CN105001338B - A kind of diazepam lipocalin analog antibodies - Google Patents
A kind of diazepam lipocalin analog antibodies Download PDFInfo
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- CN105001338B CN105001338B CN201410157737.4A CN201410157737A CN105001338B CN 105001338 B CN105001338 B CN 105001338B CN 201410157737 A CN201410157737 A CN 201410157737A CN 105001338 B CN105001338 B CN 105001338B
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- lipocalin
- diazepam
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Abstract
The invention discloses a kind of diazepam lipocalin analog antibodies, the DNA sequence dna for expressing the antibody is the sequence in sequence table shown in SEQ ID NO.1.The diazepam lipocalin analog antibodies of the present invention lay a good foundation quickly detecting for diazepam to realize.In lipocalin, embedding rate of the recognition site in albumen that can be interacted with target molecules is up to 95%, and embedding rate of the binding site in antibody molecule is only 66%.This is because there is the big face that contacts with each other that can be combined with target molecules in lipocalin intramoleculars（Ingo,2003）.Lipocalin is simple in structure, and molecular weight is small, have it is larger can stablize with the recognition site and skeleton that target molecules are combined, have unique advantage compared with antibody.
Description
Technical field
The present invention relates to a kind of diazepam analog antibody.Belong to biological technical field, which can be used for developing
The quick detection remaining immunity detection reagent of diazepam.
Background technology
Diazepam（Diazepam, DZP）, alias stabilizes or benzodiazepine, and chloro- 1, the 3- dihydros -1- methyl of chemical name 7- -
5- phenyl -2H-1,4- Benzodiazepine -2- ketone, molecular formula C16H13ClN2O, molecular weight 284.76.It belongs to long-acting Benzodiazepine
Class anxiolytic, has antianxiety, calmness, hypnosis, anticonvulsion, anti-epileptic, relaxation skeletal muscle and eliminates the effect such as memory.Extensively
Applied to clinical medicine, long-term use has additive and dependence.Diazepam is national contraband, is only used as prescription medicine and sells,
With calm, syngignoscism, offender often implements crime using this feature of DZP, causes very great society harm.And throw
Poison, wrongly take a large amount of DZP and poisoning also happens occasionally.Therefore, it is that strike is such to establish quick, sensitive DZP detection methods
Criminal activity, a kind of important means of control DZP abuses.
The detection method of DZP focuses mostly in clinical medicine, criminal investigation context of detection, in blood, urine, stomach Dissolve things inside, liver
The detection of diazepam.Common detection method has：Instrumental method, including high performance liquid chromatography（HPLC）, thin layer chromatography scanning
（TLC）, Photochemical Fluorimetry（PCFA）, differential spectrophotometry.Every kind of detection method also has the shortcoming of oneself.Greatly
Type instrument detection method sensitivity is higher, can solve part practical problem.But its sample pre-treatments is cumbersome, expensive equipment, inspection
Survey it is of high cost, need professional technician to operate, and during operating cost, analysis efficiency is low, and technology popularization is difficult, and most of is used for method
To the identification of poisoning symptom on doctor, be not suitable for the remaining detection of diazepam in edible animal tissue, be not also suitable for live quick
A large amount of detections.
Ribosomal display technology（Ribosome display, RD）It is a kind of simple and effective novel in vitro screening and molecule
The biological libraries technology of evolution function protein, it is by amplifiable genomic information DNA or RNA and selectable protein
Link together.MRNA lacks terminator codon and by appropriate modification in RD technologies, and ribose is known from experience in translation process in vitro
The 3' ends of mRNA are rested on, mRNA is not disintegrated down with translation product from ribosomes, forms sufficiently stable " mRNA-
Ribosomes-protein " ternary complex, this ternary complex form connect together protein with mRNA information, application
RD can carry out peptide and the protein screening of specific function.Lipocalin is from unicellular lower eukaryote（Bacterium）To high mammal
The general name of the one group of extracellular lipophilic protein all contained, is to be widely present in nature small protein family vdiverse in function,
From unicellular lower eukaryote（Bacterium）To high mammal（Including the mankind）Find its trace.Lipocalin is from bilitrien
Associated proteins（BBP）Middle discovery, it is present in large white butterfly（Pieris brassicaed）In, its openend be it is wide and
Shallow β-barrel structure, is easier to combine with small-molecule substance.The Lipocalin libraries reported at present are all with bilitrien knot
Hop protein comes for the support reformation.Although lipocalin protein plays different physiologic functions in different biologies,
Transport and storage material with low solubility or chemosensitivity material, such as fragrant element, vitamin, steroids, a variety of two levels
Metabolite and pheromones are their most typical biological functions.Due to lipocalin protein family higher structure feature with
Antibody is similar, and lipocalin protein is but also with the unexistent advantage of some antibody, therefore, using Lipocalin as framework construction
Mutated library be more and more widely used.Lipocalin mutant by design simulation has similar antibody
Binding specificity, therefore analog antibody can be become.At present, using ribosomal display technology screening small molecular antibody research also
Fewer, the screening for diazepam analog antibody has not been reported.
The content of the invention
The purpose of the present invention is obtain a kind of diazepam lipocalin analog antibodies.
Technical scheme is summarized as follows：
A kind of diazepam lipocalin analog antibodies, the DNA sequence dna for expressing the antibody are SEQ ID in sequence table
Sequence shown in NO.1.
Advantages of the present invention：
, by taking turns biological screening, the diazepam lipocalin simulations obtained for diazepam high specific resist more for we
Body.To realize that the quick detection to diazepam is laid a good foundation., can be with the knowledge of target molecules interaction in lipocalin
Embedding rate of the other site in albumen is up to 95%, and embedding rate of the binding site in antibody molecule is only 66%.This is because
There is the big face that contacts with each other that can be combined with target molecules in lipocalin intramoleculars（Ingo,2003）.Lipocalin is tied
Structure is simple, and molecular weight is small, have it is larger can stablize with the recognition site and skeleton that target molecules are combined, the tool with antibody compared with
There is unique advantage.(Flower DR,1996).
Brief description of the drawings
Fig. 1 is the amplifiable property identification in the ribosomal display analog antibody library of structure.
Fig. 2 is for the qualification figure after the wheel screening of target small molecule diazepam five in analog antibody library.
Fig. 3 identifies for diazepam analog antibody positive colony daughter colony PCR.
Fig. 4 is the identification of diazepam analog antibody positive clone molecule.
Fig. 5 is the expression and purifying of diazepam analog antibody.
Fig. 6 is the competition binding activity that indirect competitive ELISA detects antibody.
Embodiment
Diazepam lipocalin analog antibodies provided by the invention are from ribosomal display Lipocalin analog antibodies storehouse
It is that middle screening obtains and carried out soluble functional expression.
Embodiment 1
The amplifiable property identification in lipocalin analog antibodies library：
With a member of lipocalin families, bilitrien associated proteins（BBP）For skeleton, 16 mutation positions are randomly incorporated into
Point, builds lipocalin ribosomal-display libraries.Using the lipocalin libraries of structure as template, using T7F and PDR as upstream and downstream
Primer carries out PCR amplification, and purpose band is about 900bp.
T7 fragments such as Fig. 1 is expanded from pET32a plasmids by upstream and downstream primer T7F and RT7（A）It is shown, with primer PDF
With the T20-V109 sequences in the glutelin D of PDR amplification Lambda bacteriophages as intervening sequence (i.e. P fragments), such as Fig. 1（B）
It is shown.As can be seen from the figure there is purpose band at 102bp and 300bp, with T7 fragments and the size of P protein gene fragments
It is consistent, illustrates to expand successfully.By sense primer FBBP and anti-sense primer Rtail, the BBP antibody libraries built are expanded
Increase, agarose gel electrophoresis is identified shown in its result such as Fig. 1 (C), there is bright band at 558bp sizes, this is with building mould
The size in lectin library is suitable.Thus, the amplifiable property in the ribosomal display analog antibody library of structure is good.
Primer sequence：T7F:5’-ATACGAAATTAATACGACTCAC-3’（SEQ ID NO.2）
PDR:5’-CCGCACACCAGTAAGGTGTGCGGTAACGATGCTGATTGCCGTTCCG-3’（SEQ ID NO.3）
RT7:5’-CATGGATATATCTCCTTCTTAAAG-3’（SEQ ID NO.4）
PDF:5’-GGTGGAGGTGGCTCTGGCGGTGGCGGATCTA-3’（SEQ ID NO.5）
FBBP：5’-GAAGGAGATATATCCATGAACGTGTACCACGACGGTGC-3’（SEQ ID NO.6）
Rtail:5’-CGCCAGAGCCACCTCCACCTGAACCGCCTCCACCATTGTTGACTTTGCAGGCG-3’（SEQ
ID NO.7）
Embodiment 2
The screening of diazepam lipocalin analog antibodies
1 in-vitro transcription and translation
E.coli S30 of the in-vitro transcription with translation using Promega（Escherichia coli S30Extract
System）Linear die system, it will be transcribed together with translation coupling.
2 solid-phase screenings
(1) it is coated with：ELISA Plate takes the comlete antigen DZP-OVA/DZP-BSA after coupling, uses after the immersion drying of DEPC water
Carbonate buffer solution is diluted to 10 μ g/mL, and per 200 μ L are added in hole, 8h is incubated under the conditions of 4 DEG C.
(2) second days, coating buffer of inclining, washed coating hole 3 times, each 3min with the PBS after sterilizing；Then hole will be coated with
In plus 200 μ L confining liquid（0.5% BSA or 1%OVA）, close 1h.The liquid of falling deblocking, PBS are eluted 2 times, each 2min,
The WBT of precooling is used again（150mmol/L NaCl, 50mmol/L Tris-acetate pH7.5,0.1%Tween, 50mmol/L
magnesium acetate）Washing 2 times, each 2min, WBT is then filled in screen holes, places 20min on ice.Experiment is adopted
The mode for taking secondary screening to select, adds one during closing and does not contain envelope antigen, containing only the screen holes of carrier protein BSA or OVA.
(3) product after the translation placed on ice is gone to be ready for contain only carrier protein（BSA or OVA）Screening
Kong Zhong, when shaking 1 is small under the conditions of 4 DEG C.After translation product is fully combined with carrier protein, then the liquid in hole is added to
1h is shaked in screen holes containing coating antigen, to reduce non-specific binding.
(4) liquid being incubated in screen holes of inclining, is washed 3 times, each 2min with the WBT solution of precooling.
(5) liquid in screen holes of inclining, adds the elution buffer of the prior precooling of 100 μ l in screen holes, then
The RNase inhibitor of 1 μ L is added, 4 DEG C of shaking 10min, make the big small subunit separation of ribosomes, discharge mRNA.Eluent is drawn, so
The Dnase I of 1 μ l is added afterwards, 15min is incubated under the conditions of 37 DEG C, removes DNA profiling unnecessary in reaction system.
(6) 5 steps are repeated.
(7) obtained mRNA is purified immediately.
(8) RT-PCR of mRNA
3. rebuild the ribosomal display template of next round
BBP library fragments after recycling and T7 promoters and P fragments are built into library using the method for SOE-PCR.
（1）The amplification of T fragments
（2）The amplification of P fragments
（3）T7 promoters and the connection in BBP analog antibodies library：
（4）The connection of T7+BBP fragments and P fragments：
Embodiment 3
Identification after the wheel screening of diazepam lipocalin analog antibodies five
Plasmid pMD18-T-BBP will be extracted after right-on strain culturing is sequenced, double digestion is then carried out, after digestion
The BBP fragments containing restriction enzyme site are obtained, endonuclease reaction system is the same.
Fig. 2 show it is every wheel screening after, there is EDTA to disintegrate down mRNA from triplet compound, using PM as primer into
Row reversion, obtains cDNA, then, then using FBBP and Rtail as primer amplification analog antibody fragment.Through 1.5% Ago-Gel
Electroresis appraisal, can see purposeful band in size about at 500bp.After being connected after being recycled with ribosomal display element
The library screened to the next round of 900bp.Fig. 2 is after being screened from analog antibody library for the wheel of target small molecule diazepam five
Qualification figure, as shown in Figure 2, the mRNA amounts of the acquisition after first round screening are less, obtained band after progress RT-PCR reversions
It is very weak.But with the increase of screening number, the band of RT-PCR products is increasingly brighter, after five wheel screenings, due to pin
Enrichment is obtained for the analog antibody gene of small molecule diazepam, the analog antibody gene to be become clear after RT-PCR.This
Prove by the external affine screening of ribosomal display technology, the specific analog antibody gene being combined can be carried out with diazepam
Obvious enrichment is obtained.
PM:5’-GCAAGAATGCCAACGGCAGC-3’（SEQ ID NO.8）
Embodiment 4
The sequencing of RT-PCR products after the wheel screening of diazepam lipocalin analog antibodies five
Reversion primer of the amplified production containing restriction enzyme digestion sites that will be obtained after five wheel screenings
EXPRESS and REPRESS amplifications, introduce restriction enzyme site at gene order both ends, are connected to after adding A on cloning vector pMD18-T,
Then it is transformed into E.coil.Fig. 3 is diazepam-analog antibody positive colony daughter colony PCR identifications.Single positive gram is selected at random
Send the sequencing identification of Invitrogen companies in Longzi.
EXPRESS（Introduce EcoRI restriction enzyme sites）：5′-CGCGAA TTC TAA ATG AAC GTG TAC CAC GAC
GGTG-3′（SEQ ID NO.9）
REPRESS（Introduce XhoI restriction enzyme sites）：5′-GCCCTC GAG ATT GTT GAC TTT GCA GGC
GGCG-3′（SEQ ID NO.10）
Embodiment 5
The identification of diazepam lipocalin analog antibody positive clone molecules and protein purification
In the screening to target molecules diazepam, still with bilitrien associated proteins（BBP）For the screening of framework construction
Screened in storehouse.Clone of the picking containing pTIG-TRX-BBP is incubated overnight, and EcoR I and XhoI restriction endonucleases are used after extracting plasmid
Double digestion processing is carried out to clone, product is identified with agarose gel electrophoresis after digestion.Digestion result such as Fig. 4 shows, digestion
The fragment of size about 500bp is obtained afterwards, it is and expected consistent.The dientification of bacteria the result shows that, diazepam-analog antibody gene is correctly inserted
Enter into expression pTIG-TRX.The clone strain for being accredited as the positive is induced with IPTG, 30 DEG C of induced expression 4h, bacterium solution centrifugation
After taking precipitation to be resuspended, SDS-PAGE electrophoresis detections are carried out.Protein molecular is understood according to gene order and the analysis of the size of amino acid
Measure as 20Kda or so.Due to being connected with label A nti-His tag on analog antibody, thus use the affine layers of Anti-His tag
Analysis column purifies single-chain antibody.Albumen is sequestered on chromatographic column by 6 × His labels and Ni2+ interactions, then is passed through
Elution containing imidazoles.Purification result is shown in Fig. 5 (B), as can be seen from the figure elute after purpose band go out to have one it is bright
Band, and the concentration of foreign protein is decreased obviously.Illustrate that albumen is purified.Albumen after purification carries out SDS-PAGE electricity
After swimming, such as Fig. 5（C）It is shown, further illustrate Protein expression and purification success, albumen exists in the form of soluble.
Embodiment 6
The Property Identification of diazepam lipocalin analog antibodies
The gene order that screening obtains, which is imported into expression vector, to be expressed, and albumen size is 20KDa.1 is cloudy in Fig. 5
Property（Empty plasmid bacterial strain is expressed）2,3 be the supernatant after broken containing the purpose bacterial strain expression screened, wherein 2 be that size is 470bp
Gene order imported into the albumen expressed in expression vector, 3 be the albumen that the gene order that size is 520bp is expressed.But
Size is that the albumen after the gene expression of 470bp is active without competition.4 be the strain protein expression and purification that gene is 520bp
Rear electrophoresis, 5,6 be albumen progress Western Blot identifications after purification.As can be seen from Figure 5 containing the purpose base screened
The expression system energy successful expression albumen of cause, and can successful purification.The diazepam lipocalin simulations obtained by sequencing are anti-
The DNA sequence dna of body is the sequence shown in SEQ ID NO.1 in sequence table.Its competition activity is analyzed preferably by competitive ELISA, such as
Fig. 6 is with the increase of the concentration of small molecule, and inhibiting rate is bigger, and small molecule is stronger to the competitiveness of envelope antigen.Detection range is：
0.04-17.70 μ g/mL, sensitivity are 0.41 μ g/mL.The diazepam lipocalin analog antibodies that we screen acquisition can be used
The remaining immunity detection reagent of diazepam is quickly detected in developing.
Claims (1)
- A kind of 1. diazepam lipocalin analog antibodies, it is characterized in that the DNA sequence dna for expressing the antibody is SEQ in sequence table Sequence shown in ID NO.1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103172740A (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 | Preparation method of beta-estradiol analogue antibody and use thereof |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103172740A (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 | Preparation method of beta-estradiol analogue antibody and use thereof |
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| Construction of ribosome display library based on lipocalin scaffold and screening anticalins with specificity for estradiol;Jianqing Liu等;《Analyst》;20121231;第137卷(第10期);第2470-2479页 * |
| 基于脂运载蛋白骨架的抗体库构建与农兽药残留抗体的筛选;宁保安，等;《中国药理学与毒理学杂志》;20131130;第27卷;摘要 * |
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