CN103421742B - Recombinant monoclonal antibodies H2 resisting fumonisins B1 - Google Patents
Recombinant monoclonal antibodies H2 resisting fumonisins B1 Download PDFInfo
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Abstract
The invention belongs to the technical field of plant immunity detection and particularly relates to hybridoma cell strains 1(11 capable of secreting monoclonal antibodies resisting fumonisins B1 as well as the preparation and application of recombinant monoclonal antibodies H2 of the monoclonal antibodies. The fumonisins B1 are conjugated with the carrier proteins of KLH and BSA respectively to obtain antigens; the obtained antigens FB1-KLH are used for immunizing mice; after cell fusion, selective culture is performed on the immunized mice; hybridoma cells are screened through the FB1-BSA conjugates; the hybridoma cell strains 1(11 which can stably secret the monoclonal antibodies resisting the fumonisins B1 are obtained by subcloning. The recombinant monoclonal antibodies H2 of the hybridoma cell strains 1(11 are screened according to the antibody gene pool technology and the phage display technology. According to the invention, a large quantity of the monoclonal antibodies and the recombinant monoclonal antibodies H2 are produced and purified. The obtained monoclonal antibodies 1(11 and the recombinant monoclonal antibodies H2 can be used for the immunological detection of the fumonisins B1.
Description
Technical field
The invention belongs to phytoimmunology technical field, be specifically related to preparation and the application thereof of the mono-clonal recombinant antibodies H2 of a kind of anti-fumonisins B1, described hybridoma cell strain can secrete anti-fumonisins B1 monoclonal antibody, prepare the recombinant antibodies of described monoclonal antibody further by genetic engineering technique, the recombinant antibodies of this monoclonal antibody or preparation can be applicable in the immunology detection of fumonisins.
Background technology
From fusarium moniliforme (Fusarium verticillioides (Sacc.) Nirenberg) nutrient solution, isolation identification was out first in 1988 for fumonisins (Fumonisins), that a class forms the diester compound of similar by difference many hydrogen alcohol and tricarballylic acid, the hydrophilic side-chains comprising an aliphatic chain be made up of 20 carbon and connected by two ester bonds.Up to the present, isolation identification goes out tens kinds of similar things of fumonisins or isomer, belong to A, B, C, D and P group, wherein pollute the most serious with FB1, FB2 and FB3 of B group, toxicity is also the strongest, can cause mouse liver cancer, pig pulmonary edema and horse alba malacosis, and be the potential carcinogen of the mankind, international cancer research institution (IARC) is classified as 2B class.Report has can produce various types of fumonisins more than 15 kinds of sickle-like bacteria and Alternaria alternata (Alternaria alternata (Fr.) Keissler f.sp.lycopersici) and aspergillus niger (Aspergillus niger), but fusarium moniliforme (f. verticillioides) and many births sickle-like bacteria (f. proliferatum) are topmost toxigenic bacterium strains.Fumonisins can pollute corn, Chinese sorghum, paddy rice, wheat, barley, broomcorn millet, Kidney bean and spice crop etc., and in the food and feed of wherein corn and corn source, content is maximum.At present, the pollution of fumonisins worldwide has report in portion, but pollutes the most serious with the some areas in China and South Africa.In China, investigation display corn, paddy rice and the pollution being subject to fumonisins all in various degree of wheat Three major grain crops.Therefore, in the food crop such as China's corn, the pollution of fumonisins is in the severe situation of high rate and high-content, and also obtains the extensive concern of society about the research of fumonisins, becomes the focus in food safety and mycotoxins research over nearly 20 years.
The detection method of mycotoxins is more, is mainly divided into Biological Detection, chemical detection and immunology detection.What Biological Detection was conventional has cell culture experiments, Seeds Germination Tests and skin toxicology test etc., although simple to operate, have the shortcomings such as poor specificity, error are large, application is less now.Chemical detection method has higher sensitivity, accuracy and specificity, but needs more complicated sample pretreatment process and more valuable plant and instrument, not easily promotes the use of in low developed area and rural area.Immunological method possesses simple to operate, the advantage such as expense is low, highly sensitive and specificity is good, and the quick detection kit of exploitation and test strip etc. are easy to carry, sample can be detected at any time, so immunologic detection method is more and more widely used in the detection analysis of various sample.Therefore, by preparing monoclonal antibody and the recombinant antibodies of anti-fumonisins FB1, bonding force and the specificity of antibody is detected after large-scale purification, obtain monoclonal antibody and the recombinant antibodies of high-affinity, the foundation of the antigenicity research and immunological detection method that can be fumonisins FB1 provides starting material, the immunologic detection method developed on this basis is in food safety and import and export in inspection and quarantine process, for the pollution condition detecting and investigate fumonisins in food crop, feed and the food such as corn or goods provides fast and reliable detection means.So the hybridoma cell strain that screening can secrete monoclonal antibody specific is a large amount of manufacture order clonal antibody and the basis of preparing mono-clonal recombinant antibodies.In addition, relative to polyclonal antibody and monoclonal antibody, recombinant antibodies and encoding gene thereof obtain by display technique of bacteriophage screening, then can in escherichia expression system after overexpression from extraction purification, its preparation process does not need to use animal, simple to operate, time saving and energy saving.But, due to the impact of the factor such as a large amount of existence and gene recombination of nonfunctional antibody-encoding genes in hybridoma, round pcr is directly utilized to increase from hybridoma the encoding gene of recombinant antibodies, the phenomenon such as can there is disappearance, frameshit and stop mutation, the recombinant antibodies after assembling not necessarily keeps the activity of original parental antibody.And display technique of bacteriophage is not only a high frequency zone system, be also an external ripening process, likely obtain avidity and the improved mono-clonal recombinant antibodies of specificity by Antibody geometric mean titer technology and display technique of bacteriophage.Therefore, the present invention is by the hybridoma cell strain of the anti-fumonisins monoclonal antibody of hybridoma technology screening secretion, and obtain the mono-clonal recombinant antibodies of high-affinity by display technique of bacteriophage on this basis, significant to the effective Determination of Fumonisin Mycotoxins of exploration development etc.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide the hybridoma cell strain can secreting anti-fumonisins B1 monoclonal antibody, and separation obtains mono-clonal recombinant antibodies on this basis, the recombinant antibodies of the monoclonal antibody that the present invention obtains or preparation can be applicable in the immunology detection of fumonisins.
The present invention is achieved through the following technical solutions:
The present invention is achieved in that and fumonisins B1 (FB1) is prepared antigen with keyhole chirp azurin (KLH) and bSA (BSA) by glutaraldehyde one step coupling method respectively, after gained antigen FB1-KLH conjugate immunity Ba1b/c mouse, get splenic lymphocyte and Sp2/0 myeloma cell carries out cytogamy cultivation, FB1-BSA conjugate is utilized to carry out ELISA screening to hybridoma after being cultivated by selectivity, obtaining through subclone can the hybridoma cell strain of stably excreting anti-fumonisins B1 monoclonal antibody again, applicant is by its called after 1C11.This hybridoma cell strain can utilize conventional ascites method to prepare ascites containing monoclonal antibody in a large number, monoclonal antibody 1C11 is obtained again through ammonium sulfate precipitation method purifying, utilize tiring of elisa assay antibody, utilize surface plasma resonance technology (SPR) to analyze the bonding force of antibody, biological assessment is carried out to the monoclonal antibody of gained.On this basis, build the library of antibody genes of hybridoma, by the mono-clonal recombinant antibodies of the anti-fumonisins of display technique of bacteriophage screening high-affinity, through phage ELISA, express ELISA and SPR qualification, obtain a kind of mono-clonal recombinant antibodies and encoding gene thereof of anti-fumonisins, applicant is by its called after H2.
The hybridoma cell strain 1C11 that can secrete anti-fumonisins B1 monoclonal antibody of gained is sent China on January 6th, 2012 by applicant. Wuhan. and Wuhan University's China typical culture collection center (CCTCC) preservation, its preserving number is: CCTCC NO:C2011122.
H2 mono-clonal recombinant antibodies encoding gene is by 840 nucleotide codings, and its sequence is for shown in SEQ ID NO:1.Variable region of heavy chain V
hby 387 nucleotide codings, its sequence is for shown in SEQ IDNO:3.Variable region of light chain V
lby 330 nucleotide codings, its sequence is for shown in SEQ ID NO:5.
H2 mono-clonal recombinant antibodies sequence is for shown in SEQ ID NO:2, and be made up of 279 amino acid, the mono-clonal recombinant antibodies of anti-fumonisins of the present invention is primarily of antibody heavy chain variable region V
h, antibody chain variable region V
lwith connection peptides composition, antibody heavy chain variable region V
hwith antibody chain variable region V
lby connection peptides (Gly
4ser)
3connect the identification and combination that jointly complete fumonisins molecule.Its variable region of heavy chain V
hbe made up of 129 amino acid, sequence for shown in SEQ ID NO:4, variable region of light chain V
lbe made up of 110 amino acid, its sequence is for shown in SEQ ID NO:6.
The application of 1C11 monoclonal antibody in fumonisins detects, its application process is:
The 1C11 monoclonal antibody of producing purifying in mouse peritoneal is reacted by competitive ELISA or prepares the detection that detection kit or protein chip etc. are applied to fumonisins.
In elisa plate hole, add 100 μ L (1 μ g) sheep anti-mouse antibody (purchased from Hangzhou Long Ji Bioisystech Co., Ltd), spent the night by 2h or 4 DEG C in 37 DEG C of bags; 3 times are washed with 200 μ L phosphate buffered saline buffers (PBS); Add bSA (BSA) solution of 150 μ L1% (w/v) concentration, 37 DEG C of closed 2h; 3 times are washed with 200 μ LPBS; Add the monoclonal antibody of the present invention (0.2 μ g/mL) of 100 μ L purifying, 37 DEG C of bags are by 2h; 3 times are washed respectively with 200 μ LPBST (PBS solution containing the Tween-20 of 0.1% (v/v) concentration) and PBS; Add FB1-HRP (conjugate of fumonisins FB1 and horseradish peroxidase HRP) and FB1 toxin standard substance (available from Sigma) or the sample extracting solution of 100 μ L, 1: 2000 dilution by volume, 37 DEG C are reacted 1h; 3 times are washed respectively with 200 μ LPBST and PBS; Add 100 μ L solubilities single-component tmb substrate (purchased from TIANGEN Biotech (Beijing) Co., Ltd.), under dark condition, react 15 ~ 30min; Adding 50 μ L concentration is the H of 2M
2sO
4solution termination reaction, surveys OD by microplate reader
450nmread value.Competitive ELISA result shows monoclonal antibody of the present invention and fumonisins specific binding, can according to the OD of the FB1 toxin standard substance of different concns
450nmread value and carry out drawing standard curve, then estimate the content of the fumonisins in sample.
The application of H2 mono-clonal recombinant antibodies in fumonisins detects, its application process is:
By after the H2 mono-clonal recombinant antibodies purifying of expressing in bacterium, reacted by competitive ELISA or prepare the detection that detection kit or protein chip etc. are applied to fumonisins.
In elisa plate hole, add 100 μ L (1 μ g) sheep anti-mouse antibody (originating the same), spent the night by 2h or 4 DEG C in 37 DEG C of bags; 3 times are washed with 200 μ LPBS; Add the BSA solution of 150 μ L1% (w/v) concentration, 37 DEG C of closed 2h; 3 times are washed with 200 μ LPBS; Add the anti-His monoclonal antibody (purchased from TIANGEN Biotech (Beijing) Co., Ltd.) of 100 μ L, 1: 3000 dilution by volume, 37 DEG C of bags are by 2h; 3 times are washed respectively with 200 μ LPBST and PBS; Add the H2 mono-clonal recombinant antibodies of the present invention that 100 μ L concentration are 200nM purifying, 37 DEG C of bags are by 2h; 3 times are washed respectively with 200 μ LPBST and PBS; Add FB1-HRP and FB1 toxin standard substance or the sample extracting solution of 100 μ L, 1: 2000 dilution by volume, 37 DEG C of reaction 1h; 3 times are washed respectively with 200 μ LPBST and PBS; Add 100 μ L solubility single-component tmb substrates, under dark condition, react 15 ~ 30min; Adding 50 μ L concentration is the H of 2M
2sO
4solution termination reaction, surveys OD by microplate reader
450nmread value.Competitive ELISA result shows mono-clonal recombinant antibodies of the present invention and fumonisins specific binding, can according to the OD of the FB1 toxin standard substance of different concns
450nmread value and carry out drawing standard curve, then estimate the content of the fumonisins in sample.
Beneficial effect of the present invention:
1, one of beneficial effect of the present invention is that screening obtains the hybridoma strain 1C11 that can secrete anti-fumonisins monoclonal antibody, and this cell strain system can be used for the monoclonal antibody of producing high-affinity in a large number.
2, two of beneficial effect of the present invention is that the activated monoclonal antibody 1C11 of tool produces in a large number by ascites method, then adopts ammonium sulfate precipitation purifying.The monoclonal antibody obtained has very high avidity, can be used for the detection analysis of fumonisins.
3, three of beneficial effect of the present invention is bonding forces that the mono-clonal recombinant antibodies H2 obtained has than its parent Geng Gao, and H2 antibody is by can be used for the detection analysis of fumonisins after Prokaryotic expression, purification.
4, four of beneficial effect of the present invention be H2 antibody can in intestinal bacteria solubility expression, simple to operate, production cost is low.
Accompanying drawing explanation
Sequence table SEQ ID NO:1 is the nucleotide coding sequence of mono-clonal recombinant antibodies H2 of the present invention, sequence length 840bp, 279 amino acid of encoding.
Sequence table SEQ ID NO:2 is the aminoacid sequence of mono-clonal recombinant antibodies H2 protein of the present invention, is made up of 279 amino acid.
Sequence table SEQ ID NO:3 is antibody heavy chain variable region V
hnucleotide coding sequence, sequence length is 387bp, 129 amino acid of encoding.
Sequence table SEQ ID NO:4 is antibody heavy chain variable region V
hthe aminoacid sequence of protein, is made up of 129 amino acid.
Sequence table SEQ ID NO:5 is antibody chain variable region V
lnucleotide coding sequence, sequence length is 330bp, 110 amino acid of encoding.
Sequence table SEQ ID NO:6 is antibody chain variable region V
lthe aminoacid sequence of protein, is made up of 110 amino acid.
Fig. 1: be techniqueflow chart of the present invention.
Fig. 2: utilize ELISA method to detect immune mouse antiserum titre.
Fig. 3: utilize ELISA method to detect before purifying and the bonding force of Monoclonal Antibodies in Mice Ascites 1C11 after purifying.
Fig. 4: 1C11 and the H2 antibody utilizing SDS-PAGE electrophoresis detection purifying.Wherein: a figure in Fig. 4 is the SDS-PAGE electrophorogram of 1C11 monoclonal antibody; B figure in Fig. 4 is the SDS-PAGE electrophorogram of H2 mono-clonal recombinant antibodies.
In figure: M: protein standard; 1C11: monoclonal antibody 1C11; H2: mono-clonal recombinant antibodies H2.
Fig. 5: be carrier pHENHi structure iron of the present invention.
Fig. 6: be pHENHi-H2 plasmid DNA structure iron of the present invention.
Fig. 7: utilize surface plasma resonance technology (SPR) to analyze the bonding force of 1C11 and H2 antibody.Wherein: a figure in Fig. 7 is the SPR analysis chart of 1C11 monoclonal antibody; B figure in Fig. 7 is the SPR analysis chart of H2 mono-clonal recombinant antibodies.
Embodiment
Embodiment 1: prepared by antigen
Adopt glutaraldehyde one step coupling method by fumonisins FB1 (available from Sigma) and keyhole chirp azurin (KLH, available from Sigma) coupling (KLH-NH
2+ OHC-(CH
2)
3-CHO+FB1-NH
2→ KLH-N=HC-(CH
2)
3-CH=N-FB1) after the FB1-KLH conjugate that obtains as immunizing antigen, by fumonisins FB1 and bSA (BSA, available from Sigma) coupling (BSA-NH
2+ OHC-(CH
2)
3-CHO+FB1-NH
2→ BSA-N=HC-(CH
2)
3-CH=N-FB1) after the FB1-BSA conjugate that obtains as detection screening antigen.Specific operation process is as follows: take 1mg KLH and be dissolved in 1mL PBS (composition: the KCl of the NaCl of 137mM concentration, 2.7mM concentration, the Na of 10mM concentration
2hPO
4, the KH of 1.8mM concentration
2pO
4pH7.2 ~ 7.4) in solution, add 0.6mg FB1, abundant mixing, dropwise add the glutaraldehyde solution of equal-volume 2% (v/v) concentration, 4 DEG C of vibration 1h, then adding 0.25mL concentration is that the glycine solution of 1M closes 1h, conjugate solution dialyse in PBS packing after 72h be stored in-20 DEG C for subsequent use.
Embodiment 2: immune mouse
Antigen FB1-KLH immunity Balb/c mouse (purchased from Wuhan Virology Institute,Chinan academy of Sciences's Experimental Animal Center) prepared by embodiment 14 times.After first time immunity, 4 weeks (28 days) carry out second time immunity, and rear three booster immunization cycles are 3 weeks.Immune employing dorsal sc multi-point injection, first time gets 200 μ L immunizing antigens (100 μ g) and mixes rear immunity with equal-volume Freund's complete adjuvant, and rear 200 μ L immunizing antigens of getting for three times mix rear immune with equal-volume Freund's incomplete adjuvant.In the 10th day tail venous blood sampling after third time immunity, in the centrifugal 10min of 6000r/min after 37 DEG C of standing placement 1h, collect supernatant (antiserum(antisera)) and carry out indirect ELISA detection immune effect (see embodiment 3).
Embodiment 3: antiserum(antisera) titre detects
Concrete steps are as described below:
1) the antigen FB1-BSA of embodiment 1 preparation, by volume 1: 400 dilution, get 100 μ L/ holes bags by elisa plate, 4 DEG C are spent the night.
2) wash elisa plate hole 3 times with 200 μ LPBS, add the BSA solution of 150 μ L1% (w/v) concentration, 2h is closed in 37 DEG C of water-baths.
3) wash elisa plate hole 3 times with 200 μ LPBS, add the antiserum(antisera) of 100 μ L PBS doubling dilutions respectively, dilution initial concentration is 1: 1000,37 DEG C of water-bath 2h.
4) elisa plate hole is respectively washed 3 times with 200 μ LPBST (PBS solution containing the Tween-20 of 0.1% (v/v) concentration) and PBS, the AP adding 100 μ L, 1: 5000 dilution by volume marks sheep anti-mouse antibody (available from Sigma), 37 DEG C of water-bath 1h.
5) respectively wash elisa plate hole 3 times with 200 μ LPBST and PBS, add 4-NPP (pNPP) nitrite ion of 100 μ L0.2% (w/v) concentration, develop the color under dark condition 15 ~ 30min.
6) add the NaOH solution termination reaction that 50 μ L concentration are 3M, microplate reader reads OD
40snmabsorption value.
Get blood from the mouse after immune 3 times and prepare the titre that antiserum(antisera) detects antibody.After antigen FB1-KLH immunity, the antiserum(antisera) titer analysis of Balb/c mouse as shown in Figure 2, shows that Balb/c mouse creates higher antiserum(antisera) titre through 3 immune rear portions, tires and reach 1: 512000.
Embodiment 4: myeloma cell cultivates
Myeloma cell selects SP2/0 cell (purchased from Wuhan Neweast Biosciences Co., Ltd.), starts recovery cell about merging first two weeks, cultivates until fusion cell on the same day is in logarithmic phase.The preparation method of myeloma cell is as follows:
1) merge front 36 ~ 48h, by myeloma cell's enlarged culturing in RPMI1640 substratum (purchased from American Gibco company), put the CO of 4% concentration
2in incubator (purchased from American Thermo-Scientific company), 37 DEG C of constant temperature culture.
2) merge the same day, with connector bend dropping tube, cell is blown down gently from bottle wall, be collected in 50mL centrifuge tube.
3) the centrifugal 5min of 1500r/min, abandons supernatant.
4) add the incomplete substratum of 30mL (purchased from American Gibco company), the centrifugal 5min washing of 1500r/min once.
5) the incomplete substratum re-suspended cell of 10mL is added, mixing.
6) get myeloma cell's suspension 300 μ L, add equal-volume 1mg/mL trypan blue dye liquor, mixing, counts with blood counting chamber.
Embodiment 5: prepared by feeder layer cells
Get non-immune Balb/c mouse, extract eyeball blood sampling, and prepare serum as negative control during antibody test.Feeder layer cells is generally proposed preparation the day before yesterday or is merged preparation on the same day, and its operating process is as follows:
1) put to death mouse by neck dislocation, soak 5 minutes in the alcohol of 75% (v/v) concentration.
2) in super clean bench, cut off peritonaeum with aseptic operation, take out spleen and be placed in plate, and peel off surrounding connective tissue.
3) extrude spleen with plunger, make cell dispersal.
4) add the incomplete substratum of 10mL (originating the same), purge cell for several times, makes single cell suspension.
5) enchylema is transferred to 50mL centrifuge tube, and the centrifugal 10min of 1000r/min, abandons supernatant.
6) add the incomplete substratum of 10mL, the centrifugal 10min of 1000r/min, wash 1 ~ 2 time.
7) the incomplete substratum re-suspended cell of 10mL is added, mixing.
8) get 300 μ L cell suspensions, add the trypan blue dye liquor that equal-volume concentration is 1mg/mL, mixing, counts with blood counting chamber.
9) draw cell suspension and add 96 porocyte culture plates, every hole 100 μ L, puts the CO of 4% concentration
2in incubator, 37 DEG C of constant temperature culture.
Embodiment 6: the preparation of splenic lymphocyte
Balb/c mouse after Example 2 immunity, extract eyeball blood sampling, and separation of serum is as positive control during antibody test.The preparation method of splenic lymphocyte is identical with the preparation method of feeder layer cells, sees embodiment 5.
Embodiment 7: the selectivity of cytogamy and hybridoma is cultivated
1) by 1 × 10
8splenic lymphocyte (prepared by embodiment 6) and 2 × 10
7~ 5 × 10
7myeloma cell SP2/0 (prepared by embodiment 4) is mixed in 50mL fusion pipe, adds incomplete substratum (originating the same) and, to 30mL, fully mixes.
2) centrifugal 10min under 1000r/min, exhaustion supernatant.
3) flick at the bottom of fusion pipe, make sedimentation cell evenly loose.
4) with 1mL suction pipe in the concentration that about 1min adds 800 μ L37 DEG C preheatings be the polyoxyethylene glycol (PEG) 4000 (pH8.0) of 45% (w/v), limit edged rotates fusion pipe gently.
5) in 90s, the incomplete substratum of 20mL37 DEG C of preheating is added with 10mL suction pipe.
6) the centrifugal 8min of 1000r/min, abandons supernatant.
7) flicking at the bottom of pipe makes sedimentation cell loose, slowly adds HAT substratum (the purchased from American Gibco company) suspension cell of 60mL37 DEG C of preheating.
8) cell suspending liquid is transferred in large bottle, adds HAT substratum to 80 ~ 100mL, mixing.
9) draw 100 μ L to add in the 96 hole feeder layer cells culture plates (prepared by embodiment 5) prepared, put the CO of 4% concentration
2in incubator, 37 DEG C of constant temperature culture.
10) 1/2 substratum displaced with HAT substratum afterwards for 5 days in culture hole is cultivated.
11) cultivate and within 7 ~ 10 days, use HT substratum (purchased from American Gibco company) to displace HAT substratum in culture hole afterwards.
12) observe the growing state of hybridoma, when cell grows to hole floorage more than 1/10, get supernatant detect (method is shown in embodiment 3).
Embodiment 8: filtering hybridoma
ELISA method (concrete operations are shown in described in embodiment 3) is utilized to detect cell culture supernatant, screening hybridoma.
Embodiment 9: the subclone of hybridoma
1) prepare feeder layer cells, concrete grammar is see embodiment 5.
2) hybridoma suspension to be cloned is prepared: with HT substratum (originate the same) dilution 200 hybridomas of 10mL containing the serum of 20% (v/v) concentration, be added dropwise in the feeder layer cells prepared, in hybridoma subclone to the 96 porocyte culture plate of every hole.
3) culture plate is put the CO of 4% concentration
2in incubator, cultivate 7 ~ 10 days, observe under inverted microscope, mark the hole only having single clonal growth for 37 DEG C, get supernatant and detect (experimental technique is shown in described in embodiment 3).
4) hybridoma getting monoclonal antibody test positive hole in step 3 carries out enlarged culturing, and liquid nitrogen cryopreservation.
Immune mouse splenic lymphocyte and myeloma cell are carried out fusion cultivate, hybridoma after ELISA method screening and limiting dilution assay subclone, acquisition can secrete anti-fumonisins B1's and the hybridoma cell strain 1C11 of the high monoclonal antibody of avidity.
Embodiment 10: utilize ascites method to prepare monoclonal antibody in a large number
1) Balb/c mouse peritoneal inoculation liquid of homology being grown up paraffin body, every mouse 300 ~ 500 μ L.
2) after 7 ~ 10 days, the hybridoma (3 × 10 diluted with 500 μ L PBS
5) carry out intraperitoneal inoculation.
3) interval is after 5 days, observes mouse ascites production every day, and as belly obviously expands, when touching with hand, skin has tension, and namely available No. 16 syringe needles gather ascites, can continuous acquisition 2 ~ 3 times.
4) step 3 gained ascites 3000r/min is carried out centrifugal 10min, collect supernatant.
5) antibody titer (concrete grammar is shown in described in embodiment 3) before utilizing ELISA method to detect purifying, frozen for subsequent use at-70 DEG C after packing.
ELISA method is adopted to detect the result of monoclonal antibody in mouse ascites as shown in Figure 3.As can be seen from the figure, the monoclonal antibody 1C11 in ascites has higher bonding force to fumonisins B1.
Embodiment 11: utilize ammonium sulfate precipitation method monoclonal antibody purification
1) ascites of embodiment 10 gained is added 2 times of volume PBS to dilute.
2) ammonium sulfate is joined in the ascites dilutions of step 1 according to 0.277g/mL concentration, stir under ice bath in adding procedure.
3) in 4 DEG C of standing 2h or spend the night.
4) centrifugal 10min under 12000r/min, abandons supernatant.
5) redissolve to original volume with PBS, repeating step 2 ~ 4.
6) dissolve with appropriate PBS, measure concentration, frozen for subsequent use in-70 DEG C after adding the glycerine packing of 50% (v/v) concentration.
5) bonding force (method is shown in embodiment 3) of the monoclonal antibody after ELISA method detection purifying is adopted.
ELISA method is adopted to detect the monoclonal antibody of purifying as shown in Figure 3.As can be seen from ELISA result, from ascites, the monoclonal antibody 1C11 of purifying still keeps very high bonding force to fumonisins, the curve that its gradient dilution detects and ascites detected result are consistent, and illustrate that ammonium sulfate precipitation purge process does not obviously affect the bonding force of antibody.Therefore, the monoclonal antibody 1C11 of hybridoma cell strain secretion has very high activity, can be used for the detection of fumonisins B1.
The monoclonal antibody of embodiment 12:SDS-PAGE electrophoresis detection purifying
1) monoclonal antibody of 2 μ L embodiment 11 purifying is got, add 2.5 μ L5 × sodium lauryl sulphate (SDS) sample loading buffers (composition: the Tris-HCl (pH6.8) of 250mM concentration, the SDS (electrophoresis level) of 10% (w/v) concentration, the tetrabromophenol sulfonphthalein of 0.5% (w/v) concentration, the glycerine of 50% (v/v) concentration, the beta-mercaptoethanol of 5% (w/v) concentration), abundant mixing, 5min is boiled in water-bath, is then placed in for subsequent use on ice.
2) with reference to [U.S.s] such as J. Pehanorm Brookers, " Molecular Cloning: A Laboratory guide ", Science Press, the method preparative separation glue that the third edition is introduced and concentrated glue in 2003.
3) albumen vertical electrophoresis system (purchased from BIO-RAD company) is installed, add 1 × Tris-glycine running buffer (composition: the Tris of 25mM concentration, 250mM glycine, the SDS of 0.1% (w/v) concentration), get the sample loading be placed on ice.
4) voltage 80 volts of electrophoresis 20min are first set, then run to outside separation gel to tetrabromophenol sulfonphthalein with 120 volts of electrophoresis 80 ~ 120min.
5) gel is taken off, staining fluid (composition: the coomassie brilliant blue R_250 of 0.1% (w/v) concentration is used after removing concentrated glue, the Virahol of 25% (v/v) concentration, the Glacial acetic acid of 10% (v/v) concentration) dye 30min.
6) decolour 3 ~ 5 times with destainer (composition: the methyl alcohol of 5% (v/v) concentration, the Glacial acetic acid of 7.5% (v/v) concentration), observe and take a picture.
SDS-PAGE electrophoresis detection result, as shown in a figure in Fig. 4, has the protein band of an entry as seen respectively in the position of about 25kDa and 50kDa size, and the monoclonal antibody purity of purifying is higher.
Embodiment 13: hybridoma Total RNAs extraction
1) embodiment 8 is screened and the positive hybridoma cell strain enlarged culturing in 24 porocyte culture plates obtained through embodiment 9 subclone.
2) by cell suspension, be transferred in 50mL centrifuge tube, 4 DEG C, the centrifugal 5min of 1500r/min, abandons supernatant.
3) add 10mL RPMI-1640 substratum (purchased from American Gibco company) re-suspended cell, 4 DEG C, the centrifugal 5min of 1500r/min, abandons supernatant.
4) drawing enchylema is transferred in 2mL centrifuge tube, 4 DEG C, and the centrifugal 5min of 1500r/min exhausts substratum supernatant, flicks at the bottom of pipe and make cell dispersal.
5) 2mL TRNzol-A+ (purchased from TIANGEN Biotech (Beijing) Co., Ltd.) is added, 0.7 ~ 0.8mm syringe pressure-vaccum more than 5 times is used immediately with after pipettor mixing, make lysis, homogenate is dispensed into (1mL/ pipe) in 1.5mL centrifuge tube.
6) often pipe adds 200 μ L chloroforms, at room temperature hatches 5min after firmly rocking 15s.
7) 4 DEG C, the centrifugal 15min of 11000r/min.
8) get 500 μ L supernatant liquors to transfer in another centrifuge tube, add equal-volume Virahol, under room temperature, precipitate 10min.
9) 4 DEG C, the centrifugal 15min of 11000r/min, abandons supernatant.
10) add 1mL concentration be 75% (v/v) washing with alcohol RNA precipitation once.
11) 4 DEG C, the centrifugal 5min of 8500r/min.
12) remove supernatant, drying at room temperature 10 ~ 20min, add 20 μ L and dissolve RNA without RNase ultrapure water.
Embodiment 14: reverse transcription synthesis cDNA
Take mRNA as template, with Oligo (dT) 12-18 primer and SuperScript III ThermoScript II synthesis monoclonal antibody encoding gene cDNA first chain.Its operating process is as follows:
1) in centrifuge tube, add special primer or Oligo (dT) 12-18 primer, each 1 μ L of RNA and dNTPs (10mM), add and mix without RNase ultrapure water 10 μ L.
2) 65 DEG C of water-bath 5min, take out to be placed on immediately and hatch 1min on ice.
3) add 5 × First-strand Buffer4 μ L after gentle centrifugation, 0.1mol/L DTT, RNA enzyme inhibitors and
iII ThermoScript II (200U/ μ L) each 1 μ L, mixing.
4) 50 DEG C of water-bath 1h.
5) 70 DEG C of water-bath 15min make enzyme deactivation, be stored in-70 DEG C for subsequent use.
Embodiment 15:PCR increases variable region of heavy chain (V
h), variable region of light chain (V
l) and single-chain antibody (scFv) gene
1) pcr amplification variable region of heavy chain (V
h) and variable region of light chain (V
l) gene
With the cDNA of embodiment 14 reverse transcription synthesis for template, utilize forward mix primer MVHF1 ~ 10 described in table 1 and reverse mix primer MVHB1 ~ 4 to carry out pcr amplification respectively, obtain the variable region of heavy chain fragment (V of antibody
h); Utilize forward mix primer MVLF1 ~ 5 and reverse mix primer MVLB1 ~ 3 to carry out pcr amplification, obtain kappa light chain variable district fragment (V
κ); Utilize primer pair MVLF6 and MVLB4 to carry out pcr amplification, obtain lambda light chain variable district fragment (V
λ).In 50 μ LPCR reaction solutions, containing 4 μ LcDNA, 5 μ LPCR damping fluids are (containing Mg
2+), 8 μ L concentration are the dNTPs of 1.25mM, 2 μ L forward (mixing) primers (10 μMs), 2 μ L are (mixing) primer (10 μMs) oppositely, 2.5U Taq polysaccharase (purchased from Beijing Quanshijin Biotechnology Co., Ltd).PCR reaction conditions is: 95 DEG C of 5min; 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 80sec, 30 circulations; Last 72 DEG C of 10min.
2) with DNA gel reclaim test kit (purchased from QIAGEN company, operating according to the specification sheets of this test kit) respectively purification step 1 to increase the V obtained
hand V
l(V
κor V
λ) fragment.
3) SOE-PCR amplification single-chain antibody (scFv) gene
Add the V of the volumetric molar concentration such as near
hand V
lfragment, as template, carries out SOE-PCR amplification with forward mix primer MVHF1 ~ 10 described in table 1 and reverse mix primer MVLB1 ~ 4, obtains scFv gene.SOE-PCR has reacted in two steps, and the first step is reacted in 50 μ LPCR reaction solutions, containing V
hand V
lthe each 400ng of fragment, 5 μ L PCR damping fluids are (containing Mg
2+), 8 μ L concentration are the dNTPs of 1.25mM, 2.5U Taq polysaccharase.PCR reaction conditions is: 95 DEG C of 5min, 55 DEG C of 2min, 72 DEG C of 15min, 7 circulations; Second step reaction is in 50 μ LPCR reaction solutions, and containing 10 μ L the first step reaction PCR primer, 4 μ L PCR damping fluids are (containing Mg
2+), 8 μ L concentration are the dNTPs of 1.25mM, 2 μ L forward mix primer (10 μMs), the reverse mix primer of 2 μ L (10 μMs), 2.5UTaq polysaccharase.PCR reaction conditions is: 95 DEG C of 5min; 94 DEG C of 1min, 55 DEG C of 80sec, 72 DEG C of 2min, 30 circulations; Last 72 DEG C of 10min.
4) the scFv gene of kits recycling step 3 acquisition is reclaimed with DNA gel.
Table 1. pcr amplification and the sequence table of order-checking the primer
Embodiment 16:pHENHi carrier and scFv fragment enzyme are cut, are connected
1) use SfiI and NotI restriction enzyme (purchased from NEB company respectively, operate according to the specification sheets of this enzyme) double digestion carrier pHENHi (Peschen D, etal.Fusion proteins comprising a Fusarium-specific antibody linked to antifungal peptides protect plants against a fungal pathogen.Nat.Biotechnol., 2004,22:732-738, as shown in Figure 5) and the scFv gene that obtains of embodiment 15.
2) with DNA gel reclaim test kit (purchased from QIAGEN company, operating according to the specification sheets of this test kit) purification step 1 enzyme cut after pHENHi carrier and scFv fragment.
3) by pHENHi carrier and the scFv fragment of the recovery of T4DNA ligase enzyme (purchased from NEB company, operating according to the specification sheets of this enzyme) Connection Step 2, obtain enzyme and connect product pHENHi-scFv.
4) with reference to [U.S.s] such as J. Pehanorm Brookers, " Molecular Cloning: A Laboratory guide ", Science Press, 2003, the method for third edition introduction connected product pHENHi-scFv to the enzyme that step 3 obtains and carries out alcohol settling desalination.
Embodiment 17: mono-clonal recombinant antibodies construction of gene library
1) intestinal bacteria XL1-Blue MRF ' competent cell preparation
Coli strain XL1-Blue MRF ' (purchased from Wuhan Kai Sheng Bioisystech Co., Ltd) is dipped with sterile toothpick, at LB solid medium (composition: the Tryptones of 1% (w/v) concentration, the yeast extract of 0.5% (w/v) concentration, the NaCl of 1% (w/v) concentration, the agar of 1.5% (w/v) concentration, pH7.0) flat lining out, after 16h cultivated by 37 DEG C of thermostat containers, picking one mono-clonal colony inoculation is to 5mL LB liquid nutrient medium (composition: the Tryptones of 1% (w/v) concentration, the yeast extract of 0.5% (w/v) concentration, the NaCl of 1% (w/v) concentration, pH7.0) in, 37 DEG C, 200r/min shaken overnight cultivates 16h.Getting 1.6mL bacterium liquid joins in 160mL LB liquid nutrient medium, and 37 DEG C, 230r/min shaking culture is to OD
600nmwhen reaching about 0.45, take out and cultivate after the triangular flask of bacterium is placed in cooled on ice 30min, bacterium liquid is sub-packed in 50mL centrifuge tube, 4 DEG C, the centrifugal 15min of 4000r/min, abandon supernatant, then use glycerine washing bacterial sediment twice (4 DEG C, abandon supernatant after the centrifugal 15min of 4000r/min) of 10% (v/v) concentration of 30mL precooling.GYT substratum (the composition: the Tryptones of 0.25% (w/v) concentration of 100 μ L precoolings is added in last each centrifuge tube, the yeast extract of 0.125% (w/v) concentration, the glycerine of 10% (v/v) concentration) suspension thalline, be distributed into 100 μ L/ to manage, be stored in-80 DEG C of refrigerators immediately.
2) electric saccharase connects product
-80 DEG C of intestinal bacteria XL1-Blue MRF ' competent cells preserved are taken out, put after dissolving on ice, often add 5 μ L enzymes in pipe competent cell (100 μ L) and connect product pHENHi-scFv, put after careful mixing slightly and leave standstill 3min on ice, then electricity competent cell being transferred to precooling on ice transforms in cup (0.2cm) (purchased from BIO-RAD company), with the MicroPulser of BIO-RAD company
tMelectricity conversion instrument carries out electricity and transforms (arranging Bacteria Ec2 program).1mL SOC substratum (composition: the Tryptones of 2% (w/v) concentration is added immediately after conversion, the yeast extract of 0.5% (w/v) concentration, the NaCl of 0.05% (w/v) concentration, the glucose of 20mM concentration, pH7.0) transform in cup to electricity, and bacterium liquid is transferred in centrifuge tube, 37 DEG C, 200r/min shaking culture 1h makes cell recovery.
3) Electroporation-competent cells spread plate is cultivated
Taking out the competent cell after recovery, getting the LB solid medium that 100 μ L coat containing the glucose of 1% (w/v) concentration and 100 μ g/mL penbritins (Amp) dull and stereotyped for calculating mono-clonal colony number.Remaining bacterium liquid abandons part supernatant by inhaling after the centrifugal 1min of 6000r/min, often pipe retains the resuspended thalline of about 100 ~ 150 μ L supernatant, the LB solid medium coated containing the glucose of 1% (w/v) concentration and 100 μ g/mLAmp is dull and stereotyped, is placed in 37 DEG C of thermostat container incubated overnight 12 ~ 16h to growing mono-clonal bacterium colony.
4) library of antibody genes Collection and conservation
Calculate the mono-clonal colony counts grown after Electroporation-competent cells spread plate is cultivated, estimation library of antibody genes capacity.Collect the bacterium colony that LB solid medium flat board grows, the glycerine adding equal-volume 50% (v/v) concentration is stored in-80 DEG C of refrigerators.
Embodiment 18: phage display screening mono-clonal recombinant antibodies gene library
1) the mono-clonal recombinant antibodies gene library that 500 μ L embodiments 17 are preserved is got, join 50mL containing the glucose of 1% (w/v) concentration and 2 × TY substratum (composition: the Tryptones of 1.6% (w/v) concentration of 100 μ g/mLAmp, the yeast extract of 1% (w/v) concentration, the NaCl of 0.5% (w/v) concentration, pH7.0) in, 37 DEG C, 200r/min shaking culture is to OD
600nmreach 0.5.
2) 5mL bacterium liquid is got in 50mL centrifuge tube, add 0.5 μ L M13KO7 helper phage (purchased from Amersham Biosciences company, with reference to [U.S.s] such as J. Pehanorm Brookers, " Molecular Cloning: A Laboratory guide ", Science Press, 2003, the method preparation that the third edition is introduced was preserved, wherein: the amount of phage is colibacillary 20 times), 30min is left standstill in 37 DEG C of water-baths after mixing.
3) centrifugal 10min under 4000r/min, abandons supernatant, and be then resuspended in 140mL and contain in 2 × TY substratum of 100 μ g/mLAmp and 25 μ g/mL kantlex (Kan), 30 DEG C, 200r/min shaken overnight cultivates at least 15h.
4) the bacterium liquid of incubated overnight is sub-packed in 50mL centrifuge tube, 4 DEG C, the centrifugal 30min of 4000r/min.
5) get supernatant, add the PEG/NaCl solution (composition: the PEG6000 of 20% (w/v) concentration, the NaCl of 2.5M concentration) of 1/5 volume, fully place 1h on ice after mixing.
6) 4 DEG C, the centrifugal 30min of 8000r/min, abandons supernatant, precipitation is resuspended in 40mL sterilized water, and adds the PEG/NaCl solution of 1/5 volume, and fully 20min is placed in latter 4 DEG C of mixing.
7) 4 DEG C, the centrifugal 30min of 4000r/min, abandons supernatant.
8) gentle centrifugation, removes residual PEG/NaCl solution.
9) add the resuspended precipitation of 1.6mL sterilized water, 4 DEG C, the centrifugal 10min of 12000r/min, get supernatant in 4 DEG C of preservations.
10) with 20ng/ μ L antigen FB1-BSA (second take turns be respectively 15ng/ μ L and 10ng/ μ L with concentration during third round elutriation) bag by elisa plate (totally 20 holes, every hole 100 μ L), 37 DEG C of water-bath 2h.
11) 3 times are washed with 200 μ L PBS.
12) every hole adds the BSA solution that 150 μ L concentration are 2% (w/v), and 2h is closed in 37 DEG C of water-baths.
10) 3 times are washed, each 3min with 200 μ LPBS.
11) supernatant (phage) that 100 μ L steps 9 are preserved is added in every hole, 37 DEG C of water-bath 2h.
12) 5 times are washed respectively with 200 μ LPBST and PBS, each 3min (second takes turns and increase washing times to 10 time and 15 times with during third round elutriation, and the concentration of Tween20 in third round PBST is increased to 0.5% (v/v)).
13) every hole adds 100 μ L concentration is the triethylamine solution of 100mM, and room temperature places 10min.
14) add Tris-HCl solution (pH7.4) neutralization that 50 μ L concentration are 1M immediately, and be transferred in 50mL centrifuge tube.
15) get 6mL at 37 DEG C, under 230r/min shaking culture condition, grow to OD
600nmthe intestinal bacteria XL1-Blue MRF ' reaching 0.5 ~ 0.9 joins in 50mL centrifuge tube, and 30min is infected in 37 DEG C of water-baths.
16) the centrifugal 10min of 4000r/min, abandons supernatant.
17) TYE solid medium (composition: the Tryptones of 1% (w/v) concentration is coated after adding the resuspended thalline of 800 μ L LB substratum, the yeast extract of 0.5% (w/v) concentration, the NaCl of 0.8% (w/v) concentration, the agar of 1.5% (w/v) concentration, pH7.0), on flat board, 37 DEG C of thermostat container incubated overnight 12 ~ 16h are to growing bacterium colony.
18) collect the bacterium colony that TYE solid medium flat board grows, the glycerine carrying out next round elutriation or add equal-volume 50% (v/v) concentration is stored in-80 DEG C of refrigerators.
Embodiment 19:Phage ELISA identifies elutriation antibody library
1) in 96 porocytes cultivation plate holes, 180 μ L are added containing the glucose of 1% (w/v) concentration and 2 × TY substratum of 100 μ g/mLAmp, with sterilizing toothpick random picking 48 mono-clonal inoculations from the bacterium colony of third round elutriation antibody library, 30 DEG C, 150r/min shaken overnight cultivates 16h.
2) add in another 96 well culture plate hole 180 μ L2 × TY substratum (containing the glucose of 1% (w/v) concentration, the Amp and about 10 of 100 μ g/mL concentration
8helper phage M13KO7), then add 20 μ L incubated overnight bacterium liquid, 37 DEG C, 150r/min shaking culture 2h.
3) the centrifugal 10min of 1800r/min under room temperature condition, abandons supernatant.
4) add the 2 × TY substratum of 180 μ L containing 100 μ g/mLAmp and 50 μ g/mL Kan to cultivating in plate hole, 37 DEG C, 150r/min shaken overnight cultivates 16h.
5) the centrifugal 10min of 1800r/min, substratum supernatant is used for phage ELISA and detects.
6) in elisa plate hole, add FB1-BSA or PBS (contrast) that 100 μ L concentration are 2 μ g/mL, 37 DEG C of water-bath bags are by 2h.
7) 3 times are washed with 200 μ L PBS.
8) add the BSA solution that 150 μ L concentration are 1% (w/v), 2h is closed in 37 DEG C of water-baths.
9) 3 times are washed with 200 μ L PBS.
10) add the substratum supernatant that 50 μ L steps 5 are collected, and add the BSA solution that 50 μ L concentration are 1% (w/v), 37 DEG C of reaction 2h.
11) 3 times are washed respectively with 200 μ L PBST and PBS.
12) HRP adding 100 μ L, 1: 5000 dilution by volume marks mouse-anti M13 antibody (purchased from Amersham Biosciences company), 37 DEG C of reaction 2h.
13) 3 times are washed respectively with 200 μ L PBST and PBS.
14) add 100 μ L solubilities single-component tmb substrate (purchased from TIANGEN Biotech (Beijing) Co., Ltd.), under dark condition, react 15 ~ 30min.
15) adding 50 μ L concentration is the H of 2M
2sO
4solution termination reaction, surveys OD by microplate reader
450nmread value.
The positive colony that Phage ELISA identifies delivers to Nanjing Genscript Biotechnology Co., Ltd.'s order-checking, the mono-clonal recombinant antibodies that result display derives from hybridoma cell strain 1C11 is H2, its nucleotide coding sequence is as shown in sequence table SEQ ID NO:1, and its aminoacid sequence is as shown in sequence table SEQ ID NO:2.Intestinal bacteria containing this gene are named as recombination bacillus coli XL1-Blue MRF '/pHENHi-H2 (the intestinal bacteria XL1-Blue MRF ' bacterial strain namely containing pHENHi-H2 plasmid DNA (as shown in Figure 6)), and the glycerine that Colony Culture bacterium liquid adds equal-volume 50% (v/v) concentration is stored in-80 DEG C of refrigerators.
Embodiment 20: recombination bacillus coli overexpression, purifying H2 mono-clonal recombinant antibodies
1) recombination bacillus coli XL1-Blue the MRF '/pHENHi-H2 getting 5 μ L embodiments 19 preservations is inoculated in 20mL containing in the glucose of 1% (w/v) concentration and 2 × TY substratum of 100 μ g/mLAmp, 37 DEG C, 200r/min shaken overnight cultivates 12h.
2) get 8mL incubated overnight bacterium liquid and add 160mL containing in the glucose of 1% (w/v) concentration and 2 × TY substratum of 100 μ g/mLAmp, 37 DEG C, 200r/min shaking culture is to OD
600nmreach about 0.5.
3) add the isopropyl-β-D-thiogalactoside(IPTG) (IPTG) that final concentration is 0.1mM, be placed in 30 DEG C, 200r/min abduction delivering 8h.
4) getting cultivation bacterium liquid is dispensed in 50mL centrifuge tube, and 4 DEG C, the centrifugal 10min of 5000r/min, abandons supernatant.
5) often 500 μ LPPP solution (compositions: the Tris-HCl of 30mM concentration are added in pipe, the sucrose of 20% (w/v) concentration, pH8.0) resuspended thalline, then ethylenediamine tetraacetic acid (EDTA) (EDTA) solution adding that 1 μ L final concentration is 1mM, place 15min on ice.
6) 4 DEG C, the centrifugal 15min of 5000r/min, supernatant loads in another centrifuge tube.
7) often add the resuspended thalline of MgCl2 solution that 1mL final concentration is 5mM in pipe, then add the EDTA solution that 2 μ L final concentrations are l mM, place 15min on ice.
8) 4 DEG C, the centrifugal 15min of 5000r/min, supernatant and step 6 supernatant merge.
9) get collect step 6 and 8 supernatants centrifuge tube in 4 DEG C, the centrifugal 15min of 12000r/min.
10) get supernatant PBS to dialyse 72h.
11) purification column (purchased from BIO-RAD company) is installed, adds the matrix (purchased from QIAGEN company) that 400 μ L fully mix, make its fixing more than 2h.
12) cut off lower end closure, under making liquid stream, balance with 5mL PBS.
13) post crossed by the sample (mono-clonal recombinant antibodies) added after step 10 dialysis, and Collection and conservation crosses the sample effluent liquid after post.
14) 1mL buffer B (composition: the NaH of 50mM concentration is added
2pO
4, the imidazoles of the NaCl of 300mM concentration, 20mM concentration, pH8.0) and eluting post 3 times, collecting elutriant is respectively B1, B2 and B3 (foreign protein).
15) 400 μ L damping fluid C (compositions: the NaH of 50mM concentration are added
2pO
4, the imidazoles of the NaCl of 300mM concentration, 250mM concentration, pH8.0) and eluting post 3 times, collecting elutriant is respectively C1, C2 and C3 (target protein).
16) after adding 5mL PBS balance purification column, add the alcohol of 1mL30% (v/v) concentration, preserve purification columns for 4 DEG C.
17) mono-clonal recombinant antibodies gets 10 μ L by after SDS-PAGE electrophoresis detection (concrete operations are shown in described in embodiment 12), dialyses with PBS.
SDS-PAGE electrophoresis detection result, as shown in the b figure in Fig. 4, has the target protein band of a treaty 32kDa size as seen, and the H2 mono-clonal recombinant antibodies purity of expression is higher, and integrity is good.
Embodiment 21: utilize surface plasma resonance technology (SPR) to measure the bonding force of 1C11 and H2 antibody
1) according to the operational manual chip of the BIAcore T200 of GE Healthcare company, setting working temperature is 25 DEG C, injection operation 1 × HBS-EP damping fluid (composition: the 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES of 10mM concentration, pH7.4), the NaCl of 0.15M concentration, the EDTA of 3mM concentration, the tensio-active agent P20 of 0.005% (v/v) concentration), flow velocity is 10 μ L/min, initialize sensing chip 3 ~ 4 times, obtains steady baseline after 2 ~ 3min.
2) take the speed implantation concentration of 10 μ L/min as antigen protein (FB1-BSA) 1min of 8 μ g/mL, make it be combined with chip.
3) antibody respectively with different concentration (4,16,64,256,1024nM) and the speed injection of 30 μ L/min to the surface of FB1-BSA.
4) combination and Dissociation time are respectively 4min and 10min.
5) injection concentration is glycine solution (pH2.0) 30s of 10mM, regeneration chip.
6) the software BIAcore T200evaluation software by giving as an addition at random simulates the bonding force analyzing antibody.
Adopt the result of SPR analysis as shown in Figure 7, the binding kinetics parameter of the antibody obtained after utilizing the analysis of BIAcore T200evaluation software simulation is in table 2.Kinetic constant (the K that 1C11 and H2 antibody is combined with fumonisins
d) reach 9.84 × 10 respectively
-8m and 1.20 × 10
-9m, the latter also higher than the former about 82 times, illustrate that 2C5 and H7 antibody all has very high bonding force to fumonisins, can be used for fumonisins pollute detection application.
Table 2SPR measures the kinetic constant of 1C11 and H2 antibody
Note: k
a: binding constant; k
d: dissociation constant; K
d: kinetic constant (K
d=k
d/ k
a)
Embodiment 22: the mono-clonal recombinant antibodies H2 of purifying is used for the detection of fumonisins
1) in elisa plate hole, add the FB1-BSA that 100 μ L concentration are 1 μ g/mL, 37 DEG C of water-bath bags are by 2h.
2) 3 times are washed with 200 μ L PBS.
3) in antigen coated hole and control wells, add the BSA solution that 150 μ L concentration are 1% (w/v) respectively, 2h is closed in 37 DEG C of water-baths.
4) 3 times are washed with 200 μ L PBS.
5) in antigen coated hole and control wells thereof, the H2 antibody that 100 μ L concentration are embodiment 20 purifying of 200nM is added respectively, 37 DEG C of reaction 2h.
6) 3 times are washed respectively with 200 μ L PBST and PBS.
7) every hole adds the anti-His monoclonal antibody (purchased from TIANGEN Biotech (Beijing) Co., Ltd.) of 100 μ L, 1: 5000 dilution by volume, 37 DEG C of reaction 1.5h.
8) 3 times are washed respectively with 200 μ L PBST and PBS.
9) every hole adds AP mark sheep anti-mouse igg antibody (available from Sigma) of 100 μ L, 1: 5000 dilution by volume, 37 DEG C of reaction 1.5h.
10) 3 times are washed respectively with 200 μ L PBST and PBS.
11) every hole adds the pNPP solution that 100 μ L concentration are 0.2% (w/v), reacts 15 ~ 30min under dark condition.
12) every hole adds the NaOH solution termination reaction that 50 μ L concentration are 3M, surveys OD by microplate reader
405nmread value.
After adding pNPP nitrite ion colour developing 30min, sample OD
405nmon average read value (1.225) and negative control OD
405nmon average read the ratio P/N=28.5 of value (0.043), illustrate that the H2 mono-clonal recombinant antibodies through intestinal bacteria great expression purifying still keeps very high avidity to fumonisins, can be used for the detection of fumonisins.
Claims (2)
1. the hybridoma cell strain 1C11 of anti-fumonisins B1 monoclonal antibody is secreted in a strain, and it is characterized in that, this hybridoma cell strain is deposited in China typical culture collection center (CCTCC), and its preserving number is: CCTCC NO:C2011122.
2. the mono-clonal recombinant antibodies H2 of an anti-fumonisins B1, it is characterized in that, the coding nucleotide sequence of described H2 mono-clonal recombinant antibodies is as shown in sequence table SEQ ID NO:1, and its aminoacid sequence is as shown in sequence table SEQ ID NO:2, and it also has following features:
Described H2 mono-clonal recombinant antibodies is primarily of antibody heavy chain variable region V
h, antibody chain variable region V
lwith connection peptides composition, described antibody heavy chain variable region V
hwith antibody chain variable region V
lby connection peptides (Gly
4ser)
3connect the identification and the combination that jointly complete fumonisins,
Wherein:
Described antibody heavy chain variable region VH is by 387 nucleotide codings, and its nucleotide sequence is as shown in sequence table SEQ ID NO:3;
Described antibody heavy chain variable region VH is made up of 129 amino acid, and its aminoacid sequence is as shown in sequence table SEQ ID NO:4;
Described antibody chain variable region VL is by 330 nucleotide codings, and its nucleotide sequence is as shown in sequence table SEQ ID NO:5;
Described antibody chain variable region VL is made up of 110 amino acid, and its aminoacid sequence is as shown in sequence table SEQ ID NO:6.
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| CN101955542A (en) * | 2010-05-06 | 2011-01-26 | 北京维德维康生物技术有限公司 | Immunoassay kit and special antibody for detecting fumonisins |
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| CN101955542A (en) * | 2010-05-06 | 2011-01-26 | 北京维德维康生物技术有限公司 | Immunoassay kit and special antibody for detecting fumonisins |
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