CN108567978B - The vaccine of carbon quantum dot adjuvant and carbon containing quantum dot adjuvant - Google Patents

The vaccine of carbon quantum dot adjuvant and carbon containing quantum dot adjuvant Download PDF

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Publication number
CN108567978B
CN108567978B CN201810365996.4A CN201810365996A CN108567978B CN 108567978 B CN108567978 B CN 108567978B CN 201810365996 A CN201810365996 A CN 201810365996A CN 108567978 B CN108567978 B CN 108567978B
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quantum dot
adjuvant
vaccine
carbon quantum
cqds
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CN108567978A (en
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刘建柱
程佳
刘永夏
赵鹏
成子强
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Shandong Agricultural University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/11011—Alpharetrovirus, e.g. avian leucosis virus
    • C12N2740/11034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The invention discloses a kind of carbon quantum dot adjuvant and the vaccine of carbon containing quantum dot adjuvant, i.e., contain 200-400 μ g carbon quantum dot adjuvant in every single part of vaccine dose.Present invention firstly discovers that CQDs can be used as the superior adjuvant of anti-chicken ALV-J, the study found that CQDs no cytotoxicity prepared by the present invention, will not the decline of inducing cell vigor, good biocompatibility, using being safe and reliable within the scope of immunizing dose.The vaccine that the present invention is prepared using CQDs as adjuvant can be induced more effectively relative to the vaccine of Freund's adjuvant preparation and generate antibody titers progress immunoprotection and provide more longlasting protective immunological reaction.

Description

The vaccine of carbon quantum dot adjuvant and carbon containing quantum dot adjuvant
Technical field
The present invention relates to immunological technique fields, and in particular to a kind of carbon quantum dot adjuvant and carbon containing quantum dot adjuvant Vaccine.
Background technique
Adjuvant is that one kind can induce body and generate for a long time, effectively with immunogenic substance specificity or non-specific binding Specific immune response, play the substance of booster action.Adjuvant can be reduced the dosage of immunogene, reduce being produced into for vaccine This.Recently as the fast development of modern biotechnology and genetic engineering, the development of vaccine has been made significant headway, recombination Subunit vaccine, nucleic acid vaccine and synthetic peptide vaccine etc. are developed, but due to these vaccines there are molecules small, immunogenicity Weak, the problems such as targeting is poor, it is difficult to induce body to generate effective immune response, it is therefore desirable to enhance its immunogene using adjuvant Property or enhancing host versus original immune response.
Currently available vaccines, existing vaccines adjuvant is broadly divided into Alum adjuvant, protide adjuvant, nucleic acid adjuvant, contains lipid adjuvant and mixing Several classes such as adjuvant.Although existing vaccine adjuvant is many kinds of, effect is also apparent, and there are also inevitably lack for they Point, such as safety issue, local side reaction problem；There are also oil adjuvant it is more sticky be unfavorable for injection, emulsification with great difficulty be layered；Aluminium Adjuvant is difficult to induce the immune response of poor antigen；There are stronger toxic side effects for lipopolysaccharide adjuvant；Aoxidize mannosan and antigen The problems such as combination limitation, propolis adjuvant injection site form lump.Therefore, developing novel immunologic adjuvant has weight The meaning wanted.
Carbon quantum dot (CQDs) is a kind of novel carbon nanomaterial, and size is in 10nm hereinafter, being Xu etc. in head in 2004 The unknown fluorescence carbon nanomaterial of one kind of secondary discovery.Common carbon is a kind of atrament, is typically considered to shine weak, water-soluble Property it is weak, however CQDs but has good water-soluble and bright fluorescence, referred to as carbon nanometer light.In recent years, in the conjunction of CQDs At, property, using etc. all achieve huge progress.Compared with traditional semiconductor-quantum-point and organic dyestuff, CQDs With highly-water-soluble, extensive chemical stability, it is easy to functionalization, anti-light Bleachability and excellent biological nature, good biology Compatibility has potential application prospect in bio-imaging, bio-sensing, medicament transport etc..Meanwhile CQDs has excellent photoelectricity Property can not only be used for electron donor but also as electron acceptor, this has it extensively in fields such as photoelectron, catalysis and sensings Application value.But there is not report of the CQDs as vaccine adjuvant application aspect also at present.
Summary of the invention
For the above-mentioned prior art, the object of the present invention is to provide a kind of carbon quantum dot adjuvants and carbon containing quantum dot to help The vaccine of agent.
To achieve the above object, the present invention adopts the following technical scheme:
The first aspect of the present invention provides carbon quantum dot and is preparing the application in vaccine adjuvant.
In above-mentioned application, it is preferred that the carbon quantum dot is prepared by the following method:
Humic acid is dissolved in secondary water, is transferred in the autoclave with polytetrafluoroethylene (PTFE), is added under the conditions of 160-200 DEG C Hot 4-6h, it is cooling, obtain dark brown solution；Dark brown solution is centrifuged 15min with 3000rpm, discards precipitating, supernatant dialysis Bag dialysis 48h；Then be centrifuged 15min under 12000rpm revolving speed, vacuum drying to get.
Preferably, the carbon quantum dot is spherical or subsphaeroidal, diameter 3-5nm.
In above-mentioned application, it is preferred that the vaccine adjuvant is the adjuvant of protein vaccine, polypeptide vaccine or nucleic acid vaccine； It is further preferred that the vaccine adjuvant is the adjuvant of protein vaccine；It is furthermore preferred that the vaccine adjuvant is ALV-J gp85 The adjuvant of recombinant protein vaccine.
The second aspect of the present invention provides a kind of vaccine containing carbon quantum dot adjuvant, contains in every single part of vaccine dose 200-400 μ g carbon quantum dot.
Vaccine form as one preferred, the third aspect of the present invention provide a kind of containing carbon quantum dot adjuvant ALV-J gp85 recombinant protein vaccine contains 600 μ g/ml gp85 albumen and 400 μ g/ml carbon quantum dots in the vaccine.
The present invention also provides the preparation methods of above-mentioned vaccine, comprising the following steps:
The gp85 albumen being diluted in PBS (pH=7.4) is slowly added into CQDs, and is incubated for 24 hours at 4 DEG C, it is complete At homogenizing, make the final concentration of 400 μ g/ml of CQDs.
Beneficial effects of the present invention:
(1) present invention firstly discovers that CQDs can be used as the superior adjuvant of anti-chicken ALV-J, the study found that prepared by the present invention CQDs no cytotoxicity, will not inducing cell vigor decline, good biocompatibility, within the scope of immunizing dose using be safety Reliably.
(2) CQDs can induce body to generate high IgG level as adjuvant, and assign lasting protective effect.Induction is exempted from The ability of epidemic disease memory is the important performance indexes of vaccine, and the vaccine prepared using CQDs as adjuvant resists 49 days after being immunized twice Body level reaches highest, shows that CQDs has lasting immunological memory ability.And CQDs also can induce and generate antigentic specificity Antibody response, including antigen specific T h2 allergic immune response.
(3) vaccine that the present invention is prepared using CQDs as adjuvant can be more effective relative to the vaccine of Freund's adjuvant preparation Induction generate antibody titers and carry out immunoprotection and more longlasting protective immunological reaction is provided.
Detailed description of the invention
Fig. 1: CQDs TEM figure.
Fig. 2 a: the SDS-PAGE analysis of recombination gp85 albumen；Fig. 2 b: the Western blot analysis of recombination gp85 albumen；Its In, 1 is recombination gp85 albumen.
Result is investigated in Fig. 3: the CQDs influence to chicken lymphocyte activity.
Fig. 4: influence of the different vaccines to chicken weight.
Fig. 5: influence of the different adjuvants to serum antibody；Blood serum sample of the S/P value higher than 0.6 is considered as ALV-J antibody It is positive；F.i. mean to be inoculated with for the first time；S.i. mean second of inoculation.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field The identical meanings of understanding.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool The technical solution of the application is described in detail in the embodiment of body.
The test material that test material is this field routine is not specifically described used in the embodiment of the present invention, It can be commercially available by commercial channel.
Embodiment 1: the preparation of carbon quantum dot
Humic acid (0.2g) is dissolved in 50mL secondary water, and is transferred in the autoclave with polytetrafluoroethylene (PTFE).It will be high Kettle is pressed to heat 5 hours at 180 DEG C, natural cooling obtains dark brown solution.By reaction solution with 3000rpm centrifugation 15 minutes.With Afterwards, sediment is discarded, and supernatant is dialysed 48 hours in bag filter (MWCO=1000) to remove small molecular weight impurity.It Afterwards, by supernatant with 12000rpm centrifugation 15 minutes, by the fluorescence CQDs of acquisition, the drying in vacuum drying oven, is placed in 4 DEG C It is saved in refrigerator.
By the size and form of the CQDs of transmission electron microscope (TEM) research synthesis, as shown in Figure 1.It can be with by Fig. 1 Find out, CQDs particle manufactured in the present embodiment is spherical shape, diameter 3-5nm.
The expression and identification of embodiment 2:ALV-J gp85 albumen
Recombinant plasmid (PET28a-gp85) is transformed into Rosetta (DE3) cell first, is then used at 37 DEG C 0.5mM IPTG is induced 6 hours, obtains recombination gp85 albumen.Gp85 albumen, dialysis are recombinated with high-affinity Ni-NTA column purification Afterwards with lauryl sodium sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) identification (Fig. 2 a).Under Western blot analysis Protein is set to be subjected to gp85 specificity M antibody JE9 (Fig. 2 b).It is dense that the gp85 albumen that PEG6000 is collected is measured by thin-layer chromatography Degree is 1.2mg/mL.
Embodiment 3: influence of the carbon quantum dot to chicken lymphocyte activity
Influence of the CQDs to chicken lymphocyte activity is determined using MTT analysis.It is specific as follows:
By chicken lymphocyte in the Roswell Park containing 10% fetal calf serum and 1% penicillin/streptomycin In 96 hole microtest plates of Memorial Institute culture medium, in 5%CO2Under in 37 DEG C cultivate 24 hours.It will be different Synthesis CQDs (preparation of embodiment 1) solution of concentration is loaded into each hole；Every kind of concentration is prepared in triplicate and to incubate 24 small When.Later, the 5mg/mL 3- of 20mL (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromide (MTT) solution is added Into each hole containing cell and incubate 4 hours.Culture medium is removed, 150mL dimethyl sulfoxide is added to discharge methyl.Oscillation Mixture about 15 minutes, luminosity (OD) is measured by microplate reader.Cell viability is calculated using common formula.It is obtained at 490nm Obtain the absorbance of methyl, and the dehydrogenase activity in measuring method measurement metabolic active cells.As a result see Fig. 3.
As seen from Figure 3, when cell is exposed to the CQDs of 25 μ g/mL-400 μ g/mL, with the increase of CQDs concentration, MTT analysis shows that viability increase, CQDs concentration be 400 μ g/mL when, pI level (pI=2.1) highest.When CQDs concentration increases When adding to 800 μ g/mL, cell viability is reduced, but also shows that high viability.The result shows that CQDs prepared by the present invention does not have Cytotoxicity, and certain density CQDs can also improve cell viability and viability, have extraordinary cell compatibility.
Embodiment 4: the preparation of the ALV-J gp85 recombinant protein vaccine containing carbon quantum dot adjuvant
The gp85 albumen being diluted in PBS (pH=7.4) is slowly added into CQDs, and is incubated for 24 hours at 4 DEG C, it is complete At homogenizing, makes the final concentration of 600 μ g/ml of final concentration of 400 μ g/ml, the gp85 albumen of CQDs, that is, be prepared containing carbon The anti-fowl ALV-J gp85 recombinant protein vaccine of quantum dot adjuvant, is named as gp85-CQDs.
The preparation of comparative example 1:ALV-J gp85 recombinant protein Adjuvanted vaccines
Gp85 albumen is diluted with PBS (pH=7.4), incomplete Freund's adjuvant (Sigma chemical company) mixing is added, makes The final concentration of 600 μ g/ml of gp85 albumen, the concentration of incomplete Freund's adjuvant are 400 μ g/ml, ultrasonic emulsification, emulsification condition Are as follows: power 350W, working time 2s, intermittent time 5s, for temperature setting at 25 DEG C, ALV-J is prepared to emulsifying completely in ultrasound Gp85 recombinant protein Adjuvanted vaccines, are named as gp85-F.
Embodiment 5: vaccine effect evaluation
1. test method:
This testing program is through Walter Reed ground force research institution's animal care and studies examination using the committee and ratifies, And abide by " Animal Welfare Law " and other federal regulations and regulations in relation to animal and zoopery.Also comply with " experimental animal shield Principle described in reason and guide for use ".Scheme is specific as follows:
Totally 36 1 age in days sea orchid species cocks will be bought from extra large orchid species cock department (Tai'an, China) and give sufficient food After two weeks with water raising, chicken is randomly divided into three groups, is respectively as follows: gp85-CQDs group, gp85-F group and control group.
Wherein: vaccine prepared by every chicken application embodiment 4 of gp85-CQDs group, and with 7 days interval intramuscular immunizations two It is secondary；
Vaccine prepared by every chicken application comparative example 1 of gp85-F group, and twice with 7 days interval intramuscular immunizations；
Control group is inoculated with PBS, as negative control.
Every group of chicken is weighed weekly once.
After first time vaccine inoculation, the blood serum sample of every chicken is collected weekly to 10 weeks.It is measured by ELISA to gp85 egg White antibody response.Specifically, the 96 every holes of hole microtest plate are stayed overnight with 100ng gp85 protein in 4 DEG C of coatings.With containing Have the PBS (pH 7.4) of 5% bovine serum albumin(BSA) 37 DEG C blocking antigen 1 hour.By plate with PBS-T wash three times and It is incubated together with the single chicken serum in the triplicate hole of each blood serum sample 2 hours at room temperature.Plate is washed again and in room Lower 1/5000 diluted (PBS) the incubation 1 hour anti-Chicken immunoglobulin of rabbitization with horseradish peroxidase-labeled of temperature (is always exempted from Epidemic disease Lysozyme [IgG]) Abs (Southern Biotechnology Associates).Three times and led to PBS-T washing flat board Cross the colour developing of addition ABTS substrate.Chromogenic reaction is measured by measuring the OD of 450nm using Dynatech MR5000 microplate reader. Determine opposite serum antibody titer by calculating sample and positive (S/P) ratio as follows: [(average value of sample OD)-is (negative Compare the average value of OD)]/[(average value of positive control OD)-(average value negative control OD)].Every group of serum is flat at three It is tested in row experiment, and blood serum sample of the S/P ratio higher than 0.6 is considered as ALV-J antibody positive.
Second week after booster immunization inoculation, in addition to it is immune with PBS and initially be not affected by attack six control chickens it Outside, all chickens all use 102.4Attack in the ALV-J bacterial strain peritonaeum of TCID50.After infection, the state of mind of every chicken is monitored daily With diet desire.ALV viremia virusemia is detected, before and after infecting sexual assault weekly, especially by Blood Sample Collection Into test tube of hepari pipe.Plasma sample of the CEF cell inoculation from chicken, and at 37 DEG C, 5%CO2It is lower to be incubated for 7 days.By using ALV P27 antigen ELISA test kit (IDEXX USA Inc., Beijing, China) detects depositing for virus using cell In.S/P ratio is calculated by following formula and determines that opposite antigen valence is horizontal, and plasma sample of the S/P ratio greater than 0.2 is considered It is virus-positive.
Viremia virusemia is detected by ELISA:
In the pipe of blood sample sterile collection to test tube of hepari and 4 DEG C will be stored in.Before use, sample at 4 DEG C with 2000g separates 2min.Then plasma sample is inoculated into CEF cell to detect ALV-J viremia virusemia.In brief, by 24 CEF cell inoculation plasma sample in orifice plate, then in 38.5 DEG C and 5%CO2It is lower to incubate 7 days.Then ALV P27 antigen is used ELISA test kit (IDEXX USA Inc., Beijing, China) checks cell with the presence or absence of virus.Pass through following public affairs Formula calculates S/P ratio and determines that opposite antigen titre is horizontal.All samples are tested in triplicate, and S/P ratio is higher than 0.2 Plasma sample is considered as virus-positive.
Statistics is for statistical analysis using SPSS (version 11.0, SPSS Inc., USA).All numerical value with average value ± Standard error near average value indicates.All in triplicate, P < 0.05 is considered as that significant difference 2. tries to all measurement results Test result:
(1) to the influence of chicken weight (BWs)
Two weeks after inoculation, gp85-CQDs Zu Ji group weight it is significant be higher than control group, 42 ages in days it is significant be higher than control group (P < 0.05).In gp85-F group, only at 35,42 and 56 days, BWs was significant is higher than control (Fig. 4).The result shows that relative comparison group For, chicken weight can be improved in CQDs adjuvant and F adjuvant, and from 63d and there was no significant difference (P < 0.05).
(2) ability of immunoprotection
After being inoculated with for the first time, the IgG in gp85-F group continues to increase 3 weeks, and the IgG in gp85-CQDs group continues growing 5 Week is to highest IgG level 3.08 ± 0.14 (n=12).In addition, the positive antibody result of two experimental groups continue for 7 weeks or more. The IgG level of the chicken of immunity inoculation gp85-CQDs from 42 days to 70 it is day significant be higher than those of immunity inoculation gp85-F (P < 0.05) (Fig. 5).Indicated by the positive critical value obtained by using ALV-J antibody ELISA kit (IDEXX), gp85- Antibody positive ratio in CQDs group is 100% (12/12).These results indicate that CQDs specific time more than Freund's adjuvant Effectively enhance the antibody level for gp85 albumen.
Two groups of chickens (n=12) are immunized, and at the 35th day with 102.4The ALV-J strain challenge of TCID50.Gp85-CQDs group chicken It only all survives, and most of chickens are still resistant to infecting after immune with second of gp85-CQDs vaccine.In When detecting ALV viremia virusemia, as a result as shown in table .1.
Table .1
As can be seen from Table 1, the ALV positive rate of gp85-CQDs group was 8.3% at first week, second week 8.3%, the Three weeks are 16.7%, 4th week 16.7%.In gp85-F group, these ratios are respectively 8.3%, 25%, 33.3% He 25%, the results showed that gp85-CQDs vaccine provides better protection than Freund's adjuvant vaccine.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.

Claims (6)

1. carbon quantum dot is preparing the application in ALV-J gp85 recombinant protein vaccine adjuvant.
2. application according to claim 1, which is characterized in that the carbon quantum dot is prepared by the following method:
Humic acid is dissolved in secondary water, is transferred in the autoclave with polytetrafluoroethylene (PTFE), 4- is heated under the conditions of 160-200 DEG C 6h, it is cooling, obtain dark brown solution；Dark brown solution is centrifuged 15min with 3000rpm, discards precipitating, supernatant bag filter is saturating Analyse 48h；Then be centrifuged 15min under 12000rpm revolving speed, vacuum drying to get.
3. application according to claim 2, which is characterized in that the carbon quantum dot is spherical or subsphaeroidal, diameter 3- 5nm。
4. a kind of protein vaccine containing carbon quantum dot adjuvant, which is characterized in that contain 200-400 μ g's in every single part of vaccine dose Carbon quantum dot；
The carbon quantum dot is spherical or subsphaeroidal, diameter 3-5nm.
5. a kind of ALV-J gp85 recombinant protein vaccine containing carbon quantum dot adjuvant, which is characterized in that contain in the vaccine The carbon quantum dot of the gp85 albumen of 600 μ g/ml and 400 μ g/ml；
The carbon quantum dot is spherical or subsphaeroidal, diameter 3-5nm.
6. the preparation method of vaccine described in claim 5, which comprises the following steps:
The gp85 albumen being diluted in the PBS of pH=7.4 is slowly added into carbon quantum dot, and is incubated for 24 hours at 4 DEG C, is completed It homogenizes, makes the final concentration of 400 μ g/ml of carbon quantum dot.
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