CN105214100A - A kind of pH response microcapsule-type protein vaccine carrier and preparation method - Google Patents
A kind of pH response microcapsule-type protein vaccine carrier and preparation method Download PDFInfo
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- CN105214100A CN105214100A CN201510785244.XA CN201510785244A CN105214100A CN 105214100 A CN105214100 A CN 105214100A CN 201510785244 A CN201510785244 A CN 201510785244A CN 105214100 A CN105214100 A CN 105214100A
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Abstract
A kind of pH response microcapsule-type protein vaccine carrier and preparation method, preparation method is: protein mimic antigen joins in aqueous sodium carbonate by (1), pour into isopyknic containing in the calcium chloride water of kayexalate after dissolving, stir, centrifugal, deionization is washed, and obtains the calcium carbonate granule containing analogue antigen; (2) be scattered in deionized water by the calcium carbonate granule containing analogue antigen, add tannic acid aqueous solution, vibration makes to be uniformly dispersed, add aluminum chloride aqueous solution, vibration makes to be uniformly dispersed, and adds 3-(N-morpholine) propanesulfonate salt buffer solution and regulates pH, vibration, centrifugal, granule deionized water is washed, and uses EDTA solution washing, removes calcium carbonate, by washed with de-ionized water, obtain pH response microcapsule-type protein vaccine carrier.Preparation process of the present invention is carried out under room temperature aqueous phase, mild condition, ensure that activity and the carrier safety in utilization of protein vaccine; Cheaper starting materials is easy to get, and preparation technology is simple.
Description
Technical field
The present invention relates to a kind of pH response microcapsule-type protein vaccine carrier and preparation method, belong to nano micro-material preparation field.
Background technology
Traditional vaccine is deactivation or SA pathogen, in use there is potential safety hazard.New generation vaccine (as DNA, protein vaccine etc.) is safe and effective, but easily degraded by enzymes and less immunogenic, need by other means protection vaccine and immune stimulatory response.Traditional vaccine adjuvant is invalid to these antigens, therefore anxious novel vaccine carrier to be developed.
Aluminum salt is first adjuvant going through can be used for people.Alum adjuvant (Al (OH)
3) can the humoral immune reaction of effective stimulus B cell mediation, but its concrete mechanism of action it be unclear that.Be commonly referred to be aluminum hydroxide sol to the slow releasing function of vaccine and aluminium hydroxide itself to the stimulation of macrophage.The stimulation of aluminium hydroxide to macrophage is rooted in Al (III) to have researcher to think.Therefore, based on Al (III) and the vaccine carrier with antigen slow releasing function effectively may stimulate humoral immune reaction.
But Alum adjuvant is invalid to cellular immunization, can not as the adjuvant of cellular pathogens vaccine, the vaccine of the diseases such as such as HIV, malaria and pulmonary tuberculosis.In addition, when Alum adjuvant and high-purity protein antigen use, antibody response reacts more weak.Therefore, novel vaccine adjuvant is prepared in design, realizes efficiently presenting in protein vaccine body being still a difficult task.Microparticle type adjuvant (0.1 ~ 10 μm) has imitated the size of antibacterial and virus, can interact with dendritic cell, thus dendritic cell activated improve the presented by cells of antigen.At present, the research preparing microcapsule-type protein vaccine carrier based on the coordination of Al (III) and tannic acid have not been reported.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of pH response microcapsule-type protein vaccine carrier is provided.
Second object of the present invention is to provide a kind of preparation method of pH response microcapsule-type protein vaccine carrier.
Technical scheme of the present invention is summarized as follows:
A preparation method for pH response microcapsule-type protein vaccine carrier, comprises the steps:
(1) protein mimic antigen is joined in 10mL, 0.2 ~ 0.5M aqueous sodium carbonate, analogue antigen concentration is made to be 0.5 ~ 5mg/mL, pour into isopyknic containing in the calcium chloride water of kayexalate after dissolving, the concentration of described kayexalate is 1 ~ 6mg/mL, the concentration of calcium chloride is 0.2 ~ 0.5M, stirs 20 ~ 60s, centrifugal, deionization washing 2 ~ 4 times, obtains the calcium carbonate granule containing analogue antigen;
(2) calcium carbonate granule containing analogue antigen step (1) obtained is scattered in 20mL deionized water, add 0.5 ~ 1.5mL, the tannic acid aqueous solution of 8 ~ 24mM, vibration makes to be uniformly dispersed, add and tannic acid aqueous solution equal-volume, isocyatic aluminum chloride aqueous solution, vibration makes to be uniformly dispersed, add 20mL, 3-(N-morpholine) the propanesulfonate salt buffer solution of pH=8.0 regulates pH to be 8.0, vibration 1 ~ 5min, centrifugal, granule deionized water washes 2 ~ 4 times, with 20 ~ 40mL, the EDTA solution washing of pH=8.0 3 ~ 5 times, remove calcium carbonate, by washed with de-ionized water, obtain pH response microcapsule-type protein vaccine carrier.
The preferred chicken ovalbumin of protein mimic antigen.
PH response microcapsule-type protein vaccine carrier prepared by said method.
PH response microcapsule-type protein vaccine Carrier diameters 4 ± 0.5 μm of the present invention, thickness is 17 ± 2nm about, and chicken ovalbumin is embedded in microcapsule cavity.Because Al (III) and tannic acid coordinate bond affect by pH, microcapsule is made to have pH response.Preparation process of the present invention is carried out under room temperature aqueous phase, mild condition, ensure that the activity of protein vaccine and the safety in utilization of carrier; Cheaper starting materials is easy to get, and preparation is simple.
Accompanying drawing explanation
The electron scanning micrograph of the pH response microcapsule-type protein vaccine carrier of Fig. 1 obtained by embodiment 1.
The transmission electron microscope photo of the pH response microcapsule-type protein vaccine carrier of Fig. 2 obtained by embodiment 1.
The element energy spectrogram of the pH response microcapsule-type protein vaccine carrier of Fig. 3 obtained by embodiment 1.
The atomic force microscopy of the pH response microcapsule-type protein vaccine carrier of Fig. 4 obtained by embodiment 1 and difference in height figure thereof.Wherein A is atomic force microscopy; B is difference in height figure.
The release profiles of pH response microcapsule-type protein vaccine carrier under different pH the chicken ovalbumin of Fig. 5 obtained by embodiment 2.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated.
Embodiment 1
A preparation method for pH response microcapsule-type protein vaccine carrier, comprises the steps:
(1) pouring 10mL, 0.33M aqueous sodium carbonate into equal-volume contains in the calcium chloride water of kayexalate, and the concentration of described kayexalate is 2mg/mL, and the concentration of calcium chloride is 0.33M, stir 30s, centrifugal, deionization washes 3 times, obtains calcium carbonate granule;
(2) calcium carbonate granule that step (1) obtains is scattered in 20mL deionized water, add 1mL, the tannic acid aqueous solution of 24mM the 10s that vibrates on whirlpool instrument makes it be uniformly dispersed, add and tannic acid aqueous solution equal-volume, isocyatic aluminum chloride aqueous solution, and the 10s that vibrates on whirlpool instrument makes it be uniformly dispersed, add 20mL, 3-(N-morpholine) the propanesulfonate salt buffer solution of pH=8.0 regulates pH to be 8.0, and the 1min that vibrates on whirlpool instrument, centrifugal, granule deionized water washes 3 times, with 40mL, the EDTA solution washing of pH=8.0 3 times, remove calcium carbonate, by washed with de-ionized water, obtain a kind of preparation method of pH response microcapsule-type protein vaccine carrier.
See Fig. 1,2,3 and 4.
Embodiment 2
A preparation method for pH response microcapsule-type protein vaccine carrier, comprises the steps:
(1) protein mimic antigen-chicken ovalbumin is joined in 10mL, 0.33M aqueous sodium carbonate, chicken ovalbumin concentration is made to be 2mg/mL, pour into isopyknic containing in the calcium chloride water of kayexalate after dissolving, the concentration of described kayexalate is 2mg/mL, the concentration of calcium chloride is 0.33M, stirs 30s, centrifugal, deionization washes 3 times, obtains the calcium carbonate granule containing analogue antigen;
(2) calcium carbonate granule containing analogue antigen step (1) obtained is scattered in 20mL deionized water, add 1mL, the tannic acid aqueous solution of 24mM, vibration 10s makes it be uniformly dispersed, add and tannic acid aqueous solution equal-volume, isocyatic aluminum chloride aqueous solution, and the 10s that vibrates on whirlpool instrument makes it be uniformly dispersed, add 20mL, 3-(N-morpholine) the propanesulfonate salt buffer solution of pH=8.0 regulates pH to be 8.0, and the 1min that vibrates on whirlpool instrument, centrifugal, granule deionized water washes 3 times, with 40mL, the EDTA solution washing of pH=8.0 3 times, remove calcium carbonate, by washed with de-ionized water, obtain pH response microcapsule-type protein vaccine carrier.
Embodiment 3
A preparation method for pH response microcapsule-type protein vaccine carrier, comprises the steps:
(1) protein mimic antigen-chicken ovalbumin is joined in 10mL, 0.2M aqueous sodium carbonate, chicken ovalbumin concentration is made to be 0.5mg/mL, pour into isopyknic containing in the calcium chloride water of kayexalate after dissolving, the concentration of described kayexalate is 1mg/mL, the concentration of calcium chloride is 0.2M, stirs 20s, centrifugal, deionization washes 2 times, obtains the calcium carbonate granule containing analogue antigen;
(2) calcium carbonate granule containing analogue antigen step (1) obtained is scattered in 20mL deionized water, add 0.5mL, the tannic acid aqueous solution of 12mM, vibration 10s makes it be uniformly dispersed, add and tannic acid aqueous solution equal-volume, isocyatic aluminum chloride aqueous solution, and the 10s that vibrates on whirlpool instrument makes it be uniformly dispersed, add 20mL, 3-(N-morpholine) the propanesulfonate salt buffer solution of pH=8.0 regulates pH to be 8.0, and the 3min that vibrates on whirlpool instrument, centrifugal, granule deionized water washes 2 times, with 20mL, the EDTA solution washing of pH=8.0 4 times, remove calcium carbonate, by washed with de-ionized water, obtain pH response microcapsule-type protein vaccine carrier.
Embodiment 4
A preparation method for pH response microcapsule-type protein vaccine carrier, comprises the steps:
(1) protein mimic antigen-chicken ovalbumin is joined in 10mL, 0.5M aqueous sodium carbonate, chicken ovalbumin concentration is made to be 5mg/mL, pour into isopyknic containing in the calcium chloride water of kayexalate after dissolving, the concentration of described kayexalate is 6mg/mL, the concentration of calcium chloride is 0.5M, stirs 60s, centrifugal, deionization washes 4 times, obtains the calcium carbonate granule containing analogue antigen;
(2) calcium carbonate granule containing analogue antigen step (1) obtained is scattered in 20mL deionized water, add 1.5mL, the tannic acid aqueous solution of 8mM, vibration 10s makes it be uniformly dispersed, add and tannic acid aqueous solution equal-volume, isocyatic aluminum chloride aqueous solution, and the 10s that vibrates on whirlpool instrument makes it be uniformly dispersed, add 20mL, 3-(N-morpholine) the propanesulfonate salt buffer solution of pH=8.0 regulates pH to be 8.0, and the 5min that vibrates on whirlpool instrument, centrifugal, granule deionized water washes 4 times, with 30mL, the EDTA solution washing of pH=8.0 5 times, remove calcium carbonate, by washed with de-ionized water, obtain pH response microcapsule-type protein vaccine carrier.
Embodiment 5
A kind of pH response microcapsule-type protein vaccine support dispersion embodiment 2 prepared is in 10mL, pH is respectively 5.0,6.0,7.4 and concentration is in the phosphate buffered solution of 10mM, be positioned over 37 DEG C, start timing in the concussion incubator of 200rpm, at regular intervals, sample is centrifugal and take out the supernatant of 5mL respectively, add the phosphate buffered solution of the different pH of the fresh configuration of 5mL afterwards more respectively, and be again positioned over 37 DEG C, in the concussion incubator of 200rpm.Measure protein concentration in supernatant with Coomassie Brilliant Blue and calculate the Tot Prot released, i.e. protein accumulation burst size, protein delivery rate=(in protein accumulation burst size/carrier Tot Prot) * 100%.
See Fig. 5.
Claims (3)
1. a preparation method for pH response microcapsule-type protein vaccine carrier, is characterized in that comprising the steps:
(1) protein mimic antigen is joined in 10mL, 0.2 ~ 0.5M aqueous sodium carbonate, analogue antigen concentration is made to be 0.5 ~ 5mg/mL, pour into isopyknic containing in the calcium chloride water of kayexalate after dissolving, the concentration of described kayexalate is 1 ~ 6mg/mL, the concentration of calcium chloride is 0.2 ~ 0.5M, stirs 20 ~ 60s, centrifugal, deionization washing 2 ~ 4 times, obtains the calcium carbonate granule containing analogue antigen;
(2) calcium carbonate granule containing analogue antigen step (1) obtained is scattered in 20mL deionized water, add 0.5 ~ 1.5mL, the tannic acid aqueous solution of 8 ~ 24mM, vibration makes to be uniformly dispersed, add and tannic acid aqueous solution equal-volume, isocyatic aluminum chloride aqueous solution, vibration makes to be uniformly dispersed, add 20mL, 3-(N-morpholine) the propanesulfonate salt buffer solution of pH=8.0 regulates pH to be 8.0, vibration 1 ~ 5min, centrifugal, granule deionized water washes 2 ~ 4 times, with 20 ~ 40mL, the EDTA solution washing of pH=8.0 3 ~ 5 times, remove calcium carbonate, by washed with de-ionized water, obtain pH response microcapsule-type protein vaccine carrier.
2. method according to claim 1, is characterized in that described protein mimic antigen is chicken ovalbumin.
3. the pH response microcapsule-type protein vaccine carrier prepared of the method for claim 1 or 2.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110025777A (en) * | 2019-04-30 | 2019-07-19 | 中国医学科学院生物医学工程研究所 | A kind of preparation method for the micron particles vaccine carrier being loaded with nano particle |
| CN112516070A (en) * | 2020-12-16 | 2021-03-19 | 南开大学 | Single injection vaccine of protein antigen and preparation method thereof |
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Cited By (3)
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| CN110025777A (en) * | 2019-04-30 | 2019-07-19 | 中国医学科学院生物医学工程研究所 | A kind of preparation method for the micron particles vaccine carrier being loaded with nano particle |
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