CN102552901B - Application of fullerene derivative to prepare gene transmission vector - Google Patents
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Abstract
The invention provides a fullerene derivative, which is a fullerol shown by a general formula C60OxHy (y is more than 10 and less than or equal to x, and x is more than or equal to y and less than 50) or a fullerene carboxyl derivative shown by a general formula C60(C(COOH)2)n (n is an integral number of between 1 and 4), wherein the diameter range of nano-particles of the fullerene derivative in a solvent is between 1 and 400nm. The invention also provides application of the vaccine adjuvant of the fullerene derivative in the field of vaccines, and the vaccine adjuvant of the fullerene derivative has high safety, can remarkably improve the immunogenicity of the vaccine through different immune ways, and has great application prospect.
Description
Technical field
The present invention relates to a kind of fullerene derivate, specifically, relate to richness and strangle alcohol granule C
60o
xh
y(10 < y≤x < 50) and fullerene carboxy derivatives C
60(C (COOH)
2)
nthe vaccine adjuvant of (integer that n is 1-4) and this fullerene derivate, bacterin preparation and its application in vaccine field, belong to biotechnology and vaccine field.
Background technology
In prevention and treatment major disease, especially aspect infectious disease, vaccine has been brought into play extremely important effect.In the research and development process of vaccine, mainly the design optimization by antigen and vaccine carrier or adjuvant obtains vaccine safely and effectively.At present, the adjuvant that unique approval is used is clinically aluminium adjuvant, and it can promote the humoral immunity level of body preferably, in the prevention process of disease, has brought into play important function.Yet aluminium adjuvant has suppressed the cellular immune level of body to a certain extent, it causes certain inflammation and anaphylaxis at injection part potential energy simultaneously, and these have all limited the extensive use of aluminium adjuvant in clinical to a certain extent.The traditional vaccine adjuvant of research and development is because the defect of safety or effectiveness aspect is difficult to drop into clinical at present.
In immunoreactive generating process, antigen-presenting cell is dendritic cell (Dendritic cells especially, DC) bringing into play pivotal role, by the activation of antigen-presenting cell, cellular immunization and the humoral immune reaction of body are fast and effeciently occurred.Therefore, the research and development of DC vaccine adjuvant become the emphasis that current people pay close attention to.
In recent years, the fast development of nanotechnology has infiltrated into every field.Nano material is because its particle diameter is little, specific surface area presents greatly the not available novel characteristics of block materials.Nano material is easily carried out finishing processing simultaneously, thereby realizes different objects.This injects new vitality by the research and development that are vaccine carrier or adjuvant.
Fullerene C
60be a kind of globular molecule of the nanoscale being formed by carbon atom, there are unique physicochemical properties, at biomedical and material science, there is great application prospect.Because fullerene itself belongs to lyophobic dust, this has greatly limited its application at biomedical sector.The success of water-soluble fullerene and derivant thereof in recent years synthesizes its application assurance is provided.Simultaneously fullerene derivate can be by being agglomerated into nanoparticles with the interaction of self or other materials in water environment system, and its safety issue is resolved.In numerous nano materials, fullerene derivate is metal fullerol (Gd@C especially
82(OH)
22) and richness strangle alcohol (C
60o
xh
y) demonstrate good anti-tumor activity (number of patent application: 200610152170.7,200710175456.1).Research discovery metal fullerol and Fu Le alcohol are mainly brought into play its tumor-inhibiting action by strengthening the cell immune response of mice body simultaneously, and metal fullerol can be induced the maturation of dendritic cell, and fullerene derivate has good biological safety (Ying Liu, Fang Jiao, Yang Qiu, et al.Biomaterials, 2009, 30:3934-3945.Ying Liu, Fang Jiao, Yang Qiu, et al.Nanotechnology, 2009, 20:415102.De Yang, Yuliang Zhao, Hua Guo, et al.ACS Nano, 2010, 4:1178-1186.).
Summary of the invention
The inventor finds in to the research process of fullerene derivate, and it can be used as adjuvant, significantly improves cellular immunization and the humoral immunity level of mice body.Therefore the object of the present invention is to provide a kind of fullerene derivate, on its surface, introduce hydrophilic group, as hydroxyl (OH), carboxyl (COOH) etc., can solve the problem of fullerene poorly water-soluble, for its application lays the foundation.Another object of the present invention is to provide described fullerene derivate vaccine adjuvant and bacterin preparation, and this fullerene derivate is in the application in vaccine field, it can strengthen the immunogenicity of vaccine, can solve the defect of traditional adjuvant aspect safety and/or effectiveness.
The object of the invention is to be achieved through the following technical solutions:
On the one hand, a kind of fullerene derivate provided by the invention is at the carrier for the preparation of gene delivery and/or for improving the application of the immunogenic medicine of vaccine.
Further, described fullerene derivate is the fullerene carboxy derivatives of being strangled alcohol or being represented with formula 2 by the richness of following molecular formula 1 expression:
Formula 1:C
60o
xh
y, wherein, 10 < y≤x < 50; Preferably, x=y=10-50; X=y=15-25 more preferably;
Formula 2:C
60(C (COOH)
2)
n, wherein, the integer that n is 1-4; Preferably, n=1-3; More preferably, n=2.
On the other hand, a kind of vaccine adjuvant provided by the invention, described adjuvant comprises fullerene derivate, this adjuvant carries out immunity to experimenter after can mixing with vaccine simultaneously, can also be first for experimenter injects this adjuvant, afterwards experimenter is carried out to vaccine immunity, or can carry out injecting this adjuvant after vaccine immunity to experimenter again, preferably this adjuvant carries out immunity to experimenter after mixing with vaccine simultaneously.Described adjuvant and vaccine are identical or different to the position of experimenter's immunity.
Further, the diameter range of the nanoparticles of described fullerene derivate in solvent is 1-400nm.
Another aspect, a kind of bacterin preparation provided by the invention, described preparation comprises described vaccine adjuvant.
Further, described vaccine includes, but are not limited to: the serious infectious diseases vaccines such as HIV (human immunodeficiency virus) vaccine, Hepatitis B virus vaccine and human papillomavirus's vaccine.It is object of study that the inventor be take (HIV-1 Env) expression plasmid (DNA) of coding hiv envelope protein gene, compared fullerene derivate as adjuvant on the immunogenic impact of HIV DNA vaccination, find after this adjuvant and DNA vaccination mixed immunity mice, can significantly strengthen the immunogenicity of DNA, and in identical immune time situation, the immunoreation level of low dosage DNA+ adjuvant and high dose DNA initiation is separately without significant difference.Meanwhile, the DNA+ adjuvant of same dose is in few immunity once in the situation that, and the immunoreation level causing and independent immune DNA vaccination are without significant difference.
Further, the immunization route of described bacterin preparation is selected from: subcutaneous injection, collunarium immunity, lumbar injection, intramuscular injection and intradermal injection, above-mentioned immunization route all can be brought into play the immunoadjuvant function of fullerene derivate.
A kind of fullerene derivate vaccine adjuvant provided by the invention is compared with traditional adjuvant, and its beneficial effect is as follows:
1. vaccine adjuvant safety provided by the invention is good, to injection site without obvious stimulation;
2. vaccine adjuvant provided by the invention can significantly improve cellular immunization and the humoral immunity level of body, can reduce the using dosage of antigen simultaneously or reduce immune time;
3. vaccine adjuvant provided by the invention has the function of antioxidation and removing free radical, when serving as immunological adjuvant, also may improve body antioxidative defence capability, makes body maintain stable state.
Accompanying drawing explanation
Below, describe by reference to the accompanying drawings embodiment of the present invention in detail, wherein:
Fig. 1 is the atomic force microscope sign picture that richness of the present invention is strangled alcohol;
Fig. 2 is the scanning electron microscope sign picture that richness of the present invention is strangled alcohol;
Fig. 3 is the dynamic light scattering testing result that richness of the present invention is strangled alcohol;
Fig. 4 is that richness of the present invention is strangled the impact of alcohol on HEK (Human embryonic kidney) 293 cell viabilities;
Fig. 5 is that richness of the present invention is strangled the impact of alcohol on HEK293 cellular morphology;
Fig. 6 is that richness of the present invention is strangled the impact of alcohol on NIH3T3 (Mouse embryonic fibroblast cell line) cell viability;
Fig. 7 is that richness of the present invention is strangled the impact of alcohol on NIH3T3 cellular morphology;
Fig. 8 is that richness of the present invention is strangled the transfection effect of alcohol to HEK293 cell;
Fig. 9 is that richness of the present invention is strangled the impact of alcohol on mouse cell immune level;
Figure 10 is that richness of the present invention is strangled the impact of alcohol on specific antibody titre.
Figure 11 is that while adopting different immunization route, richness of the present invention is strangled the impact of alcohol on mouse cell immune level.
The specific embodiment
Below in conjunction with specific embodiment, and comparable data describes in further detail the present invention.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.
In following embodiment, various processes and the method do not described in detail are conventional methods as known in the art.The source of agents useful for same, trade name and be necessary to list its constituent person, all indicate when occurring first, identical reagent used is if no special instructions, all identical with the content of indicating first thereafter.
embodiment 1 richness is strangled alcohol C
60
(OH)
20
synthetic
First, the C that the 40% TBAH solution (TBAH) of 1ml and 2ml sodium hydroxide solution (2.22g/ml) is added to 30ml
60in (Sigma company) toluene solution (1.5mg/ml), under vigorous stirring, react 24h; Discard organic facies, and remove water with vacuum rotary evaporator (RV06-ML 1-B, Germany); With 50ml methanol wash sample, methanol is removed by vacuum evaporation, and so cyclic washing is 3-5 time; Afterwards, 10ml deionized water is added in sample, continue stirring until solution and be transparent rufous, continue to add 20ml deionized water, and with Sephadex G-25 chromatographic column (Amersham company, Britain) purification of samples, last vacuum drying, can obtain richness and strangle alcohol nano-particle.By regulating the addition of sodium hydroxide can obtain containing the richness of varying number hydroxyl, strangle alcohol.
Utilize atomic force microscope (Atomic force microscopy, AFM) (BMT AFM3000, German) and scanning electron microscope (Scanning electron microscope, SEM) (S-4800, HIT) gained richness being strangled to alcohol characterizes, as depicted in figs. 1 and 2, the mean diameter that richness is strangled alcohol is about 120nm to result.
Utilize dynamic light scattering particle size analyser (DLS) (Zetasizer Nano, Britain Ma Erwen company) to detect the current potential that gained richness is strangled alcohol, as shown in Figure 3, richness is strangled alcohol surface band negative charge to result, be about-51.6mv of average potential.
Utilize x-ray photoelectron spectroscopy (XPS， Jeol Ltd.) to strangle the contained hydroxyl quantity of alcohol to richness and detect, the results are shown in Table 1, hydroxyl (OH) quantity is as shown in Table 1: (18.78/55.73) * 60=20.
The XPS testing result of the different carbon atoms of table 1
| Title | In conjunction with energy peak position (ev) | Area percent (%) |
| Anaerobic C | 284.80 | 55.73 |
| C-O | 286.59 | 18.78 |
| C＝O | 288.19 | 12.78 |
| O-C＝O | 289.68 | 8.94 |
| O-(C＝O)-O | 291.69 | 3.77 |
embodiment 2 richnesses are strangled the impact of alcohol on HEK293 cell (ATCC) vigor
1. containing rich preparation of strangling the complete medium of alcohol:
It is appropriate that the richness of first getting gained in embodiment 1 is strangled alcohol, is mixed with the aqueous solution of 2mM with sterile deionized water; With complete DMEM (Dulbecco ' s modification of Eagle ' s medium) culture medium, (contain 10% hyclone afterwards, Gibco company) richness is strangled to alcohol-water solution and be diluted to 500 μ M, and carry out gradient dilution with complete medium, thereby obtain concentration, be the serial solution of 500,100,20,4,0.8,0.16 μ M, standby.
2. richness is strangled the impact of alcohol on HEK293 cell viability:
First with 0.25% trypsinization HEK293 cell, stop, after digestion, with 1000rpm, carrying out centrifugal 5min.By the DMEM culture medium containing 10% hyclone (FBS), make single cell suspension, and with 5 * 10
4/ ml concentration is inoculated in 96 orifice plates, and every hole 100 μ l, are placed in incubator (37 ℃, 5%CO afterwards
2) the middle 24h that cultivates.Set up blank group, negative control group and Fu Le alcohol test group separately, establish three parallel holes for every group.Discard culture medium, by complete medium with containing the rich complete medium of strangling alcohol, add to Zhong，Mei hole, hole 100 μ l respectively afterwards, be placed in incubator (37 ℃, 5%CO
2) the middle 48h that cultivates, discard afterwards culture medium, with phosphate (PBS) buffer washing 2 times, finally add CCK-8 (the Cell counting kit-8 containing 10%, Japan Shimadzu company) containing blood serum medium, every hole 100 μ l, continue to cultivate 2h, utilize afterwards microplate reader (detection wavelength is 450nm) to detect, determine that richness strangles the affect situation of alcohol on HEK293 cell viability.
3. result: richness is strangled alcohol the impact of HEK293 cell viability is shown in to Fig. 4, result shows, compares with negative control group, and richness is strangled alcohol nano-particle and is processed after cell 48h, and there is not significant change in cell viability, and even in maximum dose level group, cell viability is still higher than 90%.Fig. 5 represents that richness strangles the impact of alcohol on HEK293 cellular morphology, and Fig. 5 A is Normal group, and Fig. 5 B represents that richness is strangled when alcohol working concentration is 500 μ M and process the cell picture after cell 48h, and visible test group cellular morphology is the same with Normal group, is spindle shape.These all illustrate, the richness of embodiment 1 gained is strangled alcohol and had good biocompatibility.
embodiment 3 richnesses are strangled the impact of alcohol on NIH3T3 cell (ATCC) vigor
With reference to the method described in embodiment 2, investigate rich the affect situation of alcohol nano-particle on NIH3T3 cell viability of strangling, the results are shown in Figure 6, similar with the result in embodiment 2, compare with negative control group, richness is strangled alcohol nano-particle and is processed after cell 48h, and significant change does not occur cell viability, even in maximum dose level group, cell viability is still higher than 90%.Fig. 7 represents that richness strangles the impact of alcohol on NIH3T3 cellular morphology, and Fig. 7 A is Normal group, and Fig. 7 B represents that richness is strangled when alcohol working concentration is 500 μ M and process the cell picture after cell 48h, and visible test group cellular morphology is the same with Normal group, is spindle shape.These all illustrate that richness strangles alcohol and have good biocompatibility.
embodiment 4 richnesses are strangled the transfection of alcohol to HEK293 cell
1. richness is strangled the preparation of alcohol-EGFP plasmid (green fluorescent protein encoding gene, Clontech company) mixture:
First getting in embodiment 1 gained richness, to strangle alcohol appropriate, is mixed with the aqueous solution of 2mM with sterile deionized water; The EGFP aqueous solution (0.325 μ g/ μ l) of respectively 6 μ l richnesses being strangled to alcohol-water solution (2mM) and 7.5 μ L with serum-free medium is diluted to 150 μ l, afterwards the richness after dilution being strangled to alcoholic solution adds in EGFP solution, mix homogeneously, hatches 30min under room temperature, standby.
2. the preparation of polyethyene diamine (PEI)-EGFP mixture:
With serum-free medium, respectively the EGFP aqueous solution of the PEI aqueous solution of 31.2 μ l (100 μ g/ml) and 7.5 μ L (0.325 μ g/ μ l) is diluted to 150 μ l, afterwards PEI solution is added in EGFP solution, mix homogeneously, hatches 30min under room temperature, standby.
3. richness is strangled alcohol transfected HEK 293:
First with 0.25% trypsinization HEK293 cell, stop, after digestion, with 1000rpm, carrying out centrifugal 5min; By the DMEM culture medium containing 10% hyclone (FBS), make single cell suspension, and with 5 * 10
4/ ml concentration is inoculated in 24 orifice plates, and every hole 500 μ l, are placed in incubator (37 ℃, 5%CO
2) the middle 24h that cultivates; The former culture medium of sucking-off afterwards, every hole adds 400 μ l containing the culture medium of serum.Set up blank group, naked EGFP plasmid matched group, richness separately and strangle alcohol-EGFP mixture test group and PEI positive controls, establish 3 parallel holes for every group.Blank group adds containing blood serum medium 100 μ l/ holes, naked EGFP plasmid matched group adds and contains the EGFP plasmid (plasmid DNA of green fluorescent protein, Biovector-08, Clontech company) containing serum DMEM culture medium (concentration of EGFP plasmid is 8 μ g/mL) 100 μ l/ holes, richness is strangled alcohol-EGFP mixture test group and is added the richness of above-mentioned preparation to strangle alcohol-EGFP mixture 100 μ l/ holes, positive controls adds PEI-EGFP mixture 100 μ l/ holes, shake up gently, be placed in incubator (37 ℃, 5%CO
2) middle continuation cultivation, and after 48h, utilize fluorescence microscope transfection effect.
4. result: richness is strangled alcohol to the transfection effect of HEK293 cell as shown in Figure 8, and Fig. 8 A is naked EGFP matched group, and Fig. 8 B is that richness is strangled alcohol-EGFP test group, and Fig. 8 C is PEI positive controls.As seen from the figure, naked EGFP plasmid is substantially without transfection effect, and richness is strangled alcohol has certain transfection effect to cell, and positive controls has good transfection effect.Because richness is strangled alcohol surface band negative charge, and DNA surface is also negative electricity, so it is difficult to combine by electrostatic adsorption, thereby DNA is carried in cell.Yet due to fullerene itself small-sized (being less than 1nm), in different dicyandiamide solutions, thereby it can be reunited and form the particulate matter of greater particle size with self or other matter interactions, therefore can obtain by controlling the difference of dicyandiamide solution the fullerene or derivatives thereof of different size, and fullerene derivate probably wraps up DNA in aggregation procedure, thereby make DNA avoid degraded, can enter easily cell interior, finally for accurate translation albumen is brought into play its biological action simultaneously.
It is object of study that (HIV-1 Env) expression plasmid (DNA) of coding hiv envelope protein gene is take in the present invention, compared fullerene derivate as adjuvant on the immunogenic impact of HIV DNA vaccination.
1. richness is strangled the preparation method of alcohol-HIV Env plasmid DNA mixture:
Getting in embodiment 1 gained richness, to strangle alcohol appropriate, is mixed with respectively the solution of 80 μ M, 400 μ M and 2mM with aseptic injection water; With aseptic injection water, HIV Env plasmid DNA (in the std aids prevention and control of Chinese Disease Control and Prevention Center, Cardioviruses and immune Research chamber provide) is diluted to 800 μ g/ml.Respectively the richness of variable concentrations is strangled afterwards to alcoholic solution 200 μ l and mix homogeneously with the HIV Env plasmid DNA solution of same volume, under room temperature, hatch 30min, standby.
2. richness is strangled the impact of alcohol on mice organism immune response:
By 40 female Balb/c mices (ages in 6-8 week, Beijing dimension tonneau China animal experiment technique company limited) be divided at random 5 groups, every group 8, be divided into blank group, naked HIV Env plasmid DNA matched group, richness and strangle alcohol-HIV Env plasmid DNA test group (low, in and high dose group); The HIV Env plasmid DNA of every mice intradermal injection 20 μ g, the dosage that richness is strangled alcohol is respectively 0.1,0.5 and 2.5 μ mol/kg; Every immunity in two weeks once, immunity volume is 50 μ l, immunity three times, and after immunity for the third time finishes, within 2 weeks, pluck eyeball and put to death mice, separating mouse serum, utilizes ELISA (Enzyme-linked immunosorbent assay) method to detect rich impact of strangling alcohol antagonist titre; Aseptic collection mouse spleen simultaneously, separated splenocyte, utilizes enzyme linked immunological spot test (ELISPOT) to detect the situation of splenocyte secretion IFN (Interferon)-γ, determines that richness strangles the impact of alcohol on mice Cellular Immunity level.
3.ELISPOT method detects the cell immune response of the rear mice of immunity:
(1) preparation of mouse spleen lymphocyte
1.. pluck eyeball and put to death mice, used afterwards for 75% alcohol-pickled a moment, be placed in aseptic superclean bench;
2.. aseptic collection mouse spleen, spleen is placed in to folding sterile gauze (2 layers), and (contain 10% hyclone at the RPMI1640 complete medium (Gibco company) containing 5mL, FBS) in plate, grind spleen, imbitition is in 15ml centrifuge tube, with the centrifugal 5min of 1000rpm afterwards;
3.. supernatant discarded, knock precipitate (cell) it is evenly suspended.Add 2ml erythrocyte cracked liquid, cracking 4-5min, adds afterwards 6ml to contain serum 1640 culture medium and stops cracking, after mixing, with the centrifugal 5min of 1000rpm;
4.. supernatant discarded, again knock precipitate it is evenly suspended, get afterwards 10 times of appropriate amount of suspension dilutions, carry out cell counting, adjust cell concentration to 1 * 10
7/ ml.
(2) ELISPOT detects Secreted by Mouse Splenic IFN-γ level:
1. the coated and sealing of .ELISPOT plank: the antibody with aseptic PBS with 1: 200 anti-IFN-γ of dilution proportion, every hole 100 μ L, are placed in CO
2incubator (5%, 37 ℃) in hatch 2h, discard afterwards coated antibody, with PBST (Phosphate buffered saline Tween-20), wash 6 times, finally, every hole adds 200 μ l containing the PBS of 1%BSA (Bovine serum albumin), is placed in incubator (5%CO
2, 37 ℃) and sealing 1h;
2.. discard confining liquid, every hole add 100 μ l polypeptide (CN54 Env albumen-CD8+T cell Dominant Epitopes peptide three, aminoacid sequence is:
CKEVHNVWATHACVPTDPNP；SELYKYKVVEIKPLGIAPTA；
QQSNLLRAIEAQQHLLQLTV ，You Venereal Disease AIDS Preventing Controlling Center China Disease Preventing Con provides, and every peptide concentration is 10 μ g/ml) and 100 μ l lymphocyte suspensions (1 * 10
7/ ml); Negative control group only adds RPMI1640 complete medium, positive controls does not add polypeptide, Ionomycin (the ionomycin that adds PMA (Phorbol-12-myristate-13-acetate) (25ng/ml) He the 1 μ l of 2.5 μ l, Sigma company) (1 μ g/ml), test group is established 2 multiple holes; Be placed in afterwards incubator (5%CO
2, 37 ℃) and cultivation 30h;
3.. discard cell, with PBST washing 8 times, each 2min, every hole adds the antibody of the biotinylated anti-IFN-γ of 100 μ l afterwards, sealer, under room temperature, lucifuge is hatched 2h;
4.. discard liquid, with PBST washing 8 times, each 2min, every hole adds GABA (γ-Aminobutyric acid) solution of 50 μ l, sealer, under room temperature, lucifuge is placed 1h;
5.. discard GABA solution, with PBST washing 6 times, each 2min.PBS washing 2 times, each 2min pats dry ELISPOT plank afterwards in absorbent paper;
6.. every hole adds 100 μ l nitrite ions (every 20 μ l developers mix with 1ml chromogenic substrate), and under room temperature, lucifuge reaction 15-40min, uses distilled water flushing, cessation reaction afterwards;
7.. under room temperature, dry, with ELISPOT, read plate instrument (Bioreader 5000, German Bio-Sys GmbH company) afterwards and detect, evaluate the cellular immune level of mice.
(3) ELISA detects the humoral immune reaction of the rear mice of immunity:
1.. with HIV-1 B/C recombinant strain CN54 restructuring gp120 antigen protein (purity is greater than 95%) coated elisa plate (0.1 μ g/ml), every hole adds 0.1ml, 4 ℃ are spent the night, inferior daily PBST washing 3 times, get rid of most residual liquid, with antibody diluent sealing 60min, PBST washing afterwards 3 times, dries rear 4 ℃ of preservations;
2.. sample to be checked (mice serum) is carried out to doubling dilution (1: 250,1: 500,1: 1000 to 1: 32000) with PBS buffer, every hole adds 100 μ l afterwards, set up blank group and negative control group simultaneously, under 37 ℃ of conditions, hatch 1h;
3.. discard serum, with PBST washing 8 times, pat dry, with PBS, ELIAS secondary antibody is carried out to dilution in 1: 5000, every hole adds 100 μ l, under 37 ℃ of conditions, hatches 1h;
4.. discard two and resist, PBST washing afterwards 8 times, pats dry, and adds TMB (tetramethyl benzidine) the substrate solution 0.1ml of interim preparation in each reacting hole, and 37 ℃ of lucifuges are reacted 10min, add the 2M sulphuric acid cessation reaction of 50 μ l after colour developing completely.
5.. utilize microplate reader in 450nm place (630nm is reference wavelength), to detect each hole OD value after blank hole zeroing, if be greater than 2.1 times of negative control OD value of regulation, be judged to be the positive.
4. result:
Fig. 9 and 10 is respectively the testing result of cellular immunization and humoral immunization.As seen from Figure 9, compare with HIVEnv plasmid DNA matched group, the secretion level that richness is strangled alcohol low dosage and middle dosetest group IFN-γ significantly strengthens, high dose group level declines to some extent, illustrate and rich strangle alcohol the impact of organism immune response is had to dose dependent, low dosage i.e. 0.1 μ mol/kg is optimal dose, and when richness, strangling alcohol dosage is 0.1 μ mol/kg, when HIV Env plasmid DNA dosage is 4 μ g/, cellular immune level during the HIV Env plasmid DNA of mice Cellular Immunity level and injection high dose (20 μ g/ only) is suitable, visible richness is strangled the using dosage that alcohol granule can reduce antigen, this will reduce the cost of following vaccine greatly, there is important practical significance.As seen from Figure 10, compare with HIV Env plasmid DNA matched group, the antibody titer that richness is strangled alcohol test group (low dose group) obviously raises.These all illustrate that richness strangles alcohol as a kind of immunologic adjuvant, can promote better the cellular immune level of body, can improve the antibody titer that body produces simultaneously.
In addition, richness is strangled alcohol and can also be realized and the similar effect of intradermal injection approach (Figure 11) by subcutaneous injection, lumbar injection, intramuscular injection and collunarium immunization route.Meanwhile, fullerene carboxy derivatives also has with richness and strangles the same immunological adjuvant effect of alcohol granule.
Claims (5)
1. the application of fullerene derivate in the carrier for the preparation of gene delivery, is characterized in that, described fullerene derivate is the fullerene carboxy derivatives of being strangled alcohol or being represented with formula 2 by the richness of following molecular formula 1 expression:
Formula 1:C
60o
xh
y, wherein, 10 < y≤x < 50;
Formula 2:C
60(C (COOH)
2)
n, wherein, the integer that n is 1-4.
2. application according to claim 1, is characterized in that, described formula 1:C
60o
xh
y, wherein, 10<x=y<50.
3. application according to claim 2, is characterized in that, described formula 1:C
60o
xh
y, wherein, x=y=15-25.
4. application according to claim 1, is characterized in that, described formula 2:C
60(C (COOH)
2)
n, wherein, n=1-3.
5. application according to claim 4, is characterized in that, described formula 2:C
60(C (COOH)
2)
n, wherein, n=2.
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| CN103789251A (en) * | 2014-01-28 | 2014-05-14 | 上海交通大学 | Application of fullerene as conditioning agent for conditioning growth or metabolism of microorganism |
| CN104483245A (en) * | 2014-09-28 | 2015-04-01 | 上海交通大学 | Method for separating C60 nanocrystalline particle size distribution by utilization of asymmetrical field flow meter |
| US10420836B2 (en) | 2015-10-08 | 2019-09-24 | Emory University | Methods of immunizing a subject and compositions related thereto |
| CN111265547A (en) * | 2020-02-26 | 2020-06-12 | 上海紫河生物科技有限公司 | Fullerene and fullerene derivative-containing disinfection spray and preparation method thereof |
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| RU2184566C1 (en) * | 2000-12-07 | 2002-07-10 | Санкт-Петербургский научно-исследовательский институт эпидемиологии и микробиологии им. Пастера | Method of vaccine preparing |
| CN1390605A (en) * | 2002-07-24 | 2003-01-15 | 中国科学院上海原子核研究所 | Medicine C-AEDP using C as carrier for target therapy of osteopath and its usage |
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