result
1. cellular protein concentration
(1) standard curve
Canonical plotting is as shown in Figure 1.
(2) protein concentration
(3) Western-blot testing result is as shown in Figure 2.
The anti-giant panda FSH β subunit monoclonal antibody of present invention exploitation, can prepare identification target protein FSH, for for FSH
The location of albumen and tissue expression information, and utilize the monoclonal antibody part to fsh receptor of FSH high specific, antagonist,
Anti-antagonist is furtherd investigate, and creates conditions for ligands specific, antagonist, the discovery of anti-antagonist.
Embodiment described above only have expressed the detailed description of the invention of the application, and it describes more concrete and detailed, but also
Therefore the restriction to the application protection domain can not be interpreted as.It should be pointed out that, for those of ordinary skill in the art
For, on the premise of conceiving without departing from technical scheme, it is also possible to make some deformation and improvement, these broadly fall into this
The protection domain of application.
Claims (5)
- The most anti-giant panda FSH β subunit monoclonal antibody hybridoma cell strain 09199, deposit number: CCTCC NO:C2016144.
- Anti-giant panda FSH β subunit monoclonal antibody hybridoma cell strain 09199 the most according to claim 1 is used for producing Anti-giant panda FSH β subunit monoclonal antibody.
- The most anti-giant panda FSH β subunit monoclonal antibody, it is characterised in that being produced by hybridoma cell strain 09199, hypotype is IgG1。
- Anti-giant panda FSH β subunit monoclonal antibody the most according to claim 3 answering in western blot technology With.
- The preparation method of the most anti-giant panda FSH β subunit monoclonal antibody, it is characterised in that step is as follows:(1) design giant panda FSH β distinguished sequence as shown in SEQ ID NO:1, and with KLH coupling, as produce specific antibody Immunogen FSH β-KLH；(2) the immunogen FSH β-KLH immune mouse prepared with step (1)；(3) cell is warm obtains anti-giant panda FSH β subunit monoclonal antibody hybridoma cell strain 09199；(4) amplification culture obtains anti-giant panda FSH β subunit monoclonal antibody.
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| CN110694056A (en) * | 2019-10-25 | 2020-01-17 | 四川农业大学 | FSH antigen, preparation method thereof and FSH vaccine containing antigen |
| CN111321121A (en) * | 2020-03-16 | 2020-06-23 | 成都大熊猫繁育研究基地 | Two-strain novel panda C-reactive protein monoclonal antibody hybridoma cell strain and application thereof |
| CN113980910A (en) * | 2021-11-19 | 2022-01-28 | 成都大熊猫繁育研究基地 | Monoclonal antibody for resisting panda hemoglobin alpha subunit, hybridoma cell strain and application thereof |
| CN114085818A (en) * | 2021-11-19 | 2022-02-25 | 蔡开来 | Anti-panda Thy1 monoclonal antibody, hybridoma cell strain and application thereof |
| CN115521376A (en) * | 2022-04-29 | 2022-12-27 | 王神飞 | Anti-panda FSH (follicle-stimulating hormone) promoting antibody, kit and FSH enzyme-linked immunosorbent assay (ELISA) detection method |
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| CN116813768A (en) * | 2023-08-23 | 2023-09-29 | 成都大熊猫繁育研究基地 | Panda TSH beta monoclonal antibody, hybridoma cell strain and application thereof |
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| CN108823172A (en) * | 2018-07-10 | 2018-11-16 | 成都大熊猫繁育研究基地 | The preparation and application of giant panda luteotropin β subunit monoclonal antibody |
| CN110694056A (en) * | 2019-10-25 | 2020-01-17 | 四川农业大学 | FSH antigen, preparation method thereof and FSH vaccine containing antigen |
| CN110694056B (en) * | 2019-10-25 | 2023-05-05 | 四川农业大学 | FSH antigen, preparation method thereof and FSH vaccine containing FSH antigen |
| CN111321121A (en) * | 2020-03-16 | 2020-06-23 | 成都大熊猫繁育研究基地 | Two-strain novel panda C-reactive protein monoclonal antibody hybridoma cell strain and application thereof |
| CN113980910A (en) * | 2021-11-19 | 2022-01-28 | 成都大熊猫繁育研究基地 | Monoclonal antibody for resisting panda hemoglobin alpha subunit, hybridoma cell strain and application thereof |
| CN114085818A (en) * | 2021-11-19 | 2022-02-25 | 蔡开来 | Anti-panda Thy1 monoclonal antibody, hybridoma cell strain and application thereof |
| CN113980910B (en) * | 2021-11-19 | 2024-02-02 | 成都大熊猫繁育研究基地 | Anti-panda hemoglobin alpha subunit monoclonal antibody, hybridoma cell strain and application thereof |
| CN115521376A (en) * | 2022-04-29 | 2022-12-27 | 王神飞 | Anti-panda FSH (follicle-stimulating hormone) promoting antibody, kit and FSH enzyme-linked immunosorbent assay (ELISA) detection method |
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| CN116375864A (en) * | 2023-05-16 | 2023-07-04 | 成都大熊猫繁育研究基地 | Panda AMH-resistant monoclonal antibody, hybridoma cell strain and application thereof |
| CN116769030A (en) * | 2023-08-21 | 2023-09-19 | 成都大熊猫繁育研究基地 | Panda relaxin 3 monoclonal antibody, hybridoma cell strain and application thereof |
| CN116769030B (en) * | 2023-08-21 | 2023-10-20 | 成都大熊猫繁育研究基地 | Panda relaxin 3 monoclonal antibody, hybridoma cell strain and application thereof |
| CN116813768A (en) * | 2023-08-23 | 2023-09-29 | 成都大熊猫繁育研究基地 | Panda TSH beta monoclonal antibody, hybridoma cell strain and application thereof |
| CN116813768B (en) * | 2023-08-23 | 2023-11-07 | 成都大熊猫繁育研究基地 | Panda TSH beta monoclonal antibody, hybridoma cell strain and application thereof |
| CN116819103A (en) * | 2023-08-28 | 2023-09-29 | 成都大熊猫繁育研究基地 | Panda TSH enzyme-linked immunosorbent assay method and monoclonal antibody |
| CN116819103B (en) * | 2023-08-28 | 2023-11-07 | 成都大熊猫繁育研究基地 | Panda TSH enzyme-linked immunosorbent assay method and monoclonal antibody |
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