CN106908610A - It is a kind of that the reagent of morphine, preparation method and application are sucked based on analgesics screening platform technology examination - Google Patents
It is a kind of that the reagent of morphine, preparation method and application are sucked based on analgesics screening platform technology examination Download PDFInfo
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- CN106908610A CN106908610A CN201710147233.8A CN201710147233A CN106908610A CN 106908610 A CN106908610 A CN 106908610A CN 201710147233 A CN201710147233 A CN 201710147233A CN 106908610 A CN106908610 A CN 106908610A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/948—Sedatives, e.g. cannabinoids, barbiturates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
Abstract
It is more particularly to a kind of that the reagent of morphine, preparation method and application are sucked based on analgesics screening platform technology examination the present invention relates to drugs technical field of measurement and test.A kind of reagent that morphine is sucked based on analgesics screening platform technology examination, the protein sequence of the reagent is as shown in sequence table SEQ NO.1；The reagent is that Ah skin's acceptor house of correction is obtained, and point mutation is done to the 138th and 386 amino acids respectively for original series, to strengthen acceptor and ligand affinity.Using technical solution of the present invention, beneficial effects of the present invention are:The present invention uses analgesics screening platform, and selectivity is good, and production cost is relatively low.
Description
Technical field
The present invention relates to a kind of method whether novel test paper rapid screening a suspect sucks morphine, more particularly to
It is a kind of that the reagent of morphine, preparation method and application are sucked based on analgesics screening platform technology examination.
Background technology
Morphine (Morphine, MOP) is the important component of opium class drugs, french chemist Ze Erdina in 1806
It is separated from opium first.Its derivative morphine hydrochloride is clinically conventional anesthetic, has extremely strong analgesia to make
With, be used for the severe pain that wound, operation, burn etc. cause, be also used for the angina pectoris that myocardial infarction causes, be alternatively arranged as analgesia,
Antibechic and antidiarrheic etc., but its disadvantage is easy habituation.This cause long-term smoker from body or it is psychological all
Serious dependence can be produced to morphine, cause serious toxicomania, so as to cause greatly harm to itself and society.Point
Minor is C17H19NO3, molecular weight 285, scientific name：17- methyl -4,5 α-epoxy -7,8- bis- oxymorphine is muttered -3,6 salmefamols.
Opiate receptor belongs to gpcr protein family member, at least there are 8 kinds of hypotypes in vivo, in central nervous system at least
In the presence of 4 kinds of hypotypes:μ、κ、δ、σ.Morphinoid drug is to the opiate receptor of different shaped, affinity and the incomplete phase of intrinsic activity
Together, each acceptor has different subtype.Morphine is tri- kinds of activators of acceptor of μ, κ, δ, to the action intensity of three receptor subtypes
Weaken successively.
Conventional method realizes that antibody producing program is complicated, relatively costly, small molecule by antigen-antibody combination technology
Compound affinity of antibody and to different subtype selectivity also be not so good as receptors ligand.
The content of the invention
It is not enough for prior art, inhaled based on the examination of analgesics screening platform technical method it is an object of the invention to provide a kind of
The method for eating morphine personnel is higher with sensitivity（10-100 times）, production cost is lower（Reduces cost more than 30%）It is excellent
Gesture.Therefore, the present invention provides following technical scheme；
A kind of reagent that morphine is sucked based on analgesics screening platform technology examination, the protein sequence such as sequence table SEQ of the reagent
Shown in NO.1；The reagent is that Ah skin's acceptor house of correction is obtained, and the 138th and 386 amino acids are done respectively for original series
Point mutation, to strengthen acceptor and ligand affinity.Determine that the protein vigor of 138 mutation is best by immunofluorescence method
（IC50 is less than 10nM）, meet research and development of products requirement.
SEQ NO.1：
1 mdssaaptna snctdalays scspapspgs wvnlshldgn lsdpcgpnrt dlggrdslcp
61 ptgspsmita itimalysiv cvvglfgnfl vmyvivrytk mktatniyif nlaladalat
121 stlpfqsvny lmgtwpfmti lckivisidy ynmftsiftl ctmsvdryia vchpvkaldf
181 rtprnakiin vcnwilssai glpvmfmatt kyrqgsidct ltfshptwyw enllkicvfi
241 fafimpvlii tvcyglmilr lksvrmlsgs kekdrnlrri trmvlvvvav fivcwtpihi
301 yviikalvti pettfqtvsw hfcialgytn sclnpvlyaf ldenfkrcfr efciptssni
361 eqqnstrirq ntrdhpstan tvdrtnhqle nleaetaplp。
A kind of kit that morphine is sucked based on analgesics screening platform technology examination, the kit is included described in use
The opiate receptor conjugate pad that reagent is prepared into, the opiate receptor conjugate pad is assembled into instead with nitrocellulose membrane and sample suction pad
Plate is answered, the reaction plate is cut into reagent strip and is installed.
Present invention additionally comprises following methods：A kind of preparation of the reagent that morphine is sucked based on analgesics screening platform technology examination
Method, comprises the following steps：
1）Build opiate receptor expression system：
1.1）Double digestion, by respectively by original series, 138 and 386 mutant clons enter PET32 expression vectors.
1.2）It is transformed into amplification in DH5 α competence bacteriums, upgrading grain.
1.3）Plasmid is transformed into expression bacterial strain, bacterium detection and conservation, expression bacterial strain such as Bl21 is chosen（DE3）Albumen is lured
Lead expression.
1.3.1）Expression bacterial strain is shaken to OD=0.6 or so in 3ml LB culture mediums, IPTG is added, concentration gradient is from 25
μM to 1m M.37 degree of overnight inductions (general more than 3h has great expression).
1.3.2）The expression of SDS-PAGE electrophoresis detection destination proteins.
1.3.3）It is sufficient that having the glycerine conservation of bacterial strain 10% of expression, preservation 1ml or so, and record IPTG concentration models
Enclose.With 0.22 μm of filtration sterilization, storage concentration is usually 30%-60% to glycerine, and oneself calculates consumption when using.
1.3.4）10ml is induced to express bacterium, 18 degree, the slow-speed of revolution with the minimum of above-mentioned IPTG concentration ranges（140-
180rpm）, overnight induction is used as inclusion body detection sample.
2）138 mutation opiate receptors of expression：
2.1）The expression bacterial strain planted of going bail for first shakes 10ml, and 37 degree, 300rpm is shaken to OD>=1.5, about 5h or so, depending on the work of strain
Property and it is different, also can overnight shake bacterium.
2.2）By 37 degree in the 8ml addition 300ml culture mediums in previous step, 250rpm is shaken to OD=1.0 or so（About
2.5h~3h）, then add IPTG（The concentration that concentration is used in being detected with inclusion body）.Bacterium is overnight shaken, the temperature detected using inclusion body
Degree（18 ° or so）, rotating speed 140rpm or so.
2.2.1）Bacterium solution 6000rpm, 4min, 4 degree are collected by centrifugation thalline.20mM PBS are added, with balance after washing one time
Buffer solution is resuspended.Per 250ml bacterium solutions with 30 ml to 50ml level pads, depending on the concentration of bacterium solution.Can be with 4 50ml's
Centrifuge tube is centrifuged simultaneously, but, centrifuge tube will be reused, and cleaned after being finished and preserved.
2.3）Product to previous step carries out ultrasonic treatment, uses 6mm ultrasonic transformers, 35% power, 3.5s work, 7s to stop
Breath, 50min.
2.4）Take the product supernatant after ultrasonic treatment.The solution that will have been crushed is collected into 50ml centrifuge tubes,
10000rpm, 15min, 4 ° of centrifugation.It is precipitated as inclusion body, cell fragment and unbroken cell.Supernatant being poured out gently, it is most
Amount should not pour out precipitation.（PH value can be now measured, preferably 7.5 or so, the pH for balancing buffer is 7.8 left to pH value
The right side, supernatant will be 7.5 or so after cracking.）
2.5）Purifying supernatant --- touch elution requirement.
2.5.1）The treatment of nickel post.By in 1ml nickel post suction dead level analysis pipe, add balance slow after liquid flow therein is complete
Fliud flushing 3ml.
2.5.2）10ml supernatants are added on equilibrated NI posts, and the sample of post will be crossed and repeat loading 3 times.（If
It is that flow velocity can add a syringe needle below pillar slowly, or is dispelled colloid with the rifle of 1ml.）The sample that 20 μ l are crossed after post is taken,
In case running glue.
2.5.3）Respectively plus 20mM, 40mM, 60mM, the imidazole gradient of 80 mM elute foreign protein.Each gradient is distinguished
Applied sample amount be 4ml, 2ml, 2ml, 2ml.The liquid point of each time loading above 1ml and during 1ml collects EP pipes respectively below,
In case running glue.
2.5.4）Plus elution buffer（Elution buffer）Destination protein is eluted.Applied sample amount is 4.5ml（By above
0.5ml is independently accessed EP pipes, then 4ml below is accessed into two other EP pipes）, stay race glue sample.
2.6）Run glue checking.
2.6.1）Run 2 pieces of PAGE of 0.75mm（Polyacrylamide gel electrophoresis）Glue, all adds up all of sample,
The dense segregation ratio of two pieces of glue wants identical two pieces of glue just can be than more consistent.
2.6.2）Run glue sequentially：Negative control, positive control, excessively supernatant, post sample, the quick moulding 5min of 300V or so, 160V
Separate sample 50min or so.（Can also 300V, 30min, simply scheme no 160V good-looking.）
2.6.3）Coomassie brilliant blue staining.Ordinary stain 2h.
2.6.4）Decolourize.15min first is taken off with decolorising agent 1, then it is de- overnight with decolorising agent 2.
2.7）Purifying supernatant --- collect destination protein
2.7.1）The nickel post lived again is balanced again（I.e. plus offset eluent Equilibration Buffer 3ml）.
2.7.2）Operation in repeat step 12, is only washed when simply cleaning with the imidazole concentration touched in step 12.
2.8）Glue is run again to verify and preserve；Current glue figure 160V, keeps.
2.9）Dialysis.
2.9.1）Prepare 2L dialyzates（Formula sees appendix 1）, and be put in -20 degree refrigerator precoolings but should not freeze.
2.9.2）Bag filter（Cut 10cm）10min is boiled with bag filter treatment fluid, MILIQ H are used2O is fully cleaned.
2.9.3）Bag filter is clamped with dialysis clamp（Bag filter is folded can press from both sides more firm）, the albumen that will be eluted
Sample is added thereto, and inserts the 500ml dialyzates of precooling in advance（20mM PBS+50 mM NaCl）In.Slight magnetic force under ice bath
Liquid is changed after inserting 4 ° of refrigerator 1.5h after stirring 20min.Dialysis 3 times.
2.10）Concentration.
2.10.1）The sample dialysed is placed in white boxes together with bag filter, with the polyethylene glycol of precooling
2000 solid entrappings.
2.10.2）To be removed in starchy polyethylene glycol after water suction after 30min, add new solid polyethylene glycol.
2.10.3）Every 10min observations once, once there is muddy stopping concentration immediately.
2.10.4）After bag filter is finished, clean, be stored in 4 ° of storages in 20% ethanol.
2.11）Ultrafiltration concentration, dialysis albumen.
2.11.1）4ml is offseted during the protein solution that affords adds super filter tube.
2.12）Trim.
2.12.1）7400g, 30min, 4 ° centrifugation, at the end of 4ml be changed into 1ml or so.4 times of concentration.Can select as needed
Different centrifugation times are selected, to reach different cycles of concentration.Eluent that can be several times（Elution）Concentrate together.
2.11.2）Add the protein storage liquid for needing to 4ml, be centrifuged.Repeating can be such that Elution liquid gradually changes
Protein storage liquid for needed for.Protein storage liquid typically uses the three of 20mM PBS+*mM NaCl or debita spissitudo and pH（Hydroxyl first
Base）Aminomethane（tris-HCl）Solution.If albumen has precipitation not reach predetermined concentration can add appropriate glycerine（1%-
5%）Hydrotropy.
2.13）Collect.Solution is collected into EP pipes from super filter tube, the composition of storing liquid has been marked, protein name,
Protein concentration（After measurement）, and prepare the date.
3）Prepare opiate receptor colloidal gold pad.
3.1）Acceptor gold mark treatment：The colloidal gold solution of 40 nm-50 nm or so is prepared by reducing process, color becomes
It is scarlet it is limpid after respectively three parts tested, it is 8.0 that first part of mol/L K2CO3 of use 0.1 adjusts PH, and it is 8.5 that second part is adjusted PH,
It is 9.0 that 3rd part is adjusted PH.Then 1 mg, 1.5 mg, 2.0 mg opiate receptors are separately added into the solution, are slowly added dropwise,
After continuing to stir 35 minutes, the TWEEN20 for adding final concentration of 3% BSA and final concentration of 1% continues stirring 10 minutes, so
4 DEG C afterwards, 12000 rpm are centrifuged 45 minutes, and careful supernatant of drawing is discarded；100ml is settled to again with colloidal gold solution, by 1ml
The ratio uniform that solution spreads 30 cm2 is layered on glass fibre or non-woven fabrics, is placed in drying room, 37 DEG C, is done under the conditions of humidity 25%
Dry 3 hours, it is made colloidal gold pad.
3.2）The treatment of sample pad：Glass fibre or non-woven fabrics are soaked in the phosphorus of the mol/L PH=7.4 of 50 ml 0.01
In phthalate buffer（Containing the BSA of final concentration 3%, 0.05% TWEEN20）In 1 hour, 37 DEG C drying, Vacuum Package, 4 DEG C guarantor
Deposit standby.
3.3）Morphine is coated with：Morphine solution is diluted to the BSA mixed liquors of carbonate buffer solution and final concentration of 3%
0.5mg/ml, 1mg/ml and 1.5mg/ml, are drawn onto nitrocellulose membrane coating buffer with a film machine, 37 DEG C of dryings 4 hours, 4 DEG C
Save backup.
3.4）Cutting assembling：
Loading pad, the opiate receptor conjugate pad of colloid gold label, the nitrocellulose membrane of morphine, sample suction pad are assembled into reaction
Plate, 4mm reagent strips are cut into cutting machine, after being installed, are loaded aluminium foil bag and are made finished product.
Detection method and result judge：50ul-100ul or so urines to be measured or saliva are taken on loading pad, about 1 is reacted
Minute or so, according to color reaction with the naked eye direct observed result, T lines and C lines to develop the color and be free of morphine in explanation sample simultaneously, such as
Fruit contains morphine in there was only C lines colour developing explanation sample, if C lines do not have appearance, explanation Product checking is invalid.
Parameter determination：Shown according to last testing result, reagent paper marks PH to be about 9.0 recently；Opiate receptor collaurum
The mg/100 ml colloidal gold solutions of labelled antibody optimised quantity 1.5, the optimum mark amount of morphine is 1 mg/ml, optimal collaurum
Working solution is 9.0 containing 0.01% gold chloride and 1% sodium citrate aqueous solution, and to add 3% BSA and 1% for PH
TWEEN20, calibrating Best Times are 30 seconds.
As long as human body sucked morphine, morphine will enter human body by intestines and stomach or blood circulation, and the change of morphine
Learning material will remain in a period of time in human body, and the former chemical substance or its metabolite of morphine will inhale in morphine
Presented in the saliva of anthropophagy person or in urine.According to this feature, drug addict determines series can be by testing staff's
Saliva or urine accurately detect whether once to suck certain type of morphine within a certain period of time.For example：Human body one
Denier sucked morphine, will continue that morphine residual is presented in 24 after hour, in the urine of this person, as long as the suction of this project
Malicious personnel determine morphine tests positive in series, you can judge that this person had the behavior for sucking morphine in 24 hours.
Meanwhile, medicinal health anti-epidemic department and other physical examination examinations, such as detection of military service personnel and entry and exit people
Member is checked, it is also possible to the measure series of personnel is sucked using the morphine of this project.
Using technical solution of the present invention, beneficial effects of the present invention are:The present invention uses analgesics screening platform, selectivity
Good, production cost is relatively low.In being conducive to public security department, drug control organ to the examination of suspicious crowd, drug addict is differentiated in time, from
And be that public security department, the counterdrug operation of drug control organ win the important time.
Because operation and detection are without the participation of any professional person, and with it is quick, be easy to carry the characteristics of, this is
Row product is equally applicable for being monitored in the treatment of anti-drug institution, and in the process of construction of drug-free community.
Brief description of the drawings
Fig. 1 is that clone identification of the present invention adjusts electrophoretogram.
Fig. 2 is present invention difference mutain combination vigour.
Fig. 3 is fast protein liquid chromatography of the present invention（FPLC）Protein purification UV absorption figure.
Specific embodiment
As illustrated, a kind of kit that morphine is sucked based on analgesics screening platform technology examination, the kit includes
The opiate receptor conjugate pad being prepared into using described reagent, the opiate receptor conjugate pad and nitrocellulose membrane and suction sample
Pad is assembled into reaction plate, and the reaction plate is cut into reagent strip and is installed.
1）Build opiate receptor expression system：
1.1）Double digestion, by respectively by original series, 138 and 386 mutant clons enter PET32 expression vectors.
1.2）It is transformed into amplification in DH5 α competence bacteriums, upgrading grain.
1.3）Plasmid is transformed into expression bacterial strain, bacterium detection and conservation, expression bacterial strain such as Bl21 is chosen（DE3）Albumen is lured
Lead expression.
1.3.1）Expression bacterial strain is shaken to OD=0.6 or so in 3ml LB culture mediums, IPTG is added（Isopropylthio half
Lactoside), concentration gradient is from 25 μM to 1m M.37 degree of overnight inductions (general more than 3h has great expression).
1.3.2）The expression of SDS-PAGE electrophoresis detection destination proteins.
1.3.3）It is sufficient that having the glycerine conservation of bacterial strain 10% of expression, preservation 1ml or so, and record IPTG concentration models
Enclose.With 0.22 μm of filtration sterilization, storage concentration is usually 30%-60% to glycerine, and oneself calculates consumption when using.
1.3.4）10ml is induced to express bacterium, 18 degree, the slow-speed of revolution with the minimum of above-mentioned IPTG concentration ranges（140-
180rpm）, overnight induction is used as inclusion body detection sample.
2）138 mutation opiate receptors of expression：
2.1））The expression bacterial strain planted of going bail for first shakes 10ml, and 37 degree, 300rpm is shaken to OD>=1.5, about 5h or so, depending on the work of strain
Property and it is different, also can overnight shake bacterium.
2.2）By 37 degree in the 8ml addition 300ml culture mediums in previous step, 250rpm is shaken to OD=1.0 or so（About 2.5h
~3h）, then add IPTG（The concentration that concentration is used in being detected with inclusion body）.Bacterium is overnight shaken, the temperature detected using inclusion body
（18 ° or so）, rotating speed 140rpm or so.
2.2.1）Bacterium solution 6000rpm, 4min, 4 degree are collected by centrifugation thalline.20mM PBS are added, with balance after washing one time
Buffer solution is resuspended.Per 250ml bacterium solutions with 30 ml to 50ml level pads, depending on the concentration of bacterium solution.Can be with 4 50ml's
Centrifuge tube is centrifuged simultaneously, but, centrifuge tube will be reused, and cleaned after being finished and preserved.
2.3）Product to previous step carries out ultrasonic treatment, uses 6mm ultrasonic transformers, 35% power, 3.5s work, 7s to rest,
50min.
2.4）Take the product supernatant after ultrasonic treatment.The solution that will have been crushed is collected into 50ml centrifuge tubes,
10000rpm, 15min, 4 ° of centrifugation.It is precipitated as inclusion body, cell fragment and unbroken cell.Supernatant being poured out gently, it is most
Amount should not pour out precipitation.（PH value can be now measured, preferably 7.5 or so, the pH for balancing buffer is 7.8 left to pH value
The right side, supernatant will be 7.5 or so after cracking.）
2.5）Purifying supernatant --- touch elution requirement.
2.5.1）The treatment of nickel post.By in 1ml nickel post suction dead level analysis pipe, add balance slow after liquid flow therein is complete
Fliud flushing 3ml.
2.5.2）10ml supernatants are added on equilibrated NI posts, and the sample of post will be crossed and repeat loading 3 times.（If
It is that flow velocity can add a syringe needle below pillar slowly, or is dispelled colloid with the rifle of 1ml.）The sample that 20 μ l are crossed after post is taken,
In case running glue.
2.5.3）Respectively plus 20mM, 40mM, 60mM, the imidazole gradient of 80 mM elute foreign protein.Each gradient is distinguished
Applied sample amount be 4ml, 2ml, 2ml, 2ml.The liquid point of each time loading above 1ml and during 1ml collects EP pipes respectively below,
In case running glue.
2.5.4）Plus elution buffer（Elution buffer）Destination protein is eluted.Applied sample amount is 4.5ml（By above
0.5ml is independently accessed EP pipes, then 4ml below is accessed into two other EP pipes）, stay race glue sample.
2.6）Run glue checking.
2.6.1）2 pieces of PAGE glues of 0.75mm are run, all of sample is all added up, the dense segregation ratio of two pieces of glue wants phase
Just can be than more consistent with two pieces of glue.
2.6.2）Run glue sequentially：Negative control, positive control, excessively supernatant, post sample, the quick moulding 5min of 300V or so, 160V
Separate sample 50min or so.（Can also 300V, 30min, simply scheme no 160V good-looking.）
2.6.3）Coomassie brilliant blue staining.Ordinary stain 2h.
2.6.4）Decolourize.15min first is taken off with decolorising agent 1, then it is de- overnight with decolorising agent 2.
2.7）Purifying supernatant --- collect destination protein
2.7.1）The nickel post lived again is balanced again（I.e. plus Equilibration Buffer 3ml）.
2.7.2）Operation in repeat step 12, is only washed when simply cleaning with the imidazole concentration touched in step 12.
2.8）Glue is run again to verify and preserve；Current glue figure 160V, keeps.
2.9）Dialysis.
2.9.1）Prepare 2L dialyzates（Formula sees appendix 1）, and be put in -20 degree refrigerator precoolings but should not freeze.
2.9.2）Bag filter（Cut 10cm）10min is boiled with bag filter treatment fluid, MILIQ H are used2O is fully cleaned.
2.9.3）Bag filter is clamped with dialysis clamp（Bag filter is folded can press from both sides more firm）, the albumen that will be eluted
Sample is added thereto, and inserts the 500ml dialyzates of precooling in advance（20mM PBS+50 mM NaCl）In.Slight magnetic force under ice bath
Liquid is changed after inserting 4 ° of refrigerator 1.5h after stirring 20min.Dialysis 3 times.
2.10）Concentration.
2.10.1）The sample dialysed is placed in white boxes together with bag filter, with the polyethylene glycol of precooling
2000 solid entrappings.
2.10.2）To be removed in starchy polyethylene glycol after water suction after 30min, add new solid polyethylene glycol.
2.10.3）Every 10min observations once, once there is muddy stopping concentration immediately.
2.10.4）After bag filter is finished, clean, be stored in 4 ° of storages in 20% ethanol.
2.11）Ultrafiltration concentration, dialysis albumen.
2.11.1）The protein solution that 4ml Elution are obtained is added in super filter tube.
2.12）Trim.
2.12.1）7400g, 30min, 4 ° centrifugation, at the end of 4ml be changed into 1ml or so.4 times of concentration.Can select as needed
Different centrifugation times are selected, to reach different cycles of concentration.Wash-out night that can be several times（Elution）Concentrate together.
2.11.2）Add the protein storage liquid for needing to 4ml, be centrifuged.Repeating can be such that Elution liquid gradually changes
Protein storage liquid for needed for.Protein storage liquid typically uses the three of 20mM PBS+*mM NaCl or debita spissitudo and pH（Hydroxyl first
Base）Aminomethane（tris-HCl）Solution.If albumen has precipitation not reach predetermined concentration can add appropriate glycerine（1%-
5%）Hydrotropy.
2.13）Collect.Solution is collected into EP pipes from super filter tube, the composition of storing liquid has been marked, protein name,
Protein concentration（After measurement）, and prepare the date.
3）Prepare opiate receptor colloidal gold pad.
3.1）Acceptor gold mark treatment：The colloidal gold solution of 40 nm-50 nm or so is prepared by reducing process, color becomes
It is scarlet it is limpid after respectively three parts tested, it is 8.0 that first part of mol/L K2CO3 of use 0.1 adjusts PH, and it is 8.5 that second part is adjusted PH,
It is 9.0 that 3rd part is adjusted PH.Then 1 mg, 1.5 mg, 2.0 mg opiate receptors are separately added into the solution, are slowly added dropwise,
After continuing to stir 35 minutes, the TWEEN20 for adding final concentration of 3% BSA and final concentration of 1% continues stirring 10 minutes, so
4 DEG C afterwards, 12000 rpm are centrifuged 45 minutes, and careful supernatant of drawing is discarded；100ml is settled to again with colloidal gold solution, by 1ml
The ratio uniform that solution spreads 30 cm2 is layered on glass fibre or non-woven fabrics, is placed in drying room, 37 DEG C, is done under the conditions of humidity 25%
Dry 3 hours, it is made colloidal gold pad.
3.2）The treatment of sample pad：Glass fibre or non-woven fabrics are soaked in the phosphorus of the mol/L PH=7.4 of 50 ml 0.01
In phthalate buffer（Containing the BSA of final concentration 3%, 0.05% TWEEN20）In 1 hour, 37 DEG C drying, Vacuum Package, 4 DEG C guarantor
Deposit standby.
3.3）Morphine is coated with：Morphine solution is diluted to the BSA mixed liquors of carbonate buffer solution and final concentration of 3%
0.5mg/ml, 1mg/ml and 1.5mg/ml, are drawn onto nitrocellulose membrane coating buffer with a film machine, 37 DEG C of dryings 4 hours, 4 DEG C
Save backup.
3.4）Cutting assembling：
Loading pad, the opiate receptor conjugate pad of colloid gold label, the nitrocellulose membrane of morphine, sample suction pad are assembled into reaction
Plate, 4mm reagent strips are cut into cutting machine, after being installed, are loaded aluminium foil bag and are made finished product.
Detection method and result judge：50ul-100ul or so urines to be measured or saliva are taken on loading pad, about 1 is reacted
Minute or so, according to color reaction with the naked eye direct observed result, T lines and C lines to develop the color and be free of morphine in explanation sample simultaneously, such as
Fruit contains morphine in there was only C lines colour developing explanation sample, if C lines do not have appearance, explanation Product checking is invalid.
Parameter determination：Shown according to last testing result, reagent paper marks PH to be about 9.0 recently；Opiate receptor collaurum
The mg/100 ml colloidal gold solutions of labelled antibody optimised quantity 1.5, the optimum mark amount of morphine is 1 mg/ml, optimal collaurum
Working solution is 9.0 containing 0.01% gold chloride and 1% sodium citrate aqueous solution, and to add 3% BSA and 1% for PH
TWEEN20, calibrating Best Times are 30 seconds.
As long as human body sucked morphine, morphine will enter human body by intestines and stomach or blood circulation, and the change of morphine
Learning material will remain in a period of time in human body, and the former chemical substance or its metabolite of morphine will inhale in morphine
Presented in the saliva of anthropophagy person or in urine.According to this feature, drug addict determines series can be by testing staff's
Saliva or urine accurately detect whether once to suck certain type of morphine within a certain period of time.For example：Human body one
Denier sucked morphine, will continue that morphine residual is presented in 24 after hour, in the urine of this person, as long as the suction of this project
Malicious personnel determine morphine tests positive in series, you can judge that this person had the behavior for sucking morphine in 24 hours.
Meanwhile, medicinal health anti-epidemic department and other physical examination examinations, such as detection of military service personnel and entry and exit people
Member is checked, it is also possible to the measure series of personnel is sucked using the morphine of this project.
SEQUENCE LISTING
<110>Shao Xingkang knows bio tech ltd
<120>It is a kind of that the reagent of morphine, preparation method and application are sucked based on analgesics screening platform technology examination
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 400
<212> PRT
<213> Human adenovirus type 1
<400> 1
Met Asp Ser Ser Ala Ala Pro Thr Asn Ala Ser Asn Cys Thr Asp Ala
1 5 10 15
Leu Ala Tyr Ser Ser Cys Ser Pro Ala Pro Ser Pro Gly Ser Trp Val
20 25 30
Asn Leu Ser His Leu Asp Gly Asn Leu Ser Asp Pro Cys Gly Pro Asn
35 40 45
Arg Thr Asp Leu Gly Gly Arg Asp Ser Leu Cys Pro Pro Thr Gly Ser
50 55 60
Pro Ser Met Ile Thr Ala Ile Thr Ile Met Ala Leu Tyr Ser Ile Val
65 70 75 80
Cys Val Val Gly Leu Phe Gly Asn Phe Leu Val Met Tyr Val Ile Val
85 90 95
Arg Tyr Thr Lys Met Lys Thr Ala Thr Asn Ile Tyr Ile Phe Asn Leu
100 105 110
Ala Leu Ala Asp Ala Leu Ala Thr Ser Thr Leu Pro Phe Gln Ser Val
115 120 125
Asn Tyr Leu Met Gly Thr Trp Pro Phe Met Thr Ile Leu Cys Lys Ile
130 135 140
Val Ile Ser Ile Asp Tyr Tyr Asn Met Phe Thr Ser Ile Phe Thr Leu
145 150 155 160
Cys Thr Met Ser Val Asp Arg Tyr Ile Ala Val Cys His Pro Val Lys
165 170 175
Ala Leu Asp Phe Arg Thr Pro Arg Asn Ala Lys Ile Ile Asn Val Cys
180 185 190
Asn Trp Ile Leu Ser Ser Ala Ile Gly Leu Pro Val Met Phe Met Ala
195 200 205
Thr Thr Lys Tyr Arg Gln Gly Ser Ile Asp Cys Thr Leu Thr Phe Ser
210 215 220
His Pro Thr Trp Tyr Trp Glu Asn Leu Leu Lys Ile Cys Val Phe Ile
225 230 235 240
Phe Ala Phe Ile Met Pro Val Leu Ile Ile Thr Val Cys Tyr Gly Leu
245 250 255
Met Ile Leu Arg Leu Lys Ser Val Arg Met Leu Ser Gly Ser Lys Glu
260 265 270
Lys Asp Arg Asn Leu Arg Arg Ile Thr Arg Met Val Leu Val Val Val
275 280 285
Ala Val Phe Ile Val Cys Trp Thr Pro Ile His Ile Tyr Val Ile Ile
290 295 300
Lys Ala Leu Val Thr Ile Pro Glu Thr Thr Phe Gln Thr Val Ser Trp
305 310 315 320
His Phe Cys Ile Ala Leu Gly Tyr Thr Asn Ser Cys Leu Asn Pro Val
325 330 335
Leu Tyr Ala Phe Leu Asp Glu Asn Phe Lys Arg Cys Phe Arg Glu Phe
340 345 350
Cys Ile Pro Thr Ser Ser Asn Ile Glu Gln Gln Asn Ser Thr Arg Ile
355 360 365
Arg Gln Asn Thr Arg Asp His Pro Ser Thr Ala Asn Thr Val Asp Arg
370 375 380
Thr Asn His Gln Leu Glu Asn Leu Glu Ala Glu Thr Ala Pro Leu Pro
385 390 395 400
Claims (10)
1. a kind of reagent that morphine is sucked based on analgesics screening platform technology examination, it is characterised in that：The protein sequence of the reagent
As shown in sequence table SEQ NO.1；The reagent is that Ah skin's acceptor house of correction is obtained, for original series respectively to the 138th and
386 amino acids do point mutation.
2. a kind of kit that morphine is sucked based on analgesics screening platform technology examination, it is characterised in that：The kit includes
The opiate receptor conjugate pad being prepared into using the reagent described in claim 1, the opiate receptor conjugate pad is fine with nitric acid
Dimension film and sample suction pad are assembled into reaction plate, and the reaction plate is cut into reagent strip and is installed.
3. a kind of preparation method of the reagent that morphine is sucked based on analgesics screening platform technology examination, it is characterised in that：Including with
Lower step：
1）Build opiate receptor expression system；
2）138 mutation opiate receptors of expression；
3）Prepare opiate receptor colloidal gold pad.
4. the preparation side of a kind of reagent that morphine is sucked based on analgesics screening platform technology examination according to claim 3
Method, it is characterised in that：The step of building opiate receptor expression system includes：
1.1）Double digestion, by respectively by original series, 138 and 386 mutant clons enter PET32 expression vectors；
1.2）It is transformed into amplification in DH5 α competence bacteriums, upgrading grain；
1.3）Plasmid is transformed into expression bacterial strain, bacterium detection and conservation is chosen, the induced expression of bacterial strain such as Bl21 albumen is expressed.
5. the preparation side of a kind of reagent that morphine is sucked based on analgesics screening platform technology examination according to claim 4
Method, it is characterised in that：The step 1.3）Specifically include：
1.3.1）Expression bacterial strain is shaken up in LB culture mediums, IPTG, overnight induction is added；
1.3.2）The expression of 0SDS-PAGE electrophoresis detection destination proteins；
1.3.3）There to be the bacterial strain conservation of expression, record IPTG concentration ranges；
1.3.4）With the minimum induced expression bacterium of above-mentioned IPTG concentration ranges, overnight induction is used as inclusion body detection sample.
6. the preparation side of a kind of reagent that morphine is sucked based on analgesics screening platform technology examination according to claim 3
Method, it is characterised in that：The step of 138 mutation opiate receptors of expression, includes：
2.1）The expression bacterial strain planted of going bail for shakes bacterium；
2.2）Bacterium is overnight shaken during product in previous step is added into culture medium；
2.3）Product to previous step carries out ultrasonic treatment；
2.4）Take the product supernatant after ultrasonic treatment；
2.5）Purifying supernatant --- touch elution requirement；
2.6）Run glue checking；
2.7）Purifying supernatant --- collect destination protein；
2.8）Glue is run again to verify and preserve；
2.9）Dialysis；
2.10）Concentration；
2.11）Ultrafiltration concentration, dialysis albumen；
2.12）Trim；
2.13）Collect：Solution is collected into EP pipes from super filter tube, the composition of storing liquid, protein name, albumen has been marked
Concentration（After measurement）, prepare the date.
7. the preparation side of a kind of reagent that morphine is sucked based on analgesics screening platform technology examination according to claim 3
Method, it is characterised in that：The step of preparing opiate receptor colloidal gold pad includes：
3.1）Acceptor gold mark treatment：Colloidal gold solution is prepared by reducing process, is divided into three parts and is tested, distinguished in the solution
The opiate receptor of different quality is added dropwise over, stirring adds BSA and TWEEN20 and continues to stir, is then centrifuged for, and removes supernatant；With
Colloidal gold solution constant volume again, is uniformly layered on glass fibre or non-woven fabrics by a certain percentage, dries, and is made colloidal gold pad；
3.2）The treatment of sample pad：Glass fibre or non-woven fabrics are soaked in middle drying in phosphate buffer, Vacuum Package,
Save backup；
3.3）Morphine is coated with：Morphine solution is diluted respectively with carbonate buffer solution and BSA mixed liquors, will be coated with a film machine
Liquid is drawn onto nitrocellulose membrane, is dried, and 4 DEG C save backup；
3.4）Cutting assembling：By loading pad, the opiate receptor conjugate pad of colloid gold label, the nitrocellulose membrane of morphine, suction sample
Pad is assembled into reaction plate, and reagent strip is cut into cutting machine, after being installed, loads aluminium foil bag and is made finished product.
8. the preparation side of a kind of reagent that morphine is sucked based on analgesics screening platform technology examination according to claim 6
Method, it is characterised in that：Purifying supernatant --- touch comprising the concrete steps that for elution requirement：
The treatment of (2.5.1) nickel post：By in nickel post suction dead level analysis pipe, level pad is added after liquid flow therein is complete；
Be added to supernatant on equilibrated NI posts by (2.5.2), and will cross the sample of post and repeat loading 3 times, takes 20 μ l and crosses post
Sample afterwards, in case running glue；
(2.5.3) is respectively plus the imidazole gradient of different gradients elutes foreign protein, the liquid point of each loading above 1ml and below
1ml is collected in EP pipes respectively, in case running glue；
(2.5.4) plus elution buffer elute destination protein, stay race glue sample.
9. the preparation side of a kind of reagent that morphine is sucked based on analgesics screening platform technology examination according to claim 6
Method, it is characterised in that：Run comprising the concrete steps that for glue checking：
(2.6.1) is all added separately to all of sample in upper 2 pieces of PAGE glues；
(2.6.2) runs glue sequentially：Negative control, positive control, excessively supernatant, post sample, moulding simultaneously separate sample；
(2.6.3) coomassie brilliant blue staining；
Ordinary stain 2h；
(2.6.4) decolourizes.
10. a kind of application of the reagent that morphine is sucked based on analgesics screening platform technology examination, it is characterised in that：By claim
Reagent any one of 1-9 is used to detect human urine or saliva, so that it is determined that whether detected person sucks morphine.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101171022A (en) * | 2005-03-07 | 2008-04-30 | 罗彻斯特大学 | Compositions and methods for inhibiting G protein signaling |
| WO2010105236A1 (en) * | 2009-03-13 | 2010-09-16 | Allergan, Inc. | Immuno-based retargeted endopeptidase activity assays |
| CN103740741A (en) * | 2014-01-22 | 2014-04-23 | 北京工业大学 | HPV18 E and E7 fusion gene mutant as well as related biological material and coding protein thereof |
| WO2014118297A1 (en) * | 2013-01-30 | 2014-08-07 | Vib Vzw | Novel chimeric polypeptides for screening and drug discovery purposes |
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2017
- 2017-03-13 CN CN201710147233.8A patent/CN106908610B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101171022A (en) * | 2005-03-07 | 2008-04-30 | 罗彻斯特大学 | Compositions and methods for inhibiting G protein signaling |
| WO2010105236A1 (en) * | 2009-03-13 | 2010-09-16 | Allergan, Inc. | Immuno-based retargeted endopeptidase activity assays |
| WO2014118297A1 (en) * | 2013-01-30 | 2014-08-07 | Vib Vzw | Novel chimeric polypeptides for screening and drug discovery purposes |
| CN105102478A (en) * | 2013-01-30 | 2015-11-25 | 弗拉芒区生物技术研究所 | Novel chimeric polypeptides for screening and drug discovery purposes |
| CN103740741A (en) * | 2014-01-22 | 2014-04-23 | 北京工业大学 | HPV18 E and E7 fusion gene mutant as well as related biological material and coding protein thereof |
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