CN108567978A - The vaccine of carbon quantum dot adjuvant and carbon containing quantum dot adjuvant - Google Patents
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Abstract
The invention discloses a kind of carbon quantum dot adjuvant and the vaccines of carbon containing quantum dot adjuvant, i.e., contain 200 400 μ g carbon quantum dot adjuvants in every single part of vaccine dose.Present invention firstly discovers that CQDs can as the superior adjuvant of anti-chicken ALV J, the study found that CQDs no cytotoxicities prepared by the present invention, will not inducing cell vigor decline, 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 vaccine prepared by Freund's adjuvant and generate antibody titers progress immunoprotection and provide more longlasting protective immunological reaction.
Description
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 technology
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 reduce 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 type is various, effect is also apparent, and there are also inevitably lack for they
Point, such as safety issue, local side reaction problem；Also oil adjuvant is more sticky is unfavorable for injection, emulsification not with great difficulty layering；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
A kind of unknown fluorescence carbon nanomaterial 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, is 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 makes it have extensively in fields such as photoelectron, catalysis and sensings
Application value.But there is not reports of the CQDs as vaccine adjuvant application aspect also at present.
Invention content
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 that：
The first aspect of the present invention provides application of the carbon quantum dot in preparing vaccine adjuvant.
In above 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 into 15min with 3000rpm, discards precipitation, supernatant dialysis
Bag dialysis 48h；Then centrifuge 15min under 12000rpm rotating speeds, vacuum drying to get.
Preferably, the carbon quantum dot is spherical or subsphaeroidal, a diameter of 3-5nm.
In above 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 dots.
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 vaccines contain 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, include 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 as the superior adjuvant of anti-chicken ALV-J, the study found that prepared by the present invention
CQDs no cytotoxicities, 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 levels 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 responses.
(3) vaccine that the present invention is prepared using CQDs as adjuvant can be more effective relative to vaccine prepared by Freund's adjuvant
Induction generate antibody titers and carry out immunoprotection and more longlasting protective immunological reaction is provided.
Description of the drawings
Fig. 1：The TEM of CQDs schemes.
Fig. 2 a：Recombinate the SDS-PAGE analyses of gp85 albumen；Fig. 2 b：Recombinate the Western blot analysis of gp85 albumen；Its
In, 1 is recombination gp85 albumen.
Fig. 3：Result is investigated in influences of the CQDs 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 values 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 implementation mode
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 waters, and is transferred in the autoclave with polytetrafluoroethylene (PTFE).It will be high
Pressure kettle heats 5 hours at 180 DEG C, and natural cooling obtains dark brown solution.Reaction solution is centrifuged 15 minutes with 3000rpm.With
Afterwards, sediment is discarded, and supernatant is dialysed 48 hours to remove small molecular weight impurity in bag filter (MWCO=1000).It
Afterwards, supernatant is centrifuged 15 minutes with 12000rpm, the fluorescence CQDs of acquisition is dry in vacuum drying oven, it is placed in 4 DEG C
It is preserved 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 particles manufactured in the present embodiment are spherical shape, a diameter of 3-5nm.
Embodiment 2：The expression and identification of 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 are induced 6 hours, obtain recombination gp85 albumen.Gp85 albumen, dialysis are recombinated with high-affinity Ni-NTA column purifications
Lauryl sodium sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) is used to identify (Fig. 2 a) afterwards.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
Influences of the CQDs to chicken lymphocyte activity is determined using MTT analyses.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 mediums, 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；Each 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 bromides (MTT) solution is added
To in each hole containing cell and incubate 4 hours.Culture medium is removed, 150mL dimethyl sulfoxide (DMSO)s are added to discharge methyl.Oscillation
Mixture about 15 minutes measures luminosity (OD) by microplate reader.Cell viability is calculated using common formula.It is obtained at 490nm
The absorbance of methyl is obtained, and measuring method measures the dehydrogenase activity in 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, in a concentration of 400 μ g/mL of CQDs, pI level (pI=2.1) highest.When CQDs concentration increases
When adding to 800 μ g/mL, cell viability reduces, 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 ALV-J gp85 recombinant protein vaccines 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 vaccines of quantum dot adjuvant, are named as gp85-CQDs.
Comparative example 1：The preparation of ALV-J gp85 recombinant protein Adjuvanted vaccines
Gp85 albumen is diluted with PBS (pH=7.4), incomplete Freund's adjuvant (Sigma chemical companies) is added and mixes, makes
The final concentration of 600 μ g/ml of gp85 albumen, a concentration of 400 μ g/ml of incomplete Freund's adjuvant, ultrasonic emulsification, emulsification condition
For：Power 350W, working time 2s, intermittent time 5s, for temperature setting at 25 DEG C, ultrasound is complete to emulsifying, and ALV-J is prepared
Gp85 recombinant protein Adjuvanted vaccines, are named as gp85-F.
Embodiment 5：Vaccine effect is evaluated
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, respectively：Gp85-CQDs groups, gp85-F groups and control groups.
Wherein：Every chicken of gp85-CQDs groups applied vaccine prepared by embodiment 4, and with 7 days interval intramuscular immunizations two
It is secondary；
Every chicken of gp85-F groups applies vaccine prepared by comparative example 1, and twice with 7 days interval intramuscular immunizations；
Control groups are 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 to gp85 eggs by ELISA
White antibody response.Specifically, 96 hole microtest plates are coated with 100ng gp85 protein at 4 DEG C overnight per hole.With containing
There is the PBS (pH 7.4) of 5% bovine serum albumin(BSA) in 37 DEG C of blocking antigens 1 hour.By tablet 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
1 hour anti-Chicken immunoglobulin of rabbitization with horseradish peroxidase-labeled is incubated under temperature with 1/5000 diluted (PBS) (always to exempt from
Epidemic disease Lysozyme [IgG]) Abs (Southern Biotechnology Associates).Three times and led to PBS-T washing flat boards
Cross the colour developing of addition ABTS substrates.Using Dynatech MR5000 microplate reader chromogenic reaction is measured by measuring the OD of 450nm.
Opposite serum antibody titer is determined by calculating sample as follows with positive (S/P) ratio：[(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 put down at three
It is tested in row experiment, and blood serum sample of the S/P ratios higher than 0.6 is considered as ALV-J antibody positives.
Second week after booster immunization inoculation, in addition to it is immune with PBS and be initially not affected by attack six control chickens it
Outside, all chickens are all with 102.4Attack in the ALV-J bacterial strains peritonaeum of TCID50.After infection, the state of mind of every chicken is monitored daily
With diet desire.ALV viremia virusemias are 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 inoculations from chicken, and at 37 DEG C, 5%CO2It is lower to be incubated 7 days.By using ALV
P27 antigen ELISAs test kit (IDEXX USA Inc., Beijing, China) detects depositing for virus using cell
.S/P ratios are calculated by following formula and determine that opposite antigen valence is horizontal, and plasma sample of the S/P ratios more 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 detaches 2min.Then plasma sample is inoculated into CEF cells to detect ALV-J viremia virusemias.In brief, by 24
CEF cell inoculation plasma samples in orifice plate, then in 38.5 DEG C and 5%CO2It is lower to incubate 7 days.Then ALV P27 antigens are used
ELISA test kits (IDEXX USA Inc., Beijing, China) check cell with the presence or absence of virus.Pass through following public affairs
Formula calculates S/P ratios and determines that opposite antigen titre is horizontal.All samples are tested in triplicate, and S/P ratios are 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 measurement results all in triplicate, P<0.05 is considered as that significant difference 2. tries
Test result：
(1) to the influence of chicken weight (BWs)
Two weeks after inoculation, the representation of gp85-CQDs Zu Ji groups, which, is higher than control group, and 42 ages in days are significant to be higher than control group (P<
0.05).In gp85-F groups, only at 35,42 and 56 days, BWs was significant higher than control (Fig. 4).The result shows that relative comparison group
For, CQDs adjuvants can improve chicken weight with F adjuvants, 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 groups continues to increase 3 weeks, and the IgG in gp85-CQDs groups continues growing 5
Week is to highest IgG levels 3.08 ± 0.14 (n=12).In addition, the positive antibody result of two experimental groups continue for 7 weeks or more.
The IgG levels of the chicken of immunity inoculation gp85-CQDs are day significant higher than those of immunity inoculation gp85-F (P from 42 days to 70<
0.05) (Fig. 5).Indicated by the positive critical value obtained by using ALV-J antibody ELISAs kit (IDEXX), gp85-
Antibody positive ratio in CQDs groups 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 challenges of TCID50.Gp85-CQDs group chickens
It only all survives, and most of chickens are still resistant to infecting after immune for the second time with gp85-CQDs vaccines.
When detecting ALV viremia virusemias, as a result as shown in table .1.
Table .1
As can be seen from Table 1, the ALV positive rates of gp85-CQDs groups were 8.3% at first week, second week 8.3%, the
Three weeks are 16.7%, 4th week 16.7%.In gp85-F groups, these ratios are respectively 8.3%, 25%, 33.3% He
25%, the results showed that gp85-CQDs vaccines provide better protection than Freund's adjuvant vaccine.
The foregoing is merely the preferred embodiments of the application, are not intended to limit this application, for the skill of this field
For art personnel, the application can have various modifications and variations.Within the spirit and principles of this application, any made by repair
Change, equivalent replacement, improvement etc., should be included within the protection domain of the application.
Claims (9)
1. application of the carbon quantum dot in preparing 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 into 15min with 3000rpm, discards precipitation, supernatant bag filter is saturating
Analyse 48h；Then centrifuge 15min under 12000rpm rotating speeds, vacuum drying to get.
3. application according to claim 2, which is characterized in that the carbon quantum dot is spherical or subsphaeroidal, a diameter of 3-
5nm。
4. application according to claim 1, which is characterized in that the vaccine adjuvant be protein vaccine, polypeptide vaccine or
The adjuvant of nucleic acid vaccine.
5. application according to claim 4, which is characterized in that the vaccine adjuvant is the adjuvant of protein vaccine.
6. application according to claim 5, which is characterized in that the vaccine adjuvant is ALV-J gp85 recombinant protein vaccines
Adjuvant.
7. a kind of vaccine containing carbon quantum dot adjuvant, which is characterized in that wanted containing 200-400 μ g rights in per single part of vaccine dose
Seek the carbon quantum dot described in 1.
8. a kind of ALV-J gp85 recombinant protein vaccines containing carbon quantum dot adjuvant, which is characterized in that contain in the vaccine
The gp85 albumen of 600 μ g/ml and 400 μ g/ml carbon quantum dots described in claim 1.
9. the preparation method of vaccine according to any one of claims 8, which is characterized in that include 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, is completed equal
Matter makes the final concentration of 400 μ g/ml of CQDs.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113456809A (en) * | 2021-06-30 | 2021-10-01 | 澳门大学 | Quantum dot modified protein vaccine and preparation method and application thereof |
| CN113476594A (en) * | 2021-06-30 | 2021-10-08 | 澳门大学 | Quantum dot enhanced tumor inactivated vaccine and preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113456809A (en) * | 2021-06-30 | 2021-10-01 | 澳门大学 | Quantum dot modified protein vaccine and preparation method and application thereof |
| CN113476594A (en) * | 2021-06-30 | 2021-10-08 | 澳门大学 | Quantum dot enhanced tumor inactivated vaccine and preparation method and application thereof |
| WO2023274298A1 (en) * | 2021-06-30 | 2023-01-05 | 澳门大学 | Quantum dot modified protein vaccine and preparation method therefor and application thereof |
| WO2023274297A1 (en) * | 2021-06-30 | 2023-01-05 | 澳门大学 | Quantum dot-enhanced tumor inactivated vaccine, preparation method, and application |
| CN113456809B (en) * | 2021-06-30 | 2024-02-23 | 澳门大学 | Quantum dot modified protein vaccine and preparation method and application thereof |
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