CN104560885A - Monoclonal antibody against natural cow gamma-interferon, hybridoma cell strain secreting antibody and application - Google Patents

Abstract

The invention discloses a monoclonal antibody against natural cow gamma-interferon, a hybridoma cell strain secreting the antibody and application. The hybridoma cell strain capable of stably secreting the monoclonal antibody against the natural cow gamma-interferon is named as 3D7 and is preserved in China General Microbiological Culture Collection Center, the strain preservation number is CGMCC No.9329, and the preservation data is June 25, 2014. According to the invention, a eukaryotic expression vector of the cow gamma-interferon is used for immunizing a mouse to screen the hybridoma cell strain capable of generating the antibody against the natural cow 1FN gamma-interferon by means of indirect immunofluorescence assay technology, the problem that the traditionally prepared IFN gamma-interferon can only react with a prokaryotic expression product and cannot react with natural IFN gamma is solved successfully, and a new technological means is provided for the detection of the natural cow gamma-interferon.

Description

A monoclonal antibody for anti-natural cattle gamma interferon, secretes hybridoma cell strain and the application of this antibody

Technical field

The present invention relates to a kind of monoclonal antibody of anti-cattle gamma interferon, secrete hybridoma cell strain and the application of this antibody, resist the monoclonal antibody of natural cattle gamma interferon in particular to a kind of and secrete the hybridoma cell strain of this antibody, also relating to monoclonal antibody in this and detecting the application in natural cattle gamma interferon (IFN-γ).The invention belongs to biocytology field.

Background technology

Interferon, rabbit (IFN) is the glycoprotein by a class of emiocytosis under specific inducer effect with biologic activity such as antiviral, antitumor and immunoloregulation functions, molecular weight is about 20 ~ 100kDa, generally be made up of 150 ~ 170 amino acid, containing 17 kinds with upper amino acid, wherein aspartic acid, L-glutamic acid and leucine content higher (Iiaacs etc., 1957).According to the difference of gene order and acceptor, IFN can be divided into I, II and III type 3 type.I type mainly comprises IFN-α, β, IFN-ε, ψ, κ, δ, τ, ξ etc. of finding afterwards in addition, is produced (Sentsui etc., 2001) by inoblast and epithelial cell by secretion after antigenic stimulation.Interferon type Ⅱ and IFN-γ are formed, and are by the activating immune cell such as antigen, mitogen, comprise CD ⁴⁺th1, CD ⁸⁺secreted by T cell and NK cell, be therefore also called type II interferon.III type Interferon, rabbit is a newfound type cytokines, in close relations with interferon type Ⅰ, is called IFN-λ (Donnelly etc., 2010).Interferon type Ⅰ main manifestations is antiviral, antineoplastic biologic activity; And interferon type Ⅱ main manifestations is immunoregulatory biologic activity (Kenji etc., 2001).

IFN-γ gene is all containing 3 introns and 4 exons, and the exon of different animals and intron Nucleotide different amts, but all belong to secreted protein, be made up of 5 ' end non-coding region, signal peptide coding region, polypeptid coding area and 3 ' end non-coding region.People, pig and ox IFN-γ precursor are 166 amino-acid residues, and wherein front 23 amino acid are signal peptide, and maturation protein size is 143 amino acid.Various animals IFN glycosylation site number is different, and natural ox IFN-γ exists 2 glycosylation sites (Joseph C, 1998) at the l-asparagine of the 25th and 97.Although IFN-γ whether glycosylation does not affect its biologic activity in immunomodulatory, its transformation period (Thiel etc., 2000) in animal body can be affected.IFN-γ forms dimeric forms usually, and monomer whose form does not have biologic activity.The active condition of ox IFN-γ generally exists with dimer or tetrameric form.

Research shows, the height of endogenous IFN-γ level can reflect the cellular immunity of body to a great extent, and specific antigens stimulates the IFN-gamma reaction caused then can as the immune indexes of body for certain specific exotic antigen.At present, the test of IFN-γ vitro detection not only serves fundamental research, and be widely used in clinical immunology (Rodriguz MF, 1998), immune effect of vaccine assessment (Agger EM, 2001), organ transplantation (Tary-Lehmann M, 1998) diagnosis (Benyoucef S, 1997) of, anaphylaxis (Kubota Y, 1999) and multiple pathogen infection.Wherein most important application is diagnosis (Walravens K, 2002 of cattle and sheep tuberculosis and johne's disease; Kalis CH, 2003).The IFN-gamma reaction application in diagnosis of tuberculosis of many scholar's human peripheral blood T lymphocytes to tubercule bacillus specific antigens is studied, result shows, antigen-specific IFN-γ tests to test with traditional tuberculin skin test compared with (TST) many advantages, because IFN-γ test is external carrying out, without the need to observing skin hyperplasia, the subjective factor comprised in the judgement of result is little, and whole testing process only need with tested animal contact once (Lalvani A, 2004).

Detection technique for IFN-γ has established multiple method, as enzyme-linked immunosorbent assay, and enzyme-linked immunospot assay, (Liebana E, 1998 such as flow cytometry; Asai T, 2000; Numa Y, 1991), most popular in current quantitative detecting method is double-antibody sandwich elisa.The key of double-antibodies sandwich ELISA detection sensitivity is the avidity size of the monoclonal antibody for IFN-γ used and natural IFN-gamma reaction.What tradition preparation IFN-γ monoclonal antibody adopted usually is the IFN-γ purified product immune mouse that prokaryotic expression system obtains, carry out the screening of hybridoma cell line, thus obtained most antibody capable well and prokaryotic expression product react, but with natural IFN-γ anergy.This mainly due to the not glycosyafated modification of IFN-γ that prokaryotic expression system obtains, has larger difference to cause with in natural IFN-γ structure.This phenomenon makes traditional method for preparing monoclonal antibody efficiency in the preparation process of IFN-gamma antibodies very low.The present invention adopts DNA immunization mouse, with indirect immunofluorescene assay technology, screening can produce the hybridoma cell strain of the antibody for natural ox IFN-γ, successfully solve the problem that above-mentioned screening efficiency is low, also for other have and to methylate or the preparation of glycosylation modified protein specific antibody provides new technological method in eukaryote.

Summary of the invention

In order to solve in prior art the monoclonal antibody that the glycosylation modified albumen of preparing eukaryotic expression obtains, the antibody overwhelming majority obtained can only be reacted with prokaryotic expression product, can not react with natural glycosylated protein, limit the problem such as avidity and using value of obtained antibody, the invention provides a kind of utilize eukaryotic expression product immune animal after carry out the preparation method of monoclonal antibody, and provide a kind of monoclonal antibody of anti-natural cattle gamma interferon, secrete the hybridoma cell strain of this antibody.

The present invention adopts TRIZOL method to extract ox lymphocyte total serum IgE, and then adopt the number of the writing to peptide fragment (called after d-bIFN) of Access RT-PCR kit (Promega Products) one step amplification ox IFN-γ (bIFN-γ) gene coding region, d-bIFN fragment is connected to pET30b (﹢) carrier, recombinant plasmid called after p30b-bIFN.Get the spleen of mouse, use blood/cell/tissue genome DNA extracting reagent kit to extract genomic dna, operate to specifications.With mouse cell genome for template, the signal peptide sequence (called after MIE) of amplification IFN-γ, with recombinant plasmid p30b-bIFN for template, the number of the writing to peptide fragment d-bIFN of amplification ox IFN-γ.The PCR primer of MIE and d-bIFN two fragments amplification obtained carries out purifying with Omega purification kit respectively.Purified product is utilized to carry out over-lap PCR amplification as template, two fragments to be connected.Fragment ME-bIFN rubber tapping after agarose gel electrophoresis is separated is reclaimed, and with T4DNA ligase enzyme, ME-bIFN fragment is connected to pcDNA3.1 carrier.Spend in intracellular toxin and carry plasmid kit and prepare immune recombinant plasmid p3.1-MbIFN, immune BALB/c mouse, get its splenocyte and SP2/0 myeloma cell is merged.By eukaryon expression plasmid p3.1-MbIFN transient transfection Chinese hamster ovary celI, establish the control group of transfection empty carrier pcDNA3.1 simultaneously.Set up indirect ELISA detection method to screen positive hybridoma cell, all monoclonal antibody strains of screening all can detect the reactivity with eukaryotic expression ox IFN-γ by Western-blotting method, but only have 3D7 strain can only with the ox IFN-γ of eukaryotic expression, and it is reactionless with ox IFN-γ protokaryon product, described can only with the hybridoma cell strain of the ox IFN-gamma reaction of eukaryotic expression, called after 3D7, Classification And Nomenclature is: hybridoma cell strain, be deposited 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 is numbered: CGMCC No.9329, preservation date is on June 25th, 2014.Hypotype qualification result shows that the antibody subtype of monoclonal antibody 3D7 strain is IgG1/ κ.

Further, the invention allows for the monoclonal antibody of being secreted by described hybridoma cell strain.And

The monoclonal antibody of described hybridoma cell strain or its secretion detects the application in natural cattle gamma interferon reagent in preparation.

Accompanying drawing explanation

Fig. 1 is the electrophoresis result of reverse transcription PCR amplification ox IFN-γ encoding sequence;

M is DL2000Marker, and 1,2 swimming lanes are amplified production, sheet segment length 334bp;

Fig. 2 is the PAGE electrophoretic analysis result of the recombinant protein rIFN after purifying;

M is albumen Marker, and swimming lane 1 is through liquid, and swimming lane 2,3,4 is the protein sample of 500mM imidazoles three wash-out results;

Fig. 3 is the Western-bloting detected result of the recombinant protein rIFN that prokaryotic expression obtains;

The recombinant protein rIFN of purifying is carried out PAGE electrophoretic separation, and after transferring film, priority is with little mouse-anti 6His antibody as primary antibodie, and sheep anti mouse two anti-reflective of HRP mark is answered, and the recombinant protein rIFN showing band and expection is in the same size, about 18kDa;

Fig. 4 is the pcr amplification result of the signal peptide sequence (MIE) of mouse IFN-γ and the removal signal peptide fragment (d-bIFN) of ox IFN-γ encoding sequence;

M is DL2000Marker, and (A) is depicted as MIE fragment, length 113bp; (B) d-bIFN fragment is depicted as, length 333bp;

Fig. 5 is the result of overlapping pcr amplification ME-bIFN fragment;

M is DL2000Marker, and 1,2 swimming lanes are amplified production, sheet segment length 420bp;

Fig. 6 is antibody titer indirect ELISA detected result after mouse eukaryon expression plasmid p3.1-MbIFN five immunity;

Fig. 7 is indirect immunofluorescene assay result after merging first time;

Top is numbered the name being accredited as positive colony, and negative control uses SP20 cells and supernatant as primary antibodie;

Fig. 8 is reactive Western-Blotiing detected result 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 the Chinese hamster ovary celI lysate of the unloaded pcDNA3.1 of transfection, and swimming lane 3 is the Chinese hamster ovary celI lysate of transfection eukaryon expression plasmid p3.1-MbIFN;

Fig. 9 is the hypotype qualification of monoclonal antibody 3D7.

The hypotype of the reaction result representative in every hole has marked the left side in figure, and result shows that the hypotype of monoclonal antibody 3D7 strain is IgG1.

Embodiment

Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.

In the following example, method therefor is ordinary method if no special instructions.

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

1, the separation of ox peripheral blood lymphocyte and activation

Bovine jugular vein is aseptic adopts anticoagulation, adds monoploid and amasss Hank ' s liquid, mix gently, be carefully added to slowly on isopyknic ox lymphocyte separation medium.The centrifugal 20min of 2000r/min, draws the white cellular layer on separation surface, is collected in another centrifuge tube, washes the centrifugal 10min of twice, 1500r/min with RPMI-1640.The RPMI-1640 nutrient solution of collected cell containing 10 μ g/ml concanavalin As (ConA, Sigma Products) is suspended, cell density about 10 ⁷individual, ml, in 37 DEG C, CO ₂incubator stimulates cultivates 8h.After collected by centrifugation stimulates, lymphocyte is in centrifuge tube, and PBS washes three times, for extracting total serum IgE.

2, the extraction of ox lymphocyte total serum IgE

Centrifugation after the lymphocyte PBS cultivated washes three times, dissolves 2.5 × 10 according to 1ml ⁶the amount of individual cell adds TRIzol, pressure-vaccum mixing gently.Room temperature places 10min makes nucleoprotein complex dissolve completely, adds 200 μ l chloroforms, cover tightly lid, concuss 15s by every milliliter of TRIzol, in 4 DEG C of centrifugal 10min of 12000r/min after room temperature placement 2 ~ 3min.

The phase transition of careful absorption upper water is to new centrifuge tube, and add equal-volume Virahol, room temperature leaves standstill 10min, in 4 DEG C of centrifugal 10min of 12000r/min.After abandoning most supernatant, precipitation washes twice with 75% ethanol, and room temperature dries up precipitation (generally placing 5 ~ 10min), with DEPC water dissolution RNA, the quality of RNA is extracted in agarose gel electrophoresis analysis, and ultraviolet spectrophotometer mensuration concentration is placed on ﹣ 80 DEG C and saves backup.

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

Adopt the number of the writing to peptide fragment (called after d-bIFN) of Access RT-PCR kit (Promega Products) one step amplification ox IFN-γ (bIFN-γ) gene coding region.Primer sequence: IF-F (BamHI): 5 '-catGGATCCccagggccaattttttagag; IF-R (SalI): 5 '-catGTCGACcgttgatgctctccggc (capitalization part represents restriction enzyme enzyme recognition site sequence).Amplification system is: DEPC process water 11 μ l, AMV/TFI 5 × Buffer 5 μ l, primer I F-F 1 μ l (50pmol), primer I F-R1 μ l (50pmol), 2.5mM dNTP 2 μ l, 25mM MgSO ₄1 μ l, AMV ThermoScript II 0.5 μ l, TFI archaeal dna polymerase 0.5 μ l, total serum IgE 3 μ l (0.5 μ g).RT-PCR amplification program is: 48 DEG C of reverse transcription 45min, enters circulation (94 DEG C of 1min after 94 DEG C of 2min; 56 DEG C of 1min; 72 DEG C of 0.5min), increase 40 circulation after, 72 DEG C of 5min, rear 4 DEG C of preservations of having increased, product with agarose gel electrophoresis analytical results as Fig. 1.

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

The d-bIFN fragment Omega purification kit that RT-PCR amplification obtains is carried out purifying, purified product restriction enzyme (Thermo Products) BamHI and SalI double digestion process, prokaryotic expression carrier pET30b (﹢) does same double digestion process.Enzyme is cut system and is prepared according to product description, in 37 DEG C of digestion 4h.Agarose gel electrophoresis reclaims digestion products, with T4DNA ligase enzyme (Thermo Products), d-bIFN fragment is connected to pET30b (﹢) carrier, recombinant plasmid called after p30b-bIFN.Linked system, with reference to specification sheets preparation, connects 4h in 22 DEG C.

Connect product conversion DH5 α, picking list bacterium colony is cultivated, transformant is identified with BamHI and SalI double digestion after extracting plasmid, what enzyme cut out about 333bp size fragment is positive colony, selecting two positive colonies send order-checking company to carry out sequencing, the clone that sequence conforms to expection 100% remains, recombinant plasmid called after p30b-bIFN.By this recombinant plasmid transformed E. coli expression strains BL21 (DE3), picking list colony inoculation in LB substratum, 37 DEG C of 200r/min jolting overnight incubation.Get overnight culture and be forwarded to 200ml fresh LB in the ratio of 1 ︰ 100,37 DEG C of 200r/min joltings are cultured to bacterium liquid OD ₆₀₀when=0.6, add the IPTG induction target protein expression that final concentration is 1mM, cultivate 24h in 16 DEG C of 200r/min joltings.

Collected by centrifugation induction bacterium liquid, with 10ml binding buffer liquid (20mM Tris-HCl, 150mM NaCl) resuspended thalline, utilizes the broken cracking thalline of pressure breaking instrument (pressure parameter is set to 26Kpsi).Lysate, in 4 DEG C of centrifugal 20min of 12000r/min, shifts supernatant, 0.45 μm of frit supernatant.Get 2ml nickel ion affinity chromatograph resin (Ni Sepharose 6Fast Flow, GE Products) homogenate (resinous 1ml) is in clean void column (BioRad Products), add 5 times of column volume deionized waters and flow through resin, then balance resins with 5 times of column volume binding buffer liquid.Add the Sample supernatants after filtration, after it flows to end, flow through resin with the binding buffer liquid of 20 times of column volumes, wash away the foreign protein in sample.With elution buffer (20mM Tris-HCl, 150mM NaCl, 500mM imidazoles) the wash-out recombinant protein rbIFN-γ of 3 times of column volumes, point 3 wash-outs, collect elution samples respectively, each sample of SDS-PAGE electrophoretic analysis, result as shown in Figure 2.

The anti-6His antibody of rbIFN-γ purified product is verified: the fusion rotein of purifying is carried out SDS-PAGE electrophoretic separation, with half-dried electrophoretic blotting instrument, albumen is gone to nitrocellulose filter from gel electricity, and transferring film condition is voltage 15V, time 30min.The nitrocellulose filter turning upper albumen is closed with the skimming milk of 10%, TBST washes film, add anti-His tag monoclonal antibody (5000 times of dilutions) 37 DEG C, 70r/min in conjunction with 2h, wash film, add the goat anti-rabbit igg (5000 times of dilutions) of horseradish peroxidase-labeled afterwards, 37 DEG C, 70r/min washes film in conjunction with 2h, TBST, abundant washing with the colour developing of DAB substrate solution, identifies expression product afterwards.Result as shown in Figure 3.

Embodiment 2: construction of eukaryon expression plasmid for expressing

The genomic extraction of mouse cell: the spleen getting mouse, uses blood/cell/tissue genome DNA extracting reagent kit (Tian Gen biochemical technology company limited product) to extract genomic dna, operates to specifications.

With mouse cell genome for template, the signal peptide sequence (called after MIE) of amplification IFN-γ, primer is: EXIF-U:5 '-gatGGTACCatgaacgctacacactgcatc; EXIF-OR:5 '- ttcttcaaATGATGATGATGATGATGaatgactgtgccgtggcag (underscore part is the complementary sequence of over-lap PCR), uses Pfu high-fidelity DNA polymerase (Fermentas Products).Amplification system is: aqua sterilisa 26 μ l, 5 × Buffer 5 μ l, primer EXIF-U 2 μ l (20umol), primer EXIF-OR 2 μ l (20umol), dNTP (2.5mM) 4 μ l, archaeal dna polymerase 1 μ l, genomic dna 5 μ l.Amplification program is: enter circulation (95 DEG C of 30sec after 95 DEG C of denaturation 5min; 56 DEG C of 30sec; 72 DEG C of 18sec), after 30 circulations of increasing, 72 DEG C of 5min, rear 4 DEG C of preservations of having increased.

The amplification of the removal signal peptide fragment (called after d-bIFN) of ox IFN-γ encoding sequence: with recombinant plasmid p30b-bIFN (embodiment 1 builds) for template, the number of the writing to peptide fragment d-bIFN of amplification ox IFN-γ.Amplification the primer be EXIF-OF:5 '- cATCATCATCATCATCATttgaagaattggaaagatgaaagtg (underscore part is the complementary sequence of over-lap PCR); EXIF-D:5 '-gatCTCGAGttacgttgatgctctccggc, use Pfu high-fidelity DNA polymerase (Fermentas Products), amplification system is: aqua sterilisa 26 μ l, 5 × Buffer 5 μ l, primer EXIF-OF 2 μ l (20umol), primer EXIF-D 2 μ l (20umol), dNTP (2.5mM) 4 μ l, archaeal dna polymerase 1 μ l, plasmid DNA (1ug/ml) 1 μ l.Amplification program is: enter circulation (95 DEG C of 30sec after 95 DEG C of denaturation 5min; 54.5 DEG C 30sec; 72 DEG C of 45sec), after 30 circulations of increasing, 72 DEG C of 5min, rear 4 DEG C of preservations of having increased.The PCR primer agarose gel electrophoresis analytical results of MIE and d-bIFN fragment is shown in Fig. 4.

The PCR primer of MIE and d-bIFN two fragments amplification obtained carries out purifying with Omega purification kit respectively.Purified product is utilized to carry out over-lap PCR amplification as template, two fragments to be connected.The Outside primer used that increases is EXIF-U and EXIF-D.Amplification system divides two parts, (1) aqua sterilisa 33.5 μ l, 5 × Buffer5 μ l, dNTP (2.5mM) 3 μ l, MIE fragment (30ug/ml) 1.5 μ l, d-bIFN fragment (27ug/ml) 6 μ l, archaeal dna polymerase 1 μ l.(2) aqua sterilisa 31 μ l, 5 × Buffer 5 μ l, primer EXIF-U 4 μ l (20umol), primer EXIF-D 4 μ l (20umol), dNTP (2.5mM) 5 μ l, archaeal dna polymerase 1 μ l.Amplification program is: system (1) first increased, and enters circulation (95 DEG C of 30sec after 95 DEG C of denaturation 1min; 55 DEG C of 30sec; 72 DEG C of 1min 10sec), after 5 circulations of increasing, 72 DEG C extend 10min.Then the system prepared (2) is added in system (1) and fully mixes, be further divided into two pipes and continue amplification, after 95 DEG C of denaturation 1min, enter circulation (95 DEG C of 30sec; 56.5 DEG C 30sec; 72 DEG C of 1min 20sec), after 25 circulations of increasing, 72 DEG C extend 10min, rear 4 DEG C of preservations of having increased.PCR primer (called after ME-bIFN) the agarose gel electrophoresis analytical results that amplification obtains is shown in Fig. 5.

Fragment ME-bIFN rubber tapping after agarose gel electrophoresis is separated is reclaimed (Omega glue reclaims test kit), reclaim product endonuclease (Thermo Products) KpnI and XhoI double digestion process, the same restriction endonuclease process of eukaryotic expression vector pcDNA3.1, enzyme is cut system and is prepared according to product description, in 37 DEG C of digestion 4h.Agarose gel electrophoresis reclaims digestion products, with T4DNA ligase enzyme (Thermo Products), ME-bIFN fragment is connected to pcDNA3.1 carrier.Linked system, with reference to specification sheets preparation, connects 4h in 22 DEG C.

Connect product conversion DH5 α, picking list bacterium colony is cultivated, transformant is identified with KpnI and XhoI double digestion after extracting plasmid, what enzyme cut out about 420bp size fragment is positive colony, selecting two positive colonies send order-checking company to carry out sequencing, the clone that reservation queue conforms to expection 100%, recombinant plasmid called after p3.1-MbIFN.

Embodiment 3: animal immune

Docking blood sampling before mouse immune, separation of serum, as negative control.Spend in intracellular toxin and carry plasmid kit (Omega Products) and prepare immune recombinant plasmid p3.1-MbIFN, immunizing dose is every mouse 100 μ g plasmid DNA, and left and right thigh respectively injects 50 μ g.Concrete operations are as follows: first drenched by the hair alcohol of mouse femoribus internus muscle, with microsyringe (Town in Shanghai booth microsyringe factory, specification is 100 μ l) draw appropriate volume remove intracellular toxin plasmid solution (containing 100 μ g plasmids), carry out injecting (depth of penetration is 2mm ~ 2.5mm, notes avoiding muscle to prick the degree of depth of wearing or pricking inadequate) in mouse left and right inboard leg muscle each point 2.Want during injection slowly, to stop after injection to take out syringe needle again in several seconds, in case leak-stopping liquid.After injection, the hair of large for mouse leg outer side is drenched, by living gene importing equipment (NingBo XinZhi Biology Science Co., Ltd's product, model WJ-2002) electricity be clipped in the both sides that thigh drenches, voltage is adjusted to 100V, touch shock button, can be observed mouse whole body shake because of electric shock, namely complete and once shock by electricity.The positive and negative electrode of introducing apparatus is exchanged, again touches shock button, carry out second time electric shock.The thigh that opposite side has injected plasmid carries out same shock treatment by above operation.

Immunity carried out booster immunization after 15 days, and the operating process of immunity is the same, after this carries out a booster immunization at interval of 15 days.Three exempt to carry out docking blood sampling to mouse, separation of serum in latter 14th day, and detection is tired, and after this within the 14th day, take a blood sample and separation of serum after each immunity, the indirect ELISA method utilizing the ox IFN-γ of prokaryotic expression to set up as coating antigen detects immunizing potency.After the 5th immunity, immunizing potency reaches expection (shown in Fig. 6), and spleen cell and the murine myeloma cell (SP2/0) of getting mouse carry out cytogamy.

Embodiment 4: cytogamy

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

From liquid nitrogen container, taking out myeloma cell's cryopreservation tube, putting into 37 DEG C of water-baths to melting completely; Centrifugal 3 ~ the 5min of 1000r/min; Abandon supernatant liquor, with perfect medium (RPMI-1640, add 20% foetal calf serum and 1% mycillin dual anti-) cell of sinking is dispelled suspension, add in the Tissue Culture Flask having adequate nutrition liquid, mix rearmounted 37 DEG C, 5%CO ₂cultivate in incubator.Observe the growing state of myeloma cell next day, as circular, bright and in slight adherent growth, then illustrate that myeloma cell growth is good; As found, some cell shades, and floats in liquid, then can change liquid, discards floating dead cell, and the cell major part of survival can adherent growth breeding.1 ︰ 3 Secondary Culture when cell covers with individual layer, (each fusion needs about 1.5 × 10 to cell concentration needed for enlarged culturing to cytogamy always ⁵individual SP2/0 cell).

Before fusion by PEG, 40ml stop buffer (basis 1640) and HAT substratum (RPMI-1640, the foetal calf serum of interpolation 20%, the mycillin of 1% dual anti-and 1% HAT) put into 37 DEG C of pre-hot reserves of incubator.

2, the preparation of feeder cell:

Get a non-immune BALB/c mouse, eye socket bloodletting, collection serum is negative serum.After four limbs are fixing, tear initiation skin, exposes peritonaeum, peritonaeum is cut an osculum (in belly central authorities), then use suction pipe 2 ~ 3ml (depending on mouse size) RPMI-1640 basal liquid to inject mouse peritoneal, inhale and beat several times, liquid sucking-off is put in 50ml centrifuge tube.Repeat to wash one's hands and face once with RPMI-1640 basal liquid, containing Turnover of Mouse Peritoneal Macrophages in the liquid of collection.The centrifugal 10min supernatant discarded of 1000r/min, sedimentation cell is placed in 37 DEG C, 5%CO after suspending with HAT substratum ₂stand-by in incubator.

3, the preparation of immune spleen cell:

Get the BALB/c mouse one of immunity, eye socket sacrificed by exsanguination (collect serum, be positive serum), soaks 5 ~ 10min sterilization in 75% alcohol.Be fixed on by the mouse disinfected and dissect on plate, cut off skin and expose peritonaeum, more carefully cut off peritonaeum, expose spleen, tweezers press from both sides out spleen, remove the fatty tissue of adhesion on spleen with scissors, break spleen adventitia, put into the homogenizer of sterilizing.In homogenizer, add 5ml RPMI-1640 basal liquid, grinding, squeezes out splenocyte, adds 10ml RPMI-1640 basal liquid.After leaving standstill 2min, draw 5ml upper strata cell suspension in another aseptic 50ml centrifuge tube.Add 10ml RPMI-1640 liquid again in homogenizer, after pressure-vaccum mixing, leave standstill 2min, draw 10ml upper strata cell suspension in centrifuge tube, then repeat above step once.The liquid collected contains immune mouse spleen cell, in the centrifugal 10min of 1000r/min, and supernatant discarded, the resuspended rear counting of cell precipitation.

4, cytogamy:

By 1 × 10 ⁷~ 2 × 10 ⁷individual SP2/0 cell and 1 × 10 ⁸individual immunocyte (both ratios are 1 ︰ 5 ~ 1 ︰ 10) mixes, the centrifugal 8min of 1000r/min in 50ml centrifuge tube.Abandon most supernatant, and blot residual liquid with the filter paper of sterilizing, knock at the bottom of pipe gently, make cell precipitation loosening slightly, be convenient to the even action of PEG to cell.To be dipped in 37 DEG C of water-baths bottom the centrifuge tube that cell mixture is housed, in 1min, slowly add the 50%PEG solution (Sigma Products) of the pre-temperature of 0.8ml to 37 DEG C, limit edged stirs with pipette tip gently.Continue to stir 30s after adding PEG solution.After leaving standstill 30s, slowly add the RPMI-1640 basal liquid of the pre-temperature of 10ml.Add speed strictly to control: within the 1st minute, dropwise instill 1ml, within the 2nd minute, add l ml, within 3rd ~ 4 minutes, add 3ml, within the 5th minute, add 5ml, at the uniform velocity add, and constantly stir gently.Finally add 30ml RPMI-1640 liquid again.In the centrifugal 5min of 1000r/min, abandon supernatant, the cell suspension after this being merged with the HAT substratum containing feeder cell, adds appropriate HAT substratum as required, divides and plant in 96 well culture plates, about 250 μ l/ holes.Single cell fusion is inoculated in 4 piece of 96 orifice plate.In 37 DEG C, cultivate in 5%CO2 incubator.

Embodiment 5: filtering hybridoma and cloning

1, the screening of cell strain:

Within after cytogamy second day, start observe, have pollution-free, within the 4th day, suck 100 μ l substratum, add 100 μ lHT substratum (RPMI-1640, add 20% foetal calf serum, 1% mycillin dual anti-and 1% HT).When the single colony of cell is high-visible, colonization by there being the hole of multiple colony to carry out list in a hole.Concrete operations are as follows, by inverted microscope (the EVOS fl of band display screen, AMG Co. of U.S. product) remove in Bechtop, observe the distribution of colony in 96 porocyte culture plates, select after having the hole of many colonies, with capillary pipet by the whole sucking-off as far as possible of the cell of certain colony in the visual field.Capillary pipet should burn camber by one in advance, with handled easily, and carries out autoclaving before use.The cell of sucking-off is transferred in the hole of new 96 porocyte culture plates, and Kong Zhongxu adds 200 μ l HT substratum in advance.So repeatedly, the colony in many colonies hole is shifted respectively, to ensure each colony grown out can detect separately whether secrete required antibody when first time screens.Treat that cell colony grows to 1/4 ~ 1/3 of culture hole, when substratum slightly turns yellow, draw culture supernatant and carry out IIF detection.

2, indirect immunofluorescene assay positive cell strain:

Chinese hamster ovary celI (Chinese hamster ovary cell) is cultivated with the perfect medium of DMEM in high glucose (Gibco Products) (add 10% foetal calf serum and 1% mycillin is dual anti-), when being cultured to desired number, after being digested, paving 96 porocyte culture plates, make cell about be paved with 60 ~ 70% of hole.After growing about 12h, when cell covers with 80% of hole, by eukaryon expression plasmid p3.1-MbIFN transient transfection Chinese hamster ovary celI, establish the control group of transfection empty carrier pcDNA3.1 simultaneously.The transfection reagent used is SignaGen Products PolyJet DNA transfection in vitro reagent, operation steps to specifications in the flow process of general transfection carry out.After transfection, 12h changes maintain base (DMEM in high glucose, adds 2% foetal calf serum and 1% mycillin is dual anti-), continues to cultivate supernatant discarded after 24h, with PBS solution washed cell twice.In room temperature fixed cell 10min, discard stationary liquid with 80% acetone (﹣ 20 DEG C of precooling 30min), wash three times with PBS, soak 5min at every turn.The cell of transfection recombinant plasmid p3.1-MbIFN and empty carrier pcDNA3.1 adds hybridoma culture supernatant to be checked respectively; If one group of Positive control wells, add serum (the i.e. foregoing positive serum of immune mouse respectively; 1 ︰ 50 times dilution); One group of negative control hole, adds (the i.e. foregoing negative serum of the serum before mouse immune respectively; 1 ︰ 50 times dilution).After application of sample, cell plate are placed in 37 DEG C of incubation 30min, and after supernatant discarded, PBS washes three times; Add the sheep anti-mouse igg (Sigma Products, 1 ︰ 100 times dilution) that fluorescein isothiocyanate (FITC) marks, 37 DEG C of incubation 30min, PBS washes three times, observation of cell fluorescence phenomenon under fluorescent microscope.If negative control group is without visible fluorescence phenomenon, the empty carrier pcDNA3.1 hole of positive controls is without visible fluorescence, and obvious fluorescence appears in the recombinant plasmid p3.1-MbIFN hole of positive controls, then experimental result is set up; Culture supernatant makes the Chinese hamster ovary celI of transfection recombinant plasmid p3.1-MbIFN show fluorescence, and the hybridoma cell strain simultaneously reacting not aobvious fluorescence with the Chinese hamster ovary celI of transfection empty carrier pcDNA3.1 is positive colony (as shown in Figure 7).

3, the cloning of hybridoma

Select the cell in positive hole according to detected result, carry out subclone with limiting dilution assay.The peritoneal macrophage getting healthy mice according to aforesaid method before clone prepares feeder layer, prepares feeder cell, with HT perfect medium resuspended be placed in incubator for subsequent use.The hybridoma that will clone is blown down gently in culture hole, calculates viable count with blood cell counting plate.According to count results, cell HT perfect medium is diluted to 5,10,50 cell/ml.The cell suspension of above three concentration is added 96 well culture plates respectively, 100 μ l/ holes, make every hole respectively containing 0.5,1 and 5 cell.Again feeder cell are added drop-wise in hole after hybridoma paving hole completes.

Cultivate and add HT perfect medium one on the 4th day, within 5th ~ 6 days, examine the growing state of each hole inner cell, and record.After clone 7th ~ 9 days, when cell clone covers with 1/4 ~ 1/3 of hole, then detect by above-mentioned indirect immunofluorescence.If detect Growth of Cells hole have corresponding antibodies, antibody titer can be selected high, in single colony growth, the hole that cellular form is good, continues same method subclone again.This time after subclone, the hole indirect immunofluorescence detected result growing cell if all be all the positive, then strain is built in the hole can selecting single colony, cellular form good.

Embodiment 6: hybridoma frozen

The hybridoma determining strain is transferred to 24 hole enlarged culturing respectively from 96 holes, after cell covers with, collecting cell is in centrifuge tube, cell counting is carried out after mixing, in the centrifugal 8min of 1000r/min, supernatant discarded, according to count results, add the cells frozen storing liquid (40%DMEM of 4 DEG C of appropriate precoolings, 50% foetal calf serum, 10%DMSO) re-suspended cell, make final concentration of cells be 2 ~ 3 × 10 ⁶individual/ml.Add 1ml cell suspension in each cell cryopreservation tube, put into cell cryopreservation box (U.S. NALGENE Products), then spend the night in ﹣ 70 DEG C of Temperature drop in refrigerators, finally proceed to the medium-term and long-term preservation of liquid nitrogen container.

Embodiment 7: the preparation of ascites

Get the mouse in 8 ~ 10 week age, abdominal injection Freund's incomplete adjuvant, injected dose 0.5ml/ only.Within 7 ~ 10 days, pneumoretroperitoneum injects each hybridoma determining strain respectively, and injection volume is 5 × 10 ⁵~ 10 ⁶/ only, after the cell of general 24 porocyte plates covers with, the cell concentration of collection is enough.Injection observes mouse state one day after, and after 7 ~ 10 days, after seeing the obvious bulging of mouse web portion, extract ascites, 1000r/min centrifugal 8min centrifuging and taking supernatant, is the mouse ascites that each strain monoclonal antibody is corresponding.

Embodiment 8: ascites and eukaryotic expression ox IFN-γ (natural ox IFN-γ) reactivity are verified (Western-blotting)

Chinese hamster ovary celI spreads six orifice plates, and distinguish transfection recombinant plasmid p3.1-MbIFN and empty carrier pcDNA3.1 when cell confluency degree reaches about 80%, concrete operations as described in example 5 above.36h after transfection, abandons culture supernatant, with PBS washed cell one time, each hole adds 100ul cell pyrolysis liquid (the green skies, Shanghai), with cell sleaker, cell is scraped, inhale in 1.5ml centrifuge tube, in leaving standstill the foam cancellation making sample produce on ice after repeatedly blowing and beating.Each sample adds 25ul albumen Loading Buffer (Fermentas Products), boiling water bath 10min after mixing.Until sample be placed on ice completely cooling after in the centrifugal 1min of 10000r/min, get supernatant and carry out PAGE electrophoresis.With half-dried electrophoretic blotting instrument, albumen is gone to nitrocellulose filter from gel electricity, transferring film condition is voltage 15V, time 30min.The nitrocellulose filter turning upper albumen is closed with the skimming milk of 10%, TBST washes film, add mouse ascites corresponding to each strain monoclonal antibody (1000 times of dilutions), in 37 DEG C, 70r/min is in conjunction with 2h, TBST washes film, adds anti-(the KPL Products of sheep anti mouse two of Dylight680 mark afterwards; 10000 times of dilutions), in 37 DEG C, 70r/min is in conjunction with 1h, TBST washes film, Infrared fluorescence scanner (ODYSSEY is used after abundant washing, U.S. Li-cor Products) observe and record result, all monoclonal antibody strains of screening all can detect the reactivity with eukaryotic expression ox IFN-γ by Western-blotting method, but only have 3D7 strain can only with the ox IFN-γ of eukaryotic expression, and reactionless with ox IFN-γ protokaryon product, the Western-blotting detected result of 3D7 strain is as shown in Figure 8.

Described 3D7 strain is deposited 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 is numbered: CGMCC No.9329, and preservation date is on June 25th, 2014.

Embodiment 9: hypotype qualification (ELISA) of monoclonal antibody 3D7 strain

With antibody subtype identification kit SBA Clonotyping System-HRP (article No. 5300-05) of SouthernBiotech company, monoclonal antibody 3D7 strain is carried out to the identification and analysis of hypotype.Method is as follows: dissolve 15mg ABTS substrate pulvis with 1ml distilled water and be prepared into ABTS substrate conserving liquid, keep in Dark Place in 2 ~ 8 DEG C.Capture antibody PBS damping fluid (pH7.4) is diluted to the concentration of 5 ~ 10ug/ml, gets 0.1ml and add in the hole of elisa plate.In 4 DEG C of moistening environment after incubation 12h, discard liquid in hole, wash three times with the PBS containing 0.05% tween.Discard in hole after washings, the PBS solution containing 1%BSA is filled it up with in every hole, and incubated at room is 1h at least, keeps slight oscillatory when hatching.After discarding liquid, wash three times.Every hole adds the culture supernatant of hybridoma, 0.1ml, incubated at room 1h, keeps slight oscillatory when hatching.After discarding liquid, wash three times.Dilute the enzyme labelled antibody that each hypotype is corresponding, Nei Mei hole, hole corresponding in elisa plate adds 0.1ml enzyme labelled antibody.Incubated at room 1h, keeps slight oscillatory when hatching, and discards in plate and washs five times after liquid.Add 0.2ml ABTS substrate conserving liquid in 10ml citrate buffer (pH4.0) and be made into substrate solution, every hole adds 0.1ml substrate solution and develops the color at room temperature placement 10 ~ 20min.Absorbance is read in OD450.

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

Hypotype qualification (OD450) of table 1. monoclonal antibody 3D7.

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

Sequence table

Claims (4)

1. a strain can resist the hybridoma of natural cattle gamma interferon monoclonal antibody by stably excreting, called after 3D7, be deposited 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 is numbered: CGMCC No.9329, and preservation date is on June 25th, 2014.

2. the monoclonal antibody of being secreted by hybridoma cell strain according to claim 1.

3. hybridoma cell strain according to claim 1 detects the application in natural cattle gamma interferon reagent in preparation.

4. monoclonal antibody according to claim 2 detects the application in natural cattle gamma interferon reagent in preparation.