A nicotine protein conjugate, preparation method thereof and application in vaccines

Abstract

The present invention discloses a nicotine protein conjugate, a preparation method thereof and an application in a vaccine, and belongs to the field of immunotechnology. The nicotine protein conjugate is prepared by the following method: dissolving a carrier protein in N, N-dimethylformamide, vortexing and ultrasonicating until there are no large particles, adding water, and continuing to ultrasonicate until there are no particles; then adding an emulsifier and a reducing agent, stirring at room temperature; after stirring evenly, adding an antigen, stirring and reacting at room temperature; after the reaction is completed, adding PBS buffer to reduce the concentration of N, N-dimethylformamide; centrifuging the reaction solution to obtain a nicotine protein conjugate. The vaccine prepared by the nicotine protein conjugate has a higher antibody titer, is not addictive, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of immunotechnology, and specifically relates to a nicotine protein conjugate, a preparation method thereof and an application thereof in vaccines. Background Art

[0002] Nicotine is the main component of tobacco and a key factor in smokers' addiction. According to the World Health Organization, more than 1 billion people become addicted to smoking each year, and 5 million people die from tobacco-related diseases. The death toll is rising year by year. Smoking accounts for the highest proportion of all preventable causes of death, and it can cause a variety of diseases, such as lung cancer, respiratory diseases, heart diseases, reproductive system diseases, etc. Even smokeless tobacco contains a large amount of nicotine, and long-term use can also lead to nicotine addiction. Although it is well known that smoking is harmful to health, its addictive nature makes it impossible for users to stop smoking, and a series of withdrawal syndromes will occur if smoking is suddenly stopped due to quitting smoking.

[0003] Currently, the U.S. Food and Drug Administration has approved six smoking cessation drugs, five of which are nicotine replacement products (gum, patch, lozenge, nasal spray and inhaler). Each drug can release nicotine. Because nicotine is a factor that causes tobacco dependence, it can reduce nicotine withdrawal symptoms and cravings for cigarettes when people quit smoking to some extent. Some other methods to prevent tobacco use include non-nicotine treatments (bupropion, varenicline) and other drugs (clonidine, desmoking). However, current drug and non-drug smoking cessation treatments have limited effects and have not been widely promoted to the majority of smokers (Solberg et al., 2001; Thorndike et al., 1998). Despite progress in behavioral and drug treatments in recent years, the vast majority of smokers who try to quit smoking have failed (Maurer et al., 2005). For most smokers, quitting smoking is not just a matter of choice, but a struggle. Tobacco use is often intertwined with daily life and has a physiological, psychological and social reinforcing effect. Many factors, including media coverage, cultural and social aspects of tobacco use, combined with the addictive nature of tobacco, make it difficult to quit smoking.

[0004] The core of nicotine addiction mechanism lies in its action on neurotransmitter systems in the brain, especially on the dopamine system. When nicotine enters the brain, it binds to nicotinic acetylcholine receptors (nAChRs) on the surface of neurons (Benowitz, 1996). These receptors are widely distributed in the brain and are involved in a variety of physiological functions. When nicotine binds to nAChRs, it causes sodium and calcium channels to open, neuronal membranes to depolarize and action potentials to occur, leading to the release of neurotransmitters. Nicotine's effect on the dopamine system plays a key role in the addiction mechanism. Nicotine stimulates dopamine release in the brain's reward circuit, especially in two key areas, the ventral tegmental area (VTA) and the nucleus capsid (NAc). Nicotine not only stimulates dopamine secretion, but also inhibits monoamine oxidase B, which is important for the catabolism of dopamine, resulting in smokers having an average dopamine concentration much higher than non-smokers (Cerny and Cerny, 2009). Dopamine plays an important role in reward and pleasure, and when dopamine levels rise, it produces a sense of pleasure. The pleasure that smokers experience when smoking makes them more susceptible to addictive behavior.

[0005] Long-term smoking may lead to neuroadaptive changes in the brain, such as regulation of nAChRs expression and function, and reduced sensitivity of dopamine receptors. These changes can make individuals tolerant to nicotine, requiring more nicotine to obtain the same sense of pleasure, thereby exacerbating addictive behavior. At the same time, when quitting smoking, the decrease in dopamine levels may lead to withdrawal symptoms such as anxiety, depression, and irritability, making it more difficult to quit smoking.

[0006] The main effect of nicotine vaccine is to prevent smokers from feeling the pleasure brought by nicotine. Its mechanism of action is based on the fact that after nicotine molecules enter the human body through the lungs, anti-nicotine antibodies bind to nicotine molecules, and the antibody molecules cover the nicotine molecules, so that the binding of nicotine to its receptors is damaged or no longer possible. The minimum molecular weight of a molecule that causes a specific immune response is 10kDa, and nicotine molecules are too small (molecular weight is 167 Da) to cause an immune response. Therefore, people who smoke frequently do not produce antibodies to it. Nicotine vaccine is formed by combining the nicotine molecule itself or a structurally related hapten with an immunogenic carrier protein to form an immunogenic immunogen.

[0007] In terms of nicotine vaccine research, the following types of nicotine vaccines have entered preclinical stages, and they are all composed of conjugates and adjuvants. Nicotine is linked to a carrier protein so that it can be recognized by the immune system and cause an immune response, while the adjuvant can enhance the production of antibodies against nicotine.

[0008] The first vaccine is TA-NIC, which was developed by Xenova and is now funded by the British pharmaceutical company Celtic. The vaccine is a nicotine vaccine with tert-butyl acid attached to the N1 position. The recombinant cholera toxin-B subunit is used as the carrier protein of the nicotine vaccine. Its effectiveness in quitting smoking has been proven through several different experiments, but the results of several experiments have not yet been announced.

[0009] The second vaccine is Nic-Qb (NIC002), which was developed by Cytos. It uses virus-like particles (VLPs) as carrier proteins and couples the 3' position of the nicotine pyrrolidine ring to the virus-like particles of bacteriophage Qβ. The capsid protein of bacteriophage Qβ is recombinantly expressed in Escherichia coli, and 180 subunits self-assemble to form highly ordered VLPs with a diameter of about 25-30 nm. Due to the repeated antigen presentation and T helper cell epitopes on the VLP, the Qβ-based vaccine has strong immunogenicity and can achieve a 100% response rate in humans after a single immunization. In the Nic-Qb vaccine, approximately 585 nicotine molecules are covalently coupled to each Qβ VLP.

[0010] The third vaccine is NicVax, which was developed by Nabi Pharmaceuticals. It uses the amino group modified at the 3' position of nicotine and the Pseudomonas aeruginosa exoprotein A modified with succinic anhydride to obtain a protein conjugate under the EDC condensation agent. Injection of this vaccine in a rat model can induce high titers of nicotine-specific antibodies, reduce the extent of nicotine distribution to the brain, and reduce the physiological and behavioral effects of nicotine. In addition, human trials have shown that nicotine vaccines are safe and well tolerated. The immunogenicity of the vaccine is dose-related, and the highest dose causes an increase in antibody levels within the expected efficacy range. NicVAX's two Phase III clinical trials have not yet been published. Although the first and second phase trials were successful, both trials failed to show the efficacy of the vaccine compared with the control group.

[0011] The fourth vaccine is Niccine, which is developed by an independent Swedish pharmaceutical company. It is a vaccine that is modified at the 6th position of nicotine and coupled with tetanus toxoid. The results of preclinical studies on this vaccine have not yet been disclosed. However, Tonstad et al. conducted a Phase II trial to evaluate the preventive effect of Niccine on smoking relapse. The results showed that Niccine was not effective in preventing smoking relapse, and had no effect on withdrawal symptoms, exhaled carbon monoxide levels, relapse time, and withdrawal success rate.

[0012] In the nicotine vaccines that have been reported and have entered clinical trials, the coupling of nicotine haptens to carrier proteins usually adopts a direct condensation reaction in a buffer. However, the coupling reaction in a buffer usually takes several days, and the protein is prone to precipitation or cross-linking after the conformation changes. In addition, the coupling reaction uses the amino or carboxyl sites on the protein for coupling, so the sites are highly random and difficult to control accurately. Summary of the invention

[0013] The present invention provides a new method for coupling a protein carrier with a hapten in an organic solvent, using the disulfide bonds of the protein itself, breaking the disulfide bonds by adding a reducing agent to expose the thiol group, and modifying the nicotine hapten with a maleimide group. The click reaction between the thiol group and maleimide is used to construct a nicotine protein conjugate with a protein as a carrier, thus laying the foundation for the preparation of a nicotine vaccine.

[0014] The technical solution of the present invention is as follows:

[0015] The present invention provides a method for preparing a nicotine protein conjugate, comprising the following steps:

[0016] The carrier protein was dissolved in N,N-dimethylformamide, vortexed and sonicated until there were no large particles, water was added, and sonication was continued until there were no particles; then an emulsifier and a reducing agent were added, and the mixture was stirred at room temperature; after stirring evenly, the antigen was added, and the mixture was stirred at room temperature for reaction; after the reaction was completed, PBS buffer was added to reduce the concentration of N,N-dimethylformamide; the reaction solution was centrifuged to obtain a nicotine protein conjugate.

[0017] In the above-mentioned method for preparing the nicotine protein conjugate, each raw material is selected from the following quantities:

[0018] Carrier protein 1~5mg, N,N-dimethylformamide 0.5~2mL, water 50~200μL, emulsifier 0.5~2μL, reducing agent 1~3μL, antigen 1~6mg.

[0019] In practical applications, the amount of each raw material can be increased or decreased according to the actual scale and the above-mentioned proportions.

[0020] In the above-mentioned method for preparing the nicotine protein conjugate, the carrier protein is selected from freeze-dried carrier protein; and the protein is selected from CRM197 protein or BSA protein.

[0021] In the above method for preparing nicotine protein conjugate, the emulsifier is selected from Tween, preferably Tween-20.

[0022] In the above method for preparing the nicotine protein conjugate, the reducing agent is trimethyl phosphine.

[0023] In the above method for preparing the nicotine protein conjugate, the antigen is selected from nicotine, nicotine hapten 1, nicotine hapten 2 or nicotine hapten 3; preferably nicotine hapten 2.

[0024] The structure of the above-mentioned nicotine hapten 1 is as follows:

[0025]

[0026] The structure of the above-mentioned nicotine hapten 2 is as follows:

[0027]

[0028] The structure of the above nicotine hapten 3 is as follows:

[0029]

[0030] In the above method for preparing the nicotine protein conjugate, the PBS buffer is selected from a PBS buffer having a concentration of 50 mM and a pH of 6.5.

[0031] In the above-mentioned method for preparing the nicotine protein conjugate, after adding the PBS buffer, the concentration of N,N-dimethylformamide is reduced to below 5%.

[0032] In the above-mentioned method for preparing the nicotine protein conjugate, the stirring time at room temperature is selected from 0.5 to 5 hours.

[0033] The present invention provides a nicotine protein conjugate prepared by the above method.

[0034] The present invention provides the use of the nicotine protein conjugate in the preparation of a smoking cessation vaccine.

[0035] The present invention provides a nicotine vaccine, which contains the above-mentioned nicotine protein conjugate; and may also contain other components required for preparing the vaccine, such as physiological saline, neutral liposomes, etc.

[0036] The beneficial effects of the present invention are:

[0037] The present invention provides a new method for coupling a protein carrier with a hapten in an organic solvent, using the disulfide bond of the protein itself, breaking the disulfide bond by adding a reducing agent (trimethylphosphine) to expose the thiol group, modifying the nicotine hapten with a maleimide group, and then using the click reaction of the thiol group and the maleimide to construct a nicotine protein conjugate with the protein as a carrier. The present invention uses click chemistry to optimize the coupling step, and can develop a nicotine vaccine. Click chemistry has the advantages of high specificity, fast reaction rate, wide tolerance of reaction conditions, high biocompatibility of reaction components, and multiple selectable reaction types. In this method, the protein conformation will become more flexible and easier to be acquired by antigen presenting cells (APCs), so that the prepared nicotine vaccine has a higher antibody titer. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is the MALDI-TOF-MS spectrum of the unmodified CRM protein;

[0039] Figure 2 is the MALDI-TOF-MS spectrum of CRM-N (4) conjugate;

[0040] Figure 3 is the MALDI-TOF-MS spectrum of unmodified BSA protein;

[0041] Figure 4 is the MALDI-TOF-MS spectrum of BSA-N (24) conjugate;

[0042] Figure 5 is the MALDI-TOF-MS spectrum of BSA-N (35) conjugate;

[0043] Figure 6 For monitoring of mouse body weight during the vaccination regimen;

[0044] Figure 7 The titer of anti-nicotine IgG antibodies elicited by nicotine vaccines;

[0045] Figure 8 is the anti-nicotine IgG subtype antibody titer elicited by nicotine vaccine. DETAILED DESCRIPTION

[0046] Before the present invention was made, it was based on the following ideas:

[0047] In an organic solvent, the disulfide bonds of the protein are broken, the secondary structure of the protein is unfolded, and the small molecule nicotine is combined with the unfolded protein using click chemistry (Michael addition reaction) between thiol and maleimide. Since the reaction is carried out in an organic solvent, and small molecules such as nicotine also have good solubility in organic solvents, the reaction speed is greatly increased.

[0048] The present invention provides a method for synthesizing nicotine hapten 2, and the reaction formula thereof is as follows:

[0049]

[0050] The synthesis steps are as follows:

[0051] 100 mg of nicotine hapten 1 (1 eq) and 54.3 mg of succinic anhydride (1.2 eq) were dissolved in 10 mL of anhydrous methanol, and then 200 μL of DIPEA (2 eq) was added. The reaction was stirred until TLC (DCM / MeOH, 10 / 1, v / v) showed that the starting material disappeared (about 4 h). The reaction solution was evaporated to dryness and purified by column (petroleum ether / ethyl acetate system) to obtain 78 mg of nicotine hapten 2 with a yield of 53.8%.

[0052] The H NMR and C NMR spectra of nicotine hapten 2 are as follows:

[0053] 1 H NMR (500 MHz, MeOD) δ 8.17–8.13 (m, 1H, H-8), 8.07 (s, 1H, H-7), 7.58 (d, J = 2.3 Hz, 1H, H-6), 4.11 (d, J = 10.6, 6.3, 4.3 Hz, 1H, H-9 A ),4.04 (dt, J = 10.3, 5.3 Hz, 1H, H-9 B ), 3.74 (t, J = 8.3 Hz, 1H, H-5), 3.57–3.48 (m, 2H, H10), 3.48–3.40 (m, 1H, H-2 A , 2.72 (q, J = 8.9 Hz, 1H, H-2 B ),2.50–2.32 (m, 7H, H-11, H-12, H-1), 2.32–2.23 (m, 1H, H-4 a ), 2.11–1.93 (m,3H, H3, H-4 b ). 13C NMR (126 MHz, MeOD) δ 141.49, 138.31, 121.22, 70.18, 69.94,68.72, 57.97, 57.97, 38.44, 37.95, 37.71, 34.53, 30.87, 30.62, 21.10.

[0054] The present invention provides a method for synthesizing nicotine hapten 1, and the reaction formula thereof is as follows:

[0055]

[0056] The synthesis steps are as follows:

[0057] 9.25 mmol of bis(pinacol)diboron (0.75 eq), 320 μmol of 1,10-phenanthroline (25.9 eq) and 499 μmol of methoxy(cyclooctadiene)iridium dimer (40.5 eq) were dissolved in 20 mL of anhydrous n-heptane, and 1.98 mL of nicotine (compound 7, 1 equiv) was added dropwise at 90°C. Stirring was continued until TLC thin layer chromatography (DCM / MeOH, 10 / 1, v / v) showed that the starting material disappeared (about 14 h), and the reaction solution was evaporated to dryness and concentrated to obtain compound 8.

[0058] 3.55 g of compound 8 (1 eq) was dissolved in 35 mL of anhydrous THF, and 98.5 mmol H2O2 (3 eq, concentration 30%) and 24.6 mmol glacial acetic acid (2 eq) were slowly added to the above solution, stirring while adding until TLC thin layer chromatography (DCM / MeOH, 10 / 1, v / v) showed that the starting material disappeared (about 4 h). The reaction solution was poured into a separatory funnel, then diluted and extracted with ethyl acetate, first washed with saturated sodium thiosulfate, and the aqueous phase was back-extracted with ethyl acetate 2-3 times, then washed with saturated brine 2-3 times, and the aqueous phase was back-extracted with ethyl acetate 2-3 times, and finally dried with anhydrous sodium sulfate, filtered, evaporated and concentrated to obtain compound 9.

[0059] 2.2 g of compound 9 (1 eq) and 14.1 g of Cs2CO3 (3.5 eq) were dissolved in 20 mL of anhydrous DMF. After stirring at room temperature for 20 minutes, 5.65 g of 2-(Boc-amino)ethyl bromide (2 equiv) was added to the mixture and heated to 90°C in an oil bath until TLC (DCM / MeOH, 7 / 1, v / v) showed that the starting material disappeared (about 11 h). The DMF in the reaction solution was evaporated to dryness, and the product was filtered out with THF, concentrated and evaporated to dryness, and purified by column to obtain 3.43 g of compound 10 with a yield of 86.2%.

[0060] 3 g of compound 10 (1 eq) and 9 mL of trifluoroacetic acid (3 eq) were dissolved in 45 mL of DCM solution and stirred for reaction until TLC (DCM / MeOH, 10 / 1, v / v) showed that the starting material disappeared (about 1.5 h). The DMF in the reaction solution was evaporated and purified by column (dichloromethane / methanol system) to obtain 2 g of compound 1 with a yield of 97%.

[0061] The present invention provides a method for synthesizing nicotine hapten 3, and the reaction formula thereof is as follows:

[0062]

[0063] The synthesis steps are as follows:

[0064] 1.74 g maleimidopropionic acid (1.5 eq) and 5.22 g HATU (2 eq) were dissolved in 80 mL anhydrous DMF and stirred at room temperature for 10 min. 1.5 g nicotine hapten 1 (1 eq) was added to the above mixed reaction solution, and then 598 μL DIPEA (0.5 eq) was added dropwise to the reaction solution until TLC thin layer chromatography (DCM / MeOH, 10 / 1, v / v) showed that the starting material disappeared (about 6 h). The DMF in the reaction solution was evaporated and purified by column (dichloromethane / methanol system) to obtain 1.25 g nicotine hapten 3 with a yield of 49.8%.

[0065] The nuclear magnetic resonance hydrogen and carbon spectra of nicotine hapten 3 are as follows:

[0066] 1 H NMR (500 MHz, MeOD) δ 8.34 (d, J = 2.6 Hz, 1H, H-9), 8.29 (s, 1H,H-8), 7.67 (t, J = 2.3 Hz, 1H, H-7), 6.72 (d, J = 1.4 Hz, 2H, H-14, H-15), 4.39 (t, J = 8.9 Hz, 1H, H-5), 4.14 (hept, J = 5.0 Hz, 2H, H-10), 3.83 (dt, J= 12.2, 6.4 Hz, 1H, H-2 A ), 3.75 (t, J = 6.8 Hz, 2H, H-13), 3.55 (t, J = 5.5Hz, 2H, H-11), 3.31–3.24 (m, 1H, H-2 B ), 2.75 (s, 3H, H-1), 2.56 (dq, J =12.8, 6.8 Hz, 1H, H-4A ), 2.47 (t, J = 6.7 Hz, 2H, H-12), 2.40–2.31(m,1H, H-4 B ), 2.34-2.23 (m, 2H, H-3). 13 C NMR (126 MHz, MeOD) δ 141.16, 139.20, 133.83,120.65, 120.65, 69.36, 66.68, 55.69, 55.45, 37.86, 37.13, 35.91, 34.20,33.71, 30.54, 30.29, 20.77.

[0067] In the present invention, the freeze-dried BSA protein is prepared by the following method: take 20 μL of 50 mg / mL BSA protein, add 100 μL of 10% imidazole (adjusting the pH to 7.2 with HCl) and 880 μL of water, freeze in a -80°C refrigerator for 0.5 h, and freeze-dry overnight with a freeze dryer.

[0068] In the present invention, the lyophilized CRM197 protein was prepared by the following method: 91 μL of 11 mg / mL CRM197 protein was taken, 100 μL of 10% imidazole (pH adjusted to 7.2 using HCl) and 810 μL of water were added, the mixture was frozen in a -80°C refrigerator for 0.5 h, and lyophilized overnight using a lyophilizer.

[0069] The present invention carries out the coupling reaction of hapten and protein in an organic solvent, first dissolving the lyophilized protein in an organic solvent, then adding trimethyl phosphide to break the disulfide bond of the protein, and then adding the hapten modified by maleimide to carry out a click reaction. In the process of preparing lyophilized BSA protein, the present invention initially tried a BSA protein concentration of 10 mg / mL, however, we found that too low an initial protein concentration would affect the lyophilization effect, so a concentration of 50 mg / mL was finally adopted. In order to improve the solubility, we tried a variety of cosolvents, including sucrose, cyclodextrin, and imidazole with pH adjusted by acetic acid. The results show that imidazole with pH adjusted by hydrochloric acid has the best lyophilization effect. A series of attempts were also made to break the disulfide bond of the protein in an organic solvent. DMSO solvent and TCEP reagent were initially selected, but no successful results were obtained. Later, we replaced the reagent for breaking the disulfide bond of the protein with trimethyl phosphide, and added an appropriate amount of water in the reaction, successfully breaking the disulfide bond of the protein. The final reaction conditions were to first dissolve the freeze-dried protein in 1 mL of DMF, vortex and sonicate to dissolve the protein, then add 10% water for sonication, then add 0.1% Tween-20 and trimethylol phosphine, react for 30 minutes, and finally add an antigen that can modify the maleimide group for reaction.

[0070] Other materials used in the present invention, if not otherwise stated, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way. Example 1

[0071] The preparation steps of nicotine protein conjugate CRM-N (4) are as follows:

[0072] Dissolve 3.5 mg of freeze-dried CRM197 protein in 1 mL of DMF, vortex, and sonicate at 200 W power until no large particles appear. After adding 100 μL of water, continue to sonicate the reaction solution until no particles appear. Add 1 μL of Tween-20 and 2 μL of trimethyl phosphine to the reaction solution. After stirring at room temperature for 30 minutes, add 1 mg of nicotine hapten 3. After stirring at room temperature for 5 hours, add 19 mL of PBS buffer (50 mM, pH 6.5) to the reaction solution to reduce the concentration of DMF to less than 5%. The reaction solution was transferred to a 15 mL 30 kDmwco Amicon ultrafiltration tube and centrifuged for 30 min; then 13 mL PBS buffer (50 mM, pH 6.5) was added and centrifuged at 4500 g for 30 min at 20 °C; then 13 mL PBS buffer (50 mM, pH 6.5) was added and centrifuged at 4500 g for 30 min at 20 °C; then 13 mL PBS buffer (10 mM, pH 6.5) was added and centrifuged at 4500 g for 30 min at 20 °C; finally, the liquid in the ultrafiltration tube was transferred to a 2 mL centrifuge tube, and the ultrafiltration tube was rinsed twice with 200 μL PBS buffer (10 mM, pH 6.5), and the rinse solution was added to the above centrifuge tube to obtain the nicotine protein conjugate stock solution. MALDI-TOF mass spectrometry analysis showed (such as Figure 1 and Figure 2 ), the number of nicotine on each CRM197 molecule is 4, named CRM-N (4). Example 2

[0073] The preparation steps of nicotine protein conjugate BSA-N (24) are as follows:

[0074] Dissolve 3 mg of freeze-dried BSA protein in 1 mL of DMF, vortex, and sonicate at 200 W power until no large particles appear. After adding 100 μL of water, continue to sonicate the reaction solution until no particles appear. Add 1 μL of Tween-20 and 2 μL of trimethyl phosphine to the reaction solution. After stirring at room temperature for 30 minutes, add 4.8 mg of nicotine hapten 3. After stirring at room temperature for 5 hours, add 19 mL of PBS buffer (50 mM, pH 6.5) to the reaction solution to reduce the concentration of DMF to less than 5%. The reaction solution was transferred to a 15 mL 30 kDmwco Amicon ultrafiltration tube and centrifuged for 30 min; then 13 mL PBS buffer (50 mM, pH 6.5) was added and centrifuged at 4500 g for 30 min at 20 °C; then 13 mL PBS buffer (50 mM, pH 6.5) was added and centrifuged at 4500 g for 30 min at 20 °C; then 13 mL PBS buffer (10 mM, pH 6.5) was added and centrifuged at 4500 g for 30 min at 20 °C; finally, the liquid in the ultrafiltration tube was transferred to a 2 mL centrifuge tube, and the ultrafiltration tube was rinsed twice with 200 μL PBS buffer (10 mM, pH 6.5), and the rinse solution was added to the above centrifuge tube to obtain the nicotine protein conjugate stock solution. MALDI-TOF mass spectrometry analysis showed (such as Figure 3 and Figure 4 ), the number of nicotine on each BSA molecule is 24, named BSA-N (24). Example 3

[0075] The preparation steps of nicotine protein conjugate BSA-N (35) are as follows:

[0076] Dissolve 2 mg of freeze-dried BSA protein in 1 mL of DMF, vortex, and sonicate at 200 W power until no large particles appear. After adding 100 μL of water, continue to sonicate the reaction solution until no particles appear. Add 1 μL of Tween-20 and 2 μL of trimethyl phosphine to the reaction solution. After stirring at room temperature for 30 minutes, add 6 mg of nicotine hapten 3. After stirring at room temperature for 5 hours, add 19 mL of PBS buffer (50 mM, pH 6.5) to the reaction solution to reduce the concentration of DMF to less than 5%. The reaction solution was transferred to a 15 mL 30 kD mwcoAmicon ultrafiltration tube and centrifuged for 30 min; 13 mL PBS buffer (50 mM, pH 6.5) was added and centrifuged at 4500 g for 30 min at 20 °C; then 13 mL PBS buffer (50 mM, pH 6.5) was added and centrifuged at 4500 g for 30 min at 20 °C; then 13 mL PBS buffer (10 mM, pH 6.5) was added and centrifuged for 30 min; finally, the liquid in the ultrafiltration tube was transferred to a 2 mL centrifuge tube, and the ultrafiltration tube was rinsed twice with 200 μL PBS buffer (10 mM, pH 6.5), and the rinse solution was added to the above centrifuge tube to obtain the nicotine protein conjugate stock solution. MALDI-TOF mass spectrometry analysis showed (such as Figure 3 and Figure 5 ), the number of nicotine on each BSA molecule is 35, named BSA-N (35).

[0077] The nicotine protein conjugate stock solution in the above example was mixed with physiological saline, MPL and QS-21 liposomes for vaccine preparation. The concentrations of CRM-N (4) stock solution, BSA-N (24) stock solution and BSA-N (35) stock solution were 5 mg / mL, 2 mg / mL and 1 mg / mL, respectively; MPL and QS-21 liposomes were purchased from Chengdu Anyikang Company, China, with concentrations of 0.215 mg / mL and 2.222 mg / mL, respectively.

[0078] The vaccine formulations are shown in Table 1:

[0079] Table 1

[0080] Grouping Seedlings Nicotine content (μg / mL) CRM-N(4) vaccine 499μL of saline + 698μL of neutral liposome MPL + 34μL of QS-21 + 269μL of antigen, vortex mix for 30s 20.0 BSA-N(24) vaccine 535 μL of saline + 698 μL of neutral liposome MPL + 34 μL of QS-21 + 233 μL of antigen, vortex mix for 30 seconds 40.0 BSA-N(35) vaccine 328 μL of saline + 605 μL of neutral liposome MPL + 29 μL of QS-21 + 338 μL of antigen, vortex mix for 30 seconds 39.9 Comparison 3 mL 10 mM PBS buffer, pH 7.4 none

[0081] The experimental mice were adaptively fed to 6-8 weeks of age, and restrained once a day for three days before vaccination (restraint means stroking the back of the neck of the mouse every day to make it accustomed so that it will not be too manic when the vaccine is officially injected). The mice were divided into 9 groups (5 female mice and 5 male mice in each group). A total of 20 mice (10 female and 10 male mice) in the negative control group were treated with 0.1 mL PBS only on the 1st, 15th and 29th days. The experimental group mice were subcutaneously injected in the back of the neck on 0d, 14d, and 28d, respectively, and the mouse antigen dose was 2μg per mouse each time (the sample concentrations were 20μg / mL, 40μg / mL, and 39.9μg / mL, respectively). Serum was collected at 0d, 13d, 27d, and 42d (blood was collected from the eyeball, 100 μL, stored in a 1.5mL sterile EP tube, centrifuged at room temperature for 4h or at 4°C overnight, and the supernatant was stored in a -80°C refrigerator) for ELISA detection of antibody titers.

[0082] Meanwhile, mice were observed daily and monitored for signs of vaccine-related toxicity, including changes in gait, posture, and behavior. As part of basic toxicity-related measurements, mice were weighed every three days for 8 weeks starting on day 0 (first vaccination).

[0083] ELISA antibody titer detection can be performed using conventional methods. The present invention provides a detection method as follows:

[0084] (1) Antigen coating: dilute the coating solution BSA-nicotine (3.4 mg / mL) with 1×PBS (pH=7.4) to 1 μg / mL, add 3.5 μL BSA-nicotine to 11 mL 1×PBS (pH=7.4), and coat with 100 μL / well. Place the ELISA plate with the sealing film in a 4°C refrigerator and incubate overnight.

[0085] (2) Blocking: Take out the coated ELISA plate and restore it to room temperature, then pour off the coating solution. Use a plate washer to wash the ELISA plate, add 250 μL of PBST washing solution to each well, and wash three times.

[0086] (3) Add blocking solution: Add 200 μL of PBS solution containing 2% BSA to each well, seal with sealing film, and incubate at room temperature for 2 hours.

[0087] (4) Sample dilution: Dilute the serum to be tested by using a diluent (PBST solution containing 1% BSA, 1× PBS / 2% BSA diluted with 1× PBST) in a gradient dilution ratio of 1:400 (2 μL serum plus 398 μL diluent), 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, and 1:51200.

[0088] (5) Incubate samples: Pour off the blocking solution, add 100 μL / well of diluted serum, and incubate at room temperature for 2 h.

[0089] (6) Incubation with secondary antibody: Repeat the washing step in (2). Dilute HRP-labeled goat anti-mouse IgG (H+L), IgG1, IgG2a, IgG2b and IgG3 at a dilution ratio of 1:5000, 1:10000, 1:8000, 1:8000, and 1:5000, respectively, add 100 μL / well, and incubate at room temperature for 1 hour.

[0090] (7) TMB color development: Repeat the washing steps in (2) for 5 times, add TMB color development solution, 100 μL / well, and incubate in the dark for 10 min.

[0091] (8) Stop the color reaction: Add 1 M HCl to stop the color reaction, 100 μL / well.

[0092] (9) Plate reading: Read OD within 15 minutes 450 value.

[0093] The test results are as follows:

[0094] 1. Mouse weight monitoring results

[0095] like Figure 6 As shown, the weight of all mice did not decrease significantly, indicating that the nicotine vaccine prepared by the present invention has no toxicity.

[0096] 2. Antibody titer test results

[0097] (1) IgG antibody titer

[0098] Figure 7 The titers of anti-nicotine IgG antibodies elicited by nicotine vaccines are shown. Figure 7 It can be seen that BSA-N (24) and BSA-N (35) induced a strong immune response. After the second booster immunization, the IgG titer reached 5×10 5 After the final boost, the IgG titers of the two BSA-coupled vaccines were similar, up to 15×10 5 However, CRM-N (4) did not induce significant IgG production. This shows that the amount of nicotine conjugated is the key to the immune effect of the vaccine.

[0099] (2) IgG subtype antibody titer

[0100] Figure 8 The titers of anti-nicotine IgG subtype antibodies elicited by nicotine vaccines are shown. Figure 8It can be seen that the prepared BSA-N (24) and BSA-N (35) mainly induce the production of IgG1, and the amount of IgG3 is relatively small. It can be seen that the vaccines prepared by the present invention are mainly based on Th1 type antibody response.

[0101] The conditioned place preference experimental apparatus (Ningbo Anlai Instrument Co., Ltd., Anilabv1.02-CPP, mice) is a three-chamber CPP system automatically controlled by a computer. There is a shuttle door between the black and white boxes and the middle box. The bottom pattern of the left box is a grid pattern, and the surrounding walls are black. The bottom pattern of the right box is a grid pattern with round holes, and the surrounding walls are white. The walls of the middle box are white and the bottom surface is smooth. The size of the middle box is different. The bottom texture of the black and white boxes (17.38cm×13.5cm×15cm) helps mice further distinguish between the two environments. The middle compartment is neutral (9.8cm×13.5cm×15cm). The CPP apparatus consists of three boxes, and the PC is equipped with an automatic monitoring system. The rewarding effect of nicotine was evaluated using the CPP paradigm, which used previous studies and was modified to a certain extent (Maldonado, 1997).

[0102] The BSA-N (24) group mice, the BSAN (35) group mice with higher antibody titers, and 10 control group mice were selected for CPP experiments.

[0103] Pretreatment stage: The experiment began after the mice had adapted to the environment in the SPF animal room for three days. The skin on the back of the mouse's neck was gently rubbed with an injection needle every day, and then the mouse was placed in the middle box of the CPP instrument and allowed to move freely between the three equipment boxes. After 3 minutes, the mouse was taken out and put back into the rat cage. This process lasted for 3 days.

[0104] Experimental basal value: The test of mouse CPP basal value began one day after the end of pretreatment. Each mouse was placed in the middle box of the CPP instrument and moved freely among the three boxes for 15 minutes. The computer software automatically recorded the time the mouse spent in each room and determined the initial preference. Mice that stayed in a specific box for more than 540 seconds were screened out. The basal value was measured for three days.

[0105] Modeling phase: During this period, the middle chamber was blocked, the animals could not move between the three chambers, and nicotine (0.5 mg / kg, sc) was used for modeling. Starting at 8:00 am every day, the module mice were subcutaneously injected with nicotine, and the control mice were subcutaneously injected with an equal amount of saline, and immediately placed in the companion box after injection. At 15:00 pm, all mice were subcutaneously injected with saline into the non-companion medicine box, and the modeling time was 20 min. The modeling process lasted for 5 days, and the mice were returned to the mouse cage at the end of each experiment (Kota et al, 2007).

[0106] Test phase: 24 hours after the last modeling treatment, the partition of the middle box was removed and the mice were allowed to freely pass through the three chambers. The test time was 15 minutes, and the time the mice stayed in each chamber was recorded. The CPP score was expressed as the time in the companion box minus the initial time after nicotine injection. Positive numbers indicated preference for the companion box, while negative numbers indicated dislike for the companion box. A score equal to or close to zero indicated no preference for either party. If the CPP score was ≥50 seconds, it indicated successful modeling.

[0107] The test results are shown in Table 2:

[0108] Table 2

[0109] vaccine Total number of mice Number of addictions Addiction Rate BSA-N(24) 8 2 25% BSA-N(35) 10 2 20% Negative control 9 3 33.3%

[0110] As shown in Table 2, the addiction rate of the nicotine vaccine prepared by the present invention is lower than that of the control group, indicating that the nicotine vaccine of the present invention is not addictive.

[0111] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a nicotine protein conjugate, characterized in that: Here are the steps: 3 mg of freeze-dried BSA protein was dissolved in 1 mL of N,N-dimethylformamide, vortexed, and sonicated at 200 W power until no large particles appeared. After adding 100 μL of water, the reaction solution was sonicated again until no particles appeared. 1 μL of Tween-20 and 2 μL of trimethylphosphine were added to the reaction solution. After stirring at room temperature for 30 minutes, 4.8 mg of nicotine hapten 3 was added. After stirring at room temperature for 5 hours, 19 mL of PBS buffer with a pH of 6.5 and a concentration of 50 mM was added to the reaction solution to reduce the concentration of N,N-dimethylformamide to less than 5%. The reaction solution was transferred to an ultrafiltration tube and centrifuged for 30 minutes. Then 13 mL of PBS buffer with a pH of 6.5 and a concentration of 50 mM was added, and centrifuged at 4500 g for 30 minutes at 20°C. Then 13 mL of PBS buffer with a pH of 6.5 and a concentration of 50 mM was added, and centrifuged at 4500 g for 30 minutes at 20°C. Then 13 mL PBS buffer with a pH of 6.5 and a concentration of 10 mM was centrifuged at 4500 g for 30 min at 20°C; finally, the liquid in the ultrafiltration tube was transferred to a 2 mL centrifuge tube, and the ultrafiltration tube was rinsed twice with 200 μL of PBS buffer with a pH of 6.5 and a concentration of 10 mM, and the rinse liquid was added to the above centrifuge tube to obtain a nicotine protein conjugate stock solution; or, 2 mg of freeze-dried BSA protein was dissolved in 1 mL of N,N-dimethylformamide, vortexed, and sonicated at 200 W power until no large particles appeared. After adding 100 μL of water, the reaction solution was sonicated again until no particles appeared. 1 μL of Tween-20 and 2 μL of trimethylphosphine were added to the reaction solution. After stirring at room temperature for 30 minutes, 6 mg of nicotine hapten 3 was added. After stirring at room temperature for 5 hours, 19 mL of PBS buffer with a pH of 6.5 and a concentration of 50 mM was added to the reaction solution to reduce the concentration of N,N-dimethylformamide to less than 5%. The reaction solution was transferred to an ultrafiltration tube and centrifuged for 30 minutes. 13 mL of PBS buffer with a pH of 6.5 and a concentration of 50 mM was added, and centrifuged at 4500 g for 30 minutes at 20°C. Then 13 mL of PBS buffer with a pH of 6.5 and a concentration of 50 mM was added, and centrifuged at 4500 g for 30 minutes at 20°C. Then 13 mL PBS buffer with a pH of 6.5 and a concentration of 10 mM, centrifuged for 30 minutes; finally, the liquid in the ultrafiltration tube was transferred to a 2 mL centrifuge tube, and the ultrafiltration tube was rinsed twice with 200 μL of PBS buffer with a pH of 6.5 and a concentration of 10 mM, and the rinse liquid was added to the above centrifuge tube to obtain a nicotine protein conjugate stock solution; The structure of the nicotine hapten 3 is as follows: 。 2. The nicotine protein conjugate prepared by the method of claim 1.

3. A nicotine vaccine, characterized in that: The vaccine contains the nicotine protein conjugate according to claim 2.

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