CN110229791A - A kind of hybridoma for secreting anti-blue tongue virus NS3 protein monoclonal antibody - Google Patents
A kind of hybridoma for secreting anti-blue tongue virus NS3 protein monoclonal antibody Download PDFInfo
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- CN110229791A CN110229791A CN201810180525.6A CN201810180525A CN110229791A CN 110229791 A CN110229791 A CN 110229791A CN 201810180525 A CN201810180525 A CN 201810180525A CN 110229791 A CN110229791 A CN 110229791A
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- blue tongue
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/14—Reoviridae, e.g. rotavirus, bluetongue virus, Colorado tick fever virus
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Abstract
The application belongs to field of biotechnology, relates to, but are not limited to a kind of hybridoma for secreting anti-blue tongue virus NS3 protein monoclonal antibody.The application obtains the hybridoma cell strain that can secrete the monoclonal antibody for having group specificity to blue tongue virus and the monoclonal antibody secreted by it using blue tongue disease serum 1 type virus as immunogene.The monoclonal antibody that the application generates can replace rabbit source blue tongue virus antibody and blue tongue virus NS3 protein binding.
Description
Technical field
The application belongs to field of biotechnology, more particularly to a kind of hybridization for secreting anti-monoclonal antibody of bluetongue virus (BTV)
Oncocyte and the monoclonal antibody secreted by it.
Background technique
Blue tongue disease is caused by blue tongue virus infection, viral by the ruminant of insect (mainly Storehouse midge) propagation
Infectious disease.Blue tongue virus belongs to Reoviridae, Orbivirus blue tongue virus subgroup.
Blue tongue disease was most found earlier than 1876 in South Africa, after its infection animal, the oral mucosa of animal and tongue meeting
Become blue, so it was named as " blue tongue disease " in 1906.Before 1940, blue tongue disease is only propagated in Africa.However,
1948, the U.S. also reported this disease.China found blue tongue disease in Yunnan Province Shizong County in 1979 for the first time, and isolated
Blue tongue virus.
The monoclonal antibody of anti-blue tongue virus is tool very handy in blue tongue disease diagnosis.However, blue tongue virus
Having 24 serotypes, (recent years is external and finds 3 new serotypes, but the specific research to this 3 new serotypes successively
Just start to walk), and common monoclonal antibody is only sensitive to a kind of or at most several blue tongue virus serotypes, therefore is difficult
Meet the research needs of the group specificity diagnosis aspect of blue tongue disease.It has been reported that the blue tongue virus Dan Ke with group specificity
Acquisition is immunized not by with natural viral in grand antibody, but prepared after the protein immunization by manually expressing.This
Kind of monoclonal antibody and by cannot be completely equivalent with the immune monoclonal antibody prepared of natural viral.
Therefore, this field needs to have blue tongue virus group specificity, by being prepared with natural viral is immune
Monoclonal antibody.
Summary of the invention
The purpose of the application be obtain can secrete to blue tongue virus have group specificity and to its NS3 albumen it is quick
The hybridoma of the monoclonal antibody of sense.
The NS3 albumen of blue tongue virus (BTV) is encoded by the S10 gene of blue tongue virus, and is highly conserved.
NS3 albumen includes NS3 albumen and NS3A albumen, and the sequence of amino acid of both albumen of same serotype is identical, only
The latter has lacked 13 amino acid.If the monoclonal antibody sensitive to NS3 albumen can be obtained, the monoclonal antibody is exhausted
It is in most cases all sensitive to the two albumen, and be likely to group specificity.
This application provides the hybridoma that can secrete anti-blue tongue virus NS3 protein monoclonal antibody, Yi Jiyou
The anti-blue tongue virus NS3 protein monoclonal antibody of hybridoma secretion.
Particularly, the application provides a kind of hybridoma, the Dan Ke of the hybridoma secretion in first aspect
Grand antibody has group specificity to blue tongue virus.In this application, the monoclonal antibody of the hybridoma secretion is to indigo plant
Glossopathy 1-24 serotype has group specificity.In this application, the hybridoma is by utilizing blue tongue disease serum 1 type
Virus is prepared as immunogene.In this application, the deposit number of the hybridoma is CCTCC:C2014202.
The application provides a kind of monoclonal antibody in second aspect, and it includes the hybridoma secretions by the application
Monoclonal antibody.
The application is provided in the third aspect by the anti-blue tongue virus NS3 albumen list of the hybridoma secretion of the application
Clonal antibody.In this application, the monoclonal antibody can replace rabbit source blue tongue virus antibody and blue tongue virus NS3 egg
White combination.
The application provides kit of the monoclonal antibody of the application in preparation for blue tongue disease diagnosis in fourth aspect
In purposes.
Compared with prior art, it is derived from by the monoclonal antibody that the hybridoma of the application is secreted with natural blue tongue disease
Virion is immunized and has excellent group specificity to blue tongue virus.Since evidence show the diseases manually expressed
The conformation of toxalbumin and the conformation of native viral protein are identical, therefore, the immune generation of the virus protein manually expressed
Antibody certainly will not be identical with the antibody that generation is immunized with native viral protein.It is secreted due to the hybridoma of the application
Monoclonal antibody is obtained using blue tongue disease natural viral particle, so the combination of itself and blue tongue virus is closer to natural shape
State.
Other features and advantage will illustrate in the description that follows, also, partly become from these descriptions
It obtains obviously, or be appreciated that and implementing the application.The other purposes and feature of the application can be by wanting in specification, right
Specifically noted structure is sought in book and attached drawing to realize and obtain.
Detailed description of the invention
Attached drawing is only used to provide to further understand technical scheme, and constitutes part of specification, with
Embodiments herein is used to explain the technical solution of the application together, without constituting the limitation to technical scheme.
Fig. 1 is the monoclonal antibody by the hybridoma secretion of the application to the special of blue tongue disease 1-24 serotype
Property measurement result.Wherein, " yin to " refers to negative control, i.e., using the serum for being free of blue tongue virus antibody." sun to " refers to the positive
Control, that is, use the serum containing blue tongue virus polyclonal antibody.Wherein number 1-24 respectively represents blue tongue disease 1-24 serum
Type virus.
Fig. 2 is the monoclonal antibody by the hybridoma secretion of the application to peripheral Ah card's pinta poison (AKAV), ox
The specific detection result of epizootic fever virus (BEFV), middle mountain viral (CHUV).Wherein " AKAV " is represented to Ah card's pinta poison
Detection；" BEFV " represents the detection to bovine epizootic fever virus；" CHUV " represents the detection of centering mountain virus；"AKAV+"
Represent the positive control of A Ka spot antiviral antibody；" BEFV+ " represents the positive control of bovine epizootic fever virus antibody；"CHUV+"
The positive control of mountain antiviral antibody in representative；" yin to " represents negative control, i.e., using it is corresponding respectively without for AKAV,
The negative serum of the antibody of BEFV or CHUV.
Fig. 3 is the testing result that Western Blot is carried out to the anti-monoclonal antibody of bluetongue virus (BTV) of the application.Wherein
" BTV " refers to blue tongue virus；" cell " refers to that bhk cell compares；" M " refers to molecular weight of albumen marker.
Specific embodiment
To keep the purposes, technical schemes and advantages of the application clearer, below in conjunction with drawings and examples to this Shen
It please be described in detail.It should be noted that in the absence of conflict, the feature in the embodiment of the present application can be mutually any
Combination.
The application is carried out 3 times using BALB/c female mice of the highly purified blue tongue disease serum 1 type virus to 4-6 week old
It is immune, extract bone-marrow-derived lymphocyte in the spleen for the mouse being immunized, by its with it is thin with the processed mouse myeloma of 8 azaguanines
Born of the same parents SP2/0 is merged, and carries out scoreboard culture with HAT selective medium.After hybridoma cell clone is formed, pass through
ELISA method detects blue tongue virus antibody.Periodic replacement culture medium and the antibody titer for detecting each clone, are selected
It is capable of each hybridoma cell clone of stably excreting antibody, and is cloned again respectively, while continuing to supervise with ELISA method
Survey antibody-secreting situation.All hybridomas for capableing of continuous release bluetongue antibody are all finally subjected to expansion training respectively
It supports, then freezes and collected correspondingly as the monoclonal antibody secreted by them.Needle is carried out to each monoclonal antibody
To the specific detection of blue tongue disease 1-24 serotype.To all have binding specificity to blue tongue disease 1-24 serotype
Hybridoma cell strain corresponding to monoclonal antibody carries out separate marking, and carries out the other correlations in periphery to these monoclonal antibodies
The specific detection of virus.When obtained monoclonal antibody is all sensitive and viral not to periphery to 24 kinds of blue tongue disease serotypes
When sensitive, Western Blot detection is carried out.If detecting the band of 25KD-26KD, illustrate the monoclonal antibody to blue tongue
Sick virus NS albumen is sensitive, which is desired monoclonal antibody, generates the hybridoma of the monoclonal antibody
It is then desired hybridoma.
Desired hybridoma provided by the present application has been preserved in China typical culture collection center (CCTCC),
Address: Wuhan University, Wuhan, China city, cell name: HYBASVIGL-1；Deposit number: C2014202；Preservation date: 2015 1
The moon 18.
Embodiment
The hybridoma of anti-blue tongue virus NS3 protein monoclonal antibody is secreted in preparation
Following reagent and material are common commercial goods unless otherwise specified.
One, experimental material
1, reagent:
The blue tongue disease serum 1 type virus Yunnan strain of purifying, our unit laboratory is isolated and saves；
1 × RPMI1640 culture solution (article No.: SH30809.01B), HyClone product；
Fetal calf serum (article No.: 10099-141), GIBCO product；
Guanozola (article No.: 1001510707), HAT (H-hypoxanthine (hypoxanthine), A-
, HT (H- aminopterin (aminopterin), T-thymidine (thymidine), article No.: 1002424466)
, 50%PEG hypoxanthin (hypoxanthine), T-thymidine (thymidine), article No.: 1001506328)
(article No.: P7181) is SIGMA product；
0.85% ammonium chloride solution, 75% alcohol, RPMI1640 complete culture solution (that is, containing 10% fetal calf serum 1 ×
RPMI1640 culture solution), acquisition is prepared by a conventional method；
Western wet process transferring film liquid (article No.: B400020), SDS-page electrophoretic buffer (article No.: B300020),
For Proteintech product, by specification is prepared；
Sheep anti mouse HRP ELIAS secondary antibody (article No.: HS201), Quan Shi King Company product；Goat-anti rabbit HRP ELIAS secondary antibody (article No.:
A00098), GenScript product；
Peripheral virus BEFV, AKAV and CHUV, BEFV provide finished product kit, AKAV and CHUV by Lanzhou veterinary institute
It is separated and is saved by this laboratory, and finished product kit is provided；
3,3', 5,5'- tetramethyl benzidine (TMB) substrate solution, PBS buffer solution (0.01M, pH7.2-7.4), PBST drift
Wash buffer (0.01M, pH7.2-7.4), terminate liquid (2M sulfuric acid), all in accordance with People's Medical Officer Press's March the 1st edition in 2000
The method preparation of " immunology common experimental method " the 356-358 pages introduction；
TMB one-component HRP microporous substrate (TMB One Component HRP Microwell Substrate, article No.:
TMBW-1000-01), U.S. SurModics product；Cell pyrolysis liquid (article No.: P00131), 5 × SDS sample solution (article No.:
P0015), BeyoECL Plus chemiluminescence detection kit (article No.: P0018) is green skies product；Protease inhibits
Agent, Bimake product；Dehydrated alcohol, western Gansu Province product.
2, consumptive material: T25 culture bottle, Corning product；2.5ml syringe；50ml centrifuge tube；Stainless steel cell screen clothes；96
Porocyte culture plates (Haimen Boyang)；Eye scissors；Ophthalmic tweezers；Scalpel；Cell counting board；0.45 micron membranes (goods of PVDF
Number: IPVH00010), Millipore product；Elisa plate, Costar product；Preservative film etc..
3, cell: murine myeloma cell SP2/0 is purchased from Kunming cell bank.
4, experimental animal: the BABL/c female mice of 4-6 week old is purchased from Kunming Medical University's Experimental Animal Center.
5, equipment: Biohazard Safety Equipment, carbon dioxide incubator, inverted microscope, centrifuge, water-bath, cell counter
(Roche Cedex XS), microplate reader, electrophoresis apparatus, chemiluminescence imaging instrument (diligent Xiang ChemiScope 3300mini) etc..
Two, animal is immunized
1, the BABL/c female mice for selecting 3 4-6 week old, to carrying out 75% alcohol disinfecting under its abdomen；
2, blue tongue disease serum 1 type purified virus (BTV-1) is diluted with PBS buffer solution, about 50 μ g/ml of final concentration, with every
It is immune that mouse 0.4ml carries out intraperitoneal injection to the above mouse, is spaced booster immunization 1 time again in 2 weeks；
3, reinforced immunological 1 time again in 3 days before cell fusion.
Three, the selection culture of murine myeloma cell (SP2/0)
1, SP2/0 cell that 90% or more converges will be grown in T25 culture bottle with containing guanozola
The RPMI1640 complete culture solution operation instruction of guanozola (preparation method referring to), routinely cultural method secondary culture
1 week, then using the culture of RPMI1640 complete culture solution 2 weeks without guanozola；
2, it merges cell to be assigned to 4 T25 culture bottles in preceding 3 days and continue to cultivate to 90% or more and converge.
Four, the preparation of mouse spleen lymphocyte
1,3 days after the 2nd reinforced immunological, 3 mouse are put to death through cervical dislocation, after alcohol disinfecting, in ultra-clean work
Sterile taking-up spleen in platform；
2, the culture dish of 1 diameter 9cm is taken, is put into 1 stainless steel cell screen clothes thereto, 3 spleens are placed on plate
It is careful to reject fat and other connective tissues in interior stainless steel cell screen clothes；
3,1 × RPMI1640 culture solution of 5ml pre-cooling is added in the spleen in stainless steel cell screen clothes, with scalpel and eye
The cooperation of section's tweezer splits each spleen along the longitudinal axis, flows into cell liquid in the culture dish below stainless steel cell screen clothes, then with 1 ×
RPMI1640 culture solution rinses spleen several times, is flushed the lymphocyte inside each spleen fully
Come, then discards spleen；
4, the cell liquid that above step 3 obtains is transferred to 50ml centrifuge tube, is settled to 1 × RPMI1640 culture solution
30ml is centrifuged 5min in 1000RPM, abandons supernatant；
5,0.85% ammonium chloride solution of 5ml pre-cooling is added into the cell of precipitating, and ice bath places 5min, with dissolution
Then red blood cell is added 15ml RPMI1640 complete culture solution and terminates dissolution；
6, cell is dispelled with suction pipe, is centrifuged 10min then at 1000RPM revolving speed, abandon supernatant, it is complete with 10ml RPMI1640
Full nutrient solution suspension cell；
7, counting and vitality test are carried out to the cell suspension with cell counter, and carries out volume adjustment appropriate, really
It is about 1.0 × 10 that guarantor, which finally obtains sum,8The suspension of a lymphocyte living.
Five, the collection of murine myeloma cell
1, when the cell of step 3 grows to 90% or more and converges, the cell is made with 1 × RPMI1640 culture solution outstanding
Liquid, and it is centrifuged 5min in 1000RPM, abandon supernatant；
2, cell is suspended with 10ml RPMI1640 complete culture solution, and carries out counting and vitality test；
3, when cell survival rate reaches >=90%, according to count results, about 2 × 10 are drawn7A cell move into 50ml from
Heart pipe is spare.
Six, cell fusion
1, hybridoma HAT culture solution (the preparation side that 40ml exempts from feeder cells is prepared with 1 × RPMI1640 culture solution
Method is shown in HAT reagent specification and patent document CN201611218063X), with 1 × RPMI1640 of 15ml culture solution and 1.0ml
50%PEG is put into pre-temperature in 37 DEG C of water-baths together；
2, the resulting splenic lymphocytes of aspiration step four be added the ready 50ml with myeloma cell of step 5 from
Heart pipe, and be uniformly mixed；
3, mixed cell is centrifuged 10min at 1,000 rpm, then carefully outwelled and the supernatant that exhausts；
4, centrifuge tube is put into 37 DEG C of water, after the 50%PEG that 1ml pre-temperature is drawn with suction pipe, tip is inserted into centrifuge tube
Bottom stirs while adding in 1min, so that cell is become uniform suspension, is then allowed to stand 2-4min；
5, it is slowly added into 1 × RPMI1640 culture solution of 15ml pre-temperature, terminates PEG reaction, and be gently agitated for cell；
6, it adds 1 × RPMI1640 culture solution to mix to 40ml, 1000RPM is centrifuged 10min, abandons supernatant；
7, the HAT culture solution that 40ml step 1 prepares is added, is added in 4 piece of 96 porocyte culture plates after mixing, often
Hole 0.1ml is subsequently placed in 37 DEG C, 5%CO2It is cultivated under saturated humidity environment.
Seven, the selectivity culture of fused cell
1,4 piece of 96 orifice plate prepared by step 6 is persistently cultivated 7 days；
2, complete culture solution containing HAT is stopped using after 7 days, is changed to that (preparation method is shown in that HT reagent is said containing 1 × HT culture solution
Bright book and patent document CN201611218063X)；
3, it is changed to RPMI1640 complete culture solution after 7 days, continues culture until being visually able to observe that hybridoma colonies.
Eight, the detection of specific antibody.
1,50 μ l culture solutions are drawn out of each plate hole with Growth of Hybridoma Cell, 1.5 ml centrifuge tubes are added, make
For measuring samples；
2, all measuring samples (" are exempted from March the 1st edition in 2000 by indirect ELISA method referring to People's Medical Officer Press
Epidemiology common experimental method ", the 352-355 pages) titre of the anti-blue tongue virus antibody of detection, and it is right with the positive to set negative control
According to.Negative control negative serum, the working solution of the polyclonal antibody of positive control blue tongue virus.
Nine, clone's culture of hybridoma
1, the hole for selecting value >=0.5 OD450 through detecting anti-blue tongue virus antibody concentration, will be therein thin with pipettor
Born of the same parents dispel, and 100 μ l cell suspensions of absorption are added in RPMI1640 complete culture solution of the 900 μ l containing 1 × HT and are uniformly mixed, and then count
Number；
It 2, is 3- with the RPMI1640 complete culture solution preparation concentration containing hybridoma containing 1 × HT according to count results
The suspension 10ml of 10/ml；
3, suspension is added in 1 piece of 96 well culture plate, every 100 μ l of hole is placed in 37 DEG C, 5%CO2, in saturated humidity environment
Culture；
4, it when the diameter of cell colony is 1-2mm, draws part supernatant and carries out antibody test, and calculate positive rate；
5, the highest hole of antibody titer is marked, and the cell selection in monoclonal growth is come out, carried out simultaneously
Expand culture and clones again；
6, HT is not added in the 2nd time cloning and later clone, carries out anti-blue tongue virus antibody test in late stage of culture, and
Positive rate is calculated, stops clone when positive rate is 100%；
7, the hybridoma that the above step 6 filters out is expanded culture, and freezes backup.The hybridoma
Culture solution collects spare.
Ten, the hybridoma of screening secretion blue tongue disease serum 1-24 type viral monoclonal antibodies
1, the blue tongue virus Dan Ke that will be separated from the culture solution of the culture that above step nine obtains hybridoma
Grand antibody carries out the specific detection to blue tongue disease 1-24 serotype, while carrying out to the viral (bovine epizootic fever in 3 kinds of peripheries
Poison, BEFV；Ah card's pinta poison, AKAV；Middle mountain virus, CHUV) specific detection.Method be summarized as follows (detailed step referring to
People's Medical Officer Press's March the 1st edition in 2000 " immunology common experimental method ", the 357-358 pages): (1) use sheep source indigo plant tongue
Sick polyclonal antibody is coated with elisa plate, and 24 serotype one-to-one correspondence of blue tongue disease are incorporated on elisa plate；(2) simultaneously
3 kinds of periphery viruses are also coated on elisa plate；(3) to all other than the hole in the hole of positive control and negative control
Monoclonal antibody stoste to be checked is added in the hole of envelope antigen；(4) hole of heliotropism control and the hole of negative control are added and design
Corresponding positive control and negative control；(5) sheep anti mouse HRP ELIAS secondary antibody work is added in Xiang Suoyou detection zone and negative control area hole
Make liquid, heliotropism compares plate hole and corresponding HRP ELIAS secondary antibody working solution is added；(6) TMB one-component HPR micropore bottom is added
Object developing solution colour developing 5 to 10min；(7) terminate liquid is added；(8) microplate reader read plate under 450nm wavelength is used, and records knot
Fruit, i.e. OD450 value；(9) result is handled and is analyzed.
Note that every step requires reaction 1h, then board-washing for abovementioned steps (1)-(4)；For step (5), reaction
0.5h, then board-washing at least 5 times.Wherein, if positive control is all source of mouse, sheep anti mouse HRP ELIAS secondary antibody is all used, it is no
Then, corresponding HRP ELIAS secondary antibody is separately added into.
This experiment finally obtains 1 strain of hybridoma, and the monoclonal antibody of secretion is through ELISA detection and blue tongue disease 1-24
The absorbance (OD450 value) of the reaction of serotype is shown in Table 1, ties with the absorbance (OD450 value) of 3 kinds of periphery viruses reacted
Fruit is shown in Table 2:
Table 1: reactivity of the anti-monoclonal antibody of bluetongue virus (BTV) of the application to blue tongue disease 1-24 serotype
| Serotype | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 |
| OD450 | 0.77 | 0.91 | 0.62 | 0.60 | 0.65 | 0.67 | 1.25 | 0.60 | 0.72 | 0.73 | 0.91 | 0.72 | 1.23 |
| Serotype | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | Sun is right | Yin is right |
| OD450 | 0.93 | 1.24 | 1.48 | 0.91 | 0.72 | 1.20 | 1.43 | 0.75 | 1.15 | 0.65 | 0.59 | 1.00 | 0.20 |
Detection data is the average value of 2 parallel samples." sun to " represents positive control, and " yin to " represents negative control.
From in table 1 and Fig. 1 as can be seen that the list that is separated from the culture solution of the culture that step 9 obtains hybridoma
Clonal antibody, all >=0.59 to the OD450 value of the ELISA of blue tongue disease 1-24 serotype reaction, up to 1.48, it is positive
Control is 1.0, and negative control is 0.15.Thus, it will be seen that the culture obtained from step 9 hybridoma culture
The anti-monoclonal antibody of bluetongue virus (BTV) separated in liquid all exists with 24 serotypes of blue tongue disease to react, i.e., obtained by the application
The monoclonal antibody arrived has group specificity to blue tongue virus.
Table 2: reactivity of the anti-monoclonal antibody of bluetongue virus (BTV) of the application to 3 kinds of periphery virus
| Test item | AKAV | BEFV | CHUV | AKAV+ | BEFV+ | CHUV+ | Yin is right |
| OD450 | 0.07 | 0.10 | 0.07 | 1.01 | 1.85 | 1.21 | 0.06 |
" AKAV " is Ah card's spot viral antigen, and " AKAV+ " represents the control of A Ka spot virus antibody positive；
" BEFV " is bovine epizootic fever virus antigen, and " BEFV+ " represents the control of bovine epizootic fever virus antibody positive；
" CHUV " is middle mountain viral antigen, virus antibody positive control in mountain in " CHUV+ " representative；
Detection data is the average value of 2 parallel samples." yin to " represents negative control, is here all negative controls
Average value.
Can be seen that the application obtains from table 2 and Fig. 2 there is group specificity to resist blue tongue disease 1-24 serotype
The OD450 value that monoclonal antibody of bluetongue virus (BTV) is reacted with AKAV, BEFV and CHUV is very low, only 0.07-0.10.And these are peripheral
The corresponding polyclonal antibody (i.e. the positive control of the positive control of AKAV, the positive control of BEFV and CHUV) of virus though through
10,000 times of dilution is crossed, OD450 value is still very high, is 1.01 to 1.85.Blue tongue virus (BTV) antibody, AKAV antibody,
BEFV antibody and CHUV antibody are all negative serum, i.e. negative control, and OD450 value is very low, and average 0.06.It can be seen that the present embodiment
Obtained monoclonal antibody does not react to get the anti-monoclonal antibody of bluetongue virus (BTV) arrived to above-mentioned 3 kinds of peripheries peripheral virus
Virus is insensitive.
2, cultivating as a result, this hybridoma cell strain is expanded according to previous step.Method is following (with the passage of T25 culture bottle
For): (1) culture solution that the hybridoma that 90% or more converges is grown in culture bottle is poured out preservation；(2) prepare 3
It is T25 bottles an equal amount of；(3) it inhales 6ml RPMI1640 complete culture solution with suction pipe the hybridoma in T25 bottles suspends
Come, and is fifty-fifty respectively added in 3 T25 bottles；(4) with RPMI1640 complete culture solution by each T25 bottles add to
6ml；(5) 37 DEG C of cultures are put after covering tightly 3 T25 culture bottles equipped with cell suspension；(6) cell grows to 90% or more
After converging (about 48h), method it can continue to pass on according to this, until that can be used to freeze.
3, (specific method is shown in Science Press to this hybridoma liquid nitrogen Long-term Cryopreservation after will be enlarged by culture
2 months 2001 versions " philosophy and technique of in vitro culture " page 948, hybridoma freezes), while the monoclonal that will be obtained
Antibody stoste adds the thimerosal of a ten thousandth to make preservative, and packing saves backup.
11, the Western Blot identification of monoclonal antibody
In order to identify the monoclonal antibody of anti-blue tongue virus obtained has affinity to which kind of albumen of BTV, I
Carried out Western Blot experiment.
1, SDS-page electrophoresis
(1) prepare cell controls.A, prepare the bhk cell of 1 bottle of T25, cell grows at least 50% and converges in bottle；B, will
It under cell scraper, and is put into togerther in 15ml centrifuge tube together with culture solution, 2000RPM is centrifuged 10min；C, supernatant is abandoned, to centrifuge tube
100 μ l cell pyrolysis liquids (protease inhibitors containing 1%) of interior addition, fully shake rear ice bath 15min；D, centrifuge tube is taken out,
12,000RPM is centrifuged 10min；E, supernatant is carefully sucked out spare, this is bhk cell pyrolysis product.
(2) the bhk cell pyrolysis product of the unpurified blue tongue disease serum 1 type virus liquid of 40 μ l and 40 μ l is separately added into 10
μ 5 × SDS of l sample solution (green skies Products), is transferred in 1.5ml centrifuge tube respectively and is sufficiently mixed and seals, then distinguish
It is spare that taking-up cooling after boiling 10min is put into 98 DEG C of water；
(3) 10% polyacrylamide precast glue (Yi Sheng biotech firm product) is installed and is put into Vertial electrophorestic tank, is added
About 3mm above SDS-page electrophoresis liquid to gel, pulls out comb；
(4) microsyringe is used, by the ready blue tongue disease serum 1 type virus SDS lysate of step (2) and bhk cell
Lysate control is added in sample holes, every 10 μ l of hole, and last 1 hole adds 10 μ l molecular weight of albumen markers, and (green skies company produces
Product, article No.: P0076).
(5) 20min electrophoresis first is carried out with 100V DC constant voltage, then carries out 35min electrophoresis with 120V DC constant voltage.
2, transferring film
(1) by the suitable pvdf membrane of size, good front is marked, puts and impregnates 3min in dehydrated alcohol；
(2) gel after electrophoresis is carefully taken out, is put into the container for filling transferring film liquid；
(3) by outer suede, sponge, filter paper, filter paper, gel, nitrocellulose filter, filter paper, filter paper, sponge, outer in container
The sequence of suede arranges transferring film complete unit, is put into fixed clamp device, avoids generating bubble, wherein the front of pvdf membrane, i.e. egg
White transfer surface is bonded gel.
(4) it puts fixing clamp into electrophoresis tank, fills it up with transferring film liquid, 100V DC constant voltage 2h.
(5) after transferring film, film is carefully taken out, at this moment, the molecular weight of albumen marker on the visible gel of naked eyes is complete
In total transfer to film；
(6) clean about 5.5cm × 8.6cm size is taken, the square container of deep about 2.0cm (hereafter claims rectangular appearance
Device), this film is placed on the inside and is simply rinsed with PBST, is then immersed in the confining liquid of valve bag packaging (comprising 5% skimmed milk power
PBST in) overnight in 4 DEG C.
3, it is immunoreacted
(1) another square container is taken to put on shaking table, film 3 times closed with PBST rinsing, each 10min, side is gently
Side rinsing is shaken, avoids throwing away liquid；
(2) another square container is taken, injection about 3ml is resisted by the monoclonal that the hybridoma prepared in step 10 obtains
Body fluid, the residual liquid on film after blotting rinsing with filter paper, keeps blue tongue virus albumen face-down, is immersed in monoclonal antibody
In liquid, and bubble is removed, put shaking table room temperature jog 2h, then rinsed again by step (1) method；
(3) a square container separately is taken, after the dilution of sheep anti mouse HRP ELIAS secondary antibody by specification, 3ml is taken to inject the party
Describe device, the residual liquid on film after blotting rinsing with filter paper keeps blue tongue virus albumen face-down, is immersed in above-mentioned preparation
In good sheep anti mouse HRP ELIAS secondary antibody working solution, bubble removing is removed, shaking table room temperature jog 1h is put, then again by step (1) method
Rinsing；
(4) take BeyoECL Plus chemiluminescence detection reagent (green skies Products) A liquid and each 1ml of B liquid sufficiently mixed
It closes；
(5) film after rinsing is face-up placed on appropriately sized preservative film, draws 1-2ml step (4) preparation
BeyoECL Plus chemiluminescence detection reagent simultaneously equably drenches on the film, then face-up turns film at an appropriate location
Enter in chemiluminescence imaging instrument；
(6) starting chemiluminescence imaging instrument take pictures and composograph, then saves image.
Fig. 3 is to be carried out with monoclonal antibody of the blue tongue disease serum 1 type virus to the hybridoma secretion by the application
The result of the detection of Western Blot.From figure 3, it can be seen that the monoclonal antibody that the application obtains is to blue tongue virus
NS3 and NS3A protein fragments (molecular size range is 25 KD-26KD) are special.
12, the characteristic research for the monoclonal antibody that the application obtains
By further experiment, present inventor has found that the monoclonal antibody that the application as above obtains can replace rabbit source
Blue tongue virus polyclonal antibody is in conjunction with bluetongue viral antigen.Experimental method and result are as follows:
1, experimental method:
(1) the rabbit source polyclonal antibody of blue tongue virus is prepared.Using multiple spot subcutaneous inoculation method, with the blue tongue disease blood of purifying
Clear 1 type virus immunity experimental rabbit, preparing immune serum, (specific method is shown in People's Medical Officer Press's version in 2000, and " immunology is common
Experimental method " the 18-22 pages), and 0.5 μ l immune serum is taken, it is spare with 1000 times of PBST dilution；
(2) it after diluting the blue tongue disease serum 1 type of preliminary purification, 16 type virus hybrid antigens with coating buffer, is coated in
On elisa plate (specific method is shown in the version of People's Medical Officer Press 2000 " immunology common experimental method " page 353), after closing
It is spare；
(3) 100 times of PBST liquid dilution of the monoclonal antibody for obtaining the application as described above, it is spare；
(4) 1 is taken to use the coated elisa plate item of bluetongue viral antigen as described in step (2), in the hole A, B, C, D
The rabbit source polyclonal antibody of the blue tongue virus of 50 μ l steps (1) preparation is sequentially added, 50 μ l PBS liquid, H is respectively added in the hole E, F, G
The monoclonal antibody that 50 μ l steps (3) prepare is added in the hole of item, then closes the lid, and is put into 37 DEG C of incubator reaction 1h；
(5) it will be taken out with top bars, be dried after being rinsed 3 times with PBST liquid；
(6) the 50 μ l of monoclonal antibody that step (3) preparation is sequentially added in the hole A, B, C, D, then closes the lid, is put into 37
DEG C, nearly saturated humidity incubator react 20min；
(7) lath is taken out, is first gently shaken up, then one by one by the liquid of above step middle addition in the hole A, B, C, D
It draws out.Wherein, it will move in the hole E, will be moved in the hole F from the liquid drawn in the hole D, the hole A, B from the liquid drawn out in the hole C
The liquid of removal, which is retained, is used for subsequent use.Then 50 μ l PBS liquid are added in the hole A, B, C, D, G, H, closes the lid, is put into
Incubator reacts 1h；
(8) it after 1h, will be taken out with top bars, be dried after washing 3 times with PBST liquid；
(9) 50 μ l sheep anti mouse HRP ELIAS secondary antibody working solutions are separately added into the hole A, B, H, the hole C, D, E, F is separately added into
50 μ l goat-anti rabbit HRP ELIAS secondary antibody working solutions, the hole G are added each 25 μ l of both HRP ELIAS secondary antibody working solutions, then close the lid,
It is put into 37 DEG C of incubator reaction 30min；
(10) it will be taken out with top bars, be dried after washing 6 times with PBST liquid；
(11) 50 μ l TMB one-component HRP microporous substrates, color development at room temperature 5min are separately added into all holes；
(12) it is separately added into 50 μ l terminate liquids in the hole Xiang Suoyou, then reads data at 450nm wavelength using microplate reader.
2, experimental result and analysis:
It is detected through microplate reader, the porose reading of institute is as follows:
Table 3: each plate hole ELISA reacts OD450 value summary sheet
| Plate hole | A | B | C | D | E | F | G | H |
| OD450 | 1.39 | 1.22 | 1.43 | 1.42 | 0.88 | 0.76 | 0.07 | 1.61 |
By experimentation as above it is found that initial in experiment, all holes are all by blue tongue disease antigen coat, wherein A, B, C, D
Bluetongue viral antigen in four holes first in conjunction with the blue tongue virus polyclonal antibody of rabbit source, then just contacts the list of the application
Clonal antibody.
Theoretically, the monoclonal antibody of the application has no chance to combine with bluetongue viral antigen, that is to say, that A,
The OD450 value in the hole B should be very low, similar to OD450 value (0.07) in the hole negative control G；C, the OD450 value in the hole D should be very
Height, it is similar to OD450 value (1.61) in the hole positive control H.
However, as shown in table 3, the OD450 value in the hole of A, B item is also very high, this has illustrated the monoclonal antibody of the application
Through in conjunction with bluetongue viral antigen.
In addition, the reaction solution in the hole E, F detected is respectively from the hole C, D, and do not added directly from beginning to end
Rabbit source blue tongue virus polyclonal antibody.However, as shown in table 3, the HRP ELIAS secondary antibody of goat-anti rabbit can be with the hole E, F
In conjunction with, and react and obtain 0.76,0.88 the two higher OD450 values.This illustrates there is trip in the reaction solution from the hole C, D
From rabbit source blue tongue virus antibody.It follows that in the hole C, D, the rabbit source blue tongue disease that is initially combined with blue tongue virus
Viral polyclonal antibody has replaced monoclonal antibody of the part by the application, and after substitution, substituted rabbit source blue tongue disease
Viral polyclonal antibody returns in reaction solution again, is in free state.When the reaction solution in this two hole is shifted respectively to the hole E, F
Afterwards, the rabbit source blue tongue virus polyclonal antibody in reaction solution is combined with the blue tongue disease antigen generation of coating onboard again.
By analyzing above, we are tentatively concluded that the monoclonal antibody of the application has and replace rabbit source indigo plant tongue
Ability of the virus polyclonal antibody in conjunction with bluetongue viral antigen.
The available good explanation of data shown in the above table 3 as a result:
A, the average OD450 value in the hole B is higher, is about 1.31, but more slightly lower than the OD450 value 1.61 in the hole positive control H, this
It is just to resist later with the monoclonal of the application since bluetongue viral antigen is first in conjunction with the blue tongue virus polyclonal antibody of rabbit source
Body combines, and therefore, the OD450 value in the hole positive control H only combined than the monoclonal antibody of the application is slightly lower；
C, the average OD450 value in the hole D is higher, is about 1.43, is significantly higher than the OD450 value in the hole E, F, this is because the hole C, D
In bluetongue viral antigen initially combines is rabbit source blue tongue virus polyclonal antibody, although later in conjunction with rabbit source indigo plant tongue
Virus polyclonal antibody is partly replaced by the monoclonal antibody of the application, but most of rabbit source blue tongue virus Anti-TNF-α
Body is still kept in conjunction with bluetongue viral antigen, so average OD450 value is still very high；
E, the rabbit source blue tongue virus polyclonal antibody in two hole F is to be taken in two hole C, D by the monoclonal antibody of the application
The antibody that generation gets off, although the amount of this antibody is few, it is that can detecte completely that average OD450 value, which still reaches 0.82,
's；
The OD450 value in the hole G is 0.07, is negative control, any primary antibody was not added in the hole, so having used two
Kind HRP ELIAS secondary antibody participates in reaction, and since they are impossible to combine up, the OD450 value detected remains on very low；
The OD450 value in the hole H is 1.61, is positive control, has highest OD450 value in all holes, this is because should
Bluetongue viral antigen in hole binds directly the monoclonal antibody of the application, interferes without other factors.
To sum up, the monoclonal antibody of the application, which has, replaces rabbit source blue tongue virus polyclonal antibody and blue tongue virus anti-
The ability that original combines.
Although the embodiment that the application is stated is as above, the content only for ease of understanding the application and use
Embodiment is not limited to the application.Technical staff in any the application fields, not departing from, the application institute is old
Under the premise of the spirit and scope stated, any modification can be carried out in the form and details of implementation and is changed, but the application
Scope of patent protection, the range that the appended claims that must still be subject to defines.
Claims (8)
1. the monoclonal antibody of a kind of hybridoma, the hybridoma secretion has group specificity to blue tongue virus.
2. hybridoma according to claim 1, wherein the monoclonal antibody of the hybridoma secretion is to blue tongue
Sick 1-24 serotype has group specificity.
3. hybridoma according to claim 1 or 2, wherein the hybridoma is by utilizing blue tongue disease serum 1
Type virus is prepared as immunogene.
4. the deposit number of hybridoma according to any one of claim 1-3, the hybridoma is
CCTCC:C2014202.
5. a kind of monoclonal antibody, it includes the lists of the secretion of the hybridoma as described according to claim 1 any one of -4
Clonal antibody.
6. a kind of anti-blue tongue virus NS3 albumen of the secretion of the hybridoma as described according to claim 1 any one of -4
Monoclonal antibody.
7. monoclonal antibody according to claim 6, wherein the monoclonal antibody can replace rabbit source blue tongue virus
Antibody and blue tongue virus NS3 protein binding.
8. the monoclonal antibody according to any one of claim 5-7 is in the kit that preparation is diagnosed for blue tongue disease
Purposes.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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