CN104560885B - A kind of monoclonal antibody for resisting natural cattle gamma interferon, secretes hybridoma cell strain and the application of the antibody - Google Patents

Abstract

The invention discloses a kind of monoclonal antibody for resisting natural moggy gamma interferon, hybridoma cell strain and the application of the antibody are secreted.One plant of the present invention can stably excreting resist the hybridoma of natural moggy gamma interferon monoclonal antibody, be named as 3D7, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its culture presevation numbering is：CGMCC No.9329, preservation date are on June 25th, 2014.Mouse is immunized using the carrier for expression of eukaryon of moggy gamma interferon in the present invention, with indirect immunofluorescene assay technology, screening can produce the hybridoma cell strain of the antibody for natural ox IFN γ, successfully solving tradition preparation IFN γ monoclonal antibodies can only react with prokaryotic expression product, but with natural IFN γ anergies the problems such as, new technological means is provided for the detection of natural moggy gamma interferon.

Description

A kind of monoclonal antibody for resisting natural cattle gamma interferon, secretes the hybridoma of the antibody Cell line and application

Technical field

The present invention relates to a kind of monoclonal antibody of anti-cattle gamma interferon, secretes the hybridoma cell strain of the antibody and answers With more particularly to a kind of monoclonal antibody for resisting natural cattle gamma interferon and the hybridoma cell strain for secreting the antibody, further relate to Application of the monoclonal antibody in natural cattle gamma interferon (IFN-γ) is detected in this.The invention belongs to biocytology field.

Background technology

Interferon (IFN) be specific inducer effect under by cell secretion one kind have it is antiviral, antitumor and The glycoprotein of the biological activities such as immunoloregulation function, molecular weight are about 20~100kDa, generally by 150~170 amino acid Composition is higher (Iiaacs etc., 1957) with upper amino acid, wherein aspartic acid, glutamic acid and leucine content containing 17 kinds.Root Can be I, II and III type, 3 type by IFN points according to gene order and the difference of acceptor.I type mainly includes IFN-α, β, also has IFN- ε that found later, ψ, κ, δ, τ, ξ etc., are secreted and are produced after antigenic stimulus by fibroblast and epithelial cell (Sentsui etc., 2001).Interferon type Ⅱ, that is, IFN-γ is formed, and is by the activating immune cells such as antigen, mitogen, bag Include CD⁴⁺Th1、CD⁸⁺Secreted by T cell and NK cells, therefore also known as immune interferon.III type interferon is newfound one Type cytokines, it is in close relations with interferon type Ⅰ, it is known as IFN- λ (Donnelly etc., 2010).Interferon type Ⅰ is mainly shown as Antiviral, antitumor biological activity；And interferon type Ⅱ be mainly shown as immunoregulatory biological activity (Kenji etc., 2001)。

IFN-γ gene contains 3 intrones and 4 extrons, the extron and introne few nucleotide of different animals Amount is different, but belongs to secreted protein, by 5 ' end noncoding regions, signal peptide coding region, polypeptid coding area and 3 ' end non-codings District's groups into.People, pig and ox IFN-γ precursor are 166 amino acid residues, wherein preceding 23 amino acid is signal peptide, maturation protein Size is 143 amino acid.Various animals IFN glycosylation sites number is different, and natural ox IFN-γ is at the 25th and 97 Asparagine there are 2 glycosylation sites (Joseph C, 1998).Although whether IFN-γ glycosylates and has no effect on it and exempting from Biological activity in terms of epidemic disease adjusting, but its half-life period (Thiel etc., 2000) in animal body can be influenced.IFN-γ is usual Dimeric forms are formed, monomer form does not have biological activity.The activated state of ox IFN-γ is generally poly- with dimer or four The form of body exists.

Research shows that the height of endogenous IFN-γ level can largely reflect the cellular immunity shape of body State, and IFN-γ reaction caused by specific antigen stimulation can then be directed to the immune finger of certain specific exotic antigen as body Mark.At present, IFN-γ vitro detection experiment is not only served in basic research, and has been widely used in clinical immunology (Rodriguz MF, 1998), immune effect of vaccine assessment (Agger EM, 2001), organ transplant (Tary-Lehmann M, 1998), allergic reaction (Kubota Y, 1999) and the diagnosis of a variety of pathogen infections (Benyoucef S, 1997).Wherein most Important application be cattle and sheep tuberculosis and johne's disease diagnosis (Walravens K, 2002；Kalis CH,2003).Many Person's human peripheral blood T lymphocytes react the application in diagnosis of tuberculosis to the IFN-γ of tulase specific antigen and are ground Study carefully, the results showed that, the experiment of antigentic specificity IFN-γ has many advantages compared with traditional tuberculin skin test experiment (TST), Since IFN-γ experiment is to carry out in vitro, without observing skin hyperplasia, the subjective factor included in judgement as a result is small, and entirely Detection process only needs and is detected animal contact once (Lalvani A, 2004).

A variety of methods, such as enzyme linked immunosorbent assay, elisa are had been set up for the detection technique of IFN-γ Method, flow cytometry etc. (Liebana E, 1998；Asai T,2000；Numa Y, 1991), quantitative detection side at present Most popular in method is double-antibody sandwich elisa.The key of double-antibodies sandwich ELISA detection sensitivity is used Monoclonal antibody for IFN-γ and the affinity size of natural IFN-γ reaction.Tradition prepares IFN-γ monoclonal antibody Generally use is that mouse is immunized in the IFN-γ purified product that prokaryotic expression system obtains, and carries out the screening of hybridoma cell line, It is thus obtained the overwhelming majority antibody can well with prokaryotic expression product react, but with natural IFN-γ anergy.This master If due to the aglycosylated modification of IFN-γ that prokaryotic expression system obtains, with thering is larger difference to make in natural IFN-γ structure Into.This phenomenon make it that traditional method for preparing monoclonal antibody efficiency in the preparation process of IFN-γ antibody is very low.This Invention use DNA immunization mouse, and with indirect immunofluorescene assay technology, screening can produce the antibody that is directed to natural ox IFN-γ Hybridoma cell strain, successfully solves the problems, such as that above-mentioned screening efficiency is low, also methylates for other in eucaryote or glycosyl The preparation for changing the protein specific antibody of modification provides new technical method.

The content of the invention

In order to solve to prepare the monoclonal antibody that the glycosylation modified albumen of eukaryotic expression obtains in the prior art, acquisition The antibody overwhelming majority can only be reacted with prokaryotic expression product, it is impossible to reacted with natural glycosylation albumen, limited obtained antibody Affinity and application value the problems such as, the present invention provides one kind animal is immunized using eukaryotic expression product after carry out monoclonal The preparation method of antibody, and a kind of monoclonal antibody for resisting natural cattle gamma interferon is provided, the hybridoma for secreting the antibody is thin Born of the same parents' strain.

The present invention uses Access RT-PCR kits using TRIZOL methods extraction ox lymphocyte total serum IgE (Promega Products) one step amplification ox IFN-γ (bIFN- γ) gene coding region goes signal fragments of peptides (to be named as d- BIFN), d-bIFN fragments are connected to pET30b (﹢) carrier, recombinant plasmid is named as p30b-bIFN.The spleen of mouse is taken, is made With blood/cell/tissue genome DNA extracting reagent kit extraction genomic DNA, operate to specifications.With mouse cell base Because group is template, the signal peptide sequence (being named as MIE) of IFN-γ is expanded, using recombinant plasmid p30b-bIFN as template, expands ox IFN-γ removes signal fragments of peptides d-bIFN.The PCR product for expanding two fragments of MIE and d-bIFN obtained is used respectively Omega purification kits are purified.Over-lap PCR amplification is carried out as template using purified product, two fragments are connected. Fragment ME-bIFN is tapped and recovered after agarose gel electrophoresis separates, and is connected to ME-bIFN fragments with T4DNA ligases PcDNA3.1 carriers.Spend and the immune recombinant plasmid p3.1-MbIFN of plasmid kit preparation is carried in endotoxin, BALB/ is immunized C mouse, take its splenocyte to be merged with SP2/0 myeloma cell.Eukaryon expression plasmid p3.1-MbIFN is transiently transfected into CHO Cell, while set the control group of transfection empty carrier pcDNA3.1.Establish indirect ELISA detection method to positive hybridoma cell into Row screening, all monoclonal antibody strains of screening can be detected and eukaryotic expression ox IFN- with Western-blotting methods The reactivity of γ, but only 3D7 plants can only be with the ox IFN-γ of eukaryotic expression, and, institute reactionless with ox IFN-γ protokaryon product The hybridoma cell strain that can only be reacted with the ox IFN-γ of eukaryotic expression stated, is named as 3D7, Classification And Nomenclature is：Hybridoma is thin Born of the same parents' strain, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in the Chaoyang District, Beijing City North Star The institute of microbiology of the Chinese Academy of Sciences of institute of West Road 1, its culture presevation numbering are：CGMCC No.9329, preservation date are 2014 6 The moon 25.Hypotype qualification result shows that the antibody subtype of 3D7 plants of monoclonal antibody is IgG1/ κ.

Further, the invention also provides the monoclonal antibody by the hybridoma cell strain secretion.And

The hybridoma cell strain or the monoclonal antibody of its secretion are in the natural cattle gamma interferon reagent of detection is prepared Application.

Brief description of the drawings

Fig. 1 is the electrophoresis result that reverse transcription PCR expands ox IFN-γ coded sequence；

M is DL2000Marker, and 1,2 swimming lane is amplified production, piece segment length 334bp；

Fig. 2 is the PAGE electrophoretic analysis results of recombinant protein rIFN after purification；

M is albumen Marker, and swimming lane 1 is to elute the albumen sample of harvest three times through liquid, swimming lane 2,3,4 for 500mM imidazoles Product；

Fig. 3 is the Western-bloting testing results for the recombinant protein rIFN that prokaryotic expression obtains；

The recombinant protein rIFN of purifying is subjected to PAGE electrophoretic separation, successively one is used as by the use of the anti-6His antibody of mouse after transferring film Anti-, the sheep anti mouse secondary antibody reaction of HRP marks, shows band and expected recombinant protein rIFN is in the same size, about 18kDa；

Fig. 4 is the signal peptide sequence (MIE) of mouse IFN-γ and the removal signal fragments of peptides (d- of ox IFN-γ coded sequence BIFN PCR amplification result)；

M is DL2000Marker, and (A) show MIE fragments, length 113bp；(B) d-bIFN fragments, length are shown 333bp；

Fig. 5 is the result of overlapping PCR amplification ME-bIFN fragments；

M is DL2000Marker, and 1,2 swimming lane is amplified production, piece segment length 420bp；

Fig. 6 is five immune rear antibody titer indirect ELISA testing results of mouse eukaryon expression plasmid p3.1-MbIFN；

Fig. 7 is indirect immunofluorescene assay result after fusion for the first time；

Top numbering is the name for being accredited as positive colony, and negative control uses SP20 cells and supernatants as primary antibody；

Fig. 8 is the reactive Western-Blotiing testing results of odd contradictive hydroperitoneum and ox IFN；

M is albumen Marker, and swimming lane 1 is prokaryotic expression IFN-γ purified product, and swimming lane 2 is transfection zero load pcDNA3.1's Chinese hamster ovary celI lysate, swimming lane 3 are the Chinese hamster ovary celI lysate of transfection eukaryon expression plasmid p3.1-MbIFN；

The hypotype that Fig. 9 is monoclonal antibody 3D7 is identified.

The hypotype that reaction result per hole represents has been marked in the left side of figure, the results showed that the hypotype that 3D7 plants of monoclonal antibody is IgG1。

Embodiment

The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and It is apparent.But embodiment is only exemplary, does not form any restrictions to the scope of the present invention.Those skilled in the art should It should be appreciated that the details and form of technical solution of the present invention can be repaiied without departing from the spirit and scope of the invention Change or replace, but these modifications and replacement are each fallen within protection scope of the present invention.

Method therefor is conventional method unless otherwise instructed in the following example.

Embodiment 1：The structure of prokaryotic expression plasmid and the expression and purification of albumen

1st, the separation and activation of ox peripheral blood lymphocytes

Bovine jugular vein is sterile to adopt anticoagulation, adds monoploid product Hank ' s liquid, gently mixes, and the body such as is carefully slowly added to On long-pending ox lymphocyte separation medium.2000r/min centrifuges 20min, draws the white cellular layer on interface, is collected in another Centrifuge tube, is washed twice with RPMI-1640,1500r/min centrifugations 10min.Collected cell is used containing 10 μ g/ml with sword bean ball egg The RPMI-1640 nutrient solutions of white A (ConA, Sigma Products) suspend, cell density about 10⁷A, ml, in 37 DEG C, CO₂Culture Case stimulates culture 8h.Lymphocyte is in centrifuge tube after stimulation is collected by centrifugation, and PBS is washed three times, for extracting total serum IgE.

2nd, the extraction of ox lymphocyte total serum IgE

Centrifugation after the lymphocyte of culture washes three times with PBS, dissolves 2.5 × 10 according to 1ml⁶The amount of a cell adds TRIzol, gently pressure-vaccum mixing.Room temperature, which places 10min, is completely dissolved nucleoprotein complex, adds 200 μ l by every milliliter of TRIzol Chloroform, covers tightly lid, acutely shakes 15s, and room temperature places 2~3min and centrifuges 10min after 4 DEG C of 12000r/min.

Careful upper strata aqueous phase of drawing is transferred to new centrifuge tube, adds isometric isopropanol, is stored at room temperature 10min, in 4 DEG C 12000r/min centrifuges 10min.After abandoning most supernatant, precipitation washed twice with 75% ethanol, room temperature drying precipitation (generally placement 5~ 10min), RNA, the quality of agarose gel electrophoresis analysis extraction RNA, ultraviolet specrophotometer measured concentration are dissolved with DEPC water 80 DEG C of ﹣ is placed on to save backup.

3rd, the amplification of ox IFN-γ gene coding region

Using Access RT-PCR kits (Promega Products) one step amplification ox IFN-γ (bIFN- γ) base Signal fragments of peptides (being named as d-bIFN) is removed because of code area.Primer sequence：IF-F(BamHI)：5’- catGGATCCccagggccaattttttagag；IF-R(SalI)：5 '-catGTCGACcgttgatgctctccggc (capital letters Female part represents restriction enzyme enzyme recognition site sequence).Amplification system is：DEPC processing water 11 μ l, AMV/TFI 5 × 5 μ l of Buffer, 1 μ l (50pmol) of primer I F-F, primer I F-R1 μ l (50pmol), 2 μ l of 2.5mM dNTP, 25mM MgSO₄1 μ l, 0.5 μ l of AMV reverse transcriptase, 0.5 μ l of TFI archaeal dna polymerases, 3 μ l of total serum IgE (0.5 μ g).RT-PCR amplification programs For：Enter circulation (94 DEG C of 1min after 48 DEG C of reverse transcriptions 45min, 94 DEG C of 2min；56℃1min；72 DEG C of 0.5min), expand 40 After circulation, 72 DEG C of 5min, 4 DEG C of preservations, product agarose gel electrophoresis analysis result such as Fig. 1 after the completion of amplification.

4th, the structure and protein expression and purification of prokaryotic expression plasmid

The d-bIFN fragments that RT-PCR amplifications obtain are purified with Omega purification kits, purified product limitation Property the processing of restriction endonuclease (Thermo Products) BamHI and SalI double digestions, prokaryotic expression carrier pET30b (﹢) makees same double Digestion is handled.Digestion system is prepared according to product description, digests 4h in 37 DEG C.Agarose gel electrophoresis recycles digestion products, D-bIFN fragments are connected to pET30b (﹢) carrier with T4DNA ligases (Thermo Products), recombinant plasmid is named as p30b-bIFN.Linked system is prepared with reference to specification, and 4h is connected in 22 DEG C.

Connection product converts DH5 α, and picking single bacterium colony culture, is converted after extracting plasmid with the identification of BamHI and SalI double digestions Son, digestion go out about 333bp size fragments for positive colony, select two positive colonies and send sequencing company to carry out sequencing, Sequence is remained with expected 100% clone being consistent, and recombinant plasmid is named as p30b-bIFN.This recombinant plasmid transformed is big Enterobacteria expression bacterial strain BL21 (DE3), picking single bacterium colony are inoculated in LB culture mediums, and 37 DEG C of 200r/min shake overnight incubation.Take Overnight culture is forwarded to 200ml fresh LBs, 37 DEG C of 200r/min shakings cultures to bacterium solution OD in the ratio of 1 ︰ 100₆₀₀ When=0.6, the IPTG induction destination protein expression of final concentration of 1mM is added, in 16 DEG C of 200r/min shaking cultures 24h.

Induction bacterium solution is collected by centrifugation, thalline is resuspended with 10ml combination buffers (20mM Tris-HCl, 150mM NaCl), Utilize the broken cracking thalline of pressure breaking instrument (pressure parameter is arranged to 26Kpsi).Lysate is centrifuged in 4 DEG C of 12000r/min 20min, shifts supernatant, and 0.45 μm of filter filters supernatant.Take 2ml nickel ion affinity chromatographs resin (Ni Sepharose 6Fast Flow, GE Products) homogenate (resinous 1ml) is in clean void column (BioRad Products), 5 times of bed volumes of addition Deionized water flows through resin, then balances resin with 5 times of bed volume combination buffers.The Sample supernatants after filtering are added, treat it After flowing to end, resin is flowed through with the combination buffer of 20 times of bed volumes, washes away the foreign protein in sample.With 3 times of bed volumes Elution buffer (20mM Tris-HCl, 150mM NaCl, 500mM imidazoles) wash-out recombinant protein rbIFN- γ, point 3 elutions, Elution samples are collected respectively, and each sample of SDS-PAGE electrophoretic analysis, the results are shown in Figure 2.

RbIFN- γ purified products are verified with anti-6His antibody：The fusion protein of purifying is subjected to SDS-PAGE electrophoresis point From albumen being gone to nitrocellulose filter from gel electricity with half-dried electrophoretic blotting instrument, transferring film condition is voltage 15V, the time 30min.The nitrocellulose filter for turning upper albumen is closed with 10% skimmed milk, and TBST washes film, adds anti-His tag monoclonal antibodies 37 DEG C of (5000 times of dilutions), 70r/min combination 2h, wash film, add the goat anti-rabbit igg of horseradish peroxidase-labeled afterwards (5000 times dilution), 37 DEG C, 70r/min combination 2h, TBST wash film, fully developed the color after washing with DAB substrate solutions, identification expression is produced Thing.The results are shown in Figure 3.

Embodiment 2：Construction of eukaryon expression plasmid for expressing

The extraction of mouse cell genome：The spleen of mouse is taken, uses blood/cell/tissue extracting genome DNA reagent Box (Tiangeng biochemical technology Co., Ltd product) extracts genomic DNA, operates to specifications.

Using mouse cell genome as template, the signal peptide sequence (being named as MIE) of IFN-γ is expanded, primer is：EXIF- U：5’-gatGGTACCatgaacgctacacactgcatc；EXIF-OR：5’-ttcttcaaATGATGATGATGATGATGAatgactgtgccgtggcag (underscore part is the complementary series of over-lap PCR), Use Pfu high-fidelity DNA polymerases (Fermentas Products).Amplification system is：26 μ l of aqua sterilisa, 5 × Buffer, 5 μ L, 2 μ l (20umol) of primer EXIF-U, 2 μ l (20umol) of primer EXIF-OR, 4 μ l of dNTP (2.5mM), 1 μ l of archaeal dna polymerase, 5 μ l of genomic DNA.Amplification program is：Enter circulation (95 DEG C of 30sec after 95 DEG C of pre-degeneration 5min；56℃30sec；72℃ 18sec), after expanding 30 circulations, 72 DEG C of 5min, 4 DEG C of preservations after the completion of amplification.

The amplification of the removal signal fragments of peptides (being named as d-bIFN) of ox IFN-γ coded sequence：With recombinant plasmid p30b- BIFN (embodiment 1 is built) is template, and amplification ox IFN-γ removes signal fragments of peptides d-bIFN.Amplification the primer is EXIF- OF：5’-CATCATCATCATCATCATttgaagaa(underscore part is the complementary sequence of over-lap PCR to ttggaaagatgaaagtg Row)；EXIF-D：5 '-gatCTCGAGttacgttgatgctctccggc, use Pfu high-fidelity DNA polymerases (Fermentas Products), amplification system is：26 μ l of aqua sterilisa, 5 × Buffer, 5 μ l, 2 μ l (20umol) of primer EXIF-OF, primer 2 μ l (20umol) of EXIF-D, 4 μ l of dNTP (2.5mM), 1 μ l of archaeal dna polymerase, 1 μ l of Plasmid DNA (1ug/ml).Amplification program is： Enter circulation (95 DEG C of 30sec after 95 DEG C of pre-degeneration 5min；54.5℃30sec；72 DEG C of 45sec), after expanding 30 circulations, 72 DEG C 5min, 4 DEG C of preservations after the completion of amplification.The PCR product agarose gel electrophoresis analysis result of MIE and d-bIFN fragments is shown in Fig. 4.

The PCR product for expanding two fragments of MIE and d-bIFN obtained is carried out with Omega purification kits respectively pure Change.Over-lap PCR amplification is carried out as template using purified product, two fragments are connected.Expanding Outside primer used is EXIF-U and EXIF-D.Amplification system is divided to two parts, 33.5 μ l of (1) aqua sterilisa, 5 × Buffer5 μ l, 3 μ l of dNTP (2.5mM), MIE 1.5 μ l of fragment (30ug/ml), 6 μ l of d-bIFN fragments (27ug/ml), 1 μ l of archaeal dna polymerase.(2) 31 μ l of aqua sterilisa, 5 × 5 μ l of Buffer, 4 μ l (20umol) of primer EXIF-U, 4 μ l (20umol) of primer EXIF-D, 5 μ l of dNTP (2.5mM), DNA gather 1 μ l of synthase.Amplification program is：System (1) is first expanded, circulation (95 DEG C of 30sec are entered after 95 DEG C of pre-degeneration 1min；55℃ 30sec；72 DEG C of 1min 10sec), after expanding 5 circulations, 72 DEG C of extension 10min.Then the system prepared (2) is added to system (1) fully mixed in, be further divided into two pipes and continue to expand, circulation (95 DEG C of 30sec are entered after 95 DEG C of pre-degeneration 1min；56.5℃ 30sec；72 DEG C of 1min 20sec), after expanding 25 circulations, 72 DEG C of extension 10min, 4 DEG C of preservations after the completion of amplification.Amplification obtains PCR product (being named as ME-bIFN) agarose gel electrophoresis analysis result see Fig. 5.

Fragment ME-bIFN is tapped and recovered (Omega plastic recovery kits), recovery product after agarose gel electrophoresis separates Handled with endonuclease (Thermo Products) KpnI and XhoI double digestions, eukaryotic expression vector pcDNA3.1 is used same Inscribe enzymatic treatment, digestion system are prepared according to product description, digest 4h in 37 DEG C.Agarose gel electrophoresis recycling digestion production Thing, pcDNA3.1 carriers are connected to T4DNA ligases (Thermo Products) by ME-bIFN fragments.Linked system reference Specification is prepared, and 4h is connected in 22 DEG C.

Connection product converts DH5 α, and picking single bacterium colony culture, is converted after extracting plasmid with the identification of KpnI and XhoI double digestions Son, digestion go out about 420bp size fragments for positive colony, select two positive colonies and send sequencing company to carry out sequencing, Retain sequence and expected 100% clone being consistent, recombinant plasmid is named as p3.1-MbIFN.

Embodiment 3：Animal immune

Dock before mouse immune and take a blood sample, serum is separated, as negative control.Spend and plasmid kit is carried in endotoxin (Omega Products) prepare immune recombinant plasmid p3.1-MbIFN, and immunizing dose is every 100 μ g Plasmid DNA of mouse, Left and right thigh respectively injects 50 μ g.Concrete operations are as follows：The hair alcohol of mouse femoribus internus muscle is drenched first, with it is micro into Sample device (Town in Shanghai booth microsyringe factory, specification are 100 μ l) absorption appropriate volume goes endotoxin plasmid solution (to contain 100 μ g Plasmid), injected that (insertion depth is 2mm~2.5mm, pays attention to avoiding muscle in each point 2 points of mouse or so inboard leg muscle The depth for piercing through or pricking is inadequate).It is slow during injection, stop to further take out syringe needle in several seconds after injection, to prevent leak-stopping liquid., will after injection The hair of the big leg outer side of mouse drenches, by living gene importing equipment (NingBo XinZhi Biology Science Co., Ltd's product, model WJ-2002 electricity) is clipped in the both sides that thigh drenches, and voltage is adjusted to 100V, touches shock button, and mouse can be observed because of electric shock And whole body is shaken, that is, complete once to shock by electricity.The positive and negative anodes of introducing apparatus are exchanged, touch shock button again, carry out second of electricity Hit.The thigh that opposite side has injected plasmid operates the same shock treatment of progress more than.

Booster immunization is carried out after 15 days immune, immune operating process is same as above, and is hereafter once strengthened at interval of 15 days It is immune.Three exempt from after carry out within the 14th day docking blood sampling to mouse, separate serum, detect potency, adopted within the 14th day after hereafter immune every time Blood simultaneously separates serum, the indirect ELISA method detection immunizing potency established by the use of the ox IFN-γ of prokaryotic expression as coating antigen. After the 5th time immune, immunizing potency, which reaches, is expected (shown in Fig. 6), takes the spleen cell and murine myeloma cell of mouse (SP2/0) cell fusion is carried out.

Embodiment 4：Cell fusion

1st, the preparation of murine myeloma cell (SP2/0)：

Myeloma cell's cryopreservation tube is taken out from liquid nitrogen container, is put into 37 DEG C of water-baths to complete and melts；1000r/min from 3~5min of the heart；Supernatant is abandoned, (RPMI-1640, adds 20% hyclone and 1% mycillin with complete medium It is dual anti-) cell of sinking dispelled into suspension, addition has in the Tissue Culture Flask of adequate nutrition liquid, mixes 37 DEG C of postposition, 5%CO₂ Cultivated in incubator.Next day observes the growing state of myeloma cell, as being circular, bright and being in slight adherent growth, then says Shark bone myeloma cells well-grown；As found, some cells shade, and float in liquid, then can change liquid, discard the dead of floating Die cell, the cell of survival adherent growth and can largely breed.When cell covers with individual layer, 1 ︰, 3 secondary cultures, expand always To cell concentration needed for cell fusion, (fusion needs about 1.5 × 10 every time for culture⁵A SP2/0 cells).

By PEG, 40ml terminate liquid (basis 1640) and HAT culture mediums, (RPMI-1640, adds 20% before fusion Hyclone, 1% mycillin it is dual anti-and 1% HAT) be put into 37 DEG C of pre- stand-by heats of incubator.

2nd, the preparation of feeder cells：

A non-immune BALB/c mouse, eye socket bloodletting are taken, collection serum is negative serum.After four limbs are fixed, tear initiation Skin, exposes peritonaeum, an osculum (in belly center) is cut on peritonaeum, then with 2~3ml of suction pipe (depending on mouse size) RPMI-1640 basal liquids inject mouse peritoneal, and suction beats several times, liquid is suctioned out and is put in 50ml centrifuge tubes.Repetition RPMI-1640 Basal liquid is washed one's hands and face once, and Turnover of Mouse Peritoneal Macrophages is contained in the liquid of collection.1000r/min centrifuges 10min supernatant discardings, precipitation After cell is suspended with HAT culture mediums, 37 DEG C, 5%CO are placed in₂It is stand-by in incubator.

3rd, the preparation of immune spleen cell：

Immune BALB/c mouse one are taken, eye socket sacrificed by exsanguination (collects serum, be positive serum), in 75% alcohol Soak 5~10min disinfections.The mouse disinfected is fixed on dissection plate, skin is cut off and exposes peritonaeum, then carefully cut off abdomen Film, exposes spleen, and tweezers press from both sides out spleen, removes the adipose tissue of adhesion on spleen with scissors, breaks spleen outer membrane, is put into and goes out In the homogenizer of bacterium.5ml RPMI-1640 basal liquids are added into homogenizer, grinding, squeezes out splenocyte, add 10ml RPMI-1640 basal liquids.After standing 2min, 5ml upper cells suspension is drawn in another sterile 50ml centrifuge tubes.Add again For 10ml RPMI-1640 liquid in homogenizer, pressure-vaccum stands 2min after mixing, and draws 10ml upper cell suspensions in centrifuge tube, Repeat above step once.The liquid of collection contains immune mouse spleen cell, in 1000r/min centrifuge 10min, supernatant discarding, Cell precipitation counts after being resuspended.

4th, cell fusion：

By 1 × 10⁷~2 × 10⁷A SP2/0 cells and 1 × 10⁸A immunocyte (both ratios are 1 ︰, 5~1 ︰ 10) in Mixed in 50ml centrifuge tubes, 1000r/min centrifugations 8min.Most supernatant is abandoned, and residual liquid is blotted with the filter paper of sterilizing, gently Tube bottom is tapped, cell precipitation is loosened slightly, the even action easy to PEG to cell.By the centrifuge tube equipped with cell mixture Bottom is dipped in 37 DEG C of water-baths, and 50%PEG solution (the Sigma companies production of 0.8ml pre-temperatures to 37 DEG C is slowly added into 1min Product), side edged is gently stirred with pipette tip.Continue to stir 30s after adding PEG solution.After standing 30s, it is pre- to be slowly added to 10ml The RPMI-1640 basal liquids of temperature.Speed is added strictly to control：1ml, the 2nd minute plus l ml, the 3rd~4 are instilled within 1st minute dropwise Minute plus 3ml, the 5th minute plus 5ml, at the uniform velocity add, and be constantly gently mixed.Finally add 30ml RPMI-1640 liquid. 5min is centrifuged in 1000r/min, abandons supernatant, this cell after merging is suspended with the HAT culture mediums containing feeder cells, according to need Suitable HAT culture mediums are added, point kind is in 96 well culture plates, about 250 μ l/ holes.Single cell fusion is inoculated in 4 piece of 96 orifice plate In.Cultivated in 37 DEG C, 5%CO2 incubators.

Embodiment 5：Filtering hybridoma and cloning

1st, the screening of cell line：

Start to observe within second day after cell fusion, have pollution-free, suck within the 4th day 100 μ l culture mediums, add 100 μ lHT trainings Support base (RPMI-1640, add 20% hyclone, 1% mycillin it is dual anti-and 1% HT).Treat the single colony of cell When high-visible, the hole for there are multiple colonies in a hole is carried out single colonization.Concrete operations are as follows, by the inversion with display screen Microscope (EVOS fl, AMG Co. of U.S. product) is removed to superclean bench, observes point of colony in 96 porocyte culture plates Cloth, is selected after having the hole of more colonies, and the cell of some colony in the visual field is tried one's best with capillary syring and is all suctioned out.Capillary syring should Camber is burnt by one in advance, to facilitate operation, and carries out autoclaving before use.It is thin that the cell of sucking-off is transferred to new 96 hole In the hole of born of the same parents' culture plate, Kong Zhongxu adds 200 μ l HT culture mediums in advance.So repeatedly, the colony in more colony holes is turned respectively Move, can individually detect whether secrete required antibody when screening first time with the colony for ensureing each to grow out.Treat cell Colony length when culture medium slightly turns yellow, draws the detection that culture supernatant carries out indirect immunofluorescence to the 1/4~1/3 of culture hole.

2nd, indirect immunofluorescene assay positive cell strain：

With the complete medium of DMEM in high glucose (Gibco Products), (10% hyclone of addition and 1% mycillin are double It is anti-) culture Chinese hamster ovary celI (Chinese hamster ovary cell), when cultivating to requirement, 96 porocyte culture plates are spread after being digested, Cell is set about to be paved with the 60~70% of hole.After growing about 12h, when cell covers with the 80% of hole, by eukaryon expression plasmid p3.1- MbIFN transiently transfects Chinese hamster ovary celI, while sets the control group of transfection empty carrier pcDNA3.1.The transfection reagent used is SignaGen Products PolyJet DNA transfection in vitro reagents, operating procedure to specifications in the flow that generally transfects into OK.12h, which is changed, after transfection maintains culture medium (DMEM in high glucose, adds 2% hyclone and 1% mycillin is dual anti-), continues to cultivate Supernatant discarding after 24h, cell is washed twice with PBS solution.With 80% acetone (20 DEG C of precooling 30min of ﹣) cell is fixed in room temperature 10min, discards fixer, is washed three times with PBS, soaks 5min every time.Transfect recombinant plasmid p3.1-MbIFN and empty carrier The cell of pcDNA3.1 is separately added into hybridoma culture supernatant to be checked；If one group of Positive control wells, is separately added into immune mouse Serum (i.e. foregoing positive serum；1 50 times of ︰ dilutes)；One group of negative control hole, is separately added into the blood before mouse immune (i.e. foregoing negative serum clearly；1 50 times of ︰ dilutes).Cell plates are placed in 37 DEG C of incubation 30min after sample-adding, after supernatant discarding PBS is washed three times；The sheep anti-mouse igg (Sigma Products, 1 100 times of ︰ dilutions) of fluorescein isothiocynate (FITC) mark is added, 37 DEG C of incubation 30min, PBS is washed three times, in fluorescence microscopy Microscopic observation cell fluorescence phenomenon.If negative control group is without visible fluorescence Phenomenon, the empty carrier pcDNA3.1 holes of positive controls are without visible fluorescence, and the recombinant plasmid p3.1-MbIFN of positive controls There is obvious fluorescence in hole, then experimental result is set up；Culture supernatant makes the Chinese hamster ovary celI of transfection recombinant plasmid p3.1-MbIFN show glimmering Light, at the same the hybridoma cell strain of the not aobvious fluorescence of Chinese hamster ovary celI reaction with transfecting empty carrier pcDNA3.1 be positive colony (such as Shown in Fig. 7).

3rd, the cloning of hybridoma

The cell in positive hole is selected according to testing result, is subcloned with limiting dilution assay.According to above-mentioned side before clone Method takes the peritoneal macrophage of healthy mice to prepare feeder layer, prepares feeder cells, after being resuspended with HT complete mediums It is placed in spare in incubator.The hybridoma that will be cloned gently is blown down out of culture hole, is calculated with blood cell counting plate living thin Born of the same parents' number.Cell is diluted to 5,10,50 cell/ml with HT complete mediums according to count results.By three above concentration Cell suspension is separately added into 96 well culture plates, and 100 μ l/ holes, make every hole contain 0.5,1 and 5 cell respectively.Hybridoma spreads hole After the completion of feeder cells are added drop-wise in hole again.

Cultivate to the 4th day addition HT complete medium one and drip, the 5th~6 day growing state for examining each hole inner cell, And record.The 7th~9 day after clone, when cell clone covers with the 1/4~1/3 of hole, then with above-mentioned indirect immunofluorescence examine Survey.If there are corresponding antibodies in detection cell growth hole, it is high to may be selected antibody titer, and in single colony growth, cellular morphology is good Hole, continues same method and is subcloned again.This time be subcloned after, if all holes for growing cell with indirect immunofluorescence testing result all For the positive, then it can select the good hole of single colony, cellular morphology and build strain.

Embodiment 6：Hybridoma freezes

The hybridoma of singling is transferred to 24 holes from 96 holes respectively and expands culture, cell is collected after cell covers with In centrifuge tube, cell count is carried out after mixing, centrifuges 8min in 1000r/min, supernatant discarding, according to count results, adds Cell is resuspended in the cells frozen storing liquid (40%DMEM, 50% hyclone, 10%DMSO) of suitable 4 DEG C of precoolings, makes cell dense eventually Spend for 2~3 × 10⁶A/ml.1ml cell suspensions are added in each cell cryopreservation tube, are put into cell cryopreservation box (U.S. NALGENE Products) in, then stayed overnight in 70 DEG C of Temperature drop in refrigerator of ﹣, be finally transferred in liquid nitrogen container and preserve for a long time.

Embodiment 7：The preparation of ascites

The mouse of 8~10 week old is taken, incomplete Freund's adjuvant is injected intraperitoneally, injection dosage 0.5ml/ is only.Abdomen after 7~10 days Chamber injects the hybridoma of each singling respectively, and injection volume is 5 × 10⁵~10⁶/ only, the cell length of general 24 porocyte plates Man Hou, the cell concentration of collection are enough.Mouse state is observed in injection one day after, after 7~10 days, when seeing the obvious bulging of mouse web portion After extract ascites, 1000r/min centrifugation 8min centrifuging and taking supernatants, are the corresponding mouse ascites of each strain monoclonal antibody.

Embodiment 8：Ascites and eukaryotic expression ox IFN-γ (natural ox IFN-γ) reactivity verification (Western- blotting)

Chinese hamster ovary celI spread six orifice plates, transfected respectively when cell confluency degree reaches about 80% recombinant plasmid p3.1-MbIFN and Empty carrier pcDNA3.1, concrete operations are as described in example 5 above.36h after transfection, abandons culture supernatant, and cell one is washed with PBS Time, each hole adds 100ul cell pyrolysis liquids (the green skies in Shanghai), under cell scraper, will be inhaled in 1.5ml centrifuge tubes with cell scraping In, blow and beat and eliminated after the foam that standing on ice produces sample repeatedly.Each sample adds 25ul albumen Loading Buffer (Fermentas Products), boiling water bath 10min after mixing.Treat that sample is placed on ice cooling completely after 10000r/ Min centrifuges 1min, takes supernatant to carry out PAGE electrophoresis.Albumen is gone into nitrocellulose from gel electricity with half-dried electrophoretic blotting instrument Film, transferring film condition are voltage 15V, time 30min.The nitrocellulose filter for turning upper albumen is closed with 10% skimmed milk, TBST Film is washed, adds the corresponding mouse ascites of each strain monoclonal antibody (1000 times of dilutions), washes film in 37 DEG C, 70r/min combinations 2h, TBST, afterwards Add sheep anti mouse secondary antibody (the KPL Products of Dylight680 marks；10000 times of dilutions), in 37 DEG C, 70r/min combination 1h, TBST washes film, is fully observed after washing with IR fluorescence scanner (ODYSSEY, U.S.'s Li-cor Products) and records knot Fruit, all monoclonal antibody strains of screening can be detected and eukaryotic expression ox IFN-γ with Western-blotting methods Reactivity, but only 3D7 plants can only be and reactionless with ox IFN-γ protokaryon product with the ox IFN-γ of eukaryotic expression, 3D7 plants Western-blotting testing results are as shown in Figure 8.

The 3D7 strains are deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is in north The institute of microbiology of the Chinese Academy of Sciences of institute of Jing Shi Chaoyang Districts North Star West Road 1, its culture presevation numbering are：CGMCC No.9329, preservation Date is on June 25th, 2014.

Embodiment 9：The hypotype identification (ELISA) that 3D7 plants of monoclonal antibody

With the antibody subtype identification kit SBA Clonotyping System-HRP of SouthernBiotech companies The identification and analysis of (article No. 5300-05) to 3D7 plants of progress hypotypes of monoclonal antibody.Method is as follows：With 1ml distilled waters dissolving 15mg ABTS Substrate pulvis is prepared into ABTS substrates and preserves liquid, is kept in dark place in 2~8 DEG C.It is dilute with PBS buffer (pH7.4) antibody will to be captured Release and to the concentration of 5~10ug/ml, take 0.1ml to add in the hole of elisa plate.After incubating 12h in the environment of 4 DEG C of moistenings, discard Liquid in hole, is washed three times with the PBS containing 0.05% tween.Discard in hole after cleaning solution, it is molten to fill it up with the PBS containing 1%BSA per hole Liquid, is incubated at room temperature at least 1h, and when incubation keeps slight oscillatory.After discarding liquid, wash three times.Hybridoma is added per hole Culture supernatant, 0.1ml, is incubated at room temperature 1h, and when incubation keeps slight oscillatory.After discarding liquid, wash three times.Dilute each hypotype Corresponding enzyme labelled antibody, 0.1ml enzyme labelled antibodies are added into elisa plate per hole in corresponding hole.1h is incubated at room temperature, when incubation protects Slight oscillatory is held, discards in plate and is washed five times after liquid.0.2ml ABTS bottoms are added in 10ml citrate buffers (pH4.0) Thing preserves liquid and is made into substrate solution, and 0.1ml substrate solutions are added per hole and are developed the color in 10~20min of room temperature placement.Read in OD450 Absorbance.

As shown in Fig. 9 and table 1, testing result shows that the antibody subtype of 3D7 plants of monoclonal antibody is IgG1/ κ.

The hypotype identification (OD450) of 1. monoclonal antibody 3D7 of table.

IgG3	IgG2b	IgG2a	IgG1	IgA	IgM	κ	λ
								0.336	0.08	0.111	1.516	0.088	0.1	1.438	0.186

Claims (4)

1. one plant can stably excreting resist the hybridoma of natural cattle gamma interferon monoclonal antibody, be named as 3D7, preservation In China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of microbiology of the Chinese Academy of Sciences, its culture presevation numbering are：CGMCC No.9329, preservation date are on June 25th, 2014.

2. the monoclonal antibody secreted as the hybridoma cell strain described in claim 1.

3. the hybridoma cell strain described in claim 1 is preparing the application in detecting natural cattle gamma interferon reagent.

4. the monoclonal antibody described in claim 2 is preparing the application in detecting natural cattle gamma interferon reagent.