CN105481957A - Platanus pollen allergen Pla a 3 and monoclonal antibody thereof - Google Patents

Abstract

The invention discloses a platanus pollen allergen Pla a 3, a monoclonal antibody thereof and a preparation method of the monoclonal antibody. The platanus pollen allergen Pla a 3 is separated from phoenix tree pollen in a purification mode, and the apparent molecular weight of fusion protein SUMO-Pla a 3 of the platanus pollen allergen Pla a 3 is measured as 28 kDa through SDS-PAGE. The protein Pla a 3 is separated from the phoenix tree pollen in the purification mode, the molecular weight of the fusion protein SUMO-Pla a 3 of the platanus pollen allergen Pla a 3 is measured as 28 kDa through SDS-PAGE. The monoclonal antibody prepared through the platanus pollen allergen Pla a 3 is characterized in that the monoclonal antibody can be specifically combined with the platanus pollen allergen Pla a 3 and plays an important role in diagnosis and treatment of allergic skin diseases, asthmas and other I-type allergic diseases caused by phoenix tree pollen.

Description

Platanus pollen allergen Pla a 3 and monoclonal antibody thereof

Technical field

The invention belongs to biological technical field, be specifically related to a kind of Platanus pollen allergen Plaa3 and monoclonal antibody thereof.

Background technology

Phoenix tree is the green plants of a large amount of plantation on the market, and spring blooms, 4 ~ May of florescence, and its pollen can cause the disease such as allergic dermatosis and allergic asthma.

Pollinosis is mostly relevant with the growth and decline of allergen in surrounding environment, is a kind of Seasonal diseases, with the most susceptible disease of spring and summer, in northern China, and two peak periods mainly 4 ~ May and 8 ~ September that annual pollinosis is fallen ill ^[1].The large drying of the northern area of China wind, south is moistening rainy, and therefore pollinosis is main mainly with the north, takes second place in south.Visible, pollinosis is relevant with weather condition, therefore, concerning pollinosis patient, understands that to affect the meteorological conditions that Pollens sends out rule be very important ^[2].

There are two class patient morbiditys higher: one is the people having atopic physique.Two is repeated multiple times people being exposed to extraneous allergen.This two classes patient has allergic constitution usually, and namely antigen more easily produces the distinct antibodies IgE more than normal people to external world, has certain heredity and familial.So, this two classes patient comparatively easily simultaneously or first future trouble eczema, drug allergy and bronchial asthma etc.

Platanus hispanica pollen is important pollen in spring, is enrolled pollen sensitization group in spring in recent years, is conventionally used for desensitization treatment.But its anaphylactogen is known little about it.Now had report to have employed the method for ELISA and SDS-PAGE, to the purifying of platanus hispanica pollen anaphylactogen Plaa1 and Plaa2, clone etc. has made research ^[3], detect IgE in the serum of the patient of two kinds of pollen allergens and platanus hispanica pollen allergy respectively by ELISA method reactive, result shows two kinds of recombinant proteins all has immunologic competence, good with the reactivity of serum ^[4].In addition, the extraction of allergen in plant pollen, the existing a lot of report of Purification and property research.Comprise the anaphylactogen Cryj2 extracted from cryptomeria ^[5]; The molecular weight extracted from rape is the anaphylactogen of 43KDa ^[6]; The molecular weight extracted from tomato is the anaphylactogen of 46KDa ^[7]; The article of these pollen allergens is only clone corresponding allergen gene, builds prokaryotic expression carrier, carries out prokaryotic expression and purification and activity identification.

Immunoblotting is the method according to certain albumen in the specific binding detection of complex sample of antigen-antibody.This method is a kind of new immune biochemical technology grown up in gel electrophoresis and solid-phase immunoassay technical foundation.Because immunoblotting has the very high resolution of SDS-PAGE and the high specific of solid-phase immunoassay and susceptibility, now become a kind of routine techniques of analysis of protein.Immunoblotting is usually used in identifying certain albumen, and can carry out quantitative and semi-quantitative analysis to albumen.In conjunction with chemiluminescence detection, can the expression amount difference of more multiple sample same protein simultaneously.

Early stage is to the existing research of platanus hispanica pollen allergen.Li Yali, Sun Xiuzhen etc. adopt the methods analyst such as SDS-PAGE, immunoblotting platanus hispanica pollen allergen, and find that Platanus pollen allergen is containing 6 kinds of primary protein component, the protein content of molecular mass between 22 ~ 71kDa is high, is main Allergen; Molecular mass between 14 ~ 16kDa protein be secondary Allergen.Wherein have 4 protein bands can be combined with platanus hispanica pollen specific IgE, its molecular mass be 50,39,22,16kDa ^[8].

Summary of the invention

An object of the present invention is to provide Platanus pollen allergen Plaa3, further, the object of this invention is to provide Platanus pollen allergen Plaa3 and monoclonal antibody, and the preparation method of monoclonal antibody.

To achieve these goals, the present invention is by the following technical solutions: Platanus pollen allergen Plaa3, described allergen Plaa3 is that separation and purification obtains from platanus hispanica pollen, and measuring its fusion protein S UMO-Plaa3 apparent molecular weight by SDS-PAGE is 28kDa.

Allergen Plaa3 according to claim 1, the aminoacid sequence of described allergen Plaa3 is as shown in SEQIDN0.1.

The DNA of allergen Plaa3, the nucleotide sequence of described DNA is as shown in SEQIDN0.1.

The monoclonal antibody of Platanus pollen allergen Plaa3, described monoclonal antibody prepares for allergen with Plaa3 in platanus hispanica pollen, has the ability with Platanus pollen allergen Plaa3 specific binding.

The preparation method of the monoclonal antibody of Platanus pollen allergen Plaa3, comprises the following steps:

(1), between StuI and XhoI site goal gene Plaa3 being connected to carrier pSUMO-Mut, the recombinant plasmid pSUMO-Mut-Plaa3 of acquisition proceeds in ArcticExpress expression strain, utilizes IPTG abduction delivering target protein;

(2) by the recombinant protein ultrasonication that obtains and low-temperature centrifugation, solution supernatant is by affinity chromatography, and wash-out obtains purification of recombinant proteins;

(3) with the recombinant protein immunity Balb/c mouse after purifying, repeatedly immunity, after tail vein blood measures serum titer, gets Mouse spleen cells with myeloma cell SP2/0 merges, and screens with HAT and obtain stable hybridoma cell strain;

(4) hybridoma cell strain is expelled to the pretreated mouse peritoneal of whiteruss, gets ascites every other week, 50% saturated ammonium sulphate method and ProteinG affinity purification monoclonal antibody, obtain monoclonal antibody.

Induced expression temperature in described step 1 is preferably 11 DEG C, and induction rotating speed is 200rpm.The IPTG concentration of induction is 0.5mmol/L.

Albumen Plaa3 of the present invention is that separation and purification obtains from platanus hispanica pollen, and measuring its fusion protein S UMO-Plaa3 molecular weight by SDS-PAGE is 28kDa.The specificity of monoclonal antibody be worth thus be can with Platanus pollen allergen Plaa3 specific binding, the Clinics and Practices of the I metallergy diseases such as the allergic dermatosis caused for platanus hispanica pollen and asthma plays an important role.

Accompanying drawing explanation

Fig. 1 is embodiment pSUMO-Mut-Plaa3 plasmid enzyme restriction figure, Marker:100,250,500,750,1000,1500,2000,3000,5000; Gene Name: Plaa3; Enzyme cuts qualification: BamHI; 1 is that enzyme cuts rear plasmid; 2 is that enzyme cuts front plasmid; OD260/OD280:1.87.

Fig. 2 is embodiment pSUMO-Mut-Plaa3 vector construction collection of illustrative plates.

Fig. 3 is the SDS-PAGE result of embodiment fusion rotein pSUMO-Mut-Plaa3 abduction delivering qualification, and wherein 1 is the plaa3 protein immunoblot band without induction, and 2 and 3 is all albumen after induction, 4 be albumen through adding 0.5mMIPTG, at the temperature of 11 degree induction after supernatant, 5 is that albumen is through adding 0.5mMIPTG, precipitation at the temperature of 11 degree after induction, M is Protein Marker, is from top to bottom respectively 100.0,65.0,50.0,40.0,30.0,25.0,20.0kDa;

Fig. 4 is the SDS-PAGE result of embodiment fusion rotein pSUMO-Mut-Plaa3 separation and purification, wherein 1 is the plaa3 albumen without induction, and 2 is the elutriants after 10mM imidazoles purifying, and 3 is the target proteins after 250mM imidazoles purifying, M is Protein Marker, from top to bottom be respectively 100.0,65.0,50.0,40.0,30.0,25.0,20.0kDa;

Embodiment

Below in conjunction with specific embodiments and the drawings, the present invention is further illustrated.

The determination of platanus hispanica pollen allergen Plaa3:

PSUMO-Mut-Plaa3 plasmid construction:

By synthetic goal gene Plaa3 complete sequence (SEQIDN0.2), and between StuI and the XhoI site being connected to carrier pSUMO-Mut, the recombinant plasmid pSUMO-Mut-Plaa3 of acquisition is proceeded in ArcticExpress expression strain.The order-checking of picking positive colony, sequencing result is as follows, and single scribe area is Plaa3 gene region.

TATTTGAGGCTCACCGCGAACAGATTGGAGGT GCCATAACATGTGGTACGGTGGTGACCAGGCTGACCCCATGCCTCACCTTCTTGCGCTCTGGTGGGGCTGTAGCGCCCGCTTGCTGCAACGGCGTGAAGGCACTCAACAACGACGCTAAAACCACCCCAGACCGTCAGGCCGCTTGTGGATGTTTGAAGACTGCCTCCACCTCCATCTCCGGGATCCAGCTCGGTAACGCTGCCTCGCTTGCCGGCAAATGTGGTGTTAATCTCCCTTACAAGATCAGCCCCACCATTGACTGCTCCAAGGTGAAGTGACTCGAGCACCTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACT

Translation rear fusion protein sequence (SEQIDN0.3) is as follows: for the purpose of single scribe area, albumen Plaa3, Plaa3 albumen theoretical molecular is about 9kDa, and fusion protein S UMO-Plaa3 theoretical molecular is about about 28kDa.

MNWSHPQFEKSSGSSGGHHHHHHGGSGGSGSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG AITCGTVVTRLTPCLTFLRSGGAVAPACCNGVKALNNDAKTTPDRQAACGCLKTASTSISGIQLGNAASLAGKCGVNLPYKISPTIDCSKVK.

By pSUMO-Mut-Plaa3 vector to intestinal bacteria ArcticExpressTM (DE3): plasmid is joined in 100 μ LArcticExpressTM (DE3) competence bacteriums, put 20min on ice; 42 DEG C of heat shock 90sec, put rapidly 5min in ice; Add 600 μ L, the LB nutrient solution of 37 DEG C of preheatings; 37 DEG C, 220rpm jolting 1h, the centrifugal rear LB all coated containing 50 μ g/mLKan is dull and stereotyped, is inverted overnight incubation for 37 DEG C.

IPTG induces the expression of pSUMO-Mut-plaa3 carrier fusion: the mono-clonal on picking transformation plate is inoculated in the test tube containing the 3mLLB nutrient solution of 50 μ g/mLKan, and 37 DEG C of 220rpm joltings are spent the night; Next day is inoculated in the 30mLLB nutrient solution of 50 μ g/mLKan by 1:100, and 37 DEG C of 220rpm joltings are 0.4 (about 2h) to thalline OD600; Take out 1mL culture, the centrifugal 2min of 10000g room temperature, abandons supernatant, with the resuspended bacterial sediment of 100 μ L1 × sample-loading buffer; In remaining culture, adding IPTG to final concentration is 0.5mM11 DEG C of 220rpm jolting 8h respectively, induced fusion protein expression.

The determination (employing western blotting method) of allergen Plaa3: take out 1mL culture, the centrifugal 2min of 12000g room temperature, abandons supernatant, with the resuspended bacterial sediment of 100 μ L1 × sample-loading buffer.Apply 12% separation gel, 5% concentrated glue is separated sample.Starting voltage 70V, enters after separation gel until tetrabromophenol sulfonphthalein forward position and voltage is risen to 120V, constant voltage electrophoresis, until tetrabromophenol sulfonphthalein forward position arrives gel lower boundary, stops electrophoresis.Take off gel and carry out transferring film.60min is quoted in constant current, is transferred on pvdf membrane by albumen.Pvdf membrane is put into the plate filling confining liquid, close 3h.Be placed in primary antibodie by the pvdf membrane after closing, 4 spend night.Band TBST is carried out washing 3*10min by next day.Put into afterwards in mouse-anti (two resist), room temperature puts 2h on shaking table, terminates rear TBST and carries out washing 3*5min, carry out substrate colour developing, treats that protein band colour developing is clear, termination reaction.The result of immunoblotting as shown in Figure 3.In figure, 1 is the plaa3 protein immunoblot band without induction, and 2 and 3 is all albumen after induction, and 4 is that albumen is through adding 0.5mMIPTG, supernatant at the temperature of 11 degree after induction, 5 be albumen through adding 0.5mMIPTG, at the temperature of 11 degree induction after precipitation, M is Protein Marker.Can see that 28KD position albumen is main allergic protein from figure result.

The separation and purification of allergen Plaa3:

After resuspended for the cultivation bacterial sediment 20mL extract Ni-IDABinding-Buffer of abduction delivering, ultrasonication (power 400W, work 4sec, interval 8sec, 20min altogether), 4 DEG C of centrifugal 20min of 10000g, get supernatant, with 0.5mL/min flow velocity loading to the Ni-IDA-SepharoseCL-6B affinity column of Ni-IDABinding-Buffer pre-equilibration; Rinse with 0.5mL/min flow velocity with Ni-IDABinding-Buffer, arrive baseline to effluent liquid OD280 value; Rinse with 1mL/min flow velocity with Ni-IDAWashing-Buffer (20mMTris-HCl, 20mM imidazoles, 0.15MNaCl, pH8.0), arrive baseline to effluent liquid OD280 value; With Ni-IDAElution-Buffer (20mMTris-HCl, 250mM imidazoles, 0.15MNaCl, pH8.0) with 1mL/min flow velocity wash-out target protein, collect effluent liquid; By dialysis buffer liquid PBS (PH7.4) dialysis of the recombinant protein after wash-out, changed a dialyzate every 12 hours, after changing liquid 3 times, take out the protein liquid after dialysis, save backup in-20 DEG C, carry out 12%SDS-PAGE analysis simultaneously, result as shown in Figure 4.

The preparation of the monoclonal antibody of allergen plaa3:

Animal immune:

Get the immunity simultaneously of 6 6-8 female BAl BIc/c mouse in age in week.Plaa3 albumen is mixed first immunisation with Freund's complete adjuvant, subcutaneous injection 100ug.

Carry out second time immunity after 2-3 week, also claim booster immunization.Promise hundred immunological adjuvant is adopted to mix with antigen, subcutaneous injection 100ug.Four immunity are taken a blood sample detection afterwards, and by indirect ELISA method determination antiserum(antisera) tiring for Plaa3 albumen, waiting to tire is greater than 1:10000, selects 1-2 mouse to carry out cytogamy arrangement.

The preparation of myeloma cell:

Merge the last week, with the DMEM substratum enlarged culturing SP2/0 cell containing 10%FBS.During to fusion, cell covers with about 6 bottles of T25 Tissue Culture Flasks, and collected SP2/0 cell in 50mL centrifuge tube the same day in fusion, 1000rpm, 5min are centrifugal.Abandon supernatant, and then add 20mLDMEM basic medium, dispel cell and then count.

The preparation of splenocyte:

Four times Post-immunisation serum ELISA tires the mouse of more than 1:10000, within first 3 days, stops immunity, abdominal injection antigen 1 00ug in fusion.Merge the mouse will merged by cervical dislocation euthanasia the same day.Get spleen with 75% alcohol-pickled 5min. is aseptic, spleen is put into the culture dish cultivated on 10mLDMEM basis.Get screen cloth and put into another plate, spleen is transferred on screen cloth, with syringe heart grinding spleen.Add DMEM on screen cloth, rinse screen cloth, splenocyte is more collected in plate.Cell is moved in 10mL centrifuge tube, wash the centrifugal 5min of splenocyte twice, 1000rpm with the DMEM not containing serum, collect splenocyte counting.

Cytogamy:

Mixing myeloma cell and splenocyte, make myeloma cell be advisable at 1:20 with splenocyte number ratio.Cell is put in 50mL centrifuge tube, with the dilution of DMEM basic medium, then centrifugal 1000rpm5min.Abandon supernatant.Shake centrifuge tube makes cell even.Slowly add 0.8mL50%PEG, react 90 seconds, then add 20-30mLDMEM substratum and stop PEG, the cell merged is put in 37 DEG C of water-baths and reacts 10 minutes.1000rpm5min, abandons supernatant and then adds HATDMEM substratum. the cell merged is taped against in 96 orifice plates, every hole 100 μ L.Then Tissue Culture Plate is put into CO ₂cultivate in incubator.Merge and check for latter 4 days, hybridoma cell clone rate is more than 50%, and have a small amount of cell debris, cell growth state is good.Merge and start to carry out selective mechanisms after 10 days.

The screening of positive colony:

Detect the day before yesterday, with PBS bag by 5ug/mL antigen in elisa plate, spend the night.Draw cell conditioned medium 100ul/ hole next day and carry out ELISA detection according to ELISA result, judge that positive hole (sample well OD value/negative hole OD value >=2.1) is then judged to be positive hole.Choose the positive hole examined whole plate and detect with single track pipettor, carry out second time and confirm to detect, confirm positive hole further.Positive porocyte after determining carries out subclone.

Subclone:

Blow and beat cell in positive hole, counting, in centrifuge tube, add N/4mLDMEM substratum, get 100 μ L cell suspensions in centrifuge tube, blow even after stay 1mL, add DMEM to 4mL, blow even, stay 100 μ L (about 2) at the bottom of pipe.DMEM to 5mL is added in centrifuge tube, the first three rows of 96 orifice plates is dropped to after mixing, about 1.8-2mL is stayed at the bottom of the dropper of every hole one, add DMEM to 5mL, blow even after drop to the D of 96 orifice plates, E, F tri-row, one, every hole, about 1.5-1.8mL is stayed at the bottom of pipe, add about DMEM to 2.8-3mL, blow even after drop to the G of 96 orifice plates, H is capable, one, every hole, examine under a microscope after 7-10 days, detect the hole having clonal growth, mark monoclonal hole, the monoclonal cell of the picking positive carries out subclone again as far as possible, detect to 100% positive, choose mono-clonal hole enlarged culturing and determine strain.

A large amount of preparation of monoclonal antibody and purifying:

A large amount of preparations of monoclonal antibody:

Select the mouse in healthy 8-10 week, hybridoma precontract one week inject 0.5mL whiteruss in every Mice Body in inoculation.Every mouse peritoneal injects about 1*10 ⁶hybridoma, after inoculation, 7-10 days mouse start to produce ascites, and close observation mouse state of health and ascites sign, introduce in test tube with syringe by mouse ascites during this period, so repeatedly several times, before mouse is at death's door, exhausts ascites, put to death mouse.

The purifying of monoclonal antibody:

50% ammonium sulfate precipitation:

Dilute by the PBS solution of 4 times of ascites volume, the adherent saturated ammonium sulphate (PH7.0) slowly adding 5 times of ascites volume in beaker, controlling ammonium sulfate rate of addition is 3-4mL/min, limit edged stirs, add relief solution left standstill 2h, then by suspension liquid in the centrifugal 30min of 12000rpm.1 abandons supernatant, with the PBS dialyzate dissolution precipitation of 0.7 times of former ascites volume.

Dialyse centrifugal:

Loaded in dialysis tubing by antibody-solutions after dissolving, with PBS damping fluid (pH7.0) dialysis, period changes 3 not good liquors, and the timed interval must not be less than 5h, then by the solution of having dialysed in the centrifugal 10min of 12000rpm.Abandon precipitation, by the frit of supernatant 0.2um, gained solution is required monoclonal antibody solution.

ProteinG affinity purification:

By dialysis after monoclonal antibody ProteinG affinity purification, collect elution peak after wash-out, namely obtain the monoclonal antibody after purifying, its specificity be can with Platanus pollen allergen Plaa3 specific binding.

Anti-indirect ELISA detects antibody titer, and concrete operations are as follows:

1 bag quilt:

With PBS coating buffer, Plaa3 albumen is pressed 5ug/ml dilution, add after mixing in lath, every hole 100ul, 4 DEG C of refrigerator overnight.

Envelope antigen: Plaa3 albumen

Bag is by concentration: Plaa3 albumen presses 5ug/ml dilution, 100ul/ hole

Bag is buffered liquid: phosphate buffered saline buffer (PBS, pH7.4)

2 close:

Wrap after being got well, discard coating buffer, wash plate 3 times, every hole adds 200ul confining liquid, 37 DEG C of thermostat container 1h.Take out enzyme plate, discard interior liquid, wash plate 1 time.

3 primary antibodie reactions:

Antibody purification presses 1/500,2 times of dilutions, every hole 100ul, 37 DEG C of thermostat container 1h.

4 two anti-reflective are answered:

Take out enzyme plate, discard interior liquid, wash plate 3 times, in every hole, add the ELIAS secondary antibody that 100ul has diluted, ELIAS secondary antibody: rabbit anti-mouse-HRP, 1/5000.37 DEG C of thermostat containers 1 hour.

5 colour developings:

Take out enzyme plate, discard interior liquid, wash plate 4 times, every hole first adds 100ulTMB nitrite ion, and the depth according to color determines developing time, general 37 DEG C, 15min.

6 termination reactions:

Every hole adds 100ul1MHCL solution, termination reaction.Namely be engraved in 450nm reading in microplate reader, OD value be greater than the extent of dilution that the hole of 2.1 times of the negative control OD value of setting is corresponding, be decided to be tiring of this sample.

Through qualification, the tiring of monoclonal antibody of the platanus hispanica pollen allergen Plaa3 of preparation is greater than 512000.

Antibody purity is identified

Antibody after purifying carries out SDS-PAGE electrophoresis, coomassie brilliant blue staining.After qualification antibody purification, purity is more than 95%.

As mentioned above, although represented with reference to specific preferred embodiment and described the present invention, it shall not be construed as the restriction to the present invention self.Under the spirit and scope of the present invention prerequisite not departing from claims definition, various change can be made in the form and details to it.

Reference:

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[4] clone of < Platanus pollen allergen plaa1 and plaa2 gene and prokaryotic expression _ journey instruct good .Pdf> [J].

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Claims (6)

1. Platanus pollen allergen Plaa3, is characterized in that: described allergen Plaa3 is that separation and purification obtains from platanus hispanica pollen, and measuring its fusion protein S UMO-Plaa3 apparent molecular weight by SDS-PAGE is 28kDa.

2. allergen Plaa3 according to claim 1, is characterized in that: the aminoacid sequence of described allergen Plaa3 is as shown in SEQIDN0.1.

3. the DNA of allergen Plaa3 according to claim 2, is characterized in that: the nucleotide sequence of described DNA is as shown in SEQIDN0.1.

4. the monoclonal antibody of Platanus pollen allergen Plaa3, is characterized in that: described monoclonal antibody prepares for allergen with Plaa3 in platanus hispanica pollen, has the ability with Platanus pollen allergen Plaa3 specific binding.

5. the preparation method of the monoclonal antibody of Platanus pollen allergen Plaa3, is characterized in that comprising the following steps:

(1), between StuI and XhoI site goal gene Plaa3 being connected to carrier pSUMO-Mut, the recombinant plasmid pSUMO-Mut-Plaa3 of acquisition proceeds in ArcticExpress expression strain, utilizes IPTG abduction delivering target protein;

(2) by the recombinant protein ultrasonication that obtains and low-temperature centrifugation, solution supernatant is by affinity chromatography, and wash-out obtains purification of recombinant proteins;

(3) with the recombinant protein immunity Balb/c mouse after purifying, repeatedly immunity, after tail vein blood measures serum titer, gets Mouse spleen cells with myeloma cell SP2/0 merges, and screens with HAT and obtain stable hybridoma cell strain;

(4) hybridoma cell strain is expelled to the pretreated mouse peritoneal of whiteruss, gets ascites every other week, 50% saturated ammonium sulphate method and ProteinG affinity purification monoclonal antibody, obtain monoclonal antibody.

6. preparation method according to claim 5, is characterized in that: the induced expression temperature in described step 1 is preferably 11 DEG C, and induction rotating speed is 200rpm, and the IPTG concentration of induction is 0.5mmol/L.