CN101500600A - Methods for ester detoxication - Google Patents

Abstract

This invention relates to a method for detoxication of inorganic or organic esters including OP nerve agents, cocaine, and respective analogs. More specifically, this invention pertains to the treatment of potentially neurotoxic esters or other ester groups by elaborating a more effective hydrolytic enzyme for therapeutic application. The structures of the synthesized OP analogs are provided. This invention also provides a diagnostic method and an Array Biosensor for detecting OP agents in biological and environmental samples.

Description

The method of ester detoxication

The cross reference of related application

The application advocates that this application is incorporated by reference in this text to be examined in the right of the U.S. Provisional Application 60/811,370 of submission on June 7th, 2006.

Technical field

The invention provides the isolated DNA molecule of monkey butyrylcholine esterase (RhBchE); The isolated DNA molecule of people's BuCh esterase (HuBchE) mutant; With HuBchE and RhBchE is the sudden change storehouse of feature; The expression vector of dna molecular that contains the HuBchE of RhBchE and modification; High level expression system based on the butyrylcholine esterase (BchE) of adenovirus (AD); Organophosphorus ester (OP) model compound of simulation OP never poison structure; And the OP model compound is used for the purposes of the antibody of array bioprobe in acquisition.The inorganic ester or the organic ester that the invention provides comprising the organophosphorus ester never poison carry out the antidotal conventional method, and the diagnostic method that detects the OP preparation in biological specimen and environmental samples.

Background technology

Drug dependence is one of main public health problem.American above 300 ten thousand is the severe user of cocaine, and the slight misuser of the artificial cocaine of the U.S. of equivalent amount is arranged.The emergency treatment relevant with cocaine of 100,000 examples arranged every year approximately.Cardiovascular and central nervous system's common complication (can cause cardiovascular emergency case and generalized epilepsy) for the cocaine excessive use also do not have effective Therapeutic Method at present.Cocaine is an example can being carried out the antidotal organic ester by the esterase that adopts the molecular evolution method to develop with therapeutic modality.

Can carry out of antidotal inorganic ester with therapeutic modality and be exemplified as organophosphorus ester (OP) never poison or insecticide.Never poison exposes the biology threat that causes and also receives very big concern, because these chemical compounds can be blocked multiple important enzyme.Especially, serine easterase and protease can be suppressed fast and irreversibly by the OP never poison.

BchE is a kind of solubility seroglycoid enzyme of serine ester enzyme family.The physiological function of this enzyme it be unclear that.But the known BchE of people's recent decades can remove the OP and the carbamate pesticide of low dosage and protect people to avoid the toxic action of these Toxics.BchE also can be used as the earlier detection labelling that OP poisons, because be exposed to the activity that OP can reduce this sero-enzyme.BchE still is the metabolism and the main enzyme of antidotal of cocaine and intravital other ester of people.BchE can become the cocaine metabolism parmacodynamics-less activity chemical compound, for example ecgonine methyl ester and benzoic acid.This enzyme has great potentiality in cocaine detoxifcation and OP poisoning processing.

In theory, BchE has represented the ideal enzyme of enzyme replacement therapy: it does not need cofactor, and is solvable, and has efficient effect under the pH of blood plasma; And its hydrolyzate is nontoxic.The BchE of purification has the stability of several years, and has after exogenous using the relatively long half-life.The PEGization of HuBchE has significantly improved its stability and half-life.The more important thing is, the HuBchE of purification in Europe many decades be used for the treatment of the patient that succinylcholine-inductive asphyxia and OP poison safely.Do not consider its theoretic potentiality, HuBchE all is not very effective enzyme to OP or cocaine hydrolysis.The irreversible fixation of enzyme and OP is defined as scavenger with the effect of this enzyme, and non-catalytic.This enzyme is to slow 2000 times of the hydrolysis of the hydrolysis of natural (-)-cocaine comparison (+)-cocaine.The mutant of the appropriate design that obtains by protein engineering shows that this enzyme can be converted into OP hydrolytic enzyme (people such as Millard, 1995 and people such as Millard, 1998, G117H and the G117H/E197Q mutant described), and cocaine hydrolysis enzyme (people such as Pancook, 2003 and people such as Gao, 2005, the A328W/Y332M/S287G/F227A mutant of description).Although it is feasible that the mutation research prompting improves the hydrolysing activity of this enzyme, the mutant affinity of up to the present being reported is lower and conversion (turnover) is slower, and it should be improved significantly.The catalysis motility of IIuBchE is for improving its OP and the cocaine catalytic efficiency provides wide space.Yet, in order to significantly improve OP and/or cocaine hydrolysis activity, may need near and/or make up away from the HuBchE sudden change of a plurality of residues of avtive spot.Owing to follow the rapid catalytic activity of multistep and seriously sterically hindered to (-)-cocaine hydrolysis of the complexity of OP enzyme interacting, be not easy to predict the combination of those sudden changes by analysis of the molecular structure.Molecular evolution technique is used for BchE, for improving the result that will obtain desirable at the catalytic activity of OP never poison and cocaine.

The treatment of poisoning for acute OP never poison at present generally includes co-administered cholinesterase reactivator (oxime), muscarinic receptor antagonist (atropine) and anticonvulsant (diazepam).These treatments are only with competitive way generation effect, and its effect is abundant inadequately, because can not prevent neuronal brain injury and anergy.In army, introduced the chemoprophylaxis treatment, comprise independent with pyridinium bromide this bright, pyridostigmine associating cholinolytic medicine and use HI-6 (Bajgar, 2004) through skin.

The cause of disease (promptly because the method purpose that these treatment cocaine toxicity and OP poison is to treat symptom, never poison and cocaine), a kind of attractive replacement therapy method is directly removed never poison or cocaine for adopt catalyst or scavenger before damage takes place.The basic part of this treatment is to diagnose the never poison of existence or the value volume and range of product of OP.The biomarker that detects the exposure of OP never poison at a low price with highly sensitive and method just seems most important, thereby can combination medicine protection resist chemical damage and other OP harm.Because it is comparatively expensive and loaded down with trivial details to detect the existing method of never poison biomarker before damage takes place in animal body, therefore present challenge is that exploitation can be carried out and strong system at the scene.The present invention has solved this problem by the biomarker that provides process for selective to detect the OP never poison, and this method can be able to carry out easily and at an easy rate more by using portable automatic to analyze the array bioprobe.

Array format can provide numerous advantages, for example can analyze a plurality of target spots simultaneously on a sample.In addition, it can comprise the positive and negative control at each searching surface, and this is more more reliable than the contrast that be arranged in parallel on independent searching surface.The DNA array technique has been brought this effort into Laboratory Instruments and has been brought the system of two kinds of novel employing fiber waveguides, comprises the system that is put goods on the market by Zeptosens (people such as Pawlak, 2002) and Illumina (Epstein and Walt, 2003).These systems can hold thousands of capture molecules, and have the sensitivity of height.Yet their design is only at the use of the lab assistant that was subjected to senior training, and can not move or be used for on-the-spot application automatically.The array bioprobe that the present invention describes combines optical waveguide technique and detects the ability of a plurality of samples simultaneously, thereby can be at a plurality of target spots, and has portability and automatization.

This bioprobe is based on slab guide, and has sufficient surface area, can hold a plurality of small-sized (mm ²) search coverage.This waveguide is a kind of microslide of improvement, and it shines by 635nm diode laser and line generator, and ray cast is at near-end.Two of first three branch of this slide provides the mode mixture district, and light can be evenly distributed in the 2.4cm near far-end relatively thus ²Search coverage (people such as Feldstein, 1999).Under normal condition, in search coverage, can realize total internal reflection and form evanescent field.Bonded fluorogen in the fadout optical excitation search coverage, and adopt Peltier-cooled CCD camera to measure emitted fluorescence (people such as Wadkins, 1997 at 90 °; People such as Golden, 2003).The classification of the target that detects has been disclosed in the fluorescence position of waveguide surface in the array.This system puts goods on the market.

For from the sample capture target, can be in lattice array waveguide surface is fixing can be in conjunction with the antibody of target or other molecule (people such as Rowe, 1999; People such as Delehanty, 2002).In array, can comprise positive simultaneously and negative control responds people such as (, 2003) Ligler to prevent false positive or false negative.In addition, multichannel and sensing point array unites use and can support to analyze simultaneously a plurality of samples.Detection method be can make up and macromole and microorganism (sandwich detection) or micromolecule (competitive assay, displacement detects) people such as (, 2002) Sapsford detected.Use near-infrared fluorescent can prevent under shorter wavelength, to have the interference of the sample component of autofluorescence, thereby need not before analysis, target to be separated (people such as Sapsford, 2001 from sample with complex; People such as Taitt, 2004).With amount susceptiveness detector (surface plasma resonance (SPR), resonant mirror or interference system (people such as Homola, 2002 for example; Kinning and Edwards, 2002; Campbell and McCloskey, 2002, people such as Barzen, 2002)) difference, need the molecule of fluorogen labelling to carry out signal based on the array bioprobe of fluorescence and generate.This makes that this detection relatively is not subjected to disturb (people such as Ligler, 2003 from the non-specific adsorption of sample component; People such as Rowe, 1999; People such as Sapsford, 2001; People such as Taitt, 2004).

The array bioprobe can be in complex samples the detection of biological labelling, and need seldom or not need sample separation, this is by adopting non-automaticization sample device to obtain embodiment.The present invention adopts this automated system to determine that this system is in laboratory and the on-the-spot never poison significant degree relevant or other biomarker that detects at this.

Summary of the invention

On the one hand, the invention provides the isolated DNA molecule of Rhesus Macacus butyrylcholine esterase (RhBchE) (macaque).

On the other hand, the invention provides the isolated DNA molecule of people's BuCh esterase (HuBchE) and mutant thereof.

On the other hand, the invention provides with HuBchE and RhBchE is the sudden change storehouse of feature, and the method for making BchE sudden change storehouse in carrier.

On the other hand, the invention provides by storehouse packing that BchE is suddenlyd change and enter carrier granular, with the method for this carrier infection cell.

On the other hand, this aspect provides the expression vector of the dna molecular of the HuBchE that comprises RhBchE and modification; And based on the high level expression system of the BchE of adenovirus (AD).

On the other hand, the invention provides and simulate for example raceme and the enantiomer-pure OP model compound of OP neural gas structures such as VX, tabun, GF, soman and sarin.

On the other hand, the invention provides in the functional screening based on cell detects and use the OP model compound, identify by the anti-OP of BchE sudden change storehouse expression and/or the method for catalytic BchE variant.

On the other hand, the invention provides the method for using the OP model compound to obtain the antibody that can be used for the array bioprobe.

On the other hand, the invention provides the diagnostic method that in biological specimen, detects the OP material.

On the other hand, the invention provides the method at the active screening of BchE OP model compound, this method is by hatching BchE and this chemical compound, and further detects the indication of the inhibition of BchE as this compound activity.

On the other hand, the invention provides the method that in the culture medium of infected cell, detects the BchE expression.

On the other hand, the invention provides by protokaryon or eukaryotic cell and in the presence of OP chemical compound or never poison, express the method that BchE detects BchE.

On the other hand, the invention provides the method based on array, this method optionally detects never poison and other OP.

The accompanying drawing summary

Figure 1A shows the RhBchE transfection.Chinese hamster ovary celI inoculated in 6 orifice plates spend the night.The plasmid transfection cell that will contain the carrier of having powerful connections (N.C.), pRC-CMV-HuBchE (hBchE), pGS-RhBchE (GS1 and GS2) and pRC-CMV-RhBchE (RC1 and RC2) by Lipofectamine is described with reference to the manufacturer.Culture medium is gathered in 24 hours and 48 hours after transfection, and to adopt with 1mM BchI be that the Ellman detection method of substrate is carried out the BchE determination of activity.Figure 1B has shown the western engram analysis result of RhBchE.The BchE unit definition is the amount of the enzyme of per minute hydrolysis 1 μ mol substrate.The polyclonal antibody that employing forms at HuBchE (B) is analyzed the sample that obtained in 48 hours after the transfection with the western blotting.

Fig. 2 A shows the purification of RhBchE.SDS-PAGE to the RhBchE of purification detects by Coomassie blue.Fig. 2 B shows the SDS-PAGE by the RhBchE of anti-HuBchE antibody test purification.

Fig. 3 A demonstration has the inhibition of the echothiophate (ETP) of indication concentration to RhBchE.Fig. 3 B demonstration has the inhibition of the echothiophate of indication concentration to HuBchE.Data are passed through the Prism match to the one-level equation.Can obtain the k of specific ETP concentration by slope _App(min ^-1).By to k _App(min ^-1) and be used for the initial ETP concentration that suppresses to hatch of ETP and map again and can obtain the data of table 5 report.

Fig. 4 A shows the inhibition of 1 couple of HuBchE of chemical compound of specific concentrations.Fig. 4 B shows the inhibition of 2 couples of HuBchE of chemical compound of specific concentrations.Fig. 4 C shows the inhibition of 3 couples of HuBchE of chemical compound of specific concentrations.Fig. 4 D shows the inhibition of the ETP of specific concentrations to HuBchE.Data are passed through the Prism match to the one-level equation.Can obtain the k of specific OP concentration by slope _App(min ^-1).Fig. 4 E shows k _App(min ^-1) and be used for the initial OP concentration that suppresses to hatch of chemical compound 1 and map.Can obtain k by slope _Inac/ K _i=4.1 * 10 ³M ^-1Min ^-1Data are carried out the data that double-reciprocal plot can obtain in the table 7 being reported.

Fig. 5 shows 8 representativeness clones that select at random of the BchE of gene vitro recombination (gene shuffled recombinant).

Fig. 6 A shows iodate Butyryl thiocholine (BchI) hydrolysing activity.The COS cell is inoculated in 6 orifice plates in advance spends the night.Then by simulated infection (contrast) or with every hole 1 * 10 ⁸Pfu and 4 * 10 ⁸PfuHuBchE-AD is at time 0 infection cell.BchI hydrolysing activity after infection in the specified time check culture medium.The BchE unit is defined as the required enzyme of hydrolysis 1 μ M/min BchI substrate.Fig. 6 B shows the Western trace result that the BchE of adenovirus (AD) expression vector mediation expresses.On SDS-PAGE, analyze the culture sample that infects the collection of back fixed time.0,1 and 2 respectively representative simulated infection (contrast) or 1 * 10 ⁸Pfu and 4 * 10 ⁸The cell that pfu HuBchE-AD infects.HuBchE in the culture medium can detect by the western engram analysis.

Fig. 7 shows the BchE activity with the 293A cell of the reorganization AD transfection of PacI linear expression.BchE activity in the culture medium that fixed time gathers after transfection is analyzed by the Ellman method.

The solid phase that adopts different solid matrixs to carry out in 0-3 hour that is presented at Fig. 8 A is expressed the BchI hydrolysis of mediation in detecting based on the HuBchE of cell.Fig. 8 B is presented at the BchI hydrolysis of hatching in 3.5-7.3 hour in the solid phase detection identical among back and Fig. 8 A.The CHO-K1 cell of stably express WT HuBchE is inoculated in 24 orifice plates and spends the night.This cell is handled with reference to described in the literary composition.Write down of the absorption of each hole in time by plate reader at the 405nm place.Pictorial representation is used to cover the solid phase material of cell (the 1st layer) and substrate coating (the 2nd layer).

Fig. 9 A shows the location of HuBchE express cell.Described in literary composition, the flat board of having inoculated the CHO-K1 cell of stably express WT HuBchE develops by the Ellman mixture with BchI substrate.The inverted image of Fig. 9 B display panel A.

Figure 10 shows in the viral separation process, the legend of isolating specific filler from the macula lutea of identifying.Infect 293A cell in 6 orifice plates with the HuBchE of serial dilution, and dyeing as described herein detects the activity of BchE.Positive filler shown in digging out from flat board, negative filler, sample filler and background filler are transferred to and carry out the virus extraction in the culture medium.

Detailed Description Of The Invention

The invention provides the dna molecular of the separation of rhesus macaque butyrylcholine esterase (RhBchE) (macaque). Obtain this RhBchE by RACE kit and RT-PCR by the RNA clone. Total length RhBchE is cloned into the HindIII/ApaI site of pRC-CMV. With the plasmid transfection Chinese hamster ovary celI of gained, by measuring the expression of the active BchE enzyme of BchI hydrolysis monitoring in the culture medium. The present invention also provides the dna molecular of the separation of HuBchE and mutant thereof.

The invention provides the method for making the BchE mutation library take HuBchE and RhBchE as the mutation library of feature, in carrier, and by BchE mutation library packing is entered carrier granular with the method for carrier infection cell. The present invention further provides the expression vector of the dna molecular of the HuBchE that comprises RhBchE and modification, and based on the high level expression system of the butyrylcholine esterase of adenovirus (AD). In the method for the invention, described carrier can be any carrier that is applicable to this purpose. In preferred embodiments, carrier is pENTRA carrier or adenovirus vector. Cell can be any cell, the preferred mammal cell.

In order to create the BchE storehouse, can adopt a saturation mutation technology. This sudden change can be carried out in optional ad-hoc location (for example, the G117 in HuBchE storehouse and E197 position). The purpose of some saturation mutation is to integrate NNK random mutation codon (N=A, T, C or G, and K=G or T) to replace the ad-hoc location of HuBchE by two-step pcr. Then can this PCR product cloning be advanced the pENTRA1 carrier by the KpnI/XhoI site. DNA from the pENTRA-HuBchE clone who compiles can be used for recombinating with the pAD/CMV/V5/DEST carrier. Can with the plasmid in PacI enzymic digestion pAD-HuBchE mutation library pond, then adopt Lipofectamine 2000 that described plasmid transfection is carried out the AD packing in the 293A cell. Can collect restructuring AD-HuBchE virus base from cell conditioned medium liquid. Then can screen (embodiment 4) with elementary high flux solid phase functional screening and secondary liquid phase screening active ingredients to this storehouse simultaneously.

Use with mutant of enumerate in the table 1 of realizing by rite-directed mutagenesis or other method a kind of or RhBchE that all RhBchE amino acid change or HuBchE should be able to provide the enzyme with larger hydrolysing activity. These residues can cause higher Binding Capacity compatibility and the speed seen in the native enzyme of purifying alone or in combination. Different residues can change albumen folding and/or albumen posttranslational modification (namely, N-glycosylation, phosphorylation), thus changed cocaine or other ester to the identification of the binding site of this enzyme and enter, the actual hydrolysis of cocaine or the release of hydrolysate. Exist multiple different residue may change dimerization or four dimerizations of this albumen in the C end portion of protein, thereby change Enzymic stability. Therefore, the HuBchE sudden change based on the information that is obtained by RhBchE can generate the enzyme with high hydrolysing activity. Variation in the elementary sequence of RhBchE may provide some selective advantages to monkey. Adopt the amino acid that exists among the RhBchE to change and at random or the selective sudden change of introducing carry out the HuBchE molecular evolution, can provide high selectivity and effective catalyst, from blood flow, to remove potential toxicity ester (or its chemical homolog). Functional screening system provided herein is verified fully, guarantees the successful molecular evolution of this kind product.

The present invention further provides and in the culture medium of infected cell, detected the method that BchE expresses. This cell culture can be any cell culture that is suitable for the inventive method. In preferred embodiments, described cell culture is mammalian cell cultures. The expression system of BchE can be any expression system that is suitable for this purpose. In preferred embodiments, described expression system expresses and is suitable for the high level expression system based on adenovirus (AD) of high throughput format functional screening for integrating mutation library. AD can infect multiple mammalian cell, and allows various albumen to express (embodiment 10) in different divisions and Unseparated Cell system.

The invention provides simulation such as the OP model compound of the OP never poison structures such as VX, GF, tabun, soman and sarin. In the method for the invention, the working concentration of described compound is 0.01 to 20mM, and preferred working concentration is 0.1 to 10mM. In specific embodiment, the concentration of described compound is 0.5mM. The synthetic of described compound specifically describes in embodiment 12 and 13.

The invention provides the screening technique for the OP model compound of BchE activity, the method is by hatching BchE and described compound, and the inhibition that further detects BchE is with the indication as described compound activity. The cell of expressing BchE in the presence of OP compound or never poison can be protokaryon or eukaryotic.

The invention provides the diagnostic method that in biological specimen, detects the OP material. Because the Acute Toxicity of OP compound is closely related with the ability that their suppress AChE (the OP material suppresses AChE by forming metastable phosphoserine ester bond with essential serine hydroxyl reaction), described OP-ChE bond can be used as the selected marker of the sensitivity of OP exposure. Same, the Phosphation albumin (that is, Tyr411) can be used as the selected marker of OP or pesticide exposure. The accurate modification that brings based on specific OP compound, by adopting antibodies selective to come the independent OP-ChE of specific recognition or OP-albumin conjugates, can obtain or to expose the rear instrument with great diagnostic value of determining OP relative toxicity potentiality in exposure.

The present invention further provides and adopted the OP model compound to obtain method for the antibody of array bioprobe, wherein said bioprobe adopts the method based on array selectively to detect never poison and other OP. This is a kind of three-pronged method. At first, synthetic chemical reagent is used to obtain antibody; Secondly, obtain antibody and be integrated in the bioprobe and detect by different way, being identified for the best configuration of Sensitive Detection, and be optimized for sensitivity and fast detecting simultaneously; The 3rd, physiological fluid that buffer solution, puncture obtain, and finally blood, blood constituent or the brain tissue (described animal is processed under the physiology related concentrations) of animal show so selective after, more described antibody is carried out sensitivity and selective the detection. Final product is can the on-the-spot array bioprobe that uses, and it is for detection of the OP (embodiment 14) in environmental samples or the biological specimen (coming from the animal that is exposed to low dosage never poison or other OP).

Using method of the present invention

The invention provides the restructuring HuBchE (and/or this enzyme has more active catalysis variant) of detection of clinical, be used for before damage occurs, in body, removing never poison, cocaine, pesticide, other drug abuse as effective bioscrubbing agent (and/or enzyme catalyst). The present invention represents and a kind ofly is expected to protect army personnel, first responder and the common people to avoid OP to expose the method that threatens, and the emergency treatment to cocaine or other excessive use medicament is provided. This product also can be used for protecting the potential personnel that are exposed to pesticide. Except the clinical use of the mankind, this product also can be used as detoxifying equipment, and for example the never poison detoxifcation absorbs sponge, and it can with skin or other surperficial never poison absorbs and detoxifcation; For detection of equipment, for example be used for test-strips so that the quick and sensitive detection to never poison to be provided; And as never poison destruction and discarded cleanser.

Except final product, OP analog provided by the invention and OP hydrolysis detection method can be applied to other enzyme, for example paraoxonase, carboxylase and catalytic antibody simply; Described diversified storehouse can be used for screening other commercial vigor biopharmaceuticals (for example, cocaine hydrolysis) and industrial enzyme.

The present invention is further described by following non-limiting example.

The clone of embodiment 1:RhBchE, expression and purification

The structure of the total length expressed carrier of RhBchE.5 ' sequence of RhBchE can adopt the RACE test kit directly to be cloned by Rhesus Macacus (macaque) RNA with reference to manufacturer's step and obtain.Particularly, prepare total RNA by the sample that comprises three Rhesus Macacus livers.Behind short mRNA dephosphorylation, remove the cap-like structure of full length mRNA, and RNA RACE oligomer is connected on the full length mRNA.Obtain 5 ' sequence of RhBchE then by RT-PCR and clone.3 ' RhBchE sequence is confirmed and is disclosed in the provisional application 60/811,370 (be incorporated by reference in this text and examine) of submission on June 7th, 2006 by the sequence (NCBIBV211040) of input recently.Sequential design based on gained obtains primer.Total length RhBchE (comprising RhBchE signal peptide district and ripe BchE) can and be cloned into pRC-CMV and the HindIII/ApaI site of pGS carrier by pcr amplification.G418 selected marker in pRC-CMV was replaced by the rat glutamine synthetase, pGS carrier and pRC-CMV carrier were basic identical.The sequence of cloned plasmids has obtained affirmation.

The expression of functional reorganization RhBchE.Plasmid transfection is arrived Chinese hamster ovary celI, by monitor the expression of active BchE enzyme with the BchI hydrolysis in the Ellman reaction assay culture medium.The proteic expression of BchE also by adopt rabbit anti--the western engram analysis of HuBchE polyclonal antibody confirms.

The selection of stable cell line.Provide reorganization RhBchE the stable cell lines of high level expression in order to prepare, with pGS-RhBchE carrier transfection CHO cell.After the transfection 24 hours, with this cell trypsinized, and 10 times and 20 times of dilutions before placing the 15cm plate.Used selection culture medium is the serum-free medium that does not contain L-glutaminate, contains 25 μ M methionine sulfoxide acid imides (MSX).MSX is the specific inhibitor of the glutamine synthetase of internal representations.Therefore only carry the cell of pGS-RhBchE carrier can be under this selective conditions merisis.Cells transfected was kept for 2 weeks to allow colony formation in selective medium.Select and isolating 24 the independent colonies of other colony at random.From culture plate, remove culture medium.To soak tryptic little filter paper covers on the selected colony and at room temperature hatched 2 minutes.Filter paper is transferred in the independent micropore of 24 orifice plates then with above-mentioned selective medium.Cell grows to and converges.Measure the BchE activity of culture medium in each micropore, the stable cell lines of high level expression is provided with discriminating.

The preparation of affine resin.For purification RhBchE, to adjust slightly with reference to described step such as (Grunwald people, 1997), preparation combines the sepharose 4B post of procainamide.With the fast flow velocity post of the activated sepharose 4B of 1mM HCl cleaning down CNBr-(with 1 volume washing 15 times), the sugar that carries with removal.Then this resin is resuspended to the 0.2M NaCO that contains 0.4M NaCl and be adjusted to pH9.0 ₃The coupling buffer.Remove after the decoupling buffer, add the coupling buffer that 0.5 volume contains 0.1M gamma-amino caproic acid, and make reactant under 4 ℃, rotate through night (about 20 hours).Use H ₂O washs culture medium 5 times, and resin is resuspended to 0.1M1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride, and regulates pH to 4.5 with HCl, removes liquid then.Then resin is resuspended to comprise in the 0.1M 1-that concentration is the procainamide of 100mol/ml resin (3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride 2.5 hours of 0.5 volume, and to add 1M HCl be 4.5 to keep pH.In reaction, regulated a pH in per 5 minutes, and the scope of pH is between about pH4.45 to 4.65.To be reflected at ambient temperature overnight then and finish (about 20 hours).Then with package resin in post, with H ₂The O cleaning down, and under UV280, monitor to remove unreacted procainamide fully.Collect all effluent to detect the procainamide of not coupling.

Purification of Recombinant RhBchE from culture medium.In order to prepare the recombiant protein of purification, the stabilized cell of culture expression RhBchE in the T180 flask, and allow three layers of cell growths.After excretory two weeks of RhBchE accumulation, collect culture medium from flask.Culture medium and procainamide resin are at the uniform velocity rotated overnight incubation under 4 ℃.Deposited this resin in centrifugal 5 minutes by 2000 * g.The careful removal comprises the not supernatant of bound substances, and resin is resuspended to 50mM potassium phosphate pH7.2, among the 1mM EDTA (buffer A), and is loaded in the post.After with the buffer A thorough washing that contains 0.2M NaCl, with the RhBchE of buffer A gradient solution elution of bound on post that contains 0.05 to 0.5M procainamide.Adopt the BchI substrate to measure the active elution fraction of identifying in the eluent by Ellman.Compile described active elution fraction, and concentrate to block the Centricon that molecular weight is 30kD (Millipore).

Purifying natural BchE from serum of macaque.Serum of macaque is flow through the agarose column that combines procainamide.With the 20mM potassium phosphate pH7.0 that contains 0.2M NaCl, 1mM EDTA thoroughly washs this post.With the RhBchE of 0.1M procainamide elution of bound on post.The activity of the elution fraction of enzyme is determined by the BchI hydrolysis.Active elution fraction is pooled together, and with 20mM Tris pH8.0, the sepharose 4B gel permeation chromatography of 1mM EDTA is further purified.Based on the BchI hydrolysing activity, active elution fraction is pooled together and is loaded on the DEAE agarose column.With 20mM Tris pH8.0,1mMEDTA thoroughly washs this post, and to contain the 20mM Tris pH8.0 of 0.1M, 0.15M, 0.2M, 0.25M and 0.3M NaCl, the albumen of 1mM EDTA discontinuous gradient elution of bound on post.Organized enzyme is present in the 0.2M NaCl eluent.Protein concentration is measured with the BCA method.To the albumen in each elution fraction by SDS-PAGE, Coomassie blue stain and western trace are analyzed subsequently.

The substrate specificity of embodiment 2.RhBchE and HuBchE and the dynamic (dynamical) assessment of inhibition.

The BchI hydrolysis.Can be by the BchI hydrolysis of Ellman method enzyme analysis elution fraction.Briefly, under two (the 2-nitro benzoic acid) existence (DTNB) of 10mM 5,5 '-dithio, 5mM BchI and serum are hatched under 25 ℃ in 50mM potassium phosphate pH7.4.Hydrolysis with UV-Vis spectrophotometer continuous monitoring BchI under 412nm.Activity can be by molar extinction coefficient 13,600M ^-1Cm ^-1Calculate.For K _mMeasure, detection method has comprised the BchI of 25,33.3,50,100 and 200 μ M respectively, enzyme liquid storage, and the 50mM potassium phosphate pH7.2 buffer that contains 200 μ M DTNB.This is determined under 25 ℃ and carries out.K _mValue can be passed through the Lineweaver-Burk assay determination, and K _CatValue can be by being determined by the functional enzyme concentration of echothiophate (ETP) titration determination.The competitive inhibition constant K of (+)-cocaine, (-)-cocaine and part metabolite thereof _iMensuration can be undertaken by the hydrolysis that in the presence of (+)-cocaine of certain concentration range, (-)-cocaine, (-)-nor-cocaine, detects BchI.

Cocaine hydrolysis detects.Cocaine hydrolysis can be characterized by generating with the quantitatively specific ecgonine methyl ester (EME) of mass spectrum (MS).Highly purified RhBchE or HuBchE and cocaine (final concentration is 1,2,4,10 and 40 μ M among the 10mM kaliumphosphate buffer pH7.4) are hatched under 37 ℃.Mix the equal portions reactant with cessation reaction with 6N HCl at interval at 20,40 and 60 minutes, and the stabilization reactions product.Do not add the background response of enzyme simultaneously.The amount of the EME that generates at each time point can be by definite to Agilent MSD type MS with the direct flow injection of equal portions reactant, and undertaken quantitatively by the ion of selected ion monitoring to m/z 199.9-200.9 (EME).EME in the reactant mixture amount standard curve with EME solution generation by concentration known (125-1000nM) is compared.Can after deducting respective background speed, obtain the concrete enzyme catalysis speed under each cocaine concentration.K _mAnd V _MaxValue can be passed through the LineweaVer-Burk assay determination, and K _CatValue can be by being determined by the functional enzyme concentration of echothiophate (ETP) titration determination.

The OP chemical compound is to the inhibition kinetics of RhBchE and HuBchE.In 50mM kaliumphosphate buffer pH7.2, under 25 ℃, studied the kinetics that model OP chemical compound and ETP suppress the time dependence of the RhBchE of purification and HuBchE.The inhibition of RhBchE and HuBchE can mix the back startup with highly purified RhBchE and HuBchE with the ETP of various amounts.In the specified time, the hydrolysis that the reactant mixture that will contain 1mM BchI and 0.2mM DTNB is added into enzyme-compound mixture and detects BchI is to determine residual BchE activity.Adopted seven inhibitor concentration in this mensuration, and each inhibitor concentration has been provided with five time points.

Embodiment 3.HuBchE and the new interactional sign of OP chemical compound.

The OP analog is to the inhibition of WT and G117H/E197Q HuBchE.The chemical compound 1,2,3 of WT or G117H/E197Q HuBchE and 0.5mM (or 4-13, referring to embodiment 12) or ETP were hatched under 4 ℃ 48 hours.After initial enzyme-chemical compound mixtures incubated is carried out 100 times of dilutions, use standard substrate BchI (1mM) to measure the percentage ratio that residual enzyme is lived then by the Ellman method.

Model OP chemical compound suppresses the mensuration of the inhibition speed constant of HuBchE.In 50mM kaliumphosphate buffer pH7.2, under 25 ℃, studied the kinetics of model OP chemical compound to the time dependence inhibition of the HuBchE of purification.The inhibition of HuBchE can start by the highly purified HuBchE of 15nM and not commensurability chemical compound 1,2,3 (or 4-13, referring to embodiment 12) or ETP are mixed the back.In the specified time, the hydrolysis that the reactant mixture that will contain 1mM BchI and 0.2mM DTNB is added into enzyme-compound mixture and detects BchI is to determine residual HuBchE activity.Adopted seven inhibitor concentration in this mensuration, and each inhibitor concentration has been provided with five enzyme live time points.

Embodiment 4. people BchE mutant expression libraries make up.

The structure in HuBchE sudden change storehouse.Point saturation mutation technology is used to create the HuBchE storehouse at position G117 and E197.The purpose of some saturation mutation is to integrate NNK random mutation codon (N=A, T, C or G, and K=G or T) is replaced HuBchE by two-step pcr position G117 and E197.This PCR product can be cloned into this pENTRA1 carrier by the KpnI/XhoI site.Plasmid DNA from the pENTRA-HuBchE clone who compiles is used to recombinate with the pAD/CMV/V5/DEST carrier.To expose a left side and right viral ITR, adopt Lipofectamine 2000 transfections to pack to carry out AD with the plasmid in PacI enzymic digestion pAD-HuBchE sudden change pond, storehouse then to the 293A cell.The AD-huBuChE virus base of reorganization by transfection after 5 days cell conditioned medium liquid collect.Can screen this storehouse with elementary high flux solid phase functional screening and secondary liquid phase screening active ingredients simultaneously then.

As example, this functional screening platform can be verified by the storehouse that above-mentioned two-position (G117 and E197) some saturation mutation generates.This storehouse recombinant virus is used to infect the 293A cell, cell applies with the MEM sugar that contains 1% agar back 24 hours of infection, the chemical compound 4-13 that adds 0.4mM to the cell that applies (promptly then, chemical compound 5, sarin analog, referring to embodiment 13), thus interact with the reorganization HuBchE variant of expressing.At metainfective the 4th day, with BchI as substrate in the presence of DTNB to applying cell dyeing.The appearance of macula lutea on the monitoring flat board this means the existence of OP-hydrolytic enzyme in 3 hours.From flat board, dig out yellow agarose speckle, and in serum-free medium, in independent pipe, hatch.Get the recombinant virus that five equilibrium discharges and add the flat board of having inoculated the 293A cell in advance from culture medium.After infection, 96 hole culture plates were hatched 3 days.This time can allow the expression of the huBuChE variant of viral propagation and coding.At the 3rd day, measure OP inhibition tolerance/hydrolysing activity from the culture medium of 96 orifice plates.Based on the result of all said determinations, the sample of having selected to show the OP hydrolysing activity that improves is for gene identification.The HuBchE gene of encoding in the recombinant virus is carried out pcr amplification by the viral vector Auele Specific Primer from culture medium.To the order-checking of PCR product, and the sudden change in the evaluation HuBchE gene.

The HuBchE of AD-mediation expresses.The liquid storage titre of reorganization AD that comprises the variant (A328Y/Y332A) of HuBchE is 2.45 * 10 ¹⁰Pfu/ml.For the hydrolysis that improves cocaine has designed this double-mutant.Two kinds of variable concentrations (1 * 10 ⁸Pfu and 4 * 10 ⁸The virus of pfu (that is, be respectively 200 infection multiplicities (moi) and 800moi)) is used to infect Chinese hamster ovary celI and the COS cell that pre-inoculation is spent the night on 6 orifice plates.BchE activity in the culture medium can be that substrate is monitored by standard Ellman reaction with 1mM BchI in metainfective different time points.

The AD preparation of expression vectors of WT and G117H/E197Q HuBchE.The ViralPower AD expression system of Invitrogen is used to clone the HuBchE enzyme.This system relates to clone's target gene in entering carrier (entryvector).Target gene can adopt the Gateway technology to be transferred to pAD/CMV/V5/DEST through vitro recombination as described in the manufacturer.For the HuBchE gene clone is entered carrier (it provides adenovirus vector construct required recombination signal) to pENTRA1, can use Turbo Pfu to prepare the WT of PCR-amplification and G117H/E197Q HuBchE with in conjunction with restricted cloning site KpnI and XhoI from initial plasmid.Described PCR product is cloned in KpnI and the XhoI site of pENTRA1.The insertion of selecting the clone is checked order to confirm that the PCR step does not import sudden change.Then super spirial plasmid and pAD/CMV/V5/DEST are hatched, this AD expression vector comprises and is used to carry out CMV promoter and the negative selectivity ccdB gene that high-level target protein is expressed.After the conversion, only recombiant plasmid can be grown on the selectivity flat board.By restrictive diges-tion and sequencing analysis plasmid is analyzed, to determine the correct insertion of HuBchE gene in the pAD carrier.

The generation of HuBchE-AD and by the WT of AD expression system checking reorganization and the expression of G117H/E197QHuBchE.For WT-and the G117H/E197Q-hBchE-AD that generates reorganization, can arrive the 293A cell by the Lipofectamine transfection then by the PacI enzymic digestion with plasmid pAD-WT-hBchE and pAD-G117H/E197Q-hBchE linearisation.Cells transfected is kept three days to check the HuBchE activity, to be transferred to then that the culture medium that contains serum is to help to keep cytoactive in serum-free medium.Parallel use control vector pAD-lacZ.In the culture medium that the recombinant virus that contains packing was collected in infection in back 12 days, centrifugal removal cell debris also is stored in-80 ℃.In order to monitor the expression of infecting back reorganization BchE, add recombinant virus to the culture medium of cultivating the COS cell.The BchI hydrolysing activity of different time point monitoring culture medium after primary infection.

The structure in RhBchE and HuBchE chimera sudden change storehouse.Create the chimera storehouse with DNA extracorporeal recombination (DNAshuffling).The full length fragment of HuBchE and RhBchE is increased by PCR from corresponding clone.The PCR fragment of HuBchE and RhBchE is carried out gel-purified and merged with the ratio of 1:1.The PCR fragment that is combined is carried out limited DNA enzyme I digestion.Purification 100-200bp fragment, and carry out pcr amplification to be assembled into longer fragment without primer.Described longer fragment finally increases to obtain the total length recombinant products with terminal primer.The dna fragmentation of these vitro recombination is linked plasmid pCR2.1-TOPO.

The exploitation and the checking of embodiment 5. functional screening detection methods.

Set up solid phase cholinesterase activity detection method.With the CHO-K1 cell inoculation of stably express HuBchE WT or G117H/E197Q HuBchE in culture plate.Inoculate back 24 hours, remove culture medium and with twice of Ultraculture serum-free medium washed cell.Cover cell with the colourless MEM that contains 1% agar or 1% agarose then.After the culture medium solidifying, cell is put back to incubator spend the night.Preparation contains the Ellman reactant mixture of substrate (1mM BchI or 1mMETP) and/or 0.1mM DTNB in the colourless MEM that contains 1% agar (or 1% agarose).This reactant mixture is covered on the cell culture flat board of above-mentioned preparation, and at room temperature hatches.By the dull and stereotyped appearance of going up macula lutea of perusal monitoring, measure OD405 with plate reader simultaneously and absorb.

The active local detection of HuBchE.The CHO-K1 cell of serial dilution stably express WT HuBchE also is seeded to the 10cm culture dish with different density (that is, 4,20 and 100 cells/flat board).Cell is grown contain the small-sized colony of about 20 cells/colony 1 week with formation.With serum-free medium washed cell 2 times, apply with the MEM that contains 1% agarose then.After the overnight incubation, make this flat board colour developing with the Ellman reagent mixture that contains substrate (1mM BchI and 0.1mM DTNB) that in the colourless MEM of 1% agarose, prepares.

Detect the AD-BchE recombinant virus with solid phase BchE activity test method.Be seeded in 293A cell on 6 orifice plates in advance with the HuBchE-AD viral infection of serial dilution.Infect and agarose-MEM mixture covered on the cell in back 1 hour.Culture plate is put back to incubator.Repeat to prepare a plurality of flat boards, all can take out one flat plate and cover in metainfective every day like this with the agarose-MEM mixture that contains 1mM BchI and 200 μ M DTNB.Can be by xanchromatic appearance on the perusal flat board.Macula lutea isolated viral for from identification can dig out this filler from the agarose culture plate.Isolating filler is transferred in the pipe that contains the 0.5ml culture medium, and 4 ℃ of following overnight incubation.Use the culture medium of hatching to infect the 293A cell that is seeded in advance on 24 orifice plates then with filler.After infection, measured the BchE activity in the culture medium in 24 and 48 hours.In order to reclaim the BchE gene from isolating recombinant virus, the culture medium of hatching with 1 μ l and filler is a template, and increases with AD carrier specificity primer T7 and pAD-V5R.Measure the sequence of this PCR product.

The activity analysis of embodiment 6. macaque ChE.

Have bigger cocaine hydrolysis activity or the active new BchE of OP in order to identify, to from 3 kinds of monkeys: the blood plasma of macaque (Macaca mulatta), bruh (Macaca nemestrina) and machin (Macacafascicularis) carries out BchI and cocaine hydrolysis activity screening.Although studies show that the hydrolysis of BchI in all blood plasma is closely similar, the hydrolysis of (-)-cocaine has notable difference, and serum of macaque has maximum hydrolysing activity.Therefore, the BchE gene of macaque is cloned with this enzyme of further evaluation.

The cDNA sequence analysis of RhBchE.Adopt the cDNA of RT-PCR by macaque liver organization clone RhBchE.The global cDNA sequence of RhBchE (SEQ ID NO:1) and amino acid sequence corresponding (SEQ ID NO:2) are as follows.

M H S K V T I I C I R L L F W F L L L C M L I G K S H T E

D

1 ATGCATAGCA AAGTCACAAT CATATGCATC AGATTACTCT TTTGGTTTCT TTTGCTCTGC ATGCTTATTG GAAAGTCACA

TACTGAAGAT

D I V I A T K N G K V R G M N L T V L G G T V T A F L G I

P

91 GACATCGTAA TTGCAACAAA GAATGGAAAA GTCAGAGGGA TGAACTTAAC AGTTCTTGGT GGCACGGTAA CAGCCTTTCT

TGGAATTCCC

Y A Q P P L G R L R F K K P Q S L T K W S D I W N A T K Y

A

281 TATGCACAGC CACCTCTTGG TAGACTTCGA TTCAAAAAGC CACAGTCTCT GACCAAGTGG TCTGATATTT GGAATGCCAC

AAAATATGCA

N S C Y Q N I D Q S F P G F H G S E M W N P N T D L S E D

C

271 AATTCTTGCT ATCAGAACAT AGATCAAAGT TTTCCAGGCT TCCATGGATC AGAGATGTGG AACCCAAACA CTGACCTCAG

TGAAGACTGT

L Y L N V W I P A P K P K N A T V M I W I Y G G G F Q T G

T

361 TTATATCTAA ATGTATGGAT TCCGGCACCT AAACCAAAAA ATGCTACTGT AATGATATGG ATTTATGGTG GTGGTTTTCA

GACTGGAACA

S S L H V Y D G K F L A R V E R V I V V S M N Y R V G A L

G

451 TCATCTTTAC ATGTTTATGA TGGCAAGTTT CTGGCTCGAG TTGAAAGAGT TATTGTAGTG TCAATGAACT ATAGGGTGGG

TGCCCTTGGA

F L A L P G N P E A P G N M G L F D O O L A L Q W V Q K N

I

541 TTCTTAGCTT TGCCAGGAAA TCCTGAGGCT CCAGGGAACA TGGGTTTATT TGATCAACAG TTGGCTCTTC AGTGGGTTCA

AAAAAATATA

A A F G G N P K S V T L F G E S A G A A S V S L H L L S P

G

631 GCAGCCTTTG GTGGAAATCC TAAAAGTGTA ACTCTCTTTG GAGAAAGTGC AGGAGCAGCT TCAGTTAGCC TGCATTTGCT

TTCTCCTGGA

S H P L F T R A I L Q S G S S N A P W A V T S L Y E A R N

R

721 AGCCATCCAT TGTTCACCAG AGCCATTCTA CAAAGTGGAT CCTCTAATGC TCCTTGGGCA GTAACATCTC TTTATGAAGC

TAGGAACAGA

T L T L A K L T G C S R D N E T E I V K C L R N K D P H E

I

811 ACATTGACCT TGGCTAAATT GACTGGTTGC TCTAGAGATA ATGAGACTGA AATAGTCAAG TGCCTTAGAA ATAAAGATCC

CCACGAAATT

L L N E A F V V P Y G T L L S V N F G P T M D G D F L T E

M

901 CTTCTGAATG AAGCATTTGT TGTCCCCTAT GGGACTCTCT TGTCAGTAAA CTTCGGTCCA ACCATGGATG GTGATTTTCT

CACTGAAATG

P D I L L E L G Q F K K T Q I L V G V N K D E G T A F L V

Y

991 CCAGACATAT TACTTGAACT TGGACAATTT AAAAAAACCC AGATATTGGT GGGTGTTAAT AAAGATGAAG GGACAGCTTT

TTTAGTCTAT

C A P G F S K D N D S I Y T R N E F Q E G L K I F F P G V

S

1081 GGTGCTCCTG GCTTCAGCAA AGATAACGAT AGTATCATAA CTAGAAACGA ATTTCAGGAA GGTTTAAAAA TATTTTTTCC

AGGCGTGAGT

E F G K E S I L F H Y T D W V D D G R P E N Y R E A L D D

V

1171 GAGTTTGGAA AGGAATCCAT CCTTTTTCAT TACACAGACT GGGTAGATGA TCAGAGACCT GAAAACTACC GTGAGGCGTT

GGATGATGTT

V G D Y N I I C P A L E F T K K F S E W G N N A F F Y Y F

E

1261 GTTGGGGATT ATATATGAT ATGCCCTGCC TTGGAGTTTA CCAAGAAGTT CTCAGAATGG GGAAATAATG CCTTTTTCTA

CTATTTTGAA

H R S S K L P W P E W M G V M H G Y E I E F V F G L P L E

R

1351 CACCGATCCT CCAAACTTCC GTGGCCAGAA TGGATGGGAG TGATGCATGG CTATGAAATT GAATTTGTCT TTGGGTTACC

TCTGGAAAGA

R V N Y T K A B I E I L S R S I V K R W A N F A K Y G N P N

G

1441 AGAGTTAATT ACACAAAAGC TGAGGAAATT TTGAGTAGAT CCATAGTGAA ACGGTGGGCA AATTTTGCAA AATATGGGAA

TCCAAATGGG

T H N N S T K W P V F K S T E Q K Y L T L N T E S S R I L

T

1531 ACTCATAATA ATAGCACAAA ATGGCCTGTC TTCAAAAGCA CTGAACAAAA ATATCTAACCT TGAATACAG AGTCATCAAG

AATATTGACT

K L R A Q Q C R F W T S F F P K V L E M T G N I D E AEW

E

1621 AAACTACGTG CTCAGCAATG CCGATTCTGGACATCATTTTTTCCAAAAGTCTTGGAAATGACAGGAAATATTGATGAAGC

AGAATGGGAG

W X A G F N R W S N Y M M D W K N Q F N D Y T S K K E S C

V

1711 TGGAAAGCAG GATTCCATCG CTGGAGCAAT TACATGATGG ACTGGAAAAA TCAATTTAAC GATTACACTA GCAAGAAAGA

AAGTTGTGTG

G L

1801 GGTCTCTAA

574 amino acid whose polypeptide of the longest open reading frame coding of RhBchE, this polypeptide and HuBchE (gi:4557351) (SEQ ID NO:3) have 95% sequence identity (96% sequence similarity degree), has 91% sequence identity (94% similarity) with rabbit BchE (gi:116354) (SEQ ID NO:4), has 91% sequence identity (95% similarity) with horse BchE (gi:7381418) (SEQ ID NO:6), has 88% sequence identity (92% similarity) with cat BchE (gi:2981243) (SEQ ID NO:5), has 81% sequence identity (89% similarity) with mice BchE (gi:6857761) (SEQ ID NO:7).The peptide sequence of SEQ IDNOS:3-7 shows below.

The peptide sequence of HuBchE (SEQ ID NO:3):

MHSKVTIICIRFLFWFLLLCMLIGKSHTEDDIIIATKNGKVRGMNLTVFGGTVTAFLGI

PYAQPPLGRLRFKKPQSLTKWSDIWNATKYANSCCQNIDQSFPGFHGSEMWNPNTDLSED

CLYLNVWIPAPKPKNATVLIWIYGGGFQTGTSSLHVYDGKFLARVERVIVVSMNYRVGAL

GFLALPGNPEAPGNMGLFDQQLALQWVQKNIAAFGGNPKSVTLFGESAGAASVSLHLLSP

GSHSLFTRAILQSGSFNAPWAVTSLYEARNRTLNLAKLTGCSRENETEIIKCLRNKDPQE

ILLNEAFVVPYGTPLSVNFGPTVDGDFLTDMPDILLELGQFKKTQILVGVNKDEGTAFLV

YGAPGFSKDNNSIITRKEFQEGLKIFFPGVSEFGKESILFHYTDWVDDQRPENYREALGD

VVGDYNFICPALEFTKKFSEWGNNAFFYYFEHRSSKLPWPEWMGVMHGYEIEFVFGLPLERRDNYTKAEEILSRSIVKRWANFAKYGNPNETQNNSTSWPVFKSTEQKYLTLNTESTRIMTKLRAQQCRFWTSFFPKVLEMTGNIDEAEWEWKAGFHRWNNYMMDWKNQFNDYTSKKESC

VGL

The peptide sequence of rabbit BchE (SEQ ID NO:4):

MVTRSSHTEDVIITTKNGRIRGINLPVFGGTVTAFLGI

PYAQPPLGRLRFKKPQSLTKWSDIWNATKYANSCCQNIDQSFPGFHGSEMWNPNTDLSED

CLYLNVWIPTPKPKNATVMIWIYGGGFQTGTSSLQVYDGKFLTRVERVIVVSMNYRVGAL

GFLALPGNPEAPGNMGLFDQQLALQWVQKNIAAFGGNPKSVTLFGESAGAASVSLHLLSP

RSHPLFTRAILQSGSSNAPWEVMSLHEARNRTLTLAKFVGCSTENETEIIKCLRNKDAQE

ILLNEVFVVPFDSLLSVNFGPTVDGDFLTDMPDTLLQLGQLKKTQILVGVNKDEGTAFLV

YGAPGFSKDNTSIITRKEFQEGLKIFFPGVSEFGKESILFHYTDWVDEQRPENYREALDD

VVGDYNFICPALEFTKKFSEWGNNAFFYYFEHRSSKLPWPEWMGVMHGYEIEFVFGLPLE

RRVNYTKAEEILSRSIMKRWANFAKYGNPNGTQNNSTRWPVFKSTEQKYLTLNTESPRIY

TKLRAQQCRFWTLFFPKVLEMTGNIDEAEQEWKAGFHRWNNYMMAWKNHFNDYTSKKERC

AGF

The peptide sequence of cat BchE (SEQID NO:5):

MQSKGTIISIQFLLRFLLLWVLIGKSHTEEDIIITTKNGKVRGMNLPVLDGTVTAFLGI

PYAQPPLGRLRFKKPQFLTKWSDIWNATKHANSCYQNADQSFPGFPGSEMWNPNTDLSED

CLYLNVWSPTPKPKNATVMIWIYGGGFQTGTSSLPVYDGKFLARVERVIVVSMNYRVGAL

GFLALPGNPEIPGNMGLFDQQLALQWVQKNIAAFGGNPKSVTLFGESAGAGSVSLHLLSP

RSQPLFTRAILQSGSSNAPWAVMSLDEAKNRTLTLAKFIGCSKENDTEIIKCLRNKDPQE

ILLNELLVVPSDTLLSVNFGPVVDGDFLTDMPDTLLQLGQFKKTQILVGVNKDEGTAFLV

YGAPGFSKDNDSIITRKEFQEGLKIYFPGVSEFGREAILFYYVDLLDDQRAEKYREALDD

VLGDYNIICPALEFTTKFSELGNNAFFYYFEHRSSQLPWPEWMGVMHGYEIEFVFGLPLE

RRVNYTRAEEILSRSIMNYWANFAKYGNPNGTQNNSTRWPAFRSTDQKYLTLNAESPKVY

TKLRAQQCRFWTLFFPKVLEMTGNIDEAEREWRAGFYRWNNYMMDWKNQFNDYTSKKESC

AGL

The peptide sequence of horse BchE (SEQ ID NO:6):

MQSWGTIICIRILLRFLLLWVLIGNSHTEEDIIITTKNGKVRGMNLPVLGGTVTAFLGI

PYAQPPLGRLRFKKPQSLTKWSNIWNATKYANSCYQNTDQSFPGFLGSEMWNPNTELSED

CLYLNVWIPAPKPKNATVMIWIYGGGFQTGTSSLPVYDGKFLARVERVIVVSMNYRVGAL

GFLALSENPEAPGNMGLFDQQLALQWVQKNIAAFGGNPRSVTLFGESAGAASVSLHLLSP

RSQPLFTRAILQSGSSNAPWAVTSLYEARNRTLTLAKRMGCSRDNETEMIKCLRDKDPQE

ILLNEVFVVPYDTLLSVNFGPTVDGDFLTDMPDTLLQLGQFKRTQILVGVNKDEGTAFLV

YGAPGFSKDNNSIITAKEFQEGLKIFFPRVSEFGRESILFHYMDWLDDQRAENYREALDD

VVGDYNIICPALEFTKKFSELGNDAFFYYFEHRSTKLPWPEWMGVMHGYEIEFVFGLPLE

RRVNYTKAEEILSRSIMKRWANFAKYGNPNGTQSNSTRWPVFKSTEQKYLTLNTESPKVY

TKLRAQQCRFWTLFFPKVLELTGNIDEAEREWKAGFHRWNNYMMDWKNQFNDYTSKKESC

SDF

The peptide sequence of mice BchE (SEQ ID NO:7):

MQTQHTKVTQTHFLLWILLLCMPFGKSHTEEDFIITTKTGRVRGLSMPVLGGTVTAFLGI

PYAQPPLGSLRFKKPQPLNKWPDIHNATQYANSCYQNIDQAFPGFQGSEMWNPNTNLSED

CLYLNVWIRVPKPKNATVMVWIYGGGFQTGTSSLPVYDGKFLARVERVIVVSMNYRVGAL

GFLAFPGNPDAPGNMGLFDQQLALQWVQRNIAAFGGNPKSITIFGESAGAASVSLHLLCP

QSYPLFTRAILESGSSNAPWAVKHPEEARNRTLTLAKFTGCSKENEMEMIKCLRSKDPQE

ILRNERFVLPSDSILSINFGPTVDGDFLTDMPHTLLQLGKVKKAQILVGVNKDEGTAFLV

YGAPGFSKDNDSLITRKEFQEGLNMYFPGVSRLGKEAVLFYYVDWLGEQSPEVYRDALDD

VIGDYNIICPALEFTKKFAELENNAFFYFFEHRSSKLPWPEWMGVMHGYEIEFVFGLPLG

RRVNYTRAEEIFSRSIMKTWANFAKYGHPNGTQGNSTMWPVFTSTEQKYLTLNTEKSKIY

SKLRAPQCQFWRLFFPKVLEMTGDIDETEQEWKAGFHRWSNYMMDWQNQFNDYTSKKESC

TAL

The present invention also provides the comparison of RhBchE sequence of the present invention (SEQ ID NO:8) with M62777 (part RhBchE) (SEQ ID NO:9) and HuBchE (NCBI NM 000055) (SEQ ID NO:10).

The polymerized nucleoside acid sequence (SEQ ID NO:8) of coding RhBchE shows below.

AACAGTTGATTGCTACATTCAGTAACACTGAATGTCAGTGCAGTCCA

The polymerized nucleoside acid sequence of coded portion RhBchE (BV211040) sequence (SEQ ID NO:9):

AACATAGATCAAAGTTTTCCAGGCTTCCATGGATCAGAGATGTGGAACC

CAAACACTGACCTCAGTGAAGACTGTTTATATCTAAATGTATGGATTCCG

GCACCTAAACCAAAAAATGCTACTGTATTGATATGGATTTATGGTGGTGG

TTTTCAGACTGGAACATCATCTTTACATGTTTATGATGGCAAGTTTCTGGC

TCGAGTTGAAAGAGTTATTGTAGTGTCAATGAACTATAGGGTGGGTGCCC

TTGGATTCTTAGCTTTGCCAGGAAATCCTGAGGCTCCAGGGAACATGGGT

TTATTTGATCAACAGTTGGCTCTTCAGTGGGTTCAAAAAAATATAGCAGC

CTTTGGTGGAAATCCTAAAAGTGTAACTCTCTTTGGAGAAAGTGCAGGAG

CAGCTTCAGTTAGCCTGCATTTG

The polymerized nucleoside acid sequence of coding HuBchE (SEQ ID NO:10):

AGTAACAGTTGATTGTTACATTCAGTAACACTGAATGTCAGTGCAGTCCA

ATTTACAGGCTGGAGCAGCAGCTGCATCCTGCATTTCCCCGAAGTATTAC

ATGATTTTCACTCCTTGCAAAC-TTTGCCATCTTTGTTGCAGAGAATCGG

AAATCAATATGCATAGCAAAGTCACAATCATATGCATCAGATTTCTCTTT

TGGTTTCTTTTGCTCTGCATGCTTATTGGGAAGTCACATACTGAAGATGA

CATCATAATTGCAACAAAGAATGGAAAAGTCAGAGGGATGAACTTGACAG

TTTTTGGTGGCACGGTAACAGCCTTTCTTGGAATTCCCTATGCACAGCCA

CCTCTTGGTAGACTTCGATTCAAAAAGCCACAGTCTCTGACCAAGTGGTC

TGATATTTGGAATGCCACAAAATATGCAAATTCTTGCTGTCAGAACATAG

ATCAAAGTTTTCCAGGCTTCCATGGATCAGAGATGTGGAACCCAAACACT

GACCTCAGTGAAGACTGTTTATATCTAAATGTATGGATTCCAGCACCTAA

ACCAAAAAATGCCACTGTATTGATATGGATTTATGGTGGTGGTTTTCAAA

ACCAAAAAATGCCACTGTATTGATATGGATTTATGGTGGTGGTTTTCAAA

CTGGAACATCATCTTTACATGTTTATGATGGCAAGTTTCTGGCTCGGGTT

GAAAGAGTTATTGTAGTGTCAATGAACTATAGGGTGGGTGCCCTAGGATT

CTTAGCTTTGCCAGGAAATCCTGAGGCTCCAGGGAACATGGGTTTATTTG

ATCAACAGTTGGCTCTTCAGTGGGTTCAAAAAAATATAGCAGCCTTTGGT

GGAAATCCTAAAAGTGTAACTCTCTTTGGAGAAAGTGCAGGAGCAGCTTC

AGTTAGCCTGCATTTGCTTTCTCCTGGAAGCCATTCATTGTTCACCAGAG

CCATTCTGCAAAGTGGATCCTTTAATGCTCCTTGGGCGGTAACATCTCTT

TATGAAGCTAGGAACAGAACGTTGAACTTAGCTAAATTGACTGGTTGCTC

TAGAGAGAATGAGACTGAAATAATCAAGTGTCTTAGAAATAAAGATCCCC

AAGAAATTCTTCTGAATGAAGCATTTGTTGTCCCCTATGGGACTCCTTTG

TCAGTAAACTTTGGTCCGACCGTGGATGGTGATTTTCTCACTGACATGCC

AGACATATTACTTGAACTTGGACAATTTAAAAAAACCCAGATTTTGGTGG

GTGTTAATAAAGATGAAGGGACAGCTTTTTTAGTCTATGGTGCTCCTGGC

TTCAGCAAAGATAACAATAGTATCATAACTAGAAAAGAATTTCAGGAAGG

TTTAAAAATATTTTTTCCAGGAGTGAGTGAGTTTGGAAAGGAATCCATCC

TTTTTCATTACACAGACTGGGTAGATGATCAGAGACCTGAAAACTACCGT

GAGGCCTTGGGTGATGTTGTTGGGGATTATAATTTCATATGCCCTGCCTT

GGAGTTCACCAAGAAGTTCTCAGAATGGGGAAATAATGCCTTTTTCTACT

ATTTTGAACACCGATCCTCCAAACTTCCGTGGCCAGAATGGATGGGAGTG

ATGCATGGCTATGAAATTGAATTTGTCTTTGGTTTACCTCTGGAAAGAAG

AGATAATTACACAAAAGCCGAGGAAATTTTGAGTAGATCCATAGTGAAAC

GGTGGGCAAATTTTGCAAAATATGGGAATCCAAATGAGACTCAGAACAAT

GAATACAGAGTCAACAAGAATAATGACGAAACTACGTGCTCAACAATGTC

GATTCTGGACATCATTTTTTCCAAAAGTCTTGGAAATGACAGGAAATATT

GATGAAGCAGAATGGGAGTGGAAAGCAGGATTCCATCGCTGGAACAATTA

CATGATGGACTGGAAAAATCAATTTAACGATTACACTAGCAAGAAAGAAA

GTTGTGTGGGTCTCTAATTAATAGATTTACCCTTTATAGAACATAT—TT

TCCTTTAGATCAAGGCAAAAATATCAGGAGCTTTTTTACACACCTACTAA

AAAAGTTATTATGTAGCTGAAACAAAAATGCCAGAAGGATAATATTGATT

CCTCACATCTTT-AACTTAGTATTTTACCTAGCATTTCAAAACCCAAATG

GCTAGAACGTGTTTAATTAAATTTCACAATATAAAGTTCTACAGTTAATT

Hereinafter table 1 has shown between different plant species interested RhBchE residue has been compared.

The interested RhBchE residue of table 1. with from the enzyme of other species relatively.

The residue different among the RhBchE with HuBchE	L-17 (F), V5 (I), L21 (F), Y66 (C), M110 (L), P215 (S), 227 (F), T245 (N), D255 (E), V261 (I), H270 (Q), L285 (P), M294 (V), E301 (D), D342 (N), N348 (K), D390 (G), 1398 (F), V454 (D), G482 (E), H484 (Q), K489 (S), S508 (T), L511 (M), and 1S51 (N)
		The residue different but conservative among the RhBchE with HuBchE	L-17 (F), V5 (I), L21 (F), Y66 (C), M110 (L), T245 (N), D255 (E), V261 (I), H270 (Q), L285 (P), M294 (V), E301 (D), 1398 (F), H484 (Q), S508 (T), L511 (M), and S551 (N)
The residue of not having conservative with HuBchE among the RhBchE but having conservative with the BchE of other animal	P215(S)，S227(F)，D342(N)，D390(G)，V454(D), G482(E)，K489(S
		The residue that does not have conservative with any other species among the RhBchE	N348(K)
Conservative but do not have conservative between RhBchE and the HuBchE with other species	A7(T),T19(P)，G212(R/Q),E308(Q)，L511(Y)， S524(L)

Compare with the sequence of HuBchE, RhBchE has comprised 25 residues with different aminoacids.In these 25 different residues, 17 aminoacid are conservative similar aminoacid.Compare with the BchE enzyme amino acid sequence from different animals species (comprising people, rabbit, cat, horse and mice), RhBchE and HuBchE have maximum similarity.Between HuBchE and RhBchE, have six conservative but do not have the residue of conservative each other with other species.Yet 7 (P215, S227, D342, D390, V454, G482 and K489) in 8 non-conserved amino acids that RhBchE compares with HuBchE in fact have conservative with other animal ferment.The unique specific amino acid residue of RhBchE is N348, and is Lys on this position from the enzyme of all other species.

The expression of embodiment 7. functional reorganization RhBchE.

In the Chinese hamster ovary celI of transient transfection and stable transfection, detect the expression of reorganization RhBchE.Monitor the activity of the reorganization BchE enzyme of expression by the BchI hydrolysis in the Ellman response analysis culture medium.The proteic expression of BchE also can be confirmed by the western engram analysis, shows that reorganization RhBchE moves (Fig. 1) jointly by anti--HuBchE polyclonal antibody identification and with reorganization HuBchE.

From serum of macaque purification RhBchE.Behind three step chromatographies, be purified to about 70% purity (Fig. 2 A) according to SDS-PAGE and the visible RhBchE of Coomassie blue stain.Show that with anti--western engram analysis that the HuBchE polyclonal antibody carries out this antibody discerns RhBchE very effectively, and the HuBchE of immunoreation band and purification moves (Fig. 2 B) jointly.Can carry out polyethyleneglycol modified or other is derived to improve the stability of the arbitrary protein of identifying among the present invention described herein to BchE.

The substrate specificity of RhBchE.Adopt the Ellman method that the enzyme component of embodiment 1 described purification is carried out BchI hydrolysis determination and analysis (table 2).Table 2 has shown the RhBchE of purification and HuBchE at the 50mM potassium phosphate, among the pH7.4 under 30 ℃ to the kinetic constant of Butyryl thiocholine and (-)-cocaine.

The RhBchE of table 2. purification and the kinetic constant of HuBchE.

As show shown in the 2-4, RhBchE has shown the notable difference with HuBchE on substrate specificity.Although compare with HuBchE, the binding affinity of RhBchE and BchI hangs down 2.7 times of (K _m71.1 μ M), but the high 2.9 times of (K of affinity of RhBchE and (-)-cocaine _i=4.7 μ M) (HuBchE, K _i=13.6 μ M).

Substrate specificity to RhBchE detects, and with the cocaine hydrolysis product as activity inhibitor, measured the K of the different chemical compounds relevant with cocaine chemistry _iValue, as shown in table 3 (at the 50mM potassium phosphate, among the pH7.4,25 ℃ of BchI hydrolysis inhibition down).

The K that table 3. cocaine and related compound suppress the BchI hydrolysis _iValue.

	Monkey BchE	People BchE
			(-)-cocaine	4.7μM	13.6μM
(+)-cocaine	15.3μM	8.0μM
			(-)-nor-cocaine	119μM	43μM

As shown in table 3, (+) of RhBchE-cocaine and (-)-nor-cocaine Ki are higher about 2 times than HuBchE, and RhBchE (-)-cocaine K _iThan low about 3 times of HuBchE, prompting RhBchE has more structure selectivity to (-)-cocaine than other substrate.(-)-ecgonine methyl ester does not suppress the BchI hydrolysis under the concentration of being tested (0-100 μ M).

Table 4 has shown RhBchE and HuBchE kinetic constant (people such as Xie, 1999 to (-) cocaine; People such as Mets, 1998), and shown that RhBchE compares cocaine hydrolysis catalyst and the HuBchE mutant reported and has higher k _Cat/ K _m

Table 4.RhBchE and HuBchE are to the kinetic constant of (-) cocaine.

Natural B chE	K cat(min -1)	K m(μM)	K cat/K m (μM -1min -1)
				Monkey people hBchE 1 A328Y hBchE 2 A328F hBchE 1 15A 10mAb 2	22.3 2.4 3.9 10.2 5.8 2.3	6.71 10.6 14 9 24 220	3.33 0.23 0.28 1.13 0.24 0.01

Cocaine hydrolysis catalysic coefficient (the k of RhBchE _Cat/ K _m) be more than 10 times of human BchE.When comparing the interaction of RhBchE and HuBchE and 0P, ETP is that one-level suppresses (Fig. 3) to the inhibition of RhBchE and HuBchE.By to k _AppAgain the mapping of first order rate constant (inhibition) and ETP concentration can obtain the inhibition constant of the reaction of RhBchE and HuBchE and ETP.Table 5 has shown sensitivity differences between RhBchE and the HuBchE, and (the inhibition speed constant to ETP can carried out k with ETP concentration shown in Figure 3 _App(min ^-1) double-reciprocal plot after obtain).

The inhibition speed constant of table 5. couple ETP.

	HuBchE	RhBchE
			K 1×10 3(M -1min -1)	1860±135	764.4±40.5

The synthetic inhibition that reaches HuBchE of embodiment 8. OP analog.The 0P analog of simulating the structure of sarin, soman and VX respectively is successfully synthesized as described in embodiment 12 and 13.Interaction to described 0P analog and HuBchE and G117H/E197Q HuBchE detects.The results are shown in table 6 (chemical compound shown in HuBchE and G117H/E197Q HuBchE and 0.5 mM or contrast buffer were hatched 48 hours) under 4 ℃.

The OP of table 6. couple WT and G117H/E197Q HuBchE suppresses.

	G117H/E197Q	WT
			Chemical compound
1	74.30％	1.23％
			Chemical compound
2	74.73％	1.93％
			Chemical compound
3	55.05％	0.06％
			Echothiophate	12.77％	0.02％
Buffer	100.00％	100.00％

As shown in table 6, WT HuBchE is suppressed by all three kinds of chemical compounds, and its residual activity is less than 2%.Residual enzymatic activity uses 1mM BchI with the Ellman reaction detection.Yet G117H//E197Q HuBchE variant has still kept all three kinds of chemical compounds and has surpassed 50% activity.This finds very consistent with the report that in early days G117H/E197Q enzyme variants tolerance OP is suppressed, show the interaction of synthetic three kinds of OP analog and HuBchE enzyme be similar to other OP chemical compound of inspection before.In addition, when we checked the BchI hydrolysis in the longer time period, the WT residual activity after hatching with chemical compound 1 and 2 was comparatively obvious, but did not then observe the activity that can survey when hatching with chemical compound 3 or echothiophate.The G117H/E197Q HuBchE of Ti Xianing has shown the key character that can be used for the functional screening design to the tolerance of OP chemical compound inhibition herein.In principle, this method (replacing oxide ester with sulfur) can be used for any ester, thus exploitation HT functional screening.Other example can be referring to embodiment 13.

Model OP chemical compound suppresses the inhibition speed constant of HuBchE and measures.The present invention has studied the kinetics of model OP chemical compound to the time dependence inhibition of the HuBchE of purification, has wherein adopted seven inhibitor concentration, and each inhibitor concentration has been got five time points.All OP model compounds are one-level inhibition (Fig. 4 A has shown chemical compound 1,2,3 and the selected inhibitor concentration of echothiophate) to the inhibition of HuBchE.With k _App(one-level inhibition speed constant) maps again to the OP compound concentration can obtain the inhibition constant (table 7) of tested OP chemical compound and HuBchE reaction.The OP chemical compound can pass through with k the inhibition constant of HuBchE _App(min ^-1) OP concentration shown in Figure 4 is carried out double-reciprocal plot obtain.Fig. 4 B has shown the representative graph of chemical compound 1.

Table 7.OP chemical compound is to the inhibition constant of HuBchE.

Stability and the spontaneous hydrolysis of chemical compound in culture medium.Because the 0P model compound is used to the detection method based on cell, and because the low conversion ratio of expection and long hatching the cycle, these chemical compounds stability in the presence of the culture medium under condition determination is just extremely important.The spontaneous hydrolysis that does not rely on enzyme of chemical compound (that is, buffer mediation, H ₂0 hydrolysis mediation and culture medium mediation) monitors under 412nm by UV-Vis after hatching chemical compound in can mensuration buffer by the no enzyme in the presence of DTNB.All three kinds of chemical compounds all do not show detectable hydrolysis, show that these chemical compounds are being used for not recording spontaneous hydrolysis (or extremely limited background hydrolysis) under the condition determination of functional screening.The stability of chemical compound also can be passed through at H ₂Carrying out testing before the long term store and afterwards the ability that suppresses HuBchE in 0 under room temperature checks.The result shows that chemical compound 1 and 2 is comparatively stable under storage requirement: suppress the active ability of HuBchE and a spot of decline only occurred.And chemical compound 3 quite stable under this condition.

Embodiment 9. creates the RhBchE/HuBchE chimera by gene vitro recombination (gene shume).We have shown that RhBchE has the cocaine hydrolysis activity bigger 10 times than HuBchE.23 amino acid whose differences are arranged between RhBchE and HuBchE.Table 8 has hereinafter shown the different aminoacids residue of selecting at random to be cloned between HuBchE and the RhBchE from eight.

Different aminoacids residue between table 8.HuBchE and the RhBchE.

aa#	huBchE	RhBchE	Clone	1	Clone 2	Clone 3	Clone 4	Clone 5	Clone 6	Clone 7	Clone 8
												47	V	V	V	V	G	V	V	V	V	V
48	F	L	F	F	F	F	F	F	F	F
											54	T	T	A	T	T	T	T	T	I	T
90	A	A	A	T	A	A	A	A	A	A
											94	C	Y	Y	Y	Y	Y	C	C	C	Y
138	L	M	M	M	M	L	M	M	L	M
											155	V	X	V	V	V	V	V	V	V	V
186	G	G	R	G	G	G	G	G	G	G

243	S	P	P	S	P	S	S	S	P	P
											255	F	S	F	F	F	S	F	F	S	S
273	N	T	N	N	N	T	N	T	T	T
											283	E	D	E	D	D	D	E	E	D	E
285	E	E	E	G	G	G	E	E	G	E
											289	I	V	I	V	V	I	I	I	V	I
298	Q	Q	Q	H	H	Q	Q	Q	Q	Q
											313	P	L	P	L	L	P	L	L	P	P
322	V	M	V	V	V	V	M	V	V	V
											329	D	E	E	D	D	E	E	D	D	D
370	N	D	D	D	N	N	D	N	D	D
											376	K	N	N	N	N	K	K	N	N	N
402	T	T	T	T	T	I	T	T	T	T
											405	V	V	I	V	I	I	V	V	V	I
418	G	D	D	D	D	G	G	G	D	D
											422	G	G	G	G	G	G	G	R	G	G
426	F	I	I	F	I	F	F	F	F	I
											480	R	R	G	R	G	G	R	G	G	R
482	D	V	V	D	V	V	D	V	D	D
											486	E	E	G	E	E	G	E	G	E	E
494	S	S	S	S	P	S	S	S	S	S
											495	I	I	T	T	I	T	T	T	I	T
510	E	G	G	E	E	G	G	G	E	E
											512	Q	H	H	Q	Q	Q	H	H	Q	Q
517	S	K	K	S	S	S	K	K	S	S
											523	S	N	N	S	S	S	S	S	S	S
525	E	E	E	K	E	E	E	E	E	E
											536	T	S	S	T	T	T	S	S	T	T
539	M	L	L	M	M	M	L	L	L	M
											564	I	I	I	I	I	I	V	I	I	I
575	F	F	F	F	F	F	F	S	F	F
											577	R	R	R	R	H	R	R	R	R	R
579	N	S	S	S	S	S	S	N	N	S

Believe that part (if not whole words) these variants help to improve cocaine or other ester hydrolysing activity of RhBchE.In order to determine the best of breed of selected useful variant, made up the chimera storehouse by HuBchE and RhBchE.Use above-mentioned DNA extracorporeal recombination.Plasmid DNA is prepared by eight clones that select at random.To checking order from these eight clones' BchE coding region and analyzing.Fig. 5 has shown eight reorganization chimeras from HuBchE and RhBchE.All clones' 5 ' district coding HuBchE signal peptide.Multiple recombination event (average about 12 exchanges (crossover) of each clone) occurs in eight different loci of selecting the clone at random.Show that these eight clones have represented the height variation chimera storehouse of HuBchE and RhBchE recombinant.This variation storehouse is the basis of function selecting.Regrouping process has also been introduced low-level point mutation.Outside these eight clones that checked order, 12 new amino acid mutation bodies have also been identified.In these sequences, do not find to move frame sudden change or premature termination.This low mutation rate is very desirable, can not introduce the detrimental mutation that can form no functional enzyme too much simultaneously because it has improved the multiformity in this storehouse.

The HuBchE of embodiment 10.AD-mediation expresses.The AD system is the virus expression systems that another kind is widely used in the expressing human source protein.AD can infect multiple mammalian cell, and allows recombiant protein to express in multiple division and non-division mammal cell line.Culture medium to the cell of the different titers recombinant virus infection of using by oneself has been carried out the BchE activity analysis, and the HuBchE behind the active COS of demonstration of the BchE in the culture medium cell infection expresses has dose dependent and time dependence.Similarly the result can be obtained (data not shown) by Chinese hamster ovary celI.As shown in Figure 6, the BchE activity that accumulates in culture medium is Exponential growth in time.The AD construct has replication defective; Other virion can not be generated when the COS cell is infected.Therefore, the cell that the continuous raising prompting of enzymatic activity is infected in the culture medium generates the BchE enzyme continuously in a couple of days.When using microscopy, back the 4th and the 5th talent is comparatively obvious until infecting to infect the CPE (cell is concentrated) that causes by AD.Fig. 6 B has shown the western engram analysis of culture medium, and reflection HuBchE exists with time dependence and dose dependent mode, and has good concordance with the result shown in Fig. 6 A.

The structure of the adenovirus expression carrier of WT and G117H/E197Q HuBchE.ViralPower AD expression system is used to clone the HuBchE enzyme.Clone and recombination efficiency to the building process that is used to make up the storehouse are determined.By adopting 300ng pAD/CMV/V5/DEST plasmid and use transformation efficiency in recombining reaction is 1 * 10 ⁶The chemoreception attitude cell of cfu/ μ g, we have obtained about 50 recombinant clones in each recombining reaction.12 clones that select are at random carried out restrictive diges-tion, disclose and wherein 75% carried correct HuBchE insert.This shows for the clonal mutation storehouse, adopts the DNA of 10 μ g to carry out reconstitution steps, and more effective electricity changes competent cell (1 * 10 ¹⁰Cfu/ μ g) can generate 1 * 10 ⁶Recombinant clone.This has confirmed to use the AD construct to generate the feasibility in the large-scale sudden change storehouse of the functional screening that can effectively carry out success.

The reorganization WT of the generation of HuBchE-AD and affirmation AD expression system and the expression of G117H/E197QHuBchE.As described in embodiment 4, generated reorganization WT-and G117H/E197Q-hBchE-AD, and collected.Fig. 7 has shown that pAD-WT-hBchE and pAD-G117H/E197Q-hBchE transfection cause the active lifting of HuBchE, and the transfection of control vector pAD-lacZ only shows consistent background activity.The structure of this prompting pAD-WT-hBchE and pAD-G117H/E197Q-hBchE is successful.

Checking to the functional screening technology of efficient OP antidote.Other adopts the detection method of enzymatic determination in culture medium to have significant advantage relatively based on the insoluble enzyme detection method of cell in exploitation, and the former has the ability of handling relative large-scale sudden change storehouse with still less manpower and cost.The CHO-K1 cell of stably express HuBchE WT or G117H/E197Q HuBchE at first is used to determine testing conditions.The cell of the pre-inoculation of washing also covers with the colourless MEM that contains 1% agar.After agar solidifies, cell put back to incubator (spending the night) so that BchE enzyme secretion and being diffused in the agar.According to the observation of microscopically, cell can keep 10 days health status at least under such incubation conditions.The Ellman reactant mixture that contains 1mM BchI and 0.1mMDTNB can prepare in containing the colourless MEM of 1% agar.Reactant mixture is covered on the cell culture flat board of above-mentioned preparation, and at room temperature hatch.In the Ellman reaction, form the yellow that vision can be surveyed, and after hatching 10 minutes, scan this flat board.This flat board also can be hatched the back at 40 minutes and be detected OD405 absorption (data not shown) with flat bed reader (Victor 2, Perkin Elmer).The HuBchE that this experiment discloses cellular expression can diffuse into agar, and the yellow product that the meeting accumulation visual can be surveyed when the Ellamn reaction reagent is provided.

Agar and agarose are as the comparison of solid-phase media.In said determination, although we can be easily by the hydrolysis of the yellow BchE of detection occurring, we find that this yellow product is unstable always, and can disappear in hatching (a few hours are to spending the night) for a long time.Because we are to part ester substrate such as the longer incubation time of OP chemical compound expection, we need solve this stability problem.As shown in Figure 8, the formation of product is not applied in pro-30-60 minute in two-layer agar or agarose influence, and prompting HuBchE generates and diffuse into solid phase with similar method.Yet longer hatching in the cycle, the existence of agar can cause decolouring and absorb reducing in the solid phase.Only when all applying cell with agarose in two-layer, product just can be stablized (Fig. 8 B) in hatching for a long time.

The active local detection of HuBchE.One of advantage of exploitation solid phase screening active ingredients is its ability at little regional area evaluation functional activity.Make and to screen large-scale storehouse easily in cost-benefit mode.We adopt serial dilution stably express WT HuBchE the CHO-K1 cell detection the current detection method of having developed whether have this advantage.As shown in Figure 9, in substrate added several minutes, has tangible macroscopic macula lutea at the correspondence position of HuBchE express cell.For having more many cells clones' (about 100/ flat board) flat board, these specklees mix, and can't distinguish (data not shown).For the flat board of about 20 clone/flat boards, these macula luteas can easily be distinguished (Fig. 9).The ability prompting that about 20 reactive speckles are identified in difference on the 10cm culture dish just can be placed on large-scale storehouse on the culture plate as long as detected positive colony is less than 20 in functional screening.Positive colony can be after serial dilution purification easily.

Embodiment 11. is used to detect the AD-BchE recombinant virus with solid phase BchE activity test method.We attempt detecting the AD-recombinant virus by solid phase BchE activity test method.The use of AD system is usually directed to titration process slowly, because the formation of speckle is comparatively slow.The BchE activity test method can be developed the alternative method that is used for estimating virus titer in shorter time.The HuBchE-AD virus of serial dilution can be used for infecting the 293A cell that is seeded in advance on 6 orifice plates.Infect and agarose-MEM mixture covered on the cell in back 1 hour.Dye to the BchE activity on the flat board next day in primary infection.Although the cell that higher titre infects was dyed Huang on the 2nd day after infection, infect in the back 4 days low titre of infection and to have occurred similar remarkable macula lutea in the micropore with Fig. 9.Infected back 4 days, and formed macula lutea in 30 minutes of use BchI and DTNB.The speckle that forms is carried out labelling.In the micropore that has formed a plurality of speckles, this yellow merges very soon.Carrying out speckle in the micropore with 4 or 4 following speckles differentiates more or less freely.Adopting the detection method of exploitation herein to replace traditional plaque detection method carries out virus titer and determined can be each titration process saving general 7 days.

From the macula lutea isolated viral of identifying, and dig out filler from the agarose culture plate.Fig. 9 has shown the diagram of isolating concrete filler in experiment.Obtain positive filler by infecting through too high virus titer and showing behind the BchE vital staining that xanchromatic micropore is separable.Obtain negative filler by infecting through the highest viral dilution thing and not showing behind the BchE vital staining that xanchromatic micropore is separable.The micropore that the sample filler can form remarkable macula lutea from the back of dyeing separates, and speckle is dug out becomes the sample filler.The micropore that the background filler can form remarkable macula lutea from the back of dyeing separates, and the zone of being unstained of contiguous speckle is dug out becomes the background filler.Isolating filler is transferred in the pipe that contains the 0.5ml culture medium, and 4 ℃ of following overnight incubation.To dilute 10 times from the culture medium of positive filler and sample filler.Use the culture medium (N1 to 3, P1 to 3, S1 to 3 and B1 to 3) of hatching and diluted medium (P ' 1 to 3 and S ' 1 to 3) infection to be seeded in the 293A cell in 24 orifice plates in advance then with filler.BchE activity in the culture medium is 24 and 48 hours mensuration after infection, and table 9 has shown average activity.

The BchE activity of each filler in table 9. culture medium.

Hr	N1-3	B1-3
			24.00	023±0.04	0.36±0.07
48.00	0.26±0.03	2.06±0.51
			72.00	0.35±0.05	6.47±1.91

Hr	P1-3	P'1-3
			24.00	1.36±0.63	0.62±0.31
48.00	4.14±0.91	3.13±1.17
			72.00	6.52±0.75	5.40±0.34

Hr	S1-3	S'1-3
			24.00	0.93±0.09	0.32±0.10
48.00	4.24±0.30	2.34±0.24
			72.00	6.36±1.29	6.11±0.90

Negative filler does not cause BchE to express (N1-3).Positive and sample filler can improve BchE activity (S1-3 and P1-3).What is interesting is, from causing lower activity level (B1-3,48 hours) near the isolating background filler of sample filler.This activity level is lower than 10 times of diluents that obtain from sample or active filler, and (B1-3, S ' 1-3 and P ' are 1-3).This result discloses 1) we can dye xanchromatic agarose filler by separation and separate HuBchE-AD; 2) come the virus of self filler to diffuse near zone and may cause near filler (＜0.5cm) less than 10% pollution.Further the separation scab will significantly improve the viral purity in each filler.The important references point that these results have designed as II phase functional screening.

The checking of functional screening detection method.In order to verify that solid phase functional screening and test use this solid phase detection method to identify the feasibility of OP catalyst from the sudden change storehouse, AD-G117/E197Q recombinant virus (10pfu) and not commensurability wild type HuBchE recombinant virus (0,10,100 and 500pfu) are mixed.With blended recombinant virus infection 293A cell.Infected back 24 hours, and covered cell with the MEM that contains 1% agarose.Infected back 48 hours, and added 0.4mM chemical compound 1, interact with reorganization HuBchE that expresses and mutant to the cell that applies.At metainfective the 4th day, as mentioned above with BchI as substrate in the presence of DTNB to applying cell dyeing.In the flat board that the AD-G117H/E197Q HuBchE virus (being mixed with the AD-WT HuBchE virus that quantity increases) with equal number infects, obtain the macula lutea of similar quantity.From flat board, dig out yellow agarose speckle, and in independent pipe, hatch with serum-free medium.The recombinant virus that discharges in the pcr analysis culture medium that the AD Auele Specific Primer that use and gene insert intersect carries out.The PCR product is checked order.Sequence analysis confirm 3 flat boards of pro-(have 10pfu G117H/E197Q and 0,10 and the WT HuBchE of 100pfu) carry clean G117H/E197Q sequence in the isolating filler.For the flat board that has 10pfuG117H/E197Q mixing 500pfu WTHuBchE, can be observed the mixed sequence of G117H/E197Q and WT.These experimental results show that the expression system by AD, and the solid phase detection method can be separated the HuBchE variant to chemical compound 1 tolerance from the recombinant virus background of OP sensitivity.When background level recombinant virus level is higher, the filler purification step will improve separation efficiency.

Optimization and checking with the functional screening system in a saturation mutation storehouse.Shown that herein the functional screening system is used for the feasibility of molecular evolution method.In the high flux functional screening, this system is improved, and verified its sensitivity, disposal ability and repeatability fully.In order to realize this point, carried out the some saturation mutation at two amino acid position G117 and E197.Adopted the above some saturation mutation technology of summary.Final total length PCR product is checked order and verified the sudden change codon of integrating.Plasmid DNA is prepared by seven pAD-huBuChE clones that select at random.To checking order from these seven clones' BchE coding region and analyzing.Sequencing result discloses these seven clones and has represented at amino acid position 117 and 197 and have highly multifarious storehouse.This multiformity storehouse is as the basis of function selecting.

Functional screening.According to above result displayed, we have designed the system that uses above-mentioned exploitation carries out functional screening to OP catalytic enzyme workflow diagram (scheme 12).This functional screening relates to solid phase screening, liquid phase screening, speckle purification, active affirmation, gene amplification and order-checking.The workflow of this design can identify to have the required active BchE variant of organic or inorganic ester hydrolyzation catalysis.Similarly method can be used for separating cocaine catalytic enzyme.

To sum up, we have cloned new BchE enzyme from macaque, and substrate specificity and inhibition kinetics are identified.We have synthesized three kinds of OP similar compounds that can be used for OP catalytic enzyme functional screening.We have made up the sudden change storehouse, and have verified the AD high level expression system that is used for the BchE functional screening.And develop simultaneously and verified based on the active functional screening of solid phase.The abundant ability that technology that the present invention sets up and research tool provide molecular evolution is separated toxenzyme with the catalytic that HuBchE continues to be improved as at never poison, cocaine or other potential harmful organic or inorganic ester.

The chemosynthesis of embodiment 12. New O P analog.Designed the OP analog of analog neuron toxic agent VX, soman and sarin respectively.

The structure of the never poison that scheme 1. (A) is commonly used and (B) chemosynthesis of the model organophosphorus compounds of the corresponding never poison of simulation.

Shown in scheme 1, general step comprises that (±)-ephedrine and dichloromethyl sulfo- phosphine reaction form 2,3,4-trimethyl-5-phenyl-1,3,2-oxazole phosphine-2-thioketone.According to order 2,3,4-trimethyl-5-phenyl-1,3,2-oxazole phosphine-2-thioketone and alcohol reaction carry out hydrogenolysis then.With the alkyl hydrogen methyl Thiophosphonate of iodomethane alkylation gained so that required O-alkyl S-methyl phosphonothiolic acid ester compounds to be provided.The chemical synthesis process of this target compound comprises the evaluation of all intermediate, and shows below.

2,3,4-trimethyl-5-phenyl-1,3, synthesizing of 2-oxazole phosphine-2-thioketone: 2,3,4-trimethyl-5-phenyl-1,3, the racemic mixture of 2-oxazole phosphine-2-thioketone can be with reference to Cooper, Deng people (J.Chem.Soc.Perkin Trans.1977,17, step preparation (scheme 2) 1969-80).

Scheme 2.2,3,4-trimethyl-5-phenyl-1,3,2-oxazole phosphine-2-thioketone synthetic.

(8.0g, toluene solution 53.7mmol) (25mL) slowly are added into (±)-ephedrine, and (13.0g, 64.4mmol) (27.2g is 268mmol) and in the agitating solution in the toluene (210mL) at triethylamine with dichloromethyl sulfo-phosphine.After interpolation is finished reactant mixture dark place under room temperature and argon was stirred 24 hours, filter, wash with water, dry (MgSO ₄), and vacuum concentration obtains light yellow oil.(hexane/ethyl acetate, 3:1 v/v) obtain buttery non-corresponding mixture of isomers (5.39g, 22.4mmol, 42%) to column chromatography on silicon dioxide.TLC (hexane/ethyl acetate, 3:1, v/v) RF=0.26; 1H NMR (CDCl3) 7.24-7.37 (m, 5H), 5.64 (dd, JHP=3.0Hz, JHH=6.0Hz, 0.5H), 5.47 (dd, JHP=2.1Hz, JHH=5.7Hz, 0.5H), 3.61 (m, 1H), 2.75 (d, JHP=12.3Hz, 1.5H), 2.66 (d, JHP=12.0Hz, 1.5H) 2.04 (d, JHP=14.4Hz, 1.5H), 1.93 (d, JHP=14.0Hz, 1.5H), 0.80 (d, JHP=6.6Hz, 1.5H), 0.73 (d, JHP=6.6Hz, 1.5H); MS (ESI) [M+H]+m/z is to C ₁₁H ₁₈NOPS calculates 242, records 242.

Scheme 3. is used for the synthetic of the synthetic midbody compound 5 of isobutyl group hydrogen methyl Thiophosphonate.

Shown in scheme 3, to be contained in isobutyl alcohol (3mL) and dry methylene ethyl ketone (MEK, dried HCl saturated solution 3mL) slowly is added into 2,3,4-trimethyl-5-phenyl-1,3, (1.05g is 4.36mmol) in freezing, the agitating solution in isobutyl alcohol (7mL) and MEK (7mL) for 2-oxazole phosphine-2-thioketone.Reactant mixture was in the dark stirred 1.5 hours under the room temperature, inject 10% ice-cold Na then ₂CO ₃Aqueous solution (25mL).This aqueous mixture can not purifiedly be directly used in following hydrogenation.

Synthesizing of isobutyl group hydrogen methyl Thiophosphonate.

Synthesizing of scheme 4. isobutyl group hydrogen methyl Thiophosphonates.

Water (50mL) and ethanol (75mL) dilution are from previous reaction and comprise 5 crude mixture.Add Pd/C (160mg) to this solution, will fill H then ₂(g) balloon this round-bottomed flask of packing into.(4:1, v/v) the extraction back merges organic layer, dry (MgSO through processing and with chloroform/isopropyl alcohol ₄), filter, and the concentrated crude product oil (390mg, 53%) that provides.1H NMR(CDCl3)3.79(m，2H)，1.91(m，1H)，1.82(d，JHP＝15.6Hz，3H)0.90(d，J＝6.9Hz，6H).

Synthesizing of O-isobutyl group S-methyl Thiophosphonate.

Synthesizing of scheme 5.O-isobutyl group S-methyl Thiophosphonate.

Shown in scheme 5, to 6 (302mg, 1.79mmol) 10%Na that dilute at usefulness ethanol (25mL) ₂CO ₃(aq) add in the solution that forms in (2.5mL) iodomethane (2.54g, 17.9mmol).After 24 hours, handle this reactant and wash organic substance with water, dry (MgSO ₄), vacuum concentration is to provide light brown oil.Through ball ball distilling under reduced pressure (bulb-to-bulb distillation) is obtained transparent buttery pure products (100mg, 0.55mmol, 31%) with the Kugelrohr device.1H NMR(CDCl3)3.75(m，2H)，2.21(d，JHP＝12.9Hz，3H)，1.88(m，1H)，1.71(d，JHP＝15.6Hz，3H)。

Synthesizing of midbody compound 9.

Synthesizing of scheme 6. midbody compounds 9.

Shown in scheme 6, neopentyl alcohol (3.0mL) and the dry methylene ethyl ketone (MEK of HCl will be done, 3mL) saturated solution slowly is added into 2,3,4-trimethyl-5-phenyl-1,3,2-oxazole phosphine-2-thioketone (1.05g, 4.36mmol) (7.0g is 79.4mmol) and in freezing, the agitating solution that forms among the MEK (7mL) at neopentyl alcohol.Reactant mixture was in the dark stirred 1.5 hours under the room temperature, inject 10% ice-cold Na then ₂CO ₃In the aqueous solution (25mL).This aqueous mixture can not purifiedly be directly used in hydrogenation as described below.

Synthesizing of neopentyl hydrogen methyl Thiophosphonate.

Synthesizing of scheme 7. neopentyl hydrogen methyl Thiophosphonates.

Crude mixture with 9 is with water (50mL) and ethanol (75mL) dilution.Add Pd/C (181mg) to this solution, will fill H then ₂(g) balloon this round-bottomed flask of packing into.After the processing, merge organic substance, dry (MgSO4) filters, and the concentrated crude product oil (700mg, 3.84mmol, 91%) that provides.1HNMR(CDCl3)3.6(m，2H)，1.74(d，JHP＝15.6Hz，3H)，0.89(s，9H)。

Synthesizing of O-neopentyl S-methyl Thiophosphonate.

Synthesizing of scheme 8.O-neopentyl S-methyl Thiophosphonate.

(700mg is 3.84mmol) at 10% Na by ethanol (55mL) dilution to 10 ₂CO ₃(aq) add in the solution that forms in (5.5mL) iodomethane (5.45g, 38.4mmol).After 24 hours, distilling under reduced pressure obtains transparent buttery pure products (55mg, 0.28mmol, 7%) to ball through ball with the Kugelrohr device.1HNMR(CDCl3)3.71(m，2H)，2.29(d，JHP＝14.4Hz，3H)，1.79(d，JHP＝15.6Hz，3H)，0.95(s，9H)。

Synthesizing of midbody compound 13.

Synthesizing of scheme 9. midbody compounds 13.

(3.74g 20.6mmol) is added into 2,3 with 2-diisopropylaminoethyl-ethylate hydrochlorate, 4-trimethyl-5-phenyl-1,3, (1.05g is in freezing, the agitating solution that 4.36mmol) forms in 2-diisopropylaminoethyl-ethanol (7.0mL) and MEK (10mL) for 2-oxazole phosphine-2-thioketone.Reactant mixture was in the dark stirred 1.5 hours under the room temperature, inject 10% ice-cold Na then ₂CO ₃Aqueous solution (25mL).This aqueous mixture can not purifiedly be directly used in hydrogenation as described below.

Synthesizing of 2-diisopropylaminoethyl-ethyl hydrogen methyl Thiophosphonate.

Synthesizing of scheme 10.2-diisopropylaminoethyl-ethyl hydrogen methyl Thiophosphonate.

Crude mixture with 13 is with water (50mL) and ethanol (75mL) dilution.Add Pd/C (172mg) to this solution, will fill H then ₂(g) balloon this round-bottomed flask (scheme 10) of packing into.With after this flask emptying with hydrogen purification and stirred 18 hours.After the processing, merge organic substance, dry (MgSO ₄), filter, and the concentrated crude product oil (505mg, 2.11mmol, 91%) that provides.

Synthesizing of O-2-diisopropylaminoethyl-ethyl S-methyl Thiophosphonate.

Synthesizing of scheme 11.O-2-diisopropylaminoethyl-ethyl S-methyl Thiophosphonate.

(505mg is 2.11mmol) at the 10%Na by ethanol (30mL) dilution to crude product 14 ₂CO ₃(aq) (5g, 21.1mmol) and at room temperature stirred 24 hours the dark place to add iodomethane in the solution that forms in (3.0mL).After the processing, obtain light brown oil by organic layer.Through ball ball distilling under reduced pressure (bulb-to-bulb distillation) is obtained transparent buttery pure products (13mg, 0.05mmol, 2%) with the Kugelrohr device.1H NMR(CDCB)4.79(m，2H)，3.99-4.21(m，4H)，2.30(d，JHP＝13.2Hz，3H)，1.75(d，JHP＝15.6Hz，3H)，1.36(d，J＝6.0Hz 6H)，1.31(d，J＝6.3Hz，6H)。

Embodiment 13.

The chemosynthesis of used never poison analog in the biologic array.Below described can be used as obtain biomarker, antibody and in the molecular evolution screening enantio-selectivity of used OP as the never poison analog synthetic.But these chemical compounds are designed to provide and the actual littler analog of the similar phosphorylation ChE toxicity of never poison OP.Therefore, using of these analog can obtain identical enzyme adduct.

Scheme 12.ChE is to the hydrolysis of never poison analog

Because target OP chemical warfare chemical compound has high toxicity, design reduces its toxicity to the modification of this never poison analog when keeping structural similarity, thereby solves practical problems such as extensive synthetic, processing and biological test.Our chemosynthesis required target compound (that is, 4-13 and raceme equivalent 1-3), and it is detected as highly purified people's BuCh esterase (hBuChE) inhibitor.Result verification our method, and shown that the toxicity potentiality of target compound significantly are lower than the reagent more directly related with never poison.This is for breadboard practical term or all very useful to the final acquisition of biological specimen, because the littler material of these toxicity will provide identical phosphorylase in external or body.

It is synthetic that the OP analog is used to hapten.The present invention concentrates the synthetic of the OP analog paid close attention to the enantiomer-pure of simulating five kinds of never poisons.

Scheme 13. hapten synthesize required OP and mimic never poison thereof

(±) (±) (±)

1 2 3

Analog sarin soman vx

(Sp) (Rp) (Sp) (Rp) (Sp) (Rp)

4 5 6 7 8 9

Analog sarin sarin GF GF soman soman

(Rp) (Sp) (Rp) for analog (Sp)

10 11 12 13

Vx vx tabun tabun

We can synthesize and purification (〉 97%) all corresponding isomers of 4-13.Scheme 14 and 15 has shown synthesizing of corresponding isomery pure compound 4-11.

Scheme 14. is used the synthetic Rp organic phosphoric acid ester isomer of (-)-ephedrine

Scheme 15. is used the synthetic Sp organic phosphoric acid ester isomer of (+)-ephedrine

Because a kind of in the reaction of dichloromethyl sulfo-phosphine and ephedrine in the non-corresponding isomer of main only Xing Cheng oxazole phosphine thioketone, be necessary to use simultaneously (+)-ephedrine and (-)-ephedrine Rp oxazole phosphine thioketone and Sp oxazole phosphine thioketone with the acquisition high yield.Chemical compound 12 and 13 synthetic synthetic shown in scheme 14 and 15 carried out small modification, and be shown in scheme 16 and 17.

The stereo selectivity of the organophosphorus ester 12 that scheme 16. use (+)-ephedrines carry out is synthetic

The stereo selectivity of the organophosphorus ester 13 that scheme 17. use (-)-ephedrines carry out is synthetic

(2Rp, 4S, 5R) and (2Sp, 4S, 5R)-trimethyl-5-phenyl-1,3,2-oxazole phosphine-2-thioketone (15a and 15b) synthetic.The solution that is contained in the 7.04mL dichloromethyl sulfo-phosphine (67.1mmol) of 60mL toluene at room temperature slowly is added into the solution of 13.3g (-)-ephedrine (80.5mmol) that is contained in 46mL triethylamine and 200mL toluene.Reactant mixture is added a cover, and under room temperature and argon, stir and spend the night, then with diatomite filtration, water and this filter liquor of salt water washing.Use Na ₂SO ₄Dry organic layer filters, and vacuum concentration.(EtOAc/ hexane 1:5,1:4) the purification crude product is to obtain the main non-corresponding isomer 15a of 6.52g (40%) by flash chromatography.Less non-corresponding isomer 15b can be lower productive rate separate and to obtain, usually as with the mixture of 15a.15a: white solid; 1H NMR (500MHz, CDCl3) δ 0.75 (d, J=6.6Hz, 3H), δ 2.06 (d, J=14.5Hz, 3H), δ 2.77 (d, J=12.3Hz, 3H), δ 3.64 (m, IH), δ 5.66 (dd, J=6.1,2.1Hz, IH), δ 7.27 (m, 2H), 7.31 (m, IH), and δ 7.35 (m, 2H).15b: white solid; 1H NMR (500MHz, CDCl3) δ 0.83 (d, J=6.5Hz, 3H), δ 1.96 (d, J=14.2Hz, 3H), δ 2.69 (d, J=12.7Hz, 3H), δ 3.63 (m, IH), δ 5.48 (dd, J=5.8,3.3Hz, IH), δ 7.32 (m, IH), 7.37 (m, 4H).

(2Sp, 4R, 5S) and (2Rp, 4R, 5S)-trimethyl-5-phenyl-1,3,2-oxazole phosphine-2-thioketone (20a and 20b) synthetic.Except that using (+)-ephedrine, this prepares with mentioned above identical.20a: white solid; 1HNMR (500MHz, CDCl3) δ 0.75 (d, J=6.7Hz, 3H), δ 2.06 (d, J=14.6Hz, 3H), δ 2.77 (d, J=12.3Hz, 3H), δ 3.64 (m, IH), δ 5.67 (dd, J=6.0,1.9Hz, IH), δ 7.28 (m, 2H), δ 7.32 (m, IH), and δ 7.37 (m, 2H).20b: white solid; 1H NMR (500MHz, CDCl3) δ 0.84 (d, J=6.5Hz, 3H), δ 1.96 (d, J=14.2Hz, 3H), δ 2.69 (d, J=12.7Hz, 3H), δ 3.63 (m, IH), δ 5.48 (dd, J=5.8,3.3Hz, IH), δ 7.32 (m, IH), 7.37 (m, 4H).

The synthetic general preparation of Rp and Sp-O-alkyl S-methyl Thiophosphonate.Suitably adding the solution that the saturated alcohol of 0.7mL hydrogen chloride forms in the solution of alcohol and be warming up to room temperature in the 0.7mL methyl ethyl ketone to the 2mL methyl ethyl ketone of 200mg (0.83mmol) Shi Dang De oxazole phosphine-2-thioketone and 1.5mL under 0 ℃.After the stirring at room 2 hours, with the freezing sodium carbonate of 10mL (10%) aqueous solution cessation reaction.With mixture with 7.5mL water and 12mL ethanol dilution, then with 10% PdVC (15mg) and H ₂Balloon carries out hydrogenation.This reaction system is added a cover, mixture is at room temperature stirred spend the night then.Remove this catalyst through diatomite filtration, the vacuum concentration filter liquor is to remove ethanol then.Fully extract remaining water layer with ether, to pH4, and extract once more with the mixture of isopropyl alcohol and chloroform (1:4) with the citric acid acidify.With second group of extract Na ₂SO ₄Drying is filtered, and the concentrated yellow oil that obtains.By this crude product of preparation property TLC purification.The spectroscopic data of each isomer is identical.

(Sp)-O-isopropyl S-methyl Thiophosphonate (4).Prepare referring to generality.Purification condition 100% ether.Separate obtain 18mg 1HNMR (500MHz, CDCl3) δ 1.32 (d, J=6.1Hz, 3H), δ 1.37 (d, J=6.1Hz, 3H), δ 1.75 (d, J=15.4Hz, 3H), δ 2.29 (d, J=12.9Hz, 3H), δ 4.8 (m1H).

(Rp)-O-isopropyl S-methyl Thiophosphonate (5).Prepare referring to generality.Purification condition 100% ether.Separate obtain 25mg 1H NMR (500MHz, CDCl3) δ 1.32 (d, J=6.1Hz, 3H), δ 1.37 (d, J=6.1Hz, 3H), δ 1.75 (d, J=15.4Hz, 3H), δ 2.29 (d, J=12.9Hz, 3H), δ 4.8 (m IH).

(Sp)-O-cyclohexyl S-methyl Thiophosphonate (6).Prepare referring to generality.Purification condition ether/hexane 4:1.Separate obtain 54mg 1H NMR (500MHz, CDCl3) δ 1.23 (m, IH), δ 1.36 (m, 2H), and δ 1.51-1.56 (m, 3H), δ 1.72 (m, 2H), and δ 1.77 (d, J=15.6Hz, 3H), δ 1.92 (m, IH), δ 1.99 (m, IH), δ 2.30 (d, J=12.9Hz, 3H), and δ 4.51 (m, IH).

(Rp)-O-cyclohexyl S-methyl Thiophosphonate (7).Prepare referring to generality.Purification condition ether/hexane 4:1.Separate obtain 26mg 1H NMR (500MHz, CDCl3) δ 1.23 (m, 1H), δ 1.36 (m, 2H), and δ 1.51-1.56 (m, 3H), δ 1.72 (m, 2H), and δ 1.77 (d, J=15.6Hz, 3H), δ 1.92 (m, 1H), δ 1.99 (m, 1H), δ 2.30 (d, J=12.9Hz, 3H), and δ 4.51 (m, 1H).

(Sp)-and O-3,3-dimethyl-2-butyl S-methyl Thiophosphonate (8).Prepare referring to generality.Purification condition ether/dichloromethane 1:1.Separate obtain 27mg 1H NMR (500MHz, CDCl3) δ 0.91 (s, 12H), δ 1.36 (d, J=6.37Hz, 3H), δ 1.78 (d, J=15.6Hz, 3H), δ 2.33 (d, J=12.9Hz, 3H), δ 4.3 (m, IH).

(Rp)-and O-3,3-dimethyl-2-butyl S-methyl Thiophosphonate (9).Prepare referring to generality.Purification condition ether/dichloromethane 1:1.Separate obtain 12mg 1H NMR (500MHz, CDCl3) δ 0.91 (s, 12H), δ 1.36 (d, J=6.37Hz, 3H), δ 1.78 (d, J=15.6Hz, 3H), δ 2.33 (d, J=12.9Hz, 3H), δ 4.3 (m, IH).

(Sp)-and O-N, N-diisopropylaminoethyl ethyl S-methyl Thiophosphonate (10).Prepare referring to generality.Purification condition 100% ether.Separate obtain 9.6mg 1H NMR (500MHz, CDCl3) δ 1.32 (d, J=6.2Hz, 6H), δ 1.38 (d, J=6.2Hz, 6H), and δ 1.75 (d, J=15.4Hz, 3H), δ 2.30 (d, J=12.8Hz, 3H), and δ 4.1-4.2 (m, 4H), δ 4.8 (m, 2H).

(Rp)-and O-N, N-diisopropylaminoethyl ethyl S-methyl Thiophosphonate (11).Prepare referring to generality.Purification condition 100% ether.Separate obtain 9.6mg 1H NMR (500MHz, CDCl3) δ 1.32 (d, J=6.2Hz, 6H), δ 1.38 (d, J=6.2Hz, 6H), and δ 1.75 (d, J=15.4Hz, 3H), δ 2.30 (d, J=12.8Hz, 3H), and δ 4.1-4.2 (m, 4H), δ 4.8 (m, 2H).

(2S, 4R, 5S)-and (2R, 4R, 5S)-and 2-chloro-3,4-dimethyl-5-phenyl-1,3,2 oxazoles phosphine-2-thioketone (24a and 24b).25mL toluene solution to 6.9g (41mmol) thiophosphoryl chloride slowly adds the serosity that is formed by 8.6g (43mmol) (+)-ephedrine and 35mL triethylamine in 150mL toluene, and at room temperature stirs and spend the night.After the stirred overnight at room temperature this reactant is injected water, and with 3 * 150mL water washing.Use the dried over sodium sulfate organic facies, and vacuum concentration obtains yellow oil, it is leaving standstill after coagulation.Obtain Sp isomer and the Sp isomer of 2.5g and the 95:5 mixture of Rp isomer of 1.5g by column chromatography (Silicon stone 9:1 hexane/ethyl acetate) this crude product of purification.1HNMR (500MHz, CDCl3) Sp isomer δ 0.88 (d 3H), δ 2.92 (d 3H), δ 3.85 (apparent couple of quintet 1H), δ 5.83 (d 1H), δ 7.3-7.5 (m5H).Rp isomer δ 0.81 (d 3H), δ 2.73 (d 3H), δ 3.75 (apparent quintet 1H), δ 5.6 (apparent triplet 1H), δ 7.15-7.25 (m 5H).

(2S, 4R, 5S)-and 2-(N, N-dimethylamino)-3,4-dimethyl-5-phenyl-1,3,2 oxazoles phosphine-2-thioketone (25a).The dried toluene solution of the 5mL of 500mg 24a blasts anhydrous dimethyl base amine in manometer tube.After 1 minute, seal this pipe, and at room temperature stir.After 4 hours, filter this reactant mixture,, use dried over sodium sulfate, and vacuum drying is to provide quantitative 15 with 2 * 5mL water washing.This crude product can be without being further purified direct use.1HNMR (500MHz, CDCl3) δ 0.76 (d 3H), δ 2.60 (d 3H), δ 2.93 (d6H), δ 3.54 (apparent quintet 1H), δ 5.67 (d 1H), δ 7.27-7.31 (m 5H).

(Sp)-and O-ethyl S-methyl N, N-dimethyl thiophosphoryl amide (12).2mL ethanol solution to 500mg (1.9mmol) 25a adds the saturated dehydrated alcohol of 2mL hydrogen chloride.Stirring at room will be reacted after 2 hours with hydroxide aqueous solution and be alkalized to pH12 and stirring at room temperature.After stirring is spent the night, extract this reactant mixture, add excessive iodomethane (3mL) then and at room temperature continue and stirred 1 hour with 3 * 20mL ether.This reactant of dilute with water and with 4 * 20mL chloroform extraction.Merge organic layer also with 3 * 15mL water washing, carefully concentrate with dried over sodium sulfate and under coarse vacuum.With this crude product of preparation property TLC (100% ether) purification to obtain colourless oil.1HNMR (500MHz, CDCl3) δ 1.34 (apparent t 3H), δ 2.24 (d, J=14.2Hz, 3H), δ 2.74 (d, J=10.91Hz, 6H), δ 4.12 (m 2H).

(2R, 4S, 5R)-and (2S, 4S, 5R)-and 2-chloro-3,4-dimethyl-5-phenyl-1,3,2 oxazoles phosphine-2-thioketone (28a and 28b).In the 25mL toluene solution of 6.9g (41mmol) thiophosphoryl chloride, slowly add the serosity that in 150mL toluene, forms by 8.6g (43mmol) (-)-ephedrine and 35mL triethylamine, and at room temperature stir.After the stirred overnight at room temperature this reactant is injected water, and with 3 * 150mL water washing.Use the dried over sodium sulfate organic facies, and vacuum concentration obtains yellow oil, it is leaving standstill after coagulation.By Rp isomer and the Rp of 1.5g and 95: 5 mixture of Sp isomer of column chromatography (9: 1 hexane/ethyl acetate of Silicon stone) this crude product of purification to obtain 200mg.1HNMR (500MHz, CDCl3) Rp isomer δ 0.88 (d3H), δ 2.92 (d3H), δ 3.85 (apparent couple of quintet 1H), δ 5.83 (d 1H), δ 7.3-7.5 (m 5H).Sp isomer δ 0.81 (d 3H), δ 2.73 (d 3H), δ 3.75 (apparent quintet 1H), δ 5.6 (apparent triplet 1H), δ 7.15-7.25 (m 5H).

(2R, 4S, 5R)-and 2-(N, N-dimethylamino)-3,4-dimethyl-5-phenyl-1,3,2 oxazoles phosphine-2-thioketone (29a).In manometer tube, blast anhydrous dimethyl base amine in the dried toluene solution of the 2mL of 200mg28a.After 1 minute, seal this pipe, and at room temperature stir.After 4 hours, filter this reactant mixture,, use dried over sodium sulfate, and vacuum drying is to provide quantitative 20a with 2 * 5mL water washing.This crude product can be without being further purified direct use.1H NMR (500MHz, CDCl3) δ 0.76 (d 3H), δ 2.60 (d 3H), δ 2.93 (d6H), δ 3.54 (apparent quintet 1H), δ 5.67 (d 1H), δ 7.27-7.37 (m 5H).

(Rp)-and O-ethyl S-methyl N, N-dimethyl thiophosphoryl amide 13.2mL ethanol solution to 200mg (1.9mmol) 29a adds the saturated dehydrated alcohol of 2mL hydrogen chloride.Stirring at room alkalizes to pH12 with hydroxide aqueous solution reactant mixture after 2 hours and at room temperature stirs.After stirring is spent the night, extract this reactant mixture, add excessive iodomethane (3mL) then and at room temperature continue and stirred 1 hour with 3 * 20mL ether.This reactant of dilute with water and with 4 * 20mL chloroform extraction.Merge organic layer also with 3 * 15mL water washing, carefully concentrate with dried over sodium sulfate and under coarse vacuum.With this crude product of preparation property TLC (100% ether) purification to obtain colourless oil.1HNMR (500MHz, CDCl3) δ 1.34 (apparent t3H), δ 2.24 (d, J=14.2Hz, 3H), δ 2.74 (d, J=10.91Hz, 6H), δ 4.12 (m 2H).

The inhibition kinetics of synthetic never poison analog and people's BuCh esterase.The time dependence of people BuChE suppresses kinetics and can detect with highly purified people BuChE with reference to embodiment 8 is described.This kinetic constant is enumerated in following table 10.

The kinetic constant that table 10.HuBchE suppresses.

	k 1(×10 3M -1min -1)	k 2(min -1)	K d(μM)
				Echothiophate	1860±135	ND b	ND
Chemical compound
	1	4.1±0.12	>0.8	>200
Chemical compound 2					1.73±0.42	0.62±0.047	358.7±85.7
	Chemical compound 3	34.4±0.42	1.04±0.16	30.3±11.8
Chemical compound 4					0.0668±0.013	0.19±0.014	2.89±0.55 c
	Chemical compound 5	0.0145±0.003	0.16±0.020	11.42±2.40 c
Chemical compound 6					1.76±0.20	ND	ND
	Chemical compound
7		0.0137±0.003	0.031±0.0023	2.29±0.467 c

Chemical compound 8	1.29±0.1	0.53±0.02	410±30
				Chemical compound 9	0.011±0.003	0.008±0.001	720±210
Chemical compound 10	0.425±0.118	0.587±0.057	1380±360
				Chemical compound 11	1.24±0.14	0.251±0.003	202±23
Chemical compound 12	0.032±0.002	ND	ND
				Chemical compound 13	0.011±0	ND	ND

^aHighly purified people's BuCh esterase; ^bND, undetermined; ^cMM

As expection, these preparations significantly are lower than echothiophate as the effectiveness of inhibitor.The Sp enantiomer that should be understood that sarin analog and GF analog all is higher than the Rp enantiomer as the effectiveness of the inhibitor of people BuChE.This other organophosphorus ester with bibliographical information is consistent to the stereo selectivity that people BuChE suppresses.This can become useful feature in we obtain in vitro and in vivo the overall strategy of the medicament that has different attribute in the research.Handle 1-13 (table 10) with Che or albumin and can produce phosphorylated protein or polypeptide in external or body, it can be used for obtaining the antibody that uses in the array bioprobe.

The preparation and the test of the antibody of the ChE-organophosphorus ester of embodiment 14. never poisons.The ability (suppressing by forming metastable phosphoserine ester bond with necessary serine hydroxyl reaction) that the acute toxicity of OP chemical compound and they suppress AChE has good dependency.Described OP-ChE conjugate can be used as the sensitive selected marker that OP exposes.Same, phosphorylation albumin (that is, Tyr 411) can be used as the sensitive labelling of OP or insecticide exposure.Antibody has been developed and has been used to distinguish phosphorylation ChE or albumin (by making enzyme denaturation to expose avtive spot to antibody).Before, antibody is used to survey ChE structure and allostery with regard to catalytic activity.Based on the accurate modification that specific OP chemical compound is brought, adopt antibodies selective to come independent OP-ChE of specific recognition or OP-albumin conjugates.This is to determining that OP relative toxicity potentiality have great diagnostic value in exposure or after exposing.Our chemosynthesis corresponding to the OP-Che of never poison or the organophosphor acidify serine or the tyrosine octapeptide of OP-albumin conjugates.This hapten by gained can generate polyclonal antibody or monoclonal antibody.OP-ChE inactivator or the albuminous selectivity identification of OP-from different plant species are detected.Antiserum identification suppresses with enzyme, speed aging and the inductive reactivate of oxime is relevant.Described antibody is used to one group of ChE biomarker, and this labelling comprises the albumin of phosphorylation and the albumen of other phosphorylation, is used in biological specimen Chemical exposure being carried out the selectivity classification.Described antibody is placed in the array bioprobe, and it is become can the on-the-spot effective never poison OP that uses and the bioprobe of other OP sample material.

Haptenic synthetic.The serine of the organophosphor acidify that this is essential or the chemosynthesis of tyrosine octapeptide start from the selective esterification of Fmoc serine or tyrosine.The Fmoc serine that chemical compound 1-13 is produced (embodiment 13) and is used for Processing of Preparation level scale (100mg) is to provide required protected organophosphor acidify serine (scheme 18).

Scheme 18. is used for the synthetic of the synthetic phosphorylation Fmoc-serine of decapeptide

R=-CH (CH ₃) ₂-CHCH ₃C (CH ₃) ₃-cyclohexyl;-CH ₂CH ₂N[CH (CH ₃) ₂] ₂And corresponding tabun analog.

S-alkyl methyl Thiophosphonate can adopt necessary sodium alkoxide or be contained in the bromine in the alcohol and the silver nitrate that is contained in the alcohol carries out nucleophilic displacement.Alkoxide and AgNO ₃/ ROH reaction provides about 80% configuration conversion on phosphorus, and adopts Br ₂The displacement reaction of/ROH can provide 100% configuration conversion.The replacement that this bromine promotes is very fast usually and have a high yield.Yet, it is reported in some space crowded position, the alcoholysis that bromine promotes can be carried out under the situation that configuration keeps substantially.The protection of carboxylic acid may be to realize that high yield is necessary.Perhaps, use Br ₂/ Fmoc-serine or-tyrosine reaction.If this reaction does not produce gratifying productive rate, then can improve.We once can be by the quick S-oxide of attacking of nucleopilic reagent to forming with position chlorine benzylhydroperoxide (MCPBA) oxidized activating 1-13 (embodiment 13) by the NMR laboratory observation.Therefore, formed corresponding alcohol (judging), wherein this S-(O)-CH by LCMS the processing of the S-oxide of 1-13 (embodiment 13) and the interpolation fast quantification of trace water ₃Group is replaced by the OH group.At 1.2 equivalent MCPBA/CHC ₁₂Existence under 1-13 (embodiment 13) was handled 5 minutes down at 4 ℃, adding 1 normal FmocSer then will can generate bonded Fmoc serine of required OP-or tyrosine equally effectively.It is synthetic that the bonded Fmoc serine of OP-or tyrosine are integrated into described common octapeptide, will provide the required OP-that is used for immune Research in conjunction with decapeptide.

Antiserum to Che or albuminous avtive spot organophosphor acidify serine.For people, Primate and rat AchE and people, Primate and rat BuChE, the five amino acid in serine avtive spot both sides all identical (being TLFGESAGAAS) (SEQ ID NO:11).Based on this information, decapeptide LFGESAGAAC (SEQ ID NO:12) is used to develop OP-conjugate selectivity antiserum.The partial sequence (YGFQNAILVRYTQKAPQV) (SEQ ID NO:14) of the partial sequence of human albumin peptide (YKFQNALLVRYTKKVPQV) (SEQ ID NO:13), rat albumin peptide and the partial sequence (YGFQNAILVRYTQKAPQV) (SEQ ID NO:15) of mice ovalbumin peptide similarly are used to sero-fast obtaining.Adding hop protein also can directly be used.Important part is to replace terminal serine with cysteine or sulfur-bearing joint.This will provide necessary absorption chemical action (discussing hereinafter) for bioprobe.The overall use of described decapeptide will economize on resources (because it all is suitable for AChE and BuChE) and allow sero-fast bigger utilization.Following ChE will represent two kinds of enzymes (and analog, albumin) simultaneously.Code name 10S and 10SP refer to non-phosphorylating decapeptide and phosphorylation decapeptide.Anti--ChE10S and anti--ChE10SP antiserum can generate after using existing ChE10S that combines keyhole limpet hemocyanin that describes and ChE10SP peptide immunize rabbit.It should be noted that from the bonded peptide of OP-(ChE10SP) acquisition is sero-fast and obtain antiserum in contrast from natural or unbound peptide (ChE10S) simultaneously.This combination can realize by standard step.Rabbit can carry out immunity with standard step by commercial laboratory.Except the ChE10SP decapeptide comprised identical OP-conjugate from never poison 1-13 (embodiment 13), required peptide synthesized by above-mentioned standard step.Antiserum carries out chromatography purification with the blue glue post of DEAE Affi-gel.The bonded decapeptide of OP-is coupled to the Affi-gel pearl by Cys, with these this antiserums of pearl purification.The antiserum that specificity is lower passes through 1M NaSCN eluting, and decapeptide specificity elution fraction (also being buffered to pH8 rapidly with 1M glycine-HCl eluting) is confirmed specific antibody by elisa assay.The Western trace as above carries out.

In order to identify this sero-fast specificity, handle rat, Primate and people AChE and BuChE or albumin 2 hours down or drop to 1-2% at 4 ℃ until enzyme work according to the Ellman colorimetric method for determining with 0.5mM never poison analog 1-13 (embodiment 13) or carrier THF.Enzyme can be obtained by affinity purification by animal serum or recombinant sources.Our laboratory existing reorganization HuChE and Primate BuChE, rat BuChE can obtain from the rat blood serum purification.AChE can obtain by purification from animal hemocyte film.Repressed enzyme can be analyzed by the immunoblotting that adopts denaturant gel.As positive control, can resist-ChE antiserum labelled immune trace by polyclone, there is equal identification and has complete enzyme with demonstration.Competitiveness experiment between different OP-combinations and uncombined enzyme can detect to show specificity by OP-selectivity antiserum.This has specificity with we relevant resisting-ChE10SP of test to the bonded serine of OP-and also bonded OP is had specific hypothesis simultaneously.In single gel and immunoblotting, the effect of handling is compared, and the sample that contains equal protein under each treatment conditions is detected.In each sero-fast range of linearity with density analysis anti-to adopting-variation of the protein band intensity of ChE10SP antiserum and contrast antiserum labelling carries out quantitatively.Antiserum is used in the array bioprobe, to detect the deutero-Chef of OP that exposes selective probe as OP in biofluid or environmental samples.

All publications and the patent application of being quoted in this description are all quoted as a reference, and it is quoted degree and is all quoted as a reference individually by specific as each independent publication and patent application.Although aforementioned invention is described in detail by elaboration and embodiment, those of ordinary skill in the art should understand easily and can carry out certain change and adjustment to it under the situation of the spirit and scope that do not break away from accessory claim according to instruction of the present invention.

Sequence table

＜110〉John R Ka Shiman

Zhang Jun

Human Biomolecular Res Inst

＜120〉new method of ester detoxication

<130>11832-005-999

＜140〉wait to specify

<141>2007-06-05

<150>US 60/811,370

<151>2006-06-07

<160>15

＜170〉be used for the FastSEQ of windows 4.0 editions

<210>1

<211>1809

<212>DNA

＜213〉Rhesus Macacus (cDNA of RhBchE)

<400>1

<210>2

<211>602

<212>PRT

＜213〉Rhesus Macacus

<400>2

<210>3

<211>602

<212>PRT

＜213〉homo sapiens

<400>3

<210>4

<211>581

<212>PRT

＜213〉rabbit

<400>4

<210>5

<211>602

<212>PRT

＜213〉cat

<400>5

<210>6

<211>602

<212>PRT

＜213〉horse

<400>6

<210>7

<211>603

<212>PRT

＜213〉mice

<400>7

<210>8

<211>1964

<212>DNA

＜213〉Rhesus Macacus

<400>8

<210>9

<211>538

<212>DNA

＜213〉Rhesus Macacus (part RhBchE, M62777)

<400>9

<210>10

<211>2196

<212>DNA

＜213〉homo sapiens (HuBchE (NCBI NM 000055)

<400>10

<210>11

<211>11

<212>PRT

＜213〉homo sapiens's (partial sequence of serine avtive spot)

<400>11

<210>12

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉be used to develop the sero-fast peptide of OP conjugate selectivity

<400>12

<210>13

<211>18

<212>PRT

＜213〉homo sapiens's (partial sequence of human albumin)

<400>13

<210>14

<211>18

<212>PRT

＜213〉rat (partial sequence of rat albumin)

<400>14

<210>15

<211>18

<212>PRT

＜213〉mice (the albuminous partial sequence of mice)

<400>15

Claims (38)

1. detoxification, this method comprise butyrylcholine esterase (BchE) are contacted with medicine, herbicide or the insecticide of organic phosphoric acid ester formulation, abuse.

2. the method for claim 1, wherein said contact takes place in vivo.

3. the method for claim 1, wherein said contact occurs in external.

4. the method for claim 1, wherein said organic phosphoric acid ester formulation is the never poison that is selected from sarin, soman, GF, tabun and VX.

5. the method for claim 1, wherein said organic phosphoric acid ester formulation is the chemical compound that is selected from chemical compound 1,2,3,4,5,6,7,8,9,10,11,12 and 13.

6. the method for claim 1, the medicine of wherein said abuse is a cocaine.

7. method at butyrylcholine esterase (BchE) screening active ingredients chemical compound, this method may further comprise the steps:

A) BchE and described chemical compound are hatched; And

B) detect the index of the inhibition of BchE as the biology or the pharmacologically active of described chemical compound.

8. method as claimed in claim 7, wherein said BchE is Rhesus Macacus BchE or its variant.

9. method as claimed in claim 7, wherein said chemical compound are selected from chemical compound 1,2,3,4,5,6,7,8,9,10,11,12 and 13.

10. method that in carrier, prepares BchE sudden change storehouse.

11. method as claimed in claim 10, wherein said carrier are pENTRA or adenovirus vector.

12. method as claimed in claim 10, wherein said BchE is Rhesus Macacus BchE or its variant.

13. method as claimed in claim 10, wherein said BchE is people BchE or its variant.

14. method as claimed in claim 10, wherein said BchE is the mixture of Rhesus Macacus BchE and people BchE or its variant.

15. method that is packaged in usefulness recombinant adenovirus mammalian cell-infecting in the adenovirus particles by storehouse that BchE is suddenlyd change.

16. method as claimed in claim 15, wherein said BchE is Rhesus Macacus BchE or its variant.

17. method as claimed in claim 15, wherein said BchE is people BchE or its variant.

18. method as claimed in claim 15, wherein said BchE is the mixture of Rhesus Macacus BchE and people BchE or its variant.

19. the method that BchE expresses in the culture medium of a detection infected mammalian cell as claimed in claim 9, this method may further comprise the steps:

A) collect infection back culture medium; And

B) the BchE activity in the described culture medium of analysis.

20. method as claimed in claim 19, active Ellman method or the western engram analysis of adopting of wherein said BchE analyzed.

21. method as claimed in claim 19, wherein said BchE is Rhesus Macacus BchE or its variant.

22. method as claimed in claim 19, wherein said BchE is people BchE or its variant.

23. method as claimed in claim 19, wherein said BchE is the mixture of Rhesus Macacus BchE and people BchE or its variant.

24. one kind is detected the protokaryon of expression BchE or the method for the BchE in the eukaryotic cell, this method may further comprise the steps:

A) in the presence of the organophosphorus compounds that is selected from chemical compound 1,2,3,4,5,6,7,8,9,10,11,12 and 13, cover described cell with the culture medium that contains agarose;

B) mixture of step (a) is hatched different time spans; And

C) in the presence of iodate Butyryl thiocholine and DTNB, in containing the covering buffer of agarose, detect the BchE activity.

25. method as claimed in claim 24, wherein said BchE is Rhesus Macacus BchE or its variant.

26. method as claimed in claim 24, wherein said BchE is people BchE or its variant.

27. method as claimed in claim 24, wherein said BchE is the mixture of Rhesus Macacus BchE and people BchE or its variant.

28. a method that detects BchE by protokaryon or the eukaryotic cell of expression BchE, this method may further comprise the steps:

A) in the presence of never poison or insecticide, cover described cell with the culture medium that contains agarose;

B) mixture of step (a) is hatched different time spans; And

C) detect BchE activity in the covering buffer that contains agarose contain iodate Butyryl thiocholine and DTNB.

29. method as claimed in claim 28, wherein said BchE is Rhesus Macacus BchE or its variant.

30. method as claimed in claim 28, wherein said BchE is people BchE or its variant.

31. method as claimed in claim 28, wherein said BchE is the mixture of Rhesus Macacus BchE and people BchE or its variant.

32. as claim 24 or 28 described methods, wherein the cellular expression of BchE comes from the recombinant virus infection described in claim 15.

33. a method of separating the recombinant virus of expressing BchE, this method comprises step as claimed in claim 24, further comprises from the macula lutea of selectively staining and digs out the agarose filler, and this filler is transferred to aseptic culture medium.

34. method that isolating filler as claimed in claim 33 is characterized, further comprise and use the culture medium of hatching to come infection cell with described filler, and by expression as any described method monitoring BchE of claim 19,24 or 28.

35. the method for the independent recombinant virus of purification from isolating filler as claimed in claim 33, this method comprises the described culture medium of serial dilution, as as described in the claim 15 with the culture medium infection cell of dilution, as detecting BchE as described in the claim 32, and as the independent dyeing filler of separation as described in the claim 33.

36. the method to characterizing as claim 33 or 35 described isolating fillers, this method is by carrying out pcr amplification with adenovirus vector and/or the BchE gene-specific primer coded BchE gene of recombinant virus pair in the culture medium that described filler is hatched.

37. the DNA of coding Rhesus Macacus butyrylcholine esterase (RhBchE) mutant and segmental separation and purification.

38. be used to detect the array bioprobe of medicine, herbicide or the insecticide of OP material, abuse.

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