CN102002049B - Antigen and preparation method and application thereof - Google Patents
Antigen and preparation method and application thereof Download PDFInfo
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- CN102002049B CN102002049B CN2010105123777A CN201010512377A CN102002049B CN 102002049 B CN102002049 B CN 102002049B CN 2010105123777 A CN2010105123777 A CN 2010105123777A CN 201010512377 A CN201010512377 A CN 201010512377A CN 102002049 B CN102002049 B CN 102002049B
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Abstract
The invention discloses an antigen and a preparation method and application thereof. The antigen is a conjugate formed by connecting morphine-6-monoglutarate and a carrier protein through a covalent bond. The method for preparing the antigen comprises the following step of: conjugating the morphine-6-monoglutarate with the carrier protein so as to prepare the conjugate of the morphine-6-monoglutarate and the carrier protein, wherein the carrier protein is bovine serum albumin or hemocyanin. The antigen can stimulate an organism to generate a morphine antibody and specifically recognize the morphine and heroin. Mouse experiments prove that: a vaccine which takes the antibody as an active substance can prevent morphine relapse to a certain degree.
Description
Technical field
The present invention relates to a kind of antigen and preparation method thereof and application.
Background technology
Drug habit is great public health and the social concern of the healthy and social development of puzzlement global human, and the serious harm human body is healthy, spread disease, affect stabilizing and social development of family, causes huge financial loss to society.Show according to the report in 2006 of United Nations's drugs and crime affairs office, global all kinds of Drug abuse numbers reach 2.24 hundred million, and the financial loss that causes every year reaches 8,000 hundred million dollars.China's drug problem is revivable from the eighties, and situation is also very severe all the time, and by the end of the year 2005, the number of taking drugs that registers on the books is to reach 1,160,000.And according to statistics, in the newly-increased cases of infection of China's acquired immune deficiency syndrome (AIDS) in 2007, the 42%th, share virus infection and the propagation that syringe causes by the drug addict, cause great public health problem.Therefore, addictive drug abuse has become affect Chinese society Economic development, harm public health, threaten social harmony and a stable important factor, carries out the research of drug habit mechanism, seeks effective medicine for the treatment of habituation and has profound significance.
The addictive drug of whole world abuse mainly contains opiates (such as opium, morphine, heroine etc.), incitantia class (such as Cocaine, amphetamine) and halluoinogen (such as LSD, mescaline) at present.About the pharmacotherapy of opiate addiction mainly is for detoxifcation, Withrawal symptom and relapses and the treatment of keeping of Withrawal symptom.Main long-acting oral opioid drug, for example methadone, left-handed-acetylmethadol (LAAM) and the Lepetan of adopting of at present treatment.Although win initial success clinically, still there are a lot of queries.Problem is that Status In Heroin Addicts is appointed and is exposed to opiate, and may produce tolerance or Withrawal symptom to medicine.Another kind of methods for the treatment of is to stop heroine to arrive receptor binding site, such as Narlan and TREXUPONT.Although these antagonists can be used to treat heroin addiction, but there is certain limitation in their use, because in the situation that does not have exogenous opioid drug to exist, two kinds of antagonist non-activities, other also endogenous opioids medicine in the baffle simultaneously, thus impel the patient of long-term treatment that Negative Emotional is arranged.Except above-mentioned methods for the treatment of, as far back as the seventies, there are several researchs to propose to utilize autonomous immunologic mechanism to resist the effect of drug abuse (Spector, 1971; Berkowitz and Spector, 1972; Bonese etal, 1974)
Immunotherapy mainly is to use antibody with high specificity isolation medicine, and medicine is still existed in the blood system.So, the synthetic medicine of medicine-antibody can hinder medicine and enter hemato encephalic barrier, the effect of enhancing that so not only can the neutralizating medicine deposits yields, and can prevent that medicine from damaging central nervous system.In addition, when medicine freely passed through hemato encephalic barrier, the antibody of sustainable existence also can make medicine away from brain gradually in the body.1974, this vaccine technologies was employed first, and morphine-bovine serum albumin binding substances can reduce the effect (Bonese etal, 1974) of macaque heroine automedication.In addition, studies show that high heroine can reset into immunity and move ahead and be state, and can offset the effect of anti-heroine antibody.But the epoch of Bonese research also only are the initial stages of immunotherapy, and immunotherapy today after 20 years has had remarkable development.
Active immunity can produce the antibody of protectiveness by stimulating immune response, and the antibody of generation can be attacked organism and the poisonous substance with disease-related.It is by hindering or reduce the activity of exogenous material that these antibody work.
Summary of the invention
An object of the present invention is to provide a kind of compound.
Compound provided by the present invention, name is called morphine-6-glutaric acid monoester, and its chemical structural formula is as follows:
The method for preparing described compound also belongs to protection scope of the present invention.
The method for preparing morphine-6-glutaric acid monoester provided by the present invention comprises the steps:
1) Srm-Rhotaard is dissolved in the water, is 8.5 at pH value, temperature is under 37 ℃ and the condition that stirs, react suction filtration, collection solid matter 10 minutes;
2) with step 1) solid matter that obtains and Pyroglutaric acid, N, N-Dimethylamino pyridine and benzene mix, and refluxing extraction under 95 ℃ of conditions is removed benzene, collects residue;
3) with step 2) residue that obtains mixes with water, regulates pH value to 9, suction filtration, collection filtrate;
4) with step 3) the pH value of the filtrate that obtains transfers to 6, generates precipitation, and collecting precipitation namely obtains described compound.
Described step 1) proportioning of Srm-Rhotaard and water described in is the 1.3g Srm-Rhotaard: 30mL water;
The mass ratio that feeds intake of solid matter and Pyroglutaric acid described step 2) is: 1: 1.2; The time of described refluxing extraction is 4 hours.
Another object of the present invention provides a kind of antigen.
Antigen provided by the present invention is the conjugate that morphine-6-glutaric acid monoester and carrier proteins are connected to form by covalent linkage; Described covalent linkage is the amino amido linkage that forms in key carboxyl in the compound and the carrier proteins.
Described carrier proteins is human serum albumin, bovine serum albumin, hemocyanin, gamma-globulin, thyroglobulin, Parenogen, ovalbumin, Mianyang red corpuscle, flagellin or tetanus toxin.
Another object of the present invention provides the method for the described antigen of preparation.
The method of the described antigen of preparation provided by the present invention comprises the steps:
With morphine-6-glutaric acid monoester and carrier protein couplet, obtain the conjugate of morphine-6-glutaric acid monoester and carrier proteins.
Described method with morphine-6-glutaric acid monoester and carrier protein couplet comprises the steps:
1) morphine-6-glutaric acid monoester and carrier proteins are dissolved in the TE damping fluid, obtain I liquid;
2) 1-ethyl-3-3 dimethylamine propyl carbonization imines is dissolved in the TE damping fluid, obtains II liquid;
3) I liquid and II liquid are mixed, react, obtain the conjugate of morphine-6-glutaric acid monoester and carrier proteins.
The mass ratio that feeds intake of described morphine-6-glutaric acid monoester and carrier proteins and 1-ethyl-3-3 dimethylamine propyl carbonization imines is 5: 1: 2.4.
Described step 1) pH value of TE damping fluid is 8.0 in; Described step 2) pH value of TE damping fluid is 8.0 in; Described step 3) in, the temperature of reaction is 37 ℃, and the time of reaction is 2 hours; Described carrier proteins is human serum albumin, bovine serum albumin, hemocyanin, gamma-globulin, thyroglobulin, Parenogen, ovalbumin, Mianyang red corpuscle, flagellin or tetanus toxin.
The antibody that is prepared by described antigen also belongs to the scope of protection of the invention.
Another purpose of the present invention provides a kind of product that the inhibition drug habit relapses function that has.
Provided by the present invention have suppress the product that drug habit relapses function, its activeconstituents is described antigen.
Described medicine is opioid drug; Described opioid drug is morphine and/or heroine.
Described product is vaccine.
Another purpose of the present invention provides described antigen and has the application that suppresses in the product that drug habit relapses function in preparation.
Described medicine is opioid drug; Described opioid drug is morphine and/or heroine.
Described product is vaccine.
Antigen provided by the present invention can stimulate body to produce morphine antibody, and can specific recognition morphine and heroine.Vaccine take this antigen as active substance confirms to prevent to a certain extent that morphine from relapsing again through mouse experiment.
Description of drawings
Fig. 1 is artificial antigen morph-BSA/KLH building-up reactions schematic diagram.
Fig. 2 is the measurement result of morph-KLH antigen active immunity mouse antibodies titre.
Fig. 3 is the measurement result of morph-KLH antigen active immunity rat antibody titers.
Fig. 4 is the specific assay result of the antibody of morph-KLH antigen preparation.
Fig. 5 is that morph-KLH vaccine active immunity mouse prevents again Relapse behavior test result of morphine.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Srm-Rhotaard (available from company limited of Qinghai Pharmaceutic Plant); Pyroglutaric acid (available from sigma company, catalog number is G3806); N, N-Dimethylamino pyridine (available from sigma company, catalog number is Sigma-D5640); Bovine serum albumin (BSA, available from sigma company, catalog number is Sigma-A7906); Hemocyanin (KLH, available from sigma company, catalog number is Sigma-H7017); 1-ethyl-3-3 dimethylamine propyl carbonization imines (EDC is available from sigma company); BABL/C mouse (available from Department Of Medicine, Peking University's Experimental Animal Center); Freund's complete adjuvant (available from sigma-aldrich company); Freund's incomplete adjuvant (available from sigma-aldrich company); Goat-anti mouse horseradish peroxidase (available from mountain gold bridge Bioisystech Co., Ltd in Beijing); Goat-anti rat horseradish peroxidase (available from middle mountain gold bridge Bioisystech Co., Ltd, catalog number is ZB2305); TMB test kit (matching the bio tech ltd of speeding available from Beijing); SD rat (available from Department Of Medicine, Peking University's Experimental Animal Center); Morphine (available from Qinghai Pharmaceutic Plant).
The preparation of embodiment 1, morphine-6-glutaric acid monoester-bovine serum albumin antigen (morph-BSA)
Small molecules (less than the small molecules of 10KDa) medicine must carrier proteins of coupling, just can cause immune response.Therefore to produce immune response, at first will carry out the synthetic of small-molecule drug derivative, namely haptenic synthetic.
The synthetic route chart of antigen as shown in Figure 1, concrete steps are as follows:
1, preparation morphine-6-glutaric acid monoester
(1) the 1.3g Srm-Rhotaard is dissolved in the 30mL water, drips ammoniacal liquor to pH8.5, react 10min under temperature is 37 ℃ and the condition that stirs, suction filtration gets the off-white color solid, and vacuum-drying 24h obtains 1g morphine free alkali.
(2) the 1g morphine free alkali that step (1) is obtained, 1.2g Pyroglutaric acid mix with the N-Dimethylamino pyridine of catalytic amount, add 30mL benzene, and reflux is 4 hours under 95 ℃ of conditions, and the decompression distilled desolventizes benzene, collects residue; The mass ratio of described morphine free alkali and Pyroglutaric acid is: 1: 1.2.
(3) with step 2) residue that obtains adds water 50mL, transfers pH to 9 with the NaOH solution of 1mol/L, and suction filtration is collected filtrate;
(4) with step 3) filtrate that obtains transfers pH to 6 with the HCl solution of 1mol/L again, is precipitated, suction filtration, collecting precipitation, vacuum-drying obtains 100mg off-white color solid, identifies the gained white solid by ESI-MS.
The demonstration of ESI-MS analytical results, the exact mass of resulting white solid is 399.17, molecular weight is 399.14.M/e:399.17 (100%), 400.17 (25.5%), 401.17 (4.2%), its structural formula is as follows:
2, morphine-6-glutaric acid monoester and carrier proteins carry out coupling
(1) get the morphine that 100mg step 1 obtains-6-glutaric acid monoester and 20mg bovine serum albumin and be dissolved in the TE damping fluid (pH 8.0), fully concussion makes its dissolving, obtains I liquid;
(2) (1-ethyl-3-(3-dimethylaminopropyl) EDC) fully is dissolved in the TE damping fluid (pH 8.0), obtains II liquid to get 48mg 1-ethyl-3-3 dimethylamine propyl carbonization imines;
(3) I liquid is dropwise added while shaking in the II liquid, in 37 ℃ of shaking tables, react 2h, obtain the conjugate that morphine-6-glutaric acid monoester and bovine serum albumin are connected to form by covalent linkage; Described covalent linkage is the amido linkage that carboxyl in morphine-6-glutaric acid monoester and the amino in the bovine serum albumin form; The mass ratio that feeds intake of described morphine-6-glutaric acid monoester and bovine serum albumin and 1-ethyl-3-3 dimethylamine propyl carbonization imines is 5: 1: 2.4.
With Sephadex post separation coupling product and unconjugated morphine-6-glutaric acid monoester small molecules, the dialysis purifying is dialysed solution 3 days in phosphate buffered saline buffer (PBS), changes liquid twice every day, changes liquid 200ml at every turn.The low dose of packing of dialysis product saves backup in-80 ℃.
The preparation of embodiment 2, morphine-6-glutaric acid monoester-hemocyanin antigen (morph-KLH)
The synthetic route chart of antigen as shown in Figure 1, concrete steps are as follows:
1, preparation morphine-6-glutaric acid monoester
Identical with the step 1 of experiment 1.
2, morphine-6-glutaric acid monoester and carrier proteins carry out coupling
(1) get the morphine that 100mg step 1 obtains-6-glutaric acid monoester and 20mg hemocyanin and be dissolved in the TE damping fluid (pH 8.0), fully concussion makes its dissolving, obtains I liquid;
(2) (1-ethyl-3-(3-dimethylaminopropyl) EDC) fully is dissolved in the TE damping fluid (pH 8.0), obtains II liquid to get 48mg 1-ethyl-3-3 dimethylamine propyl carbonization imines;
(3) I liquid is dropwise added while shaking in the II liquid, in 37 ℃ of shaking tables, react 2h, obtain the conjugate that morphine-6-glutaric acid monoester and hemocyanin are connected to form by covalent linkage; Described covalent linkage is the amido linkage that carboxyl in morphine-6-glutaric acid monoester and the ammonia key in the hemocyanin form; The mass ratio that feeds intake of described morphine-6-glutaric acid monoester and hemocyanin and 1-ethyl-3-3 dimethylamine propyl carbonization imines is 5: 1: 2.4.
With Sephadex post separation coupling product and unconjugated morphine-6-glutaric acid monoester small molecules, the dialysis purifying was dialysed solution 3 days in phosphate buffered saline buffer, change liquid twice every day, changed liquid 200ml at every turn.The low dose of packing of dialysis product saves backup in-80 ℃.
The application of embodiment 3, antigen
One, active immunity mouse and serum antibody titer analysis
1, utilizes morph-KLH antigen Dispersal risk
Get BABL/C mouse (20-25g) as laboratory animal.Mouse is raised in the controlled rearging cage of temperature (21 ℃-24 ℃) and light (08:00 opens the pass with 20:00), and the drinking-water of freely ingesting was raised 5 days, and body weight is 25-28g approximately.Divide three groups at random, every group of each 8 mouse carry out respectively following processing: A, injection adjuvant+KLH to three groups of mouse; B, injection adjuvant+morph-KLH (50 μ g/ animal/0.2ml, namely the mixture of every injected in mice 0.2ml adjuvant and morph-KLH contains 50 μ g morph-KLH in the 0.2ml mixture); C, injection adjuvant+morph-KLH (100 μ g/ animal/0.2ml are every injected in mice 0.2ml adjuvant+moroh-KLH, contain 100 μ gmorph-KLH in the 0.2ml injection liquid).Injection system is taked subcutaneous injection, and adjuvant adopts Freund's complete adjuvant, and medicine is equally divided into 4 injections.Blood is got in later on docking in 10 days, and is centrifugal, gets serum, namely obtains morph-KLH antibody, deposits for-80 ℃, to be measured.After 21 days, for the second time injection, dosage is with for the first time identical, and adjuvant adopts Freund's incomplete adjuvant, and after 31 days, blood is got in docking again, and is centrifugal, gets serum, namely obtains morph-KLH antibody, deposits for-80 ℃.
(preparation method of 50 μ g/ animals/0.2ml) is: the morph-KLH antigen original solution (calculating with the KLH amount) of getting the concentration 5.26mg/mL that 76 μ l obtain by embodiment 2 filters with sterilizing filter adjuvant+morph-KLH in the above-mentioned treatments B, the Freund's complete adjuvant that adds 76 μ l, then add 1.5ml physiological saline, adopt communicating pipe, fully emulsified, until static profit is not stratified after the emulsification, every injected in mice 0.2ml.
(preparation method of 100 μ g/ animals/0.2ml) is: the morph-KLH antigenic solution (calculating with the KLH amount) of getting the concentration 5.26mg/mL that 152 μ l obtain by embodiment 2 filters with sterilizing filter adjuvant+morph-KLH among the above-mentioned processing C, the Freund's complete adjuvant that adds 152 μ l mL, then add 1.3ml physiological saline, adopt communicating pipe, fully emulsified, until static profit is not stratified after the emulsification, every injected in mice 0.2ml.
2, antibody effect detection
Adopt indirect Elisa method to analyze the antibody titers of immune animal.Main method is as follows: with the coated Elisa enzyme plate of morph-BSA antigen (2 μ g/100 μ l) ,-4 ℃ of overnight incubation.The diluent that employing contains with 0.5%Tween20 washs plank 3 times, pats dry, and adds serum, and serum diluting multiple is 1: 100,1: 1000,1: 10000,1: 100000, hatches 1h for 37 ℃.Wash plate, pat dry, add goat-anti mouse horseradish peroxidase (1: 2000), hatch 1h for 37 ℃.Wash plate, pat dry, the colour developing of TMB test kit, wavelength 450nm detects.Contrast is dilution buffer liquid.
Detected result is as follows: A and B show respectively for the first time and the antibody titers of immune mouse for the second time among Fig. 2.In twice immunologic process, the specific antibody titre increases gradually, and increases with vaccine dose, and titre also increases gradually, and the doses effect relation is arranged.B shows among Fig. 2, after injecting for the second time, and the vaccine of 50 μ g and 100 μ g, antibody titers all can reach 1: 100000.Show that vaccine has the ability that stimulates body the morph-KLH antigen-drug to be produced immunne response.
Two, active immunity rat and serum antibody titer analysis
1, utilizes morph-KLH antigen Dispersal risk
Get SD rat (220-250g) as laboratory animal.In the controlled rearging cage of temperature (21 ℃-24 ℃) and light (08:00 opens with 20:00 and closes), the drinking-water of freely ingesting was raised 5 days with rat feeding, and body weight is (250-280g) approximately.Divide two groups at random, every group of each 6 rat carry out respectively following processing: A, injection adjuvant+KLH to two groups of rats; B, injection adjuvant+morph-KLH (100 μ g/ animal/0.4ml, namely the mixture of every injected in mice 0.4ml adjuvant and morph-KLH contains 100 μ g morph-KLH in the 0.4ml mixture).Injection system is taked subcutaneous injection, and dosage is 100 μ g//0.4ml, and adjuvant adopts Freund's complete adjuvant, and medicine is equally divided into 4 injections, and sublingual vein is got blood after 10 days, and is centrifugal, gets serum, namely obtains morph-KLH antibody, deposits for-80 ℃, to be measured.Thereafter, the injection in 20 days of every minor tick is once injected 3 times continuously again, and per injection is after complete 10 days, and sublingual vein is got blood, and is centrifugal, gets serum, namely obtains morph-KLH antibody, deposits for-80 ℃, to be measured.Rear three injection adjuvants adopt Freunds incomplete adjuvant.
(100 μ g/ preparation method only/0.4ml) is: the morph-KLH antigenic solution (calculating with the KLH amount) of getting the concentration 5.26mg/mL that 114 μ l obtain by embodiment 2 filters with sterilizing filter adjuvant+morph-KLH in the above-mentioned treatments B, the Freund's complete adjuvant that adds 114 μ l, then add 2.2ml physiological saline, adopt communicating pipe, fully emulsified, until static profit is not stratified after the emulsification, every rat injection 0.4ml.
2, antibody effect detection
Adopt indirect Elisa method to analyze the antibody titers of immune animal.Main method is as follows: with the coated Elisa enzyme plate of morph-BSA antigen (2 μ g/100 μ l) ,-4 ℃ of overnight incubation.The diluent that employing contains with 0.5%Tween20 washs plank 3 times, pats dry, and adds serum, and serum diluting multiple is 1: 10,1: 100,1: 1000,1: 10000,1: 100000, hatches 1h for 37 ℃.Wash plate, pat dry, add goat-anti rat horseradish peroxidase (1: 2000), hatch 1h for 37 ℃.Wash plate, pat dry, the colour developing of TMB test kit, wavelength 450nm detects.Contrast is dilution buffer liquid.
Detected result as shown in Figure 3, after four immunity, maximum antibody titers reaches 1: 100000.Show that vaccine has the ability that the morph-KLH antigen-drug is produced immunne response that stimulates.
Three, antibodies specific detects
Estimate anti-morphine antibodies specific and adopt competitive Elisa method.It is the rat blood serum after the 4th immunity in the above-mentioned steps two that antibodies specific detects the serum that adopts.
Detection method is as follows: this detection method is except before hatching rat blood serum, add respectively first outside competition thing Lepetan, Narlan, TREXUPONT, heroine, morphine monomethyl ether and nalorphine and the morphine, all the other steps all method with the antibody titers of the indirect Elisa method analysis immune animal of above-mentioned steps two are identical.Add competition thing Lepetan, Narlan, TREXUPONT, heroine, morphine monomethyl ether and nalorphine and the concentration of morphine in system and be 0.0001 μ M-1000 μ M, be specially: 0.0001 μ M, 0.001 μ M, 0.01 μ M, 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M.
B represents that sample adds the absorbance of the sample well of competitive material.B0 represents not add the absorbance of competitive material positive control.
Detected result is as follows: the antibody that the E demonstration produces among Fig. 4 can not identified and dissimilar other opium compounds of morphine structure, such as Lepetan, Narlan, TREXUPONT, morphine monomethyl ether and nalorphine.The antibody that A and C demonstration produce among opposite Fig. 4 can be identified morphine and heroine.Calculate IC50 value (i.e. half inhibiting rate, when just referring to that combination rate reaches 50%, the amount of inhibition) by regression curve, morphine and heroine are respectively 0.7523 μ M (B among Fig. 4) and 158.48 μ M (D among Fig. 4).These data sheet understand that morph-KLH antigen can stimulate body to produce anti-morphine antibody, and this antibody can not only can also be identified heroine for specific identification morphine.
Heroine is the analogue that phenol 3 hydroxyls and 6 hydroxyls by the acetylize morphine obtain.Heroine by deacetylation fast in blood metabolism be 6-monoacetylmorphine (6-MAM), morphine, morphine 3 glucuronides (M-3-G) and morphine 6 glucuronides (M-6-G).
Four, the result for the treatment of of the vaccine take morph-KLH antigen as activeconstituents
The instrument of conditioned place preference training and testing is the conditioned place preference case that consists of by three casees: two side rooms and an intermediate chamber.Three Room by floor movably separately, and the floor is different.With the BABL/C mouse as laboratory animal.
The preparation method of the vaccine take morph-KLH antigen as activeconstituents as:
The morph-KLH antigenic solution (calculating with the KLH amount) of getting the concentration 5.26mg/mL that 76 μ l obtain by embodiment 2 filters with sterilizing filter, the Freund's complete adjuvant that adds 76 μ l, then add 1.5ml physiological saline, adopt communicating pipe, fully emulsified, until static profit is not stratified after the emulsification, obtaining composition is (50 μ g//0.2ml of adjuvant+morph-KLH, be every injected in mice 0.2ml adjuvant and morph-KLH mixture, contain 50 μ g morph-KLH in the 0.2ml mixture) vaccine.
The basic value test: this test is adopted without partially design.First day (D1), mouse is put into by intermediate chamber, lets alone in three Room freely movable 15 minutes, its residence time in each chamber of computer synchronous recording.The experimental animal inclusive criteria be in each case the residence time respectively the mouse of the 25%-75% of total time.
Conditioned place preference training (conditioned place preference, CPP): second day to the nine days (D2-D9), D2, D4, D6, D8 give morphine (10mg/kg) and put into and mix coyote hole 45min; D3, D5, D7, D9 gives physiological saline (1ml/kg) and puts into the non-medicine-chest 45min that mixes.Mouse is put back to rearging cage afterwards.
CPP obtains test: obtained test on the 1st day, method is tested with basic value.
What preference mark (CPP score) was defined as mouse mixes the coyote hole residence time and non-difference of mixing the coyote hole residence time.
Test design: mouse is raised in the controlled rearging cage of temperature (21 ℃-24 ℃) and light (08:00 opens the pass with 20:00), and the drinking-water of freely ingesting was raised 5 days, and body weight is (25-28g) approximately.Raise after 5 days, carry out 8 days CPP of morphine training, after morphine CPP obtains, after the test in the 10th day, have a preference for mark at random minute two groups (mean value that makes each group have a preference for mark equals 0) by CPP.One group gives adjuvant+KLH, another group give adjuvant+morph-KLH (50 μ g/ only/0.2ml), injection system is taked subcutaneous injection, adjuvant adopt Fu Shi fully/Freunds incomplete adjuvant, medicine is equally divided into 4 injections (injecting per injection interval 20 days three times).Within this time, the mouse training of disappearing, last immunity was tested after 10 days, and mouse gives the 5mg/kg morphine after 24 hours, carries out at once the CPP test.
The result shows that two groups of mouse all can form Conditioned Place Preference of Morphine (the preference score value is seen Fig. 5), and two groups of preference score values are respectively 237.17 ± 44.25 and 215.03 ± 27.01; After immunity and the training of disappearing, two groups of mouse morphine condition Place Preference memories are eliminated, and the preference score value is 30.7724 ± 56.42 and 12.96 ± 66.80.After 24 hours, dose morphine is lighted, and only gives adjuvant+KLH group, the memory of mouse condition Place Preference is aroused again, and the preference score value is 407.80 ± 124.32, and gives adjuvant+morph-KLH group, the memory of mouse condition Place Preference is not aroused, and the preference score value is 33.6 ± 44.43.Two groups of statistical analysis have significant difference (P＜0.05).
Claims (11)
1. compound, its chemical structural formula is as follows:
2. prepare the method for compound claimed in claim 1, comprise the steps:
1) Srm-Rhotaard is dissolved in the water, is 8.5 in the pH value, temperature is under 37 ℃ and the condition that stirs, react suction filtration, collection solid matter 10 minutes;
2) with step 1) solid matter that obtains and Pyroglutaric acid, N, N-Dimethylamino pyridine and benzene mix, and refluxing extraction under 95 ℃ of conditions is removed benzene, collects residue;
3) with step 2) residue that obtains mixes with water, regulates pH value to 9, suction filtration, collection filtrate;
4) with step 3) the pH value of the filtrate that obtains transfers to 6, generates precipitation, and collecting precipitation namely obtains described compound.
3. method according to claim 2 is characterized in that:
Described step 1) proportioning of Srm-Rhotaard and water described in is the 1.3g Srm-Rhotaard: 30ml water;
The mass ratio that feeds intake of solid matter and Pyroglutaric acid described step 2) is: 1: 1.2; The time of described refluxing extraction is 4 hours.
4. an antigen is the conjugate that compound claimed in claim 1 and carrier proteins are connected to form by covalent linkage; Described covalent linkage is the amido linkage that carboxyl in the described compound and the amino in the carrier proteins form;
Described carrier proteins is human serum albumin, bovine serum albumin, hemocyanin, gamma-globulin, thyroglobulin, Parenogen, ovalbumin, flagellin or tetanus toxin.
5. prepare the method for antigen claimed in claim 4, comprise the steps:
With the compound shown in the claim 1 and carrier protein couplet, obtain the conjugate of morphine-6-glutaric acid monoester and carrier proteins; Compound shown in the claim 1 is denoted as morphine-6-glutaric acid monoester;
Described method with morphine-6-glutaric acid monoester and carrier protein couplet comprises the steps:
1) morphine-6-glutaric acid monoester and carrier proteins are dissolved in the TE damping fluid, obtain I liquid;
2) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide is dissolved in the TE damping fluid, obtains II liquid;
3) I liquid and II liquid are mixed, react, obtain the conjugate of morphine-6-glutaric acid monoester and carrier proteins.
6. method according to claim 5 is characterized in that:
The mass ratio that feeds intake of described morphine-6-glutaric acid monoester and carrier proteins and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide is 5: 1: 2.4;
Described step 1) the pH value of TE damping fluid is 8.0 in; Described step 2) the pH value of TE damping fluid is 8.0 in; Described step 3) in, the temperature of reaction is 37 ℃, and the time of reaction is 2 hours; Described carrier proteins is human serum albumin, bovine serum albumin, hemocyanin, gamma-globulin, thyroglobulin, Parenogen, ovalbumin, flagellin or tetanus toxin.
7. the antibody that is prepared by antigen claimed in claim 4.
8. one kind has the product that the inhibition drug habit relapses function, and its activeconstituents is antigen claimed in claim 4, and described product is vaccine, and described medicine is opioid drug.
9. product according to claim 8, it is characterized in that: described opioid drug is morphine and/or heroine.
10. antigen claimed in claim 4 has the application that suppresses in the product that drug habit relapses function in preparation, and described product is vaccine, and described medicine is opioid drug.
11. application according to claim 10 is characterized in that: described opioid drug is morphine and/or heroine.
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| CN103149355B (en) * | 2013-01-29 | 2016-05-04 | 浙江迪恩生物科技股份有限公司 | A kind of Test paper card that utilizes sandwich method to detect Brucella abortus antibody |
| US9694069B2 (en) * | 2013-03-14 | 2017-07-04 | Alere San Diego, Inc. | 6-acetylmorphine analogs, and methods for their synthesis and use |
| CN103408656B (en) * | 2013-07-26 | 2014-12-03 | 北京大学 | Morphine/heroin vaccine formed by covalently binding hapten and carrier, and application thereof |
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| US6210677B1 (en) * | 1993-01-29 | 2001-04-03 | Robert C. Bohannon | Method to reduce the physiologic effects of drugs on mammals |
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| US6210677B1 (en) * | 1993-01-29 | 2001-04-03 | Robert C. Bohannon | Method to reduce the physiologic effects of drugs on mammals |
| EP1767221A2 (en) * | 2004-07-07 | 2007-03-28 | Instituto Nacional De Psiquiatria Ramon De La Fuente Muniz | Preparation and use of a bivalent vaccine against morphine-heroine addiction |
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| 张延军.吗啡-载体蛋白交联物的鉴定及抗吗啡单克隆抗体的药理学评价.《中国优秀博硕士学位沦为全文数据库（硕士）医药卫生科技辑》.2007,第5-41页. * |
| 李琳.吗啡疫苗及抗体制备的实验研究.《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》.2004,第11-54页. * |
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