CN105001338A - Diazepam lipocalin imitating antibody - Google Patents
Diazepam lipocalin imitating antibody Download PDFInfo
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- CN105001338A CN105001338A CN201410157737.4A CN201410157737A CN105001338A CN 105001338 A CN105001338 A CN 105001338A CN 201410157737 A CN201410157737 A CN 201410157737A CN 105001338 A CN105001338 A CN 105001338A
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- lipocalin
- diazepam
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- analog antibody
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Abstract
The present invention discloses a diazepam lipocalin imitating antibody, wherein the DNA sequence expressing the antibody is the sequence represented by SEQ ID NO.1 in the sequence table. According to the present invention, with the diazepam lipocalin imitating antibody, the foundation is established for rapid diazepam detection; in lipocalin, the encapsulation efficiency of the recognition sites capable of interacting with target molecules is up to 95% while the encapsulation efficiency of the binding sites within the antibody molecule is only 66%, wherein the large mutual contact surface (Ingo, 2003) capable of binding with target molecules exists in the lipocalin molecule so as to cause the previous conditions; and the lipocalin has characteristics of simple structure, small molecular weight, large recognition sites capable of binding with target molecules and stable skeleton, and has unique advantages compared with the antibody.
Description
Technical field
The present invention relates to a kind of diazepam analog antibody.Belong to biological technical field, this analog antibody may be used for developing the residual immunity detection reagent of rapid detection diazepam.
Background technology
Diazepam (Diazepam, DZP), another name is stabilized or diazepam, chloro-1, the 3-dihydro-1-methyl-5-phenyl-2H-1 of chemical name 7-, 4-Benzodiazepine-2-ketone, molecular formula C
16h
13clN
2o, molecular weight 284.76.It belongs to long-acting benzodiazepines anxiolytic, has anxiety, calmness, hypnosis, anticonvulsion, anti-epileptic, lax skeletal muscle and eliminates the effects such as memory.Be widely used in clinical medicine, long-term taking has additive and dependency.Diazepam is national contraband goods, only sells as prescription drugs, has calmness, syngignoscism, and offender often utilizes this feature of DZP to implement crime, causes very great society harm.And poison, wrongly take a large amount of DZP and poisoning also happens occasionally.Therefore, setting up quick, sensitive DZP detection method is a kind of important means of hitting this type of criminal activity, control DZP abuse.
The detection method of DZP focuses mostly in clinical medicine, criminal investigation context of detection, as the detection of diazepam in blood, urine, stomach Dissolve things inside, liver.Conventional detection method has: instrumental method, comprises high performance liquid chromatography (HPLC), thin layer chromatography scanning (TLC), Photochemical Fluorimetry (PCFA), difference spectrophotometry.Often kind of detection method also has oneself weak point.The sensitivity of large-scale instrument detection method is higher, can solve part practical problems.But its sample pre-treatments is loaded down with trivial details, expensive equipment, testing cost are high, need professional and technical personnel to operate, and during operational cost, analysis efficiency is low, the universal difficulty of technology, and great majority on legal medical expert to the qualification of toxicity symptom, be not suitable for the detection that in edible animal tissue, diazepam is residual, be not also suitable for on-the-spot rapid, high volume and detect.
Ribosomal display technology (ribosome display, RD) be the screening of a kind of simple and effective novel in vitro and the biological libraries technology of molecular evolution functional protein, genomic information DNA or RNA that it can increase is together with selectable protein contacts.In RD technology, mRNA lacks terminator codon and modifies through suitable, in translation process, ribose knows from experience the 3' end resting on mRNA in vitro, mRNA and translation product all do not disintegrate down from rrna, form very stable " mRNA-rrna-protein " ternary complex, protein connects together with mRNA information by this ternary complex form, and application RD can carry out the peptides and proteins screening of specific function.Lipocalin is the general name of lipophilic protein one group of born of the same parents containing from unicellular lower eukaryote (bacterium) to high mammal, be the small protein family being extensively present in nature diverse in function, all find its trace from unicellular lower eukaryote (bacterium) to high mammal (comprising the mankind).Lipocalin finds from bilitrien associated proteins (BBP), and it is present in large white butterfly (Pieris brassicaed), and its opening end is wide and shallow β-barrel structure, is easier to combine with small-molecule substance.The Lipocalin library reported at present all with bilitrien associated proteins for the support reformation.Although lipocalin protein plays different physiologic functions in different biology, but transport and store material with low solubility or chemosensitivity material, if fragrance element, VITAMIN, steroids, multiple secondary metabolism product and pheromone are their most typical biological functions.Due to the higher structure feature of lipocalin protein family and antibody class seemingly, and lipocalin protein possesses again the unexistent advantage of some antibody, therefore, is that the mutated library of framework construction obtains and applies more and more widely with Lipocalin.There is through the Lipocalin mutant of simulation the binding specificity of similar antibody, therefore can become analog antibody.At present, utilize the research of ribosomal display technology screening small molecular antibody also fewer, the screening for diazepam analog antibody have not been reported.
Summary of the invention
The object of the invention is to obtain a kind of diazepam lipocalin analog antibody.
Technical scheme of the present invention is summarized as follows:
A kind of diazepam lipocalin analog antibody, expresses the DNA sequence dna of described antibody for the sequence shown in SEQ ID NO.1 in sequence table.
Advantage of the present invention:
We, by taking turns biological screening, obtain the diazepam lipocalin analog antibody for diazepam high specific more.For realizing laying a good foundation to the rapid detection of diazepam.In lipocalin, can with the embedding rate of the interactional recognition site of target molecules in albumen up to 95%, and the embedding rate of binding site in antibody molecule is only 66%.This is owing to having the large face that contacts with each other (Ingo, 2003) that can be combined with target molecules in lipocalin molecule.Lipocalin structure is simple, and molecular weight is little, has the larger recognition site that can combine with target molecules and skeleton is stablized, and has unique advantage compared with antibody.(Flower DR,1996)。
Accompanying drawing explanation
Fig. 1 is the amplification property qualification in the ribosomal display analog antibody library built.
Fig. 2 takes turns the qualification figure after screening for target small molecules diazepam five in analog antibody library.
Fig. 3 is that diazepam analog antibody positive colony daughter colony PCR identifies.
Fig. 4 is the qualification of diazepam analog antibody positive colony.
Fig. 5 is the expression and purification of diazepam analog antibody.
Fig. 6 is the competition binding activity that indirect competitive ELISA detects antibody.
Embodiment
Diazepam lipocalin analog antibody provided by the invention be from ribosomal display Lipocalin analog antibody storehouse screening obtain and carried out soluble functional expression.
Embodiment 1
The amplification property qualification in lipocalin analog antibody library:
With a member of lipocalin family, bilitrien associated proteins (BBP) is skeleton, and random introducing 16 mutational sites, build lipocalin ribosomal-display library.With the lipocalin library built for template, with T7F and PDR for upstream and downstream primer carries out pcr amplification, object band is approximately 900bp.
From pET32a plasmid, T7 fragment is increased as shown in Fig. 1 (A) by upstream and downstream primer T7F and RT7, by the T20-V109 sequence in the glutelin D of primer PDF and PDR amplification Lambda phage as intervening sequence (i.e. P fragment), as shown in Fig. 1 (B).As can be seen from the figure there is object band at 102bp and 300bp place, conform to the size of T7 fragment with P protein gene fragment, illustrate and increase successfully.By upstream primer FBBP and downstream primer Rtail, the BBP antibody library built is increased, agarose gel electrophoresis identifies its result. as shown in Fig. 1 (C), in 558bp size, there is bright band at place, and this is suitable with the size in structure analog antibody library.Thus, the amplification 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’（SEQID 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 IDNO.7）
Embodiment 2
The screening of diazepam lipocalin analog antibody
1 in-vitro transcription and translation
That in-vitro transcription and translation adopt is the E.coli S30(Escherichia coli S30Extract System of Promega) linear die system, it will be transcribed together with translation coupling.
2 solid-phase screenings
(1) bag quilt: enzyme plate, after DEPC water soaking is dried, is got the complete antigen DZP-OVA/DZP-BSA after coupling, is diluted to 10 μ g/mL with carbonate buffer solution, add 200 μ L in every hole, hatch 8h under 4 DEG C of conditions.
(2) second days, inclined coating buffer, with the PBS washing bag after sterilizing by hole 3 times, and each 3min; Then bag is added the confining liquid (BSA or 1%OVA of 0.5%) of 200 μ L in hole, closed 1h.The liquid of falling deblocking, PBS wash-out 2 times, each 2min, use the WBT(150mmol/L NaCl of precooling again, 50mmol/L Tris-acetate pH7.5,0.1%Tween, 50mmol/Lmagnesium acetate) wash 2 times, each 2min, then fills WBT in screen holes, places 20min on ice.The mode of multiple screening is taked in experiment, adds one not containing envelope antigen, only containing the screen holes of carrier proteins BSA or OVA in closed process.
(3) product after the translation of placing on ice is gone to off-the-shelf only containing carrier proteins (BSA or OVA) screen holes in, jolting 1 hour under 4 DEG C of conditions.After translation product can be fully combined with carrier proteins, then the liquid in hole be joined in the screen holes containing coating antigen and jolt 1h, to reduce non-specific binding.
(4) liquid of hatching in screen holes of inclining, washes 3 times, each 2min with the WBT solution of precooling.
(5) liquid inclined in screen holes, adds the elution buffer of the precooling in advance of 100 μ l in screen holes, then adds the RNA enzyme inhibitors of 1 μ L, 4 DEG C of jolting 10min, the large small subunit of rrna is separated, release mRNA.Draw elutriant, under then all adding Dnase I, 37 DEG C of conditions of 1 μ l, hatch 15min, DNA profiling unnecessary in removing reaction system.
(6) 5 steps are repeated.
(7) the mRNA purifying immediately will obtained.
(8) RT-PCR of mRNA
3. rebuild the ribosomal display template of next round
The method of SOE-PCR is utilized to build library the BBP library fragments after reclaiming and T7 promotor and P fragment.
(1) amplification of T fragment
(2) amplification of P fragment
(3) connection in T7 promotor and BBP analog antibody library:
(4) connection of T7+BBP fragment and P fragment:
Embodiment 3
Diazepam lipocalin analog antibody five takes turns the qualification after screening
To extract plasmid pMD18-T-BBP after the right-on strain culturing that checks order, and then carry out double digestion, enzyme obtains the BBP fragment containing restriction enzyme site after cutting, endonuclease reaction system is the same.
After Figure 2 shows that often wheel screening, there is EDTA to be disintegrated down from triplet mixture by mRNA, be that primer reverses with PM, obtain cDNA, then, then with FBBP and Rtail for primer amplification analog antibody fragment.Through the agarose gel electrophoresis qualification of 1.5%, be about 500bp place in size and can see there is object band.The library that the next round obtaining 900bp after being connected with ribosomal display element after being reclaimed is screened.Fig. 2 takes turns the qualification figure after screening for target small molecules diazepam five from analog antibody library, and as shown in Figure 2, the mRNA amount of the acquisition after first round screening is less, and the band obtained after carrying out RT-PCR reversion is very weak.But along with the increase of screening number of times, the band of RT-PCR product is more and more brighter, after five take turns screening, because the analog antibody gene for small molecules diazepam is obtained for enrichment, obtains bright analog antibody gene after RT-PCR.This proves through the external affinity selection of ribosomal display technology, can carry out the analog antibody gene that specificity combines obtain obvious enrichment with diazepam.
PM:5’-GCAAGAATGCCAACGGCAGC-3’（SEQ ID NO.8）
Embodiment 4
Diazepam lipocalin analog antibody five takes turns the sequencing of the RT-PCR product after screening
After five take turns screening, primer EXPRESS and REPRESS of the reversion amplified production obtained containing restriction enzyme digestion sites is increased, introduce restriction enzyme site at gene order two ends, be connected to after adding A on cloning vector pMD18-T, be then transformed into E.coil.Fig. 3 is that diazepam-analog antibody positive colony daughter colony PCR identifies.The order-checking qualification of random choose single positive colony Zi Song Invitrogen company.
EXPRESS(introduces EcoRI restriction enzyme site): 5 '-CGC
gAA TTc TAA ATG AAC GTG TAC CAC GACGGTG-3 ' (SEQ ID NO.9)
REPRESS(introduces XhoI restriction enzyme site): 5 '-GCC
cTC GAGaTT GTT GAC TTT GCA GGC GGCG-3 ' (SEQ ID NO.10)
Embodiment 5
The qualification of diazepam lipocalin analog antibody positive colony and protein purification
In the screening to target molecules diazepam, the screening storehouse being still framework construction with bilitrien associated proteins (BBP) is screened.Picking is containing the clone incubated overnight of pTIG-TRX-BBP, and use EcoR I and XhoI restriction endonuclease to carry out double digestion process to clone after extracting plasmid, enzyme is cut after product agarose gel electrophoresis and identified.Enzyme is cut result such as Fig. 4 and is shown, and enzyme obtains the fragment that size is about 500bp after cutting, with expection consistent.Dientification of bacteria result shows, diazepam-analog antibody gene is correctly inserted into expresses in pTIG-TRX.Induce being accredited as positive clone strain IPTG, 30 DEG C of abduction delivering 4h, after bacterium liquid centrifuging and taking precipitation is resuspended, carry out SDS-PAGE electrophoresis detection.Be about 20Kda according to gene order and the known molecular weight of albumen of amino acid whose Analyzing on Size.Owing to analog antibody being connected with label A nti-His tag, Anti-His tag affinity column is thus adopted to carry out purifying to single-chain antibody.Albumen is interacted by 6 × His label and Ni2+ and is sequestered on chromatography column, then by the elution containing imidazoles.Purification result is shown in Fig. 5 (B), as can be seen from the figure take a bright band out of at object bar after wash-out, and the concentration of foreign protein obviously declines.Illustrate that albumen obtains purifying.After albumen after purifying carries out SDS-PAGE electrophoresis, as shown in Fig. 5 (C), further illustrate Protein expression and purification success, albumen exists with the form of solubility.
Embodiment 6
The Property Identification of diazepam lipocalin analog antibody
The gene order that screening obtains imports in expression vector to be expressed, and albumen size is 20KDa.In Fig. 5,1 is negative (expression of empty plasmid bacterial strain) 2,3 is express the supernatant after fragmentation containing the object bacterial strain screened, wherein the gene order of 2 to be sizes be 470bp imports to the albumen of expressing in expression vector, and 3 is sizes is the albumen that the gene order of 520bp is expressed.But size is the albumen after the genetic expression of 470bp does not have competition activity.4 is genes is the strain protein expression and purification rear electrophoresis of 520bp, and 5,6 is that albumen after purifying carries out Western Blot qualification.As can be seen from Figure 5 containing the expression system energy successful expression albumen of the goal gene screened, and can successful purification.DNA sequence dna through the diazepam lipocalin analog antibody obtained that checks order is the sequence shown in SEQ ID NO.1 in sequence table.Analyze its competition by competitive ELISA better active, if Fig. 6 is along with the increase of micromolecular concentration, inhibiting rate is larger, and the competitiveness of small molecules to envelope antigen is stronger.Sensing range is: 0.04-17.70 μ g/mL, and sensitivity is 0.41 μ g/mL.The diazepam lipocalin analog antibody that we screen acquisition may be used for developing the residual immunity detection reagent of rapid detection diazepam.
Claims (1)
1. a diazepam lipocalin analog antibody, is characterized in that expressing the DNA sequence dna of described antibody for the sequence shown in SEQ IDNO.1 in sequence table.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| 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 |
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| 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 |
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| JIANQING LIU等: "Construction of ribosome display library based on lipocalin scaffold and screening anticalins with specificity for estradiol", 《ANALYST》 * |
| 宁保安，等: "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", 《中国药理学与毒理学杂志》 * |
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