CN103172740A - Preparation method of beta-estradiol analogue antibody and use thereof - Google Patents
Preparation method of beta-estradiol analogue antibody and use thereof Download PDFInfo
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- CN103172740A CN103172740A CN 201110440667 CN201110440667A CN103172740A CN 103172740 A CN103172740 A CN 103172740A CN 201110440667 CN201110440667 CN 201110440667 CN 201110440667 A CN201110440667 A CN 201110440667A CN 103172740 A CN103172740 A CN 103172740A
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Abstract
The invention relates to a screening method of a beta-estradiol analogue antibody and an application thereof and belongs to the biotechnological field. The analogue antibody is screened out from a ribosome display anticalin analogue antibody library and solubility functional function is carried out onto the analogue antibody. The invention further relates to a method for screening the estradiol analogue antibody by utilizing a ribosome display technology. The screened estradiol analogue antibody can be applied to detecting estradiol illegal addition and misuse and developing an immunity quick-test kit for quickly detecting the residual estradiol.
Description
Technical field
The present invention relates to a kind of screening method of beta estradiol analog antibody, belong to biological technical field, for the preparation of the purposes of can the rapid detection beta estradiol residual immune quick testing reagent box of the efficient analog antibody of specific recognition beta estradiol and development.
Background technology
Beta estradiol (β-estradiol, β-E
2, hereinafter to be referred as E
2) be white or oyster white crystal powder, odorless is soluble in dioxane or acetone, is slightly soluble in ethanol, and is water insoluble.Its molecular formula is C
18H
24O
2Molar mass is 272.39g/mol, fusing point is 173～179 ℃, E2 is synthetic in animal and human's body, activity with endogenous hormone, on human and animal's impact be by simulation, disturb or the antagonism body in give birth to original normal synthetic, the transportation of hormone, discharge and effect, make its balance that can't keep self and regulate and realize that E2 is to the harm of biology, in the majority with hydrobiont, be mainly manifested in the impact on reproductive system, can cause the synthetic acceleration of vitellogenin in organism, the animals female phenomenon appears in the sexual organ variation.This is because E2 can play restraining effect to the enzyme in the steroid building-up process relevant with reproduction, and the synthetic of steroid hindered.The discoveries such as Irwin are obviously improved by the interior vitellogenin level of female green turtle body that E2 affects, and think and can or lay eggs to cause green turtle reproduction infringement in a way by change energy distribution, physiology.Colucci etc. think that E2 has caused the reproduction deformity of wild fish, and Purdom etc. think that the telianthus of fish also is associated with E2.In addition, E2 can pass through the approach such as diet, air intake and skin contact and invade human body, is the normal function of disturbance endocrine reproductive system to the mankind's direct harm, causes the decline of male fecundity even to lose.Peterson etc. think, the decline of mankind spermatozoon quantity, and the increase of carcinoma of testis, prostate cancer and breast cancer incidence and male genetic obstacle are all contacted directly with E2.Rajapakse etc. think that also the minimizing of mankind spermatozoon quantity is the impact that is subjected to E2.As seen E2 wide material sources, very harmful, therefore need to set up detection and the screening methods that cover is fast and convenient, science is sensitive.
Ribosomal display technology (ribosome display, RD) be a kind of external display technique, genotype (DNA sequence dna) and phenotype (protein) can connect, and the genomic information DNA that soon can increase or RNA and selectable mutein link together.In the RD technology, mRNA lacks terminator codon and modifies through suitable, ribose is known from experience the 3 ' end that rests on mRNA in the In Vitro Translation process, form very stable " mRNA-rrna-protein " ternary complex, this ternary complex form connects together protein with mRNA information, use the peptides and proteins screening that RD can carry out specific function.
Lipocalin is the general name of the outer lipophilic protein of one group of born of the same parents containing from the bacterium to people, as acceptor stronger have important biological function in conjunction with small molecules.although there is diversity in lipocalin on primary structure, but surprising similar of its secondary structure and quaternary structure: by 8 anti-phase parallel β-pleated sheet structure sheets and α is helically twisted forms a barrel-like structure, formed " Lipocalin support " structure, contain a ligand binding site, the opening end of bucket is comprised of a plurality of Loop that connect the β band, it is the more pliable and tougher part of whole molecular ratio, and the amino acid member is polare Aminosaeren, Loop1, Loop2, Loop3, Loop4 is the zone of its random mutation, studies show that recently, can be in conjunction with particular target as ligands specific by transformation Lipocalin skeleton, but that is be combined with small molecules by the support analog antibody of design lipocalin, the lipocalin mutant of these designs is called Anticalin, has the ligand specificity.
Summary of the invention
Main purpose of the present invention is to provide a kind of potential detection estradiol analog antibody.
Second purpose of the present invention provides a kind of preparation method of estradiol analog antibody.
Technical scheme of the present invention is summarized as follows:
That anti-estradiol analog antibody provided by the invention is that screening obtains from ribosomal display anticalin analog antibody storehouse and carried out the soluble functional expression.
The preparation method of anti-estradiol analog antibody provided by the invention is:
1. the structure in ribosomal display anticalin analog antibody storehouse
select bilitrien in conjunction with albumen (bilin binding protein, bbp) as the template in anticalin storehouse, design random mutation site, take synthetic BBP gene order as template, carry out overlapping primer extension pcr amplification by the primer of design and synthesize Anticalin analog antibody storehouse fragment, about 522bp, add promotor by PCR method again, intervening sequence, the needed element of the ribosomal display such as loop-stem structure, the ribosomal display anticalin analog antibody storehouse that finally successfully constructs, RNA molecule after transcribe in this storehouse comprises 5 '-loop and 3 '-loop loop-stem structure, a ribosome bind site, the anticalin storehouse of random mutation, C-terminal intervening sequence etc., total length is 950bp approximately.
Wherein the template of mutation library can be other lipocalin molecules, preferred bbp.
2. the screening of anti-estradiol analog antibody
After the in-vitro transcription translation is carried out in the ribosomal display anticalin analog antibody storehouse that builds, alternately screen the analog antibody of estradiol as the antigen solid liquid phase with E2-BSA and E2-magnetic bead, by change Mg
2+Analog antibody-rrna that concentration obtains screening-mRNA triplet dissociates, and obtains corresponding mRNA, the DNA library after being screened through RT-PCR.Then repeat in-vitro transcription-In Vitro Translation-affine screening-RT-PCR amplification procedure, obtain repeated screening several analog antibody DNA libraries of taking turns.After taking turns ribosomal display screening through eight, the obvious enrichment of analog antibody sequence of antigen positive in the analog antibody library.
3. the soluble functional of estradiol analog antibody is expressed
take turns after screening the analog antibody library carrying out Sequence Identification eight, 20 clone's of random choose, this gene and pTIG-TRX are connected into expression vector, express in e. coli bl21 (DE3), identify through SDS-PAGE and Western Blot, at the 20KDa place, the target protein band is arranged, with theoretical albumen in the same size, determine in the supernatant liquor after expression product is present in broken thalline, take the empty plasmid Host Strains as contrast, E2-BSA is coating antigen, E2 is the competition small molecules, competition ELSIA detects and induces the competition of supernatant in conjunction with activity, competition is arranged in conjunction with active further extensive abduction delivering and purifying.
4. the purifying of estradiol analog antibody and evaluation
Utilize Anti-His tag affinity column to carry out purifying to the specific junction mixture of expressing, make the purpose band obtain significantly concentrating and separating, competitive ELISA is analyzed the competition of purifying after product in conjunction with activity.The SPR method detects the avidity of the analog antibody that obtains.
The present invention can also provide a kind of most critical detection reagent---specific combination analog antibody of measuring in the residual detection kit of estradiol in environment and food, although HUMAN HEALTH in the residual serious threat of E2, but its application in livestock industry still can bring certain economic worth, this makes illegal use phenomenon remain incessant after repeated prohibition, the estradiol analog antibody of the present invention preparation can partly replace measures the required reagent of the residual mensuration of estradiol in Food and environment sample, and this analog antibody can also further be applied to the detection of estradiol in serum clinically or urine.
The advantage of the anti-estradiol analog antibody that the present invention is prepared is: (1) utilizes estradiol-BSA and estradiol-magnetic bead as antigen solid phase and alternately screening of liquid phase, can overcome the interference that BSA causes, thereby obtain the antibody of E2 high specific; (2) the ancalin-E2 sequence that obtains is clear, is convenient to genetically engineered operation, is easy in intestinal bacteria functional expression in a large number; (3) molecular weight is little, a little less than immunogenicity, for further clinical application is in the future laid a good foundation.
Description of drawings
The structure in Fig. 1 ribosomal display anticalin analog antibody storehouse, A are the process schematic diagram of building the storehouse, and B is analog antibody storehouse framework schematic diagram.
Fig. 2 builds the agarose gel electrophoresis figure in ribosomal display anticalin analog antibody storehouse, and M is DNA marker, and 1 is ribosomal display anticalin analog antibody storehouse DNA
Fig. 3 eight takes turns the volume analysis of the rear every RT-PCR of the wheel product of screening.
The evaluation of Fig. 4 anticalin-E2 abduction delivering product, wherein A figure induces the SDS-PAGE electrophorogram of broken rear supernatant, and B figure is corresponding Western-blotting figure, and 1-4 is respectively four kinds of anticalin-E2, and M is albumen Marker.
Fig. 5 Salmonella is analyzed the combination activity of anticalin-E2. and wherein E2-A, E2-B, E2-C and E2-D are respectively four kinds of anticalin-E2.
Fig. 6 indirect competitive ELISA is analyzed the competition of anticalin-E2 in conjunction with activity, and the IC50 of four kinds of anticalin-E2 detection E2 is respectively 0.05,0.92,2.52,0.41 μ g/ml.
Fig. 7 SPR analyzes the binding kinetics constant of anticalin-E2A
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment if no special instructions, is ordinary method.Reagent and material used in following embodiment if no special instructions, all can be bought from conventional reagent company and obtain.
The structure of embodiment 1 ribosomal display anticalin analog antibody
One. primer is synthetic
According to the bbp sequence and build the mutational site that Anticalin antibody library needs change and design synthetic primer, the random nucleic acid coding region is designed to NNK, and (N is G, C, A, T; K is G and T), design simultaneously the primer that is connected with the ribosomal display element, main primer is as shown in the table.
Table 1 Oligonucleolide primers
Two. the structure in ribosomal display Anticalin analog antibody storehouse
Ribosomal display Anticalin analog antibody building process schematic diagram and the primer are as shown in Figure 1A, the framework schematic diagram in this storehouse as shown in Figure 1B, shown in, PCR reaction system component is as follows: 2 * PfuPCRmix, 12.5 μ L, template, forward primer, each 1 μ L of reverse primer, ddH
2O9.5 μ L.The first step PCR reaction conditions: 94 ℃ of denaturation 3min; 94 ℃ of sex change 30s; 40 ℃ of annealing 30s; 72 ℃ are extended 1min; 10 circulations; 94 ℃ of sex change 30s; 45 ℃ of annealing 30s; 72 ℃ are extended 1min; 20 circulations; 72 ℃ are extended 5min; 4 ℃ of preservations; Second step, the 3rd step PCR reaction conditions reaction conditions: 94 ℃ of denaturation 3min; 94 ℃ of sex change 30s; 45 ℃ of annealing 30s; 72 ℃ are extended 1min; 5 circulations; 94 ℃ of sex change 30s; 60 ℃ of annealing 30s; 72 ℃ are extended 1min; 25 circulations; 72 ℃ are extended 5min; 4 ℃ of preservations.React complete after, 1.5% agarose gel electrophoresis reclaims reaction product, Promega glue reclaims the test kit purifying and reclaims product.Finally successfully construct ribosomal display anticalin storehouse, length is about 950bp (Fig. 2).
The screening of embodiment 2 estradiol analog antibodies
1. in-vitro transcription and translation
The in-vitro transcription of employing Promega company is carried out the in-vitro transcription translation of DNA library with translation coupling system intestinal bacteria (Escherichia coli) S30 Extract System, hatch 10min at 37 ℃, the outer transcription and translation of perfect aspect, then the translation product ice bath is placed, added the Mg (AC) of 1mol/L in the 50 μ L translation systems
22.6 μ L, and then the WBTH that adds 50 μ L is 100 μ L with the translation product dilution, places on ice.
2. antigen coated
Spend the night with coating antigen (E2-BSA) 100 μ L coated elisa plates, the dereaction of inclining liquid is with sterilization PBS washing 3 times, each 3min.Then use the WBT washed twice, each 3min.At last, fill with screen holes with WBT, place 20min at least on ice.
3. the affine screening of solid phase:
Getting on ice the translation product of placing, to add 1% OVA and the yeast rna of 5 μ L purifying be 100 μ L with the translation product dilution, places on ice.Get rid of and place the WBT that surpasses 20min in screen holes, translation product is transferred in the hole of envelope antigen ice bath jolting 1h.The raffinate in the hole that inclines, WBT washing 5 times, each 3min.Then add elution buffer Elution buffer 150 μ L, on ice jolting 5min wash-out.
4. the affine screening of liquid phase
Get the good magnetic bead of coupling of 100 μ L, PBS washs closed pores 3 times with sterilization, each 3min.Then use cold WBT washed twice, each 3min put into icehouse some minutes with magnetic bead, made magnetic bead ice-cold.In Vitro Translation product solution is added in ice-cold magnetic bead jolting 1 hour gently in icehouse.Remove raffinate in the hole by magnetic frame, WBT washing 5 times, each 3min.Then add elution buffer 150 μ L, on ice jolting 5min wash-out.
5. rebuilding with next round of template screened:
The mRNA that the RNeasy clean up kit purifying screening of employing Pormega company obtains, utilize the test kit of invitrogen to carry out reverse transcription, and further pcr amplification obtains the anticalin library, and with ribosomal display element coupling successively, build next round complete ribosomal display library.
Take turns with eight the RT-PCR product that obtains after screening and distinguish electrophoresis, can see, the content of DNA progressively increases, and illustrates that the DNA sequence dna of the analog antibody that E2 is special is able to highly enriched (Fig. 3).
Functional expression, purifying and the evaluation of embodiment 3 estradiol analog antibodies
One. the functional expression of anti-estradiol analog antibody
1. the amplification of anticalin-E2 gene after the screening
Obtain anticalin-E2 gene with restriction enzyme site with the primer amplification amplification that contains EcoRI, XhoI restriction enzyme site of redesign, primer sequence is as follows: forward primer 5 ' CGC
GAA TTC ATG AAC GTG TAC CAC GAC GGTG 3 ', reverse primer: 5 ' GCC
CTC GAGATT GTT GAC TTT GCA GGC GGCG 3 '.Reaction conditions: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s; 50 ℃ of annealing 30s; 72 ℃ are extended 1min20s; 10cycles; 94 ℃ of sex change 30s; 56 ℃ of annealing 30s; 72 ℃ are extended 1min20s; 20cycles; 72 ℃ are extended 7min; 4 ℃ of preservations.1.5% agarose gel electrophoresis is identified amplified production, and glue reclaims the purpose fragment.
2. restructuring anticalin-E2 prokaryotic expression plasmid builds:
With EcoRI, XhoI double digestion plasmid pTIG-TRX.Enzyme is cut system 50 μ L:EcoRI 2.5 μ L, XhoI 2.5 μ L, 10 * buffer, 5 μ L, dd H
2O 10 μ L; Plasmid pTIG-TRX 30 μ L.37 ℃ of enzymes are cut and are spent the night.1.5% agarose gel electrophoresis, the plasmid pTIG-TRX that anticalin-E2 fragment and the enzyme of being connected of EcoRI, XhoI double digestion purifying are cut connects with the T4DNA ligase enzyme, linked system: pTIG-TRX 1 μ L, anticalin-E2 DNA 3 μ L, dd H
2O 4 μ L, T4 ligase enzyme (Takara) 1 μ L, 10 * buffer, 1 μ L, 16 ℃ connect 3h.Transform Host Strains BL21 (DE3).
3.Anticalin-E2 the abduction delivering of albumen
The positive bacterium colony that step 2 is identified is inoculated in the LB liquid nutrient medium that 5ml contains Pyocianil, 30 ℃, the 180r/min overnight incubation, next day the bacterium liquid of incubated overnight is transferred again in the ratio of 1: 100 and contain the LB liquid nutrient medium of Pyocianil in 5mL, and establish the empty plasmid contrast, 30 ℃ of shaking culture are to A
600Be about 0.6-0.8, add IPTG to final concentration be 1mmol/L, then continue shaking culture, induce 4h.Thalline is carried out cytoclasis.The centrifugal rear thalline of binding buffer liquid 50mL piping and druming with dilution, carry out fragmentation with biomixer, it is 20s that the broken time is set, interval time 20s, work times 50 times is crushed to bacterium liquid limpid, take out, centrifugal at refrigerated centrifuge, the centrifugal 15min of 10000r/min centrifugally gets respectively cleer and peaceful precipitation and carries out the SDS-PAGE protein electrophoresis afterwards.Carry out the detection of WesternBlot as primary antibodie to carry out mouse-anti 6 * His tag monoclonal antibody, as shown in Figure 4, purpose expression product estradiol analog antibody mainly exists in supernatant.
Two. the evaluation of estradiol analog antibody
1.anticalin-E2 purifying
Supernatant liquor after the anticalin-E2 abduction delivering is carried out separation and purification of protein through the Ni post, through linear gradient elution, collect elution peak and carry out SDS-PAGE, merge the elution peak that target protein has single band.
2.anticalin-E2 in conjunction with active detection
Take E2-BSA as envelope antigen, and simultaneously coated BSA in contrast, Salmonella detect anticalin-E2 to E2 in conjunction with active, result such as Fig. 5, anticalin-E2 has significant difference to the combination of E2 and combination to BSA.
3.anticalin-E2 the detection of competition activity
Take E2-BSA as envelope antigen, take estradiol as the competition small molecules, competitive ELISA is analyzed the competition activity of anticalin-E2.Competition ELSIA typical curve is seen Fig. 6, and the IC50 of four kinds of anticalin-E2 detection E2 is respectively 0.05,0.92,2.52,0.41 μ g/ml.
4.anticalin-E2A the binding kinetics constant measuring
Take E2-BSA as coated gold plaque surface, with 5 gradients of anticalin-E2A dilution, measure the binding kinetics constant on surface plasma resonance instrument, as Fig. 7, the Kd that obtains anticalin-E2A based on five curve calculation is about 7.22 * 10
-8L/mol.
Claims (3)
1. the preparation method of an estradiol analog antibody mainly is comprised of following steps:
(1) structure in ribosomal display anticalin analog antibody storehouse
select bilitrien in conjunction with albumen (bilin binding protein, bbp) as the template in anticalin storehouse, design random mutation site, take synthetic BBP gene order as template, carry out overlapping primer extension pcr amplification by the primer of design and synthesize Anticalin analog antibody storehouse fragment, about 522bp, add promotor by PCR method again, intervening sequence, the needed element of the ribosomal display such as loop-stem structure, the ribosomal display anticalin analog antibody storehouse that finally successfully constructs, RNA molecule after transcribe in this storehouse comprises 5 '-loop and 3 '-loop loop-stem structure, a ribosome bind site, the anticalin storehouse of random mutation, C-terminal intervening sequence etc., total length is 950bp approximately,
(2) screening of anti-estradiol analog antibody
After the in-vitro transcription translation is carried out in the ribosomal display anticalin analog antibody storehouse that builds, alternately screen the analog antibody of estradiol as the antigen solid liquid phase with E2-BSA and E2-magnetic bead, dissociate by changing analog antibody-rrna that Mg2+ concentration obtains screening-mRNA triplet, obtain corresponding mRNA, the DNA library after being screened through RT-PCR; Then repeat in-vitro transcription-In Vitro Translation-affine screening-RT-PCR amplification procedure, obtain repeated screening several analog antibody DNA libraries of taking turns; After taking turns ribosomal display screening through eight, the obvious enrichment of analog antibody sequence of antigen positive in the analog antibody library;
(3) soluble functional of estradiol analog antibody is expressed
take turns after screening the analog antibody library carrying out Sequence Identification eight, 20 clone's of random choose, this gene and pTIG-TRX are connected into expression vector, express in e. coli bl21 (DE3), identify through SDS-PAGE and Western Blot, at the 20KDa place, the target protein band is arranged, with theoretical albumen in the same size, determine in the supernatant liquor after expression product is present in broken thalline, take the empty plasmid Host Strains as contrast, E2-BSA is coating antigen, E2 is the competition small molecules, competition ELSIA detects and induces the competition of supernatant in conjunction with activity, competition is arranged in conjunction with active further extensive abduction delivering and purifying,
(4) purifying of estradiol analog antibody and evaluation
Utilize Anti-His tag affinity column to carry out purifying to the specific junction mixture of expressing, make the purpose band obtain significantly concentrating and separating, competitive ELISA is analyzed the competition of purifying after product in conjunction with activity; The SPR method detects the avidity of the analog antibody that obtains.
2. the template in the described random mutation of claim 1 storehouse can be other lipocalin molecules, preferred bbp.
3. the coating antigen in the described screening method of claim 1 can be selected E2-BSA, can also select the E2-magnetic bead to screen, and preferred the intersection screens.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103172694A (en) * | 2011-12-26 | 2013-06-26 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Screening method of ampicillin special conjugate and use of conjugate |
| CN105001338A (en) * | 2014-04-18 | 2015-10-28 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Diazepam lipocalin imitating antibody |
| CN112981543A (en) * | 2021-03-02 | 2021-06-18 | 北京创智智能健康技术研究院有限公司 | Alpha helical structure-containing pseudoantibody screening library, and construction method and application thereof |
-
2011
- 2011-12-26 CN CN 201110440667 patent/CN103172740A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103172694A (en) * | 2011-12-26 | 2013-06-26 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Screening method of ampicillin special conjugate and use of conjugate |
| CN105001338A (en) * | 2014-04-18 | 2015-10-28 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Diazepam lipocalin imitating antibody |
| CN105001338B (en) * | 2014-04-18 | 2018-05-01 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | A kind of diazepam lipocalin analog antibodies |
| CN112981543A (en) * | 2021-03-02 | 2021-06-18 | 北京创智智能健康技术研究院有限公司 | Alpha helical structure-containing pseudoantibody screening library, and construction method and application thereof |
| CN112981543B (en) * | 2021-03-02 | 2021-11-30 | 北京创智智能健康技术研究院有限公司 | Alpha helical structure-containing pseudoantibody screening library, and construction method and application thereof |
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Application publication date: 20130626 |