CN103897060B - A kind of Aspergillus fumigatus protein monoclonal antibody and its production and use - Google Patents

Abstract

The invention discloses a kind of Aspergillus fumigatus protein monoclonal antibody and its production and use.This monoclonal antibody is CGMCC by being preserved in CGMCC preserving number? the monoclonal antibody of the hybridoma secretion of No.6606; This preparation method is by the gene of clone Aspergillus fumigatus albumen; The gene of clone is inserted expression vector, builds recombinant expression vector; Use recombinant expression vector transform competent E. coli, abduction delivering, affinity chromatography, obtain the recombinant protein of this Aspergillus fumigatus albumen of purifying; With this recombinant protein Mice Inoculated, get its splenic lymphocyte and mouse Sp2/0 myeloma cell fusion, set up hybridoma cell line, therefrom filter out this hybridoma cell strain that Aspergillus fumigatus protein antibodies is the most stable, antibody activity is the highest of secretion, the antibody that they produce is required monoclonal antibody; This monoclonal antibody can prepare aspergillosis diagnostic reagent.This monoclonal antibody there is high degree of specificity and strong avidity, preparation method is simply efficient.

Description

A kind of Aspergillus fumigatus protein monoclonal antibody and its production and use

Technical field

The present invention relates to the immunodiagnosis field of genetically engineered, cell engineering and aspergillosis, relate to a kind of Aspergillus fumigatus protein monoclonal antibody and its production and use.

Background technology

Recent research proves, larger by the serological diagnostic method potentiality measured based on fungal antigen, antibody and cyclic metabolism product ^{[1 ~ 4}].When invasive infection occurs aspergillus in vivo, become mycelia by spore state elongation growth, now in body fluid, discharge mycelian protein, and stimulate body to produce antibody.In view of this feature of aspergillus infection, it is generally acknowledged, in circulation of blood, antigen or Specific antibody rise, and imply that the development of mycelium extension and/or aspergillus infection.Because aspergillus protein component is complicated, and different its body of patient of underlying diseases, medication in early stage is in different immunological status, causes Aspergillus specific antigen in body, antibody type and amount also to have difference ^{[5 ~ 8]}.Therefore, as target antigen and the antibody of laboratory diagnosis, should select when the patient of different immunological status suffers from Aspergillosis, advantage great expression in its body also can the aspergillus proteantigen component of intense stimulus generated human antibody.

When we are with immunoproteomics method screening Aspergillus fumigatus Immunodominant Antigenic ^[9], found one with make a definite diagnosis Aspergillosis (IA) serum and strong immunoreactive new Aspergillus fumigatus albumen occur, i.e. thioredoxin reductase GliT(TR), molecular weight is about 36.2KDa.With the display of SignalP software prediction result, TR is with signal peptide (signalPprobability, 0.808); WoLFPSORT software prediction result display TR is extracellular protein (QueryProteinWoLFPSORTprediction:extr, 12.0; Cyto, 6.5; Cyto_nucl, 4.0; Mito, 3.0; Pero, 2.0); Homology analysis display is carried out with blast program, TR and humanized's protein do not have homology, also very low with the homology of other Fungal Proteins, as being 25% with the homology of Candida albicans protein, Oidium tropicale 25%, Candida glabrata 24%, monilia guilliermondii 27%, S. cervisiae 24%, Penicillium marneffei 27% ^[10], be a desirable diagnosis marker.TR can as the serodiagnosis of the specific antigens of Aspergillus fumigatus for aspergillosis especially IA.

For this reason, this albumen of our clonal expression, and establish ELISA method to detect the anti-TR level in serum, result shows, and namely aspergillus fumigatus spores occurs anti-TR antibody positive after infecting one week, and antibody titers rises fast.It is 80.9% and 96% that detection serum anti-TR antibody lacks at the relatively normal non-grain of immunologic function the Sensitivity and Specificity diagnosed in IPA patient, is obviously better than the detection (43.5%, p < 0.05) of available reagent box GM ^[10].And joint-detection anti-TR antibody and antigen TR, the susceptibility of detection can be significantly improved.

Therefore, preparing specific recognition Aspergillus fumigatus TR albumen and the monoclonal antibody of high-affinity combination with it, for preparing the reagent composition of diagnosis aspergillosis, is current biomedical sector problem demanding prompt solution.

Reference:

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2.KhanZU,AhmadS,TheyyathelAM.DetectionofAspergillusfumigatus-specificDNA,(1–3)-b-D-glucanandgalactomannaninserumandbronchoalveolarlavagespecimensofexperimentallyinfectedrats.Mycoses.2008,51(2):129-135

3.EinseleH,LoefflerJ.Contributionofnewdiagnosticapproachestoantifungaltreatmentplansinhigh-riskhaematologypatients.ClinMicrobiolInfect.2008,14(S4):37-45.

4. Li Fangqiu, Zhou Wanqing, Shi Lining, etc. the ELISA of Aspergillus fumigatus secretory protein antibody detects and diagnostic value assessment. Clinical Laboratory magazine, 2010,28(2): 107-109

5.SarfatiJ,MonodM,ReccoP,etal.Recombinantantigensasdiagnosticmarkersforaspergillosis.Diagnosticmicrobiologyandinfectiousdisease2006,55(4):279-291.

6.NguyenMH,JaberR,LeatherHL,etal.Useofbronchoalveolarlavagetodetectgalactomannanfordiagnosisofpulmonaryaspergillosisamongnonimmunocompromisedhosts.Journalofclinicalmicrobiology.2007,45(9):2787-2792.

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8.SchwienbacherM,WeigM,ThiesS,etal.AnalysisofthemajorproteinssecretedbythehumanopportunisticpathogenAspergillusfumigatusunderinvitroconditions.MedMycol.2005,43(7):623-630

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10.ShiLN,LiFQ,LuJF,etal.AntibodyspecifictoThioredoxinreductaseasanewbiomarkerforserodiagnosisofinvasiveaspergillosisinnon-neutropenicpatients.ClinChimActa.2012,413(9-10):938-943

Summary of the invention

Object of the present invention is exactly to solve the problem, and provides a kind of monoclonal antibody for Aspergillus fumigatus TR.

Another object of the present invention is to provide the preparation method of this monoclonal antibody.

A further object of the invention is to provide the application of this monoclonal antibody in preparation aspergillosis diagnostic reagent.

A kind of Aspergillus fumigatus protein monoclonal antibody, this monoclonal antibody is by being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is the monoclonal antibody of the hybridoma secretion of CGMCCNo.6606.

Monoclonal antibody of the present invention is interpreted as can being IgG or IgM, also can be the Fab fragment of this antibody, F (ab ') ₂fragment, single-chain antibody etc., its Fab keep this monoclonal antibody in conjunction with feature.

Secrete the hybridoma Anti-TR1 of monoclonal antibody of the present invention, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC), preserving number is CGMCCNo.6606, and the preservation time is on September 24th, 2012.

By gene recombination technology general in fact, obtain the antigen that Aspergillus fumigatus TR engineered protein is used for the immunization of animal and screening, qualification antibody, but preferred expression carrier pET-28a(+ of the present invention), make the expression of goal gene the most effective, be easy to purifying most.

The preparation method of monoclonal antibody of the present invention, comprises the following steps:

First Aspergillus fumigatus TR albumen is prepared with genetic engineering technique: from Aspergillus fumigatus mycelia, extract TR gene (SEQIDNO.1), this gene is cloned, and build the procaryotic cell expression carrier carrying this gene, this vector is entered intestinal bacteria, induces the escherichia coli high-level expression restructuring TR albumen be converted.Again with this engineered protein Mice Inoculated obtained, after immunne response being formed to restructuring TR albumen in animal body, splenic lymphocyte is extracted in this animal body, with can the murine myeloma cell of infinite multiplication in vitro merge, set up hybridoma cell line, from this clone, filter out all cell strains that can produce specific antibody.Repeated screening and mono-clonalization cultivation are carried out to these cell strains producing antibody, the hybridoma of ELISA method to secrete monoclonal antibody is adopted to screen, antagonist is tired and is measured, adopt western blot method, immunohistochemical method, immunocytochemical method and the specificity of blocking test to prepared monoclonal antibody are identified, optimize affinity of antibody the strongest, the monoclonal cell strain that specificity is the highest, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCCNo.6606, the monoclonal antibody of this hybridoma secretion is monoclonal antibody of the present invention.

For diagnosing a reagent composition for aspergillosis, said composition contains the said monoclonal antibody of the significant quantity as activeconstituents.

The application of monoclonal antibody of the present invention in preparation aspergillosis diagnostic reagent.

Hybridoma of the present invention ties up to the application in preparation aspergillosis diagnostic reagent.

Beneficial effect of the present invention:

First, the invention provides a kind of monoclonal antibody, this antibody capable specific binding is in the TR albumen of Aspergillus fumigatus, and this antibody also can the TR albumen of specific binding Aspergillus fumigatus in human or animal tissues, comprises the TR albumen be discharged in peripheral blood.

Monoclonal antibody of the present invention there is high degree of specificity and strong avidity, can be used for diagnosing the detection reagent of aspergillosis to prepare.

Monoclonal antibody prepared by the present invention is applicable to the TR albumen in the body fluid detecting aspergillosis patient, to help the type judging fungi.

Monoclonal antibody prepared by the present invention is applicable to detect the TR albumen that in patient's pathological tissues, aspergillus is expressed, to help the type judging fungi.

Accompanying drawing explanation

The agarose gel electrophoresis figure of Fig. 1: RT-PCR amplification Aspergillus fumigatus TRDNA fragment

Wherein: M.DNA molecular weight standard; 1. negative control 2.RT-PCR product.

Fig. 2: the enzyme that agarose gel electrophoresis analyzes recombinant plasmid pMD18-T/TR cuts qualification figure

Wherein: M, DNAmarker; 1, pMD18-T/TR plasmid double digestion; 2, pMD18-T/TR plasmid

The DNA sequencing analysis of Fig. 3: TR gene.

Fig. 4: the SDS-PAGE analysis chart of restructuring TR

Wherein: 1, the pET28a(+ of induction)/TR thalline ultrasonication supernatant; 2, non-Induction Transformation bacterium; 3, the weight of purifying

Histone matter; M, protein molecular weight marker.

The Mass Spectrometric Identification collection of illustrative plates of Fig. 5: restructuring TR albumen and peptide section match condition

Wherein: A, the Mass Spectrometric Identification figure of restructuring TR albumen; B, the peptide section (bold) of mating with TR

The specificity of Fig. 6: Westernblot trace qualification TR monoclonal antibody

Fig. 7: IsoStrip mouse monoclonal antibody subclass (type) measurement result.

The indirect immunofluorescence analysis figure of Fig. 8: TR monoclonal antibody and aspergillus: the TR of anti-TR monoclonal antibody and Aspergillus fumigatus, flavus and aspergillus niger specific binding occurs and reacts.

One of light microscopic figure of Fig. 9: IA rabbit model lung tissue immunohistochemical analysis: the TR that in IA rabbit model lung tissue, aspergillus hyphae is expressed and anti-TR monoclonal antibody generation specific binding, mycelia dyes tawny.

The TR that in light microscopic figure bis-: the IA rabbit model lung tissue of Figure 10: IA rabbit model lung tissue immunohistochemical analysis, aspergillus hyphae is expressed and the reaction of anti-TR monoclonal antibody block by the TR albumen that dissociates, there is not tawny mycelia.

One of light microscopic figure that Figure 11: IA Patients with Lung tissue immunohistochemistry is analyzed: the TR that in IA Patients with Lung tissue, aspergillus hyphae is expressed and anti-TR monoclonal antibody generation specific binding, mycelia dyes tawny.

Biomaterial preservation information

Anti-TR1, Classification And Nomenclature is the mouse hybridoma cell of anti-Sibutramine Hydrochloride thioredoxin reductase GliT (TR) monoclonal antibody of secretion, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCCNo.6606, preservation address is Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preservation date is on September 24th, 2012.

Embodiment

The invention will be further elaborated by the following examples.

Embodiment 1: the clone of Aspergillus fumigatus albumen TR gene and prokaryotic expression

Prepare TR monoclonal antibody, first will carry out clone and the prokaryotic expression of TR gene, for preparation TR monoclonal antibody lays the foundation.We utilize RT-PCR method and T/A Strategies For The Cloning, clone TR gene from Aspergillus fumigatus mycelia, after PCR, double digestion qualification, carry out DNA sequencing (SEQIDNo.1).Then, build recombinant expression vector pET-28a(+)/TR, after PCR and double digestion qualification, transformation of E. coli BL21(DE3), IPTG abduction delivering and SDS-PAGE detect restructuring TR, cobalt ion affinitive layer purification recombinant protein.

1.1 experimental specimens: Aspergillus fumigatus mycelia

1.2 preparation TR double stranded cDNA fragment

1.2.1 design of primers

With the design of Primer5 analysis software, PCR primer sequence is as follows:

TR upstream primer (primer1): 5 '-CACA cATATGtCGATCGGCAAACTAC-3 ' (SEQIDNo.2) (line place is Nde I restriction enzyme site)

TR downstream primer (primer2): 5 '-ACT gAATTCcTATAGCTCCTGATCGAGACG-3 ' (SEQIDNo.3) (line place is EcoRI restriction enzyme site).

1.2.2 the extraction of Aspergillus fumigatus total serum IgE

Get 50mg Aspergillus fumigatus mycelia and put into mortar, add appropriate liquid nitrogen immediately, fully grind under mycelia is frozen state, make it to become Powdered, be and then transferred in the centrifuge tube containing 1mlTrizolRNA extracting solution, put upside down mixing, room temperature places 15 minutes.Add 0.2ml chloroform, fully mix, centrifugal 15 minutes of 4 DEG C of 12000rpm.The colourless aqueous phase in transfer upper strata is in new centrifuge tube, and add 0.5ml Virahol, fully mix, room temperature places 10 minutes.Centrifugal 10 minutes of 4 DEG C of 12000rpm, abandon supernatant, add 1ml75% ethanol, vortex oscillation, centrifugal 5 minutes of 4 DEG C of 7500rpm.Abandon supernatant, with 50 μ lDEPC(diethylpyrocarbonates) process water dissolution RNA precipitation, 8g/L agarose gel electrophoresis detects the effect that RNA extracts, by frozen for sample RNA solution stand-by at-70 DEG C of refrigerators.

1.2.3 reverse transcription reaction

Total reaction system (20 μ l) is composed as follows:

Reverse transcription reaction is carried out: hatch RNA solution 10 minutes for 70 DEG C, immediately ice bath 5 minutes by test kit specification sheets; Add other reacted constituents, vibration mixing, brief centrifugation, incubated at room 10 minutes; 42 DEG C of reverse transcription reactions 30 minutes, 95 DEG C of heating 5 minutes, deactivation AMV reversed transcriptive enzyme ,-70 DEG C of Refrigerator stores are for subsequent use.

1.2.4PCR amplification

Total reaction system (25 μ l) is composed as follows:

PCR parameter is: 94 DEG C of denaturations 5 minutes, 95 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulations, 72 DEG C extend 7 minutes.17g/L agarose gel electrophoresis detects analyzes PCR primer, and result is as Fig. 1.

1.2.5PCR the purifying and quantitatively of product

Utilize commercially available purification kit, to specifications purifying is carried out to PCR primer, by the concentration of the quantitative DNA solution of ultraviolet spectrophotometer.

The structure of 1.3 recombinant cloning vector pMD18-T/TR and qualification

1.3.1 ligation

Total reaction system (10 μ l) is composed as follows:

Vibration mixing reaction system, 4 DEG C of ligations are spent the night.After reaction terminates, 65 DEG C are incubated 10 minutes, deactivation T4DNA ligase enzyme.

1.3.2 recombinant plasmid transformed escherichia coli jm109 competent cell

Joined in 100 μ lJM109 competent cells by 10 μ l ligation liquid, tube wall of flapping gently for several times, fully mixes content, is placed in ice bath 30 minutes.Centrifuge tube is placed in 42 DEG C of water-baths 90 seconds, goes to ice bath immediately, leave standstill 2 minutes.Slowly add LB substratum 0.5ml, 37 DEG C of 100rpm joltings cultivate 45 minutes, bacterium liquid is coated on the LB flat board containing penbritin (0.1g/L), 5 '-bromo-4-3-indoles-β-D-galactoside (0.3g/L) and isopropylthio-β-D-galactoside (0.3g/L), 37 DEG C of overnight incubation.

1.3.3 screening and amplification cultivation positive colony

Random picking 5 single bacterium colonies of white, are inoculated in 5 culture test tubes (the LB substratum containing 0.1g/L penbritin), 37 DEG C of 250rpm jolting overnight incubation respectively.

1.3.4DNA order-checking qualification recombinant plasmid

To cut the recombinant plasmid of qualification through PCR and enzyme, send Dalian precious biological company limited, carry out DNA sequencing with universal primer, by completely the same for the sequence in sequencing result and Genbank and other bibliographical informations, result is as Fig. 2, Fig. 3.

1.4 build recombinant expression vector pET-28a(+)/TR

1.4.1 tap rubber purifying enzyme cut after TRDNA fragment

1.4.2 test kit extracts plasmid pET-28a(+)

1.4.3Nde I, EcoRI double digestion plasmid pET-28a(+)

Total reaction system (100 μ l) is composed as follows:

Vibration mixing reaction system, hatches 1h for 37 DEG C, and add 10 μ l10 × Loading damping fluids and stop endonuclease reaction, 10g/L agarose gel electrophoresis analyzes digestion products.

1.4.4 tap rubber purified linear plasmid pET-28a(+).

1.4.5 ligation (reaction system is composed as follows):

Vibration mixing reaction system, 4 DEG C of ligations are spent the night.After reaction terminates, 65 DEG C are incubated 10 minutes, deactivation T4DNA ligase enzyme.

1.4.6 recombinant plasmid transformed E. coli competent BL21 (DE)

Be coated on by bacterium liquid on the LB flat board containing kantlex (0.1g/L), back-off plate is placed on overnight incubation in 37 DEG C of incubators.

1.4.7 screening, amplification cultivation positive colony

Random picking 5 single bacterium colonies, are inoculated in 5 culture test tubes (the LB substratum containing 0.1g/L kantlex), 37 DEG C of 250rpm jolting overnight incubation respectively.

1.5 recombinant plasmid pET-28a(+) abduction delivering of/TR

The positive colony that picking is identified through PCR and double digestion, is inoculated in the LB substratum containing kantlex (0.1g/L), 37 DEG C of 250rpm overnight incubation.Next day, dilute transferred species in the LB substratum containing kantlex (0.1g/L) with 1:50,37 DEG C of 300rpm thermal agitations are cultivated.As bacterium liquid OD ₆₀₀during ≈ 0.6, take out 1ml bacterium liquid and do not add inductor as negative control, in bacterium liquid, add inducer isopropylthio sulfo--β-D-galactoside (IPTG, final concentration is 1mmol/L), 37 DEG C of inducing culture 3h hour.Meanwhile, with containing empty plasmid pET-28a(+) and not containing the BL21 bacterium of plasmid as blank.Get 1ml and induce bacterium liquid, centrifugal 5 minutes of 7500rpm, abandon supernatant, the abundant resuspended bacterial precipitation of 50 μ l1 × sds gel sample-loading buffer, water proof boils 5 minutes, centrifugal 5 minutes of 12000rpm, gets 10 μ l supernatants and carries out SDS-PAGE(and concentrate glue 50g/L, separation gel 150g/L) determination and analysis expression product, result is as Fig. 4.

1.6 cobalt ion affinitive layer purification restructuring TR

Balance liquid soaks balance 2g resin 1h, abandons supernatant after resin natural sedimentation, the centrifugal supernatant of mixing cracking and balance resin, 4 DEG C of 1h that vibrate gently.Transfer suspension, in chromatography column, allows the liquid in post bed naturally drain off, balance liquid (50mmol/LTris-HCl, 300mmol/LNaCl, pH8.0) the fully washing resin of 10 column volumes.Finally, with 4ml imidazole elution buffer (50mmol/LTris-HCl, 300mmol/LNaCl, 250mmol/L imidazoles, pH8.0) wash-out target protein.In 4 DEG C of refrigerators, PBS dialysis elutriant 48h, changes liquid therebetween 8 times.The centrifugal cleer and peaceful purified product of cracking carries out SDS-PAGE(simultaneously and concentrates glue 50g/L, separation gel 120g/L), comparative analysis purification effect, is shown in Fig. 4.Coomassie brilliant G-250 method measures the concentration of recombinant protein.Purification of recombinant proteins Mass Spectrometric Identification is Aspergillus fumigatus TR, and mass spectrum is shown in Fig. 5.

The preparation of the anti-TR monoclonal antibody of embodiment 2

1. by TR protein immunization BALB/c mouse

During initial immunity, getting the restructuring TR(1.7g/L of purifying) 0.5ml mixes with equivalent Freund's complete adjuvant, be fully ground to complete emulsification (i.e. water-in-oil state) in mortar after, is expelled to (0.1ml/ is only) in mouse peritoneal.The 2nd booster immunization is carried out at interval after 2 weeks, use the fully emulsified antigen of freund 's incomplete adjuvant instead, injected dose and position the same.The 3rd booster immunization is carried out at interval after 2 weeks, directly use antigenic solution, injected dose and position the same.Interval measures the antibody titer in immune serum for 10 days afterwards.In cytogamy first 3 days, get a mouse that antibody positive is the strongest and carry out the 4th booster immunization, injected dose and position the same.

2. indirect enzyme-linked immunosorbent assay (ELISA) measures tiring of antibody in mice serum

3rd booster immunization be after 10 days, and gather antigen immune with non-immune Mouse Blood, as sample to be measured and the negative control of ELISA, separation of serum, 4 DEG C of preservations are to be measured.Diluting restructuring TR to final concentration with antigen coated liquid is 1 μ g/ml, and wrap by polystyrene board micropore (100 μ l/ hole), 4 DEG C of bags are spent the night.Next day, PBS-T washes plate 3 times, adds 50g/L skim-milk and closes, hatch 2 hours h, PBS-T for 37 DEG C and wash plate 3 times by 200 μ l/ holes.PBS dilutes test serum and negative control sera (1:1000), take PBS as blank, adds micropore by 100 μ l/ holes, hatch 1 hour h for 37 DEG C.PBS-T washes plate 3 times, adds HRP-sheep anti-mouse igg (1:4000 dilution), hatch 1 hour for 37 DEG C by 100 μ l/ holes.PBS-T washes plate 3 times, and every hole adds each 1 of substrate, developer, hatches 10 minutes for 37 DEG C.Add 2 1mol/LH ₂sO ₄termination reaction, records result in wavelength 450nm enzyme-linked immunosorbent assay instrument colorimetric.

3. the preparation of mouse boosting cell

The 4th booster immunization is after 3 days, and extract the bloodletting of immune mouse eyes, leave and take the positive control of serum as ELISA, 4 DEG C save backup.Neck is drawn to put to death mouse, 75% alcohol-pickled 5 minutes.Cut an osculum at mouse web portion, tear skin, cut off peritonaeum, tweezer mouse spleen, cuts mouse spleen, except degrease and reticular tissue, with RPMI1640 incomplete substratum washing mouse spleen.Mouse spleen is cut into 4 pieces, at stainless steel mesh (100 orders/cm ²) on, fully grind mouse spleen with glass syringe nook closing member, squeeze out splenocyte gently.By lapping liquid through stainless steel mesh (200 orders/cm ²) after filtration, be transferred to centrifuge tube, centrifugal 5 minutes of 1000rpm.Abandon supernatant, once, then with the abundant re-suspended cell of the incomplete substratum of RPMI1640, carrying out cell counting and calculating cell concn is 2 × 10 to the incomplete substratum washed cell of RPMI1640 ¹⁰individual/L, is placed in incubator (37 DEG C, 5%CO ₂) in stand-by.

4. cytogamy

Abundant mixing immune spleen cell (10 ⁸individual) and SP2/0 cell (10 ⁷individual) suspension, centrifugal 5 minutes of 1000rpm.Abandon supernatant, remove residual liquid as far as possible, finger flicks centrifuge tube makes cell precipitation comparatively loosening.Centrifuge tube is placed in 37 DEG C of water-baths, adds 1ml50%PEG4000 in 30 seconds, edged appropriateness in limit stirs, and adds rear continuation stirring 15 seconds, leaves standstill 90 seconds.Add the incomplete nutrient solution of RPMI1640, from slow to fast, concrete scheme: add 1ml, 2ml, 3ml, 4ml, 5ml and 10ml respectively in every 2 minutes continuously, stops fusion to liquid feeding speed.Centrifugal 5 minutes of 800rpm, abandons supernatant, with the incomplete nutrient solution of RPMI1640 resuspended, washed cell 1 time gently.Add 50mlHAT perfect medium, gently re-suspended cell, join 96 well culture plates containing feeder cell by 100 μ l/ holes, be placed in cell incubation case (37 DEG C, 5%CO ₂) middle cultivation.

5. the detection of antibody

With restructuring TR as envelope antigen, get culture supernatant 0.1ml in micropore to be measured and, for measuring, using immune mouse peripheral blood clearly as positive control, using the culture supernatant of SP2/0 cell as negative control, resist as two with HRP-sheep anti mouse (1:4000 dilution).

6. the colonized culture of hybridoma

Get 100 cells, add 10mlHT substratum and fully mixing, join in 96 well culture plates containing feeder cell by 0.1ml/ hole.Detecting antibody after 10 days, filter out 1 monoclonal cell strain that antibody positive is the strongest, is Anti-TR1.Then, use RPMI1640 perfect medium (containing 20% newborn calf serum) instead, repeatedly carry out subclone cultivation, what contain cell clone until all is antibody positive.Acquisition Anti-TR1 hybridoma like this, deliver the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number is CGMCCNo.6606.

7. the foundation of hybridoma cell line

By Anti-TR1 monoclonal cell (CGMCCNo.6606) enlarged culturing of antibody positive, set up hybridoma cell line, carry out an antibody test week about.When hybridoma cell line reaches certain quantity, freeze-stored cell, leaves and takes supernatant for measuring tiring of its monoclonal antibody.Hybridoma cell strain in-vitro cultivation liquid nitrogen cryopreservation after 1 month, still can stably excreting TR monoclonal antibody after recovery.

8. the preparation of ascites monoclonal antibody

Whiteruss (0.5ml/ is only) is expelled in mouse peritoneal, after 14 days by the hybridoma CGMCCNo.6606(0.5ml/ of antibody positive only) inject mouse peritoneal.When mouse web portion obviously expands, extract mouse ascites with No. 8 syringe needles, extracted, until dead mouse with method every 4 days.The centrifugal ascites of 3000rpm 10 minutes, abandons the various cell of upper strata grease and insolubles and lower floor and insolubles, draws faint yellow supernatant, adds isopyknic glycerine (containing the Na of 20g/L ₂hPO ₄) ,-80 DEG C of Refrigerator stores after packing.

The titration of the anti-TR monoclonal antibody of embodiment 3

Adopt ELISA method.With TR albumen (1mg/L) bag by polystyrene board micropore (100 μ l/ hole), 4 DEG C are spent the night.Next day, PBS-T washes plate 3 times, by 200 μ l/ holes add 50g/L skim-milk close 37 DEG C 2 hours, PBS-T washes plate 3 times.Add sample to be tested (culture supernatant of hybridoma cell strain, or the mouse ascites monoclonal antibody of serial dilution), hatch 1 hour for 37 DEG C.PBS-T washes plate 3 times, adds HRP-sheep anti-mouse igg (1:4000 dilution), hatches 1 hour for 37 DEG C.PBS-T washes plate 3 times, and every hole adds TMB-H ₂o ₂solution, hatches 10 minutes for 37 DEG C.Add 2 1mol/LH ₂sO ₄termination reaction, in wavelength 450nm enzyme-linked immunosorbent assay instrument colorimetric.Result: the culture supernatant antibody titer of hybridoma cell strain Anti-TR1 is respectively 1:64; The antibody titer of ascites is 5:10 ⁵.

Embodiment 4Westernblot blot analysis

By expression restructuring TR e. coli total protein trace on nitrocellulose filter.PBS-T containing 50g/L skim-milk closes and spends the night, PBS-T rinsing 3 times, and the monoclonal antibody (1:1000 dilution) adding embodiment 3 preparation hatches 1 hour, PBS-T rinsing 3 times, adds HRP-sheep anti-mouse igg (1:2000) and hatches 1 hour, PBS-T rinsing 3 times.Finally, add DAB nitrite ion and carry out color reaction.After color reaction, only on recombinant protein pillar location, occur brown color band, other tropina bands have no colour developing (see figure 6).

The mensuration of embodiment 5 monoclonal antibody hypotype

Test kit test is measured with IsoStrip mouse monoclonal antibody subclass (type).Leave and take hybridoma cell strain Anti-TR1(CGMCCNo.6606) culture supernatant, with PBS(pH7.4) carried out 10 times of dilutions, 150 μ l diluents are added drop-wise in test tube, vortex test tube makes blue latex completely resuspended a little, black one end antibody subtype being measured test strip is inserted in vitro bottom, when blue positive control lines occur, take out test strip, after 2 minutes, read result.Result judges: blue linear bands appears in antibody subtype reaction zone on the test strip, is positive findings, the correct hypotype reading correspondence.Result: antibody (CGMCCNo.6606) hypotype of hybridoma cell strain Anti-TR1 is IgG1, and light chain is κ type.As shown in Figure 7.

The specificity of embodiment 6 indirect immunofluorescence assay qualification monoclonal antibody

Get Aspergillus fumigatus A1, flavus F1, aspergillus niger N1, Candida albicans C1, Oidium tropicale T1, Candida glabrata G1(is China Committee for Culture Collection of Microorganisms's medical mycology central standard bacterial strain) mycelia and spore film-making, primary antibodie is monoclonal antibody TRmAb(1:1000 of the present invention), non-immune serum is negative control, 4 DEG C of overnight incubation, rinsing 4 times, add FITC-sheep anti-mouse igg (1:500), 37 DEG C of lucifuges hatch 1 hour, lucifuge rinsing 5 times, 50% glycerine mounting, fluorescence microscopy Microscopic observation is also taken pictures, observe the reactivity of monoclonal antibody and all kinds of fungi.Result show, there is specific binding and react (see figure 8) in anti-TR monoclonal anti physical efficiency and Aspergillus fumigatus, flavus, aspergillus niger mycelia TR, and not with each candidiasis generation specific reaction.

The specific reaction of embodiment 7 immunohistochemical assay qualification monoclonal antibody and natural TR

A. IA rabbit model is set up: Aspergillus fumigatus A ₁(China Committee for Culture Collection of Microorganisms's medical mycology central standard bacterial strain) inoculates husky Bao Shi slant medium, cultivate 3 ~ 4d for 28 DEG C, with the normal saline flushing bacterium colony containing 0.5%Tween-20, collect conidium, low-speed centrifugal removes mycelia and block impurity, counts spore concentration and be adjusted to 1 × 10 with cell counting count board ⁶/ ml.New Zealand white rabbit is in inoculating aspergillus fumigatus spores first 2 days (-2d), 0d and 3d, intramuscular injection every day hydrocortisone 10mg/Kg/d.Infected rabbits is through auricular vein injection 1 × 10 ⁶/ ml Aspergillus fumigatusconidia suspension 1ml.Put to death before infection by Aspergillus fumigatus animal is dying and dissect, get the organs such as lung, liver, kidney and carry out histopathological examination.

B. dissect infected rabbits and get lung tissue, formaldehyde is fixed, paraffin embedding, serial section.Paraffin section de-waxing, 0.03% methyl alcohol-H ₂o ₂hatch 30 minutes, in microwave oven, boil 1 minute.10% sheep blood serum closes 30 minutes, and PBS-T washes 3 times, and primary antibodie is monoclonal antibody TRmAb(1:1000 of the present invention), non-immune serum is negative control, 4 DEG C of overnight incubation, rinsing 3 times, add HRP-sheep anti-mouse igg (1:3000), hatch 1 hour for 37 DEG C, rinsing 3 times, DAB substrate solution develops the color 10 minutes, and Hematorylin redyes 1 minute, dehydration, transparent, mounting, observes under an optical microscope and takes pictures, and observes the reactivity (Fig. 9) of monoclonal antibody and Aspergillus fumigatus mycelia.

C. blocking test is in order to identify the specificity of monoclonal antibody of the present invention further, carry out blocking test, namely in monoclonal antibody solution, add the TR albumen (100mg/L) of purifying, after hatching 30min in 37 DEG C, immunohistochemical staining is carried out again with this monoclonal antibody solution, result shows, and the positive reaction of monoclonal antibody of the present invention and lung tissue Aspergillus fumigatus mycelia all can be blocked (Figure 10), thus the albumen in proving the mycelia that monoclonal antibody of the present invention combines is TR albumen.

D. get the lung tissue section of invasive pulmonary aspergillosis corrective surgery excision, fixedly spend the night with 10% neutral formalin 4 DEG C.Graded ethanol (25%, 50%, 75% and 100%) dewaters, and dimethylbenzene is transparent, paraffin embedding, repaiies block, does serial section, paving sheet.Paraffin section de-waxing to water, 0.03% methyl alcohol-H ₂o ₂hatch 30 minutes, in microwave oven, boil 1 minute.10% sheep blood serum closes 30 minutes, and PBS-T washes 3 times, drips the anti-TR monoclonal antibody of the present invention (1:1000 dilution), with non-immune serum for negative control, and 4 DEG C of overnight incubation.PBS-T rinsing 3 times, adds HRP-sheep anti-mouse igg (1:3000 dilution), hatches 1 hour for 37 DEG C.PBS-T rinsing 3 times, DAB develops the color 10 minutes, washing termination reaction.Hematorylin redyes 1 minute, dehydration, transparent, DPX mounting.With observation by light microscope and take pictures (see Figure 11).Result shows that monoclonal antibody of the present invention can be used for detecting the TR of aspergillus hyphae in tissue, respond well.

Immunohistochemical staining analytical results shows, and the TR that anti-TR monoclonal anti physical efficiency and the aspergillus hyphae in the aspergillus hyphae directly cultivated and people and rabbit tissue are expressed specific binding occurs and reacts, and cell dyes tawny (see Fig. 8,9,11).

The anti-TR monoclonal antibody of embodiment 8 is measuring the application in serum T R

After the Isolation and characterization of a.TR monoclonal antibody gets the thick leach protein of 5ml mouse ascites ammonium sulfate precipitation method, through G-protein affinity column antibody purification, method is as follows: ammonium sulfate precipitated protein redissolves after PBS, loading is to G-protein affinity column, with 0.1MpH2.7 glycine-HCL wash-out, collect the IgG antibody albumen of purifying.

B. double antibody sandwich ELISA is with the anti-TR monoclonal antibody of purifying for envelope antigen, with HRP-anti-TR monoclonal antibody for detecting antibody, selects optimum reaction condition by square formation volumetry.With anti-TR monoclonal antibody to the 1 μ g/ml of coating buffer dilution purifying, 100 μ l/ holes, 4 DEG C of refrigerator overnight.3 times are washed, 1min/ time with PBS-T.Next day, every hole added confining liquid (10% calf serum) 100 μ l, hatched 2h for 37 DEG C.After PBS washs 3 times, add the Aspergillus fumigatus restructuring TR antigen of suitably dilution, IA rabbit model serum, IA patients serum or normal healthy controls serum (diluent is the PBS damping fluid containing 0.1%Tween-20 and 1% calf serum), 100 μ l/ holes, each extent of dilution all establishes multiple hole, every plate all establishes the positive, feminine gender and blank, hatch 45 minutes for 37 DEG C, PBS-T washs 3 times, 1min/ time.Add HRP-anti-TR monoclonal antibody, hatch 45 minutes for 37 DEG C, PBS-T washs 3 times, 1min/ time.Add substrate solution TMB-H ₂o ₂colour developing, 37 DEG C 15 minutes, add stop buffer (2mol/LH ₂sO ₄) 50 μ l/ holes, survey 450nm wavelength absorbance (A) in enzyme connection instrument (Bio-RadMAel680 type) is upper.Result: with namely 0.3 is threshold value (cutoff value), and A ﹥ 0.3 is antigen levels rising.To be worth A>0.3 for positive criteria, the minimum inspection limit of this method can reach 2ng/ml, and in IA rabbit model, infect can antigen positive be detected on the 3rd day, all infection by Aspergillus fumigatus rabbits all detect antigen, and positive rate reaches 100%.Detect 2 examples in 200 routine normal healthy controls serum positive, specificity is up to 99%; Make a definite diagnosis in IA patient in 24 examples, detect 20 routine antigen positives, susceptibility reaches 83.3%.

Claims (6)

1. an Aspergillus fumigatus protein monoclonal antibody, this monoclonal antibody is by being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is the monoclonal antibody of the hybridoma secretion of CGMCCNo.6606.

2. monoclonal antibody according to claim 1, its Fab keep this monoclonal antibody in conjunction with feature.

3. secrete the hybridoma Anti-TR1 of monoclonal antibody described in claim 1, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCCNo.6606, and preservation date is on September 24th, 2012.

4., for diagnosing a reagent composition for aspergillosis, it is characterized in that described reagent composition contains the monoclonal antibody according to claim 1 of the significant quantity as activeconstituents.

5. the application of monoclonal antibody according to claim 1 in preparation aspergillosis diagnostic reagent.

6. hybridoma according to claim 3 ties up to the application in preparation aspergillosis diagnostic reagent.