CN110664749B - MF 59-based entrapped antigen nanoemulsion and preparation method and application thereof - Google Patents
MF 59-based entrapped antigen nanoemulsion and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to an MF 59-based entrapped antigen nanoemulsion as well as a preparation method and application thereof, wherein the entrapped antigen nanoemulsion comprises the following components in parts by weight: 2.58 parts of squalene, 850.9 parts of emulsifier span, 800.9 parts of tween, 20 parts of PBS and 0.005-0.05 part of antigen. The antigen-loaded nanoemulsion is prepared by adopting a multiple emulsion method based on reversed micelle, and comprises the following steps: the internal water phase is added into the oil phase containing the emulsifier to form reversed micelles, and then the reversed micelles are added into the external water phase to form the multiple emulsion. The invention also comprises application of the MF 59-based entrapped antigen nanoemulsion in vaccines. Its advantages are: the encapsulation rate of the nano-emulsion encapsulated macromolecular protein is improved, and the particle size is controlled within 100 nm.
Description
Technical Field
The invention relates to the technical field of pharmaceutical preparations, in particular to an MF 59-based entrapped antigen nanoemulsion and a preparation method and application thereof.
Background
The nano-emulsion is a colloid dispersion system formed by dispersing emulsion drops with the particle size of 10-100nm in another solution, wherein the nano-emulsion comprises an oil phase, a water phase, an emulsifier, an auxiliary emulsifier and the like. The nanoemulsion can not only encapsulate fat-soluble drugs, improve the solubility of the drugs, and promote the oral absorption and transdermal transportation of the drugs; can also be used for encapsulating water-soluble medicines, especially macromolecular protein medicines, and improving the oral bioavailability and transdermal transportation of the macromolecular medicines. In addition, researches find that the oil-in-water type nano emulsion can also be used as an immunologic adjuvant to enhance the immune effect of antigens. MF59 is an oil-in-water emulsion, can enhance the immunogenicity of influenza antigen, and is used as an adjuvant for influenza vaccine for people with low immunity in 90 years of the 20 th century. A large amount of data shows that MF59 is safer for use in influenza vaccines. However, most of the existing vaccines are prepared into vaccine freeze-dried powder due to stability reasons, and the vaccine freeze-dried powder needs to be refrigerated, transported and stored, so that the preparation cost is greatly increased. If the antigen can be directly entrapped in MF59, the nanoemulsion can increase the stability of the antigen and greatly reduce the storage cost. However, most antigens are macromolecular proteins, the entrapment rate in the nanoemulsion is not high, the particle size of the emulsion is increased by adopting a common W/O/W multiple emulsion preparation method, and the research shows that the adjuvant effect of the nanoemulsion is reduced by too large particle size.
In view of the above-mentioned drawbacks, there is a need for a novel method for preparing a nanoemulsion, which can improve the encapsulation efficiency of the nanoemulsion for encapsulating macromolecular proteins, and control the particle size within 100 nm. The invention provides an MF 59-based entrapped antigen nanoemulsion and a preparation method thereof, which can improve the entrapment rate of macromolecular proteins entrapped by the nanoemulsion and control the particle size within 100 nm. There is no report on this.
Disclosure of Invention
The first purpose of the present invention is to provide a nano-emulsion based on MF59 entrapped antigen, which is used for overcoming the defects of the prior art.
The second purpose of the present invention is to provide a preparation method of an antigen-entrapped nanoemulsion based on MF59, which is directed to the shortcomings of the prior art.
A third object of the present invention is to address the deficiencies of the prior art by providing the use of an MF 59-based entrapped antigen nanoemulsion as described above.
A fourth object of the present invention is to provide a vaccine against the deficiencies of the prior art.
In order to achieve the first purpose, the invention adopts the technical scheme that:
an antigen-entrapped nanoemulsion based on MF59 comprises the following components in parts by weight: 2.58 parts of squalene, 850.9 parts of emulsifier span, 800.9 parts of tween and 20 parts of PBS;
the nano-emulsion also comprises 0.05 part of antigen, and the antigen is selected from proteins or polypeptides related to infectious diseases or tumors.
As a preferred embodiment of the invention, the preparation method of the antigen-loaded nanoemulsion based on MF59 comprises the following steps:
(1) mixing squalene 2.58 parts, emulsifier span850.9 parts, and tween800.6 parts to obtain oil phase;
(2) dissolving 0.005-0.05 part of antigen in 0.3 part of pure water to prepare an inner water phase;
(3) dissolving 0.3 part of tween80 in 20 parts of PBS solution to prepare an external water phase;
(4) and (3) adding the internal water phase prepared in the step (2) into the oil phase prepared in the step (1) to form reversed micelles, and adding the reversed micelles into the external water phase prepared in the step (3) to form multiple emulsion.
As a preferred embodiment of the present invention, the nano-emulsion is prepared to have a particle size of 10nm to 90 nm; the encapsulation rate of the prepared nano-emulsion is more than or equal to 70-80%.
In order to achieve the second object, the invention adopts the technical scheme that:
the preparation method of the antigen-entrapped nanoemulsion based on MF59 comprises the following steps:
(1) mixing squalene 2.58 parts, emulsifier span850.9 parts, and tween800.6 parts to obtain oil phase;
(2) dissolving 0.005-0.05 part of antigen in 0.3 part of pure water to prepare an inner water phase;
(3) dissolving 0.3 part of tween80 in 20 parts of PBS solution to prepare an external water phase;
(4) and (3) adding the internal water phase prepared in the step (2) into the oil phase prepared in the step (1) to form reversed micelles, and adding the reversed micelles into the external water phase prepared in the step (3) to form multiple emulsion.
In order to achieve the third object, the invention adopts the technical scheme that:
use of an MF 59-based entrapped antigen nanoemulsion as described above in the production of vaccines.
As a preferred embodiment of the invention, the antigen is selected from proteins or polypeptides related to influenza, measles, chicken pox, rubella, epidemic encephalitis, mumps, tuberculosis and respiratory infectious diseases.
As a preferred embodiment of the invention, the antigen is selected from the group consisting of proteins or polypeptides associated with AIDS, hepatitis B, syphilis, gonorrhea, trichomonas vaginitis, bacterial vaginitis, amebiasis, genital herpes infections.
In order to achieve the fourth object, the invention adopts the technical scheme that:
a vaccine is prepared from the MF 59-based entrapped antigen nanoemulsion.
As a preferred embodiment of the invention, the antigen is selected from proteins or polypeptides related to influenza, measles, chicken pox, rubella, epidemic encephalitis, mumps, tuberculosis and respiratory infectious diseases.
As a preferred embodiment of the invention, the antigen is selected from the group consisting of proteins or polypeptides associated with AIDS, hepatitis B, syphilis, gonorrhea, trichomonas vaginitis, bacterial vaginitis, amebiasis, genital herpes infections.
The invention has the advantages that:
1. the defects of the prior art are overcome, the components and the proportion thereof are optimized, and the antigen-loaded nano-emulsion is prepared based on a reversed-phase micelle multiple emulsion method: the internal water phase is added into the oil phase containing the emulsifier to form reversed micelles, and then the reversed micelles are added into the external water phase to form the multiple emulsion. Because the particle size of the reversed micelle formed in the first step is very small, the particle size of the finally prepared multiple emulsion is relatively small and is within 100 nm; meanwhile, the entrapment rate of the nanoemulsion entrapped macromolecular protein is up to 70-80%. The antigen-loaded nanoemulsion prepared based on the reversed-phase micelle method has the characteristics of small particle size and high encapsulation rate, and overcomes the defects of the prior art.
2. The antigen-loaded nanoemulsion obtained by the preparation method can be used for preparing vaccines, can increase the stability of antigens and greatly reduce the storage cost, and meanwhile, the particle size of the nanoemulsion is controlled within 100nm, so that the adjuvant effect of the nanoemulsion is enhanced, the economic burden of vaccine-inoculated people is reduced, and the curative effect is good.
Drawings
FIG. 1 is a prescription screening result, wherein A shows that the influence of increasing the lipid and emulsifier concentrations in the same proportion on the characterization, the original MF59 lipid concentration is too low, and the particle size is increased after the lipid concentration is increased, so that the influence on the encapsulation efficiency is small;
b, the particle size is reduced along with the increase of the using amount of the emulsifier, the influence of the encapsulation efficiency is small, the encapsulation efficiency of the nanoemulsion prepared by the one-step emulsification method is too low, and the encapsulation efficiency of the nanoemulsion prepared by the two-step emulsification method is greatly improved;
c shows that the emulsifier HLB value in the oil phase changes with the addition of tween80 in the oil phase, the entrapment efficiency varies significantly, when the ratio of oil phase to tween80 in the water phase is 0.6/0.3, at which time the emulsifier HLB value in the oil phase is 7.1, the entrapment efficiency is the highest, which may be the case when the HLB value of the oil phase is most suitable for preparing w/o emulsions;
d, displaying the influence of the drug lipid ratio on the characterization, wherein the drug lipid ratio has almost no influence on the particle size and mainly influences the encapsulation rate;
e shows the influence of the external water phase medium on the characterization, and the external water phase medium is considered to be water (H)2O), pH7.4 Phosphate Buffer Solution (PBS), citric acid buffer salt (CAB) and the like, and the encapsulation efficiency is obviously improved by adding medium such as salt in an external water phase, which is probably because the ionic strength of the solution is increased by adding the salt, the solubility of ova in the external water phase containing the salt is reduced, so that the ova is more dissolved in the water phase in the nano-emulsion, and the factor encapsulation efficiency is improved, and the consideration is that the factor encapsulation efficiency is improvedSince proteins may be denatured for a long time by a 20% ethanol solution in a citrate buffer, PBS was selected as the external aqueous phase. The ova-NE encapsulation rate prepared by the micro-jet homogenizer can reach 70%, but the particle size is still larger than 100nm, the preparation instrument is tried to be replaced, and the particle size is reduced by adopting a high-pressure homogenization method;
f shows that the nano-emulsion is prepared by a high-pressure homogenizer, the particle size is reduced along with the increase of the high-pressure homogenizing pressure, the particle size is not obviously reduced when the pressure is increased from 900bar to 1000bar, the encapsulation efficiency is reduced, and then the nano-emulsion is prepared by high-pressure homogenizing at 900bar for 2 min.
FIG. 2 shows the ova-NE particle size distribution measured by a Malvern particle size analyzer.
Detailed Description
The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.
EXAMPLE 1 Components (A)
Weighing the following components in parts by weight:
2.58 parts of squalene, 850.9 parts of emulsifier span, 800.9 parts of tween, 20 parts of PBS and 0.05 part of egg albumin.
Example 2 component (II)
Weighing the following components in parts by weight:
2 parts of squalene, 851 parts of emulsifier span, 801 parts of tween, 15 parts of PBS and 0.1 part of egg albumin.
Example 3 vaccine (one)
The preparation method comprises the following steps:
(1) mixing squalene 2.58 parts, emulsifier span850.9 parts, and tween800.6 parts to obtain oil phase;
(2) dissolving 0.05 part of egg white protein in 0.3 part of pure water to prepare an inner water phase;
(3) dissolving 0.3 part of tween80 in 20 parts of PBS solution to prepare an external water phase;
(4) and (3) adding the internal water phase prepared in the step (2) into the oil phase prepared in the step (1) to form reversed micelles, and adding the reversed micelles into the external water phase prepared in the step (3) to form multiple emulsion.
Example 4 vaccine 2
The preparation method comprises the following steps:
(1) mixing squalene 2.58 parts, emulsifier span850.9 parts, and tween801.2 parts to obtain oil phase;
(2) dissolving 0.05 part of egg white protein in 0.3 part of pure water to prepare an inner water phase;
(3) dissolving 0.6 part of tween80 in 20 parts of PBS solution to prepare an external water phase;
(4) and (3) adding the internal water phase prepared in the step (2) into the oil phase prepared in the step (1) to form reversed micelles, and adding the reversed micelles into the external water phase prepared in the step (3) to form multiple emulsion.
Example 5 preparation of MF 59-based encapsulated chicken ovalbumin (ova) nanoemulsion and detection of encapsulation efficiency and particle size of nanoemulsion
The emulsion is prepared based on a commercially available MF59 formula, and the related materials are as follows: squalene (oil phase), tween80 and span85 (emulsifiers), ph7.4 phosphate buffer, citrate buffer, chicken ovalbumin (ova, model antigen).
Experimental materials: squalene (sigma corporation, usa), egg white protein (ova, sigma corporation, usa), tween80 (national group chemical reagent limited), span85 (national group chemical reagent limited), citric acid (national group chemical reagent limited), potassium dihydrogen phosphate (national group chemical reagent limited), sodium hydroxide (national group chemical reagent limited), BCA protein test kit (beijing solibao science and technology limited).
An experimental instrument: heating magnetic stirrer: (
WERKE RT 10POWER, IKA, Germany), high-speed homogenizer (
-TIKA, germany), a microfluidizer nanodispersion homogenizer ((Nano DeBEE, b.e.e. international), a high pressure homogenizer (APV, germany), a high speed refrigerated centrifuge (thermo Fisher, germany), an electronic balance (sartorius, germany), a Zetasizer laser particle size analyzer (Malvern, uk).
The specific preparation method of the ova-loaded nanoemulsion (ova-NE) comprises the following steps:
preparation of ova-NE
Based on a commercially available MF59 formula, ova-NE was prepared by a high pressure emulsification method:
one-step emulsification method: taking squalene and Span85 in the prescription amount, uniformly mixing to obtain an oil phase, dissolving ova in a solution of tween80 to obtain a water phase, pouring the water phase into the oil phase under high-speed shearing at 10000rpm, continuously shearing for 2min to obtain colostrum, and homogenizing under high pressure (or using a micro-jet homogenizer) to prepare the nanoemulsion.
② a two-step emulsification method: taking a prescription amount of squalene, span85 and a part of prescription amount of tween80, uniformly mixing to form an oil phase, dissolving ova in 300 mu l of pure water to form an inner water phase, magnetically stirring at 1000rpm, dropwise adding the inner water phase into the oil phase at 25 ℃, continuously stirring for 30min to prepare w/o colostrum (also called reversed micelle due to small particle size), then pouring the emulsion into an outer water phase in which the rest tween80 is dissolved under high-speed shearing at 10000rpm, continuously shearing for 2min, and then carrying out high-pressure homogenization (or a micro-jet homogenizer) to prepare the w/o/w nanoemulsion.
2. Prescription optimization
Optimizing a prescription by taking the particle size and the encapsulation rate as main factors, and hopefully preparing the high-encapsulation-rate nanoemulsion with proper lipid concentration and particle size less than 100 nm; the nanoemulsion comprises the proportion of lipid to an external water phase (w/v, 4.3% -25.8%), the proportion of an emulsifier to the external water phase (w/v, 1.5% -6%), and the preparation method comprises the steps of replacing one-step emulsification with a two-step emulsification method, wherein tween80 is distributed according to the proportion of oil phase to external water phase (w/w, detailed in table 1), ova dosage (w/w, 0.02/2.58-0.12/2.58), and external water phase medium types (pure water, PBS and citric acid buffer solution), and the nanoemulsion is prepared by adopting a micro-jet homogenizer in the optimization experiments under the condition of 20000psi and 4 cycles of homogenization; then, the nano-emulsion is prepared by high-pressure homogenization, and the high-pressure homogenization pressure is screened.
TABLE 1 Tween80 weight ratio between oil phase and external water phase
Determination of ova content
The ova content was determined using the BCA protein assay kit. According to the instruction, 100 mu l of sample is taken, added with 1ml of BCA working solution, mixed evenly, incubated at 37 ℃ for 30min, and an ultraviolet spectrophotometer is adopted to measure the absorbance at 562nm and bring the absorbance into a standard curve to calculate the concentration.
The ova determination of the loading in the preparation NE is determined by treating with ethanol precipitation extraction: taking 100 mu l of NE suspension diluted by a proper amount, adding 1.2ml of a glacial ethanol solution for demulsification and dissolution, placing in a refrigerator at the temperature of 20 ℃ below zero for 2h, centrifuging at the temperature of 4 ℃ and the speed of 15000rpm for 45min to precipitate protein, pouring off the ethanol solution, volatilizing the ethanol at room temperature, adding 100 mu l of pure water for redissolution, and determining the protein content as a sample by the BCA method.
4. Determination of encapsulation efficiency
The encapsulation efficiency of ova-NE was determined by passing through a gel chromatography column. The molecular weight of free ova is small, the retention time in a gel column is long, NE particles are large, and the ova loaded in NE is eluted out along with the short retention time of NE in the gel column, so that the encapsulated ova and the non-encapsulated ova are separated, and the encapsulation rate is measured.
Taking a proper amount of ova-NE stock solution to determine that the ova content is the total ova amount, taking the ova-NE stock solution to pass through a gel column loaded with glucan G50 to separate and collect a nano-emulsion section, and determining the nano-emulsion section as the encapsulated ova, wherein the ratio of the amount of the encapsulated drug to the total amount of the drug is the encapsulation rate.
5. Results of the experiment
The prescription screening results are shown in FIG. 1. FIG. 1.A shows that increasing the lipid and emulsifier concentrations in the same proportion has an effect on characterization, and the original MF59 lipid concentration is too low, and after the lipid concentration is increased, the particle size is increased, and the effect on the encapsulation efficiency is small. Lipid concentrations of 12.9% were selected for subsequent studies. The fixed lipid concentration was constant and the ratio of tween80 to span85 was constant, and fig. 1.B found that as the amount of emulsifier was increased, the particle size decreased and the encapsulation efficiency was less affected. The nano-emulsion prepared by the one-step emulsification method has too low entrapment rate, the nano-emulsion is prepared by the two-step emulsification method, part of tween80 is added into the oil phase, and the HLB value of the emulsifier in the oil phase is adjusted, which can be shown in Table 1. Figure 1.C shows that the entrapment efficiency varies significantly with the addition of tween80 to the oil phase, and is greatest when the ratio of oil phase to water phase tween80 is 0.6/0.3, which may be when the HLB value of the oil phase is most suitable for preparing w/o emulsions. Fig. 1.D shows the effect of the drug lipid ratio on the characterization, the drug lipid ratio has almost no effect on the particle size, mainly affecting the encapsulation efficiency. FIG. 1.E shows the effect of the external aqueous medium on the characterization, and the effect of the external aqueous medium being water, pH7.4 Phosphate Buffered Saline (PBS), citrate buffered saline (CAB) was examined. The encapsulation efficiency is obviously improved by adding a medium such as salt in the external water phase, which is probably because the ionic strength of the solution is increased by adding the salt, the solubility of ova in the salt solution is reduced, so that the solubility of ova in the external water phase is reduced, and more nano milk is encapsulated. PBS was chosen as the external aqueous phase, considering that 20% ethanol solution in citrate buffer may denature proteins for a long time. The ova-NE prepared by the micro-jet homogenizer has an encapsulation rate of 70%, but the particle size is still larger than 100nm, and the preparation instrument is tried to be replaced, and a high-pressure homogenizer is adopted to reduce the particle size. Fig. 1.F shows that the particle size decreases with increasing high pressure homogenization pressure, the particle size decrease is not significant when the pressure is increased from 900bar to 1000bar, but the encapsulation efficiency decreases. Homogenizing under high pressure of 900bar for 2min to obtain nanoemulsion.
6. Conclusion
Optimal prescription: the squalene 2.58g, Span850.9g and Tween800.6 g are mixed uniformly to form an oil phase, 50mg ova is dissolved in 300. mu.l of pure water to form an inner water phase, and 0.3g Tween80 is dissolved in 20ml of PBS solution to form an outer water phase. And (2) stirring by magnetic force at 1000rpm and 25 ℃, dropwise adding the inner water phase into the mixed oil phase, continuously stirring for 30min to prepare reversed-phase micelles, namely colostrums, then pouring the colostrums into the outer water phase dissolved with tween80 under high-speed shearing at 10000rpm, continuously shearing for 2min to obtain multiple emulsions, homogenizing at high pressure of 900bar for 2min to reduce the particle size, and preparing the w/o/w nano-emulsion. The prepared nano-emulsion has the particle size of 81.2nm and the encapsulation efficiency of 70.2 percent.
The components and the proportion thereof are optimized, and the antigen-loaded nano-emulsion is prepared based on a reversed-phase micelle multiple emulsion method: the method comprises the steps of adding an inner water phase into an oil phase containing an emulsifier to form reversed micelles, and adding the reversed micelles into an outer water phase to form the multiple emulsion. The nano-emulsion prepared by the method can improve the encapsulation rate of the nano-emulsion for encapsulating the macromolecular protein and can control the particle size within 100 nm; meanwhile, the antigen is directly entrapped in MF59, so that the stability of the antigen can be increased, and the cost of vaccine refrigeration, transportation and storage can be greatly reduced. The antigen-loaded nanoemulsion obtained by the preparation method can be used for preparing vaccines, is cheap and good, and has a good application prospect.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these should also be considered as the protection scope of the present invention.
Claims (9)
1. An antigen-entrapped nanoemulsion based on MF59 is characterized by comprising the following components in parts by weight: 2.58 parts of squalene, 850.9 parts of emulsifier span, 800.9 parts of tween and 20 parts of PBS; the nano-emulsion also comprises 0.005-0.05 part of antigen, and the antigen is selected from proteins or polypeptides related to infectious diseases or tumors; the preparation method of the antigen-entrapped nanoemulsion based on MF59 comprises the following steps:
(1) mixing squalene 2.58 parts, emulsifier span850.9 parts, and tween800.6 parts to obtain oil phase;
(2) dissolving 0.005-0.05 part of antigen in 0.3 part of pure water to prepare an inner water phase;
(3) dissolving 0.3 part of tween80 in 20 parts of PBS solution to prepare an external water phase;
(4) magnetically stirring at 1000rpm, adding the inner water phase prepared in the step (2) into the oil phase prepared in the step (1), continuously stirring for 30min to form reversed-phase micelles, and then adding the reversed-phase micelles into the outer water phase prepared in the step (3) and continuously shearing for 2min to form multiple emulsion;
(5) and (4) homogenizing the multiple emulsion formed in the step (4) at high pressure of 900bar for 2min, and reducing the particle size to obtain the compound emulsion.
2. The MF 59-based antigen-entrapped nanoemulsion of claim 1, wherein the nanoemulsion prepared by the method has a particle size of 10nm-90 nm; the encapsulation rate of the prepared nano-emulsion is 70-80%.
3. The preparation method of the MF 59-based antigen-entrapped nanoemulsion of claim 1, which comprises the following steps:
(1) mixing squalene 2.58 parts, emulsifier span850.9 parts, and tween800.6 parts to obtain oil phase;
(2) dissolving 0.005-0.05 part of antigen in 0.3 part of pure water to prepare an inner water phase;
(3) dissolving 0.3 part of tween80 in 20 parts of PBS solution to prepare an external water phase;
(4) magnetically stirring at 1000rpm, adding the inner water phase prepared in the step (2) into the oil phase prepared in the step (1), continuously stirring for 30min to form reversed-phase micelles, and then adding the reversed-phase micelles into the outer water phase prepared in the step (3) and continuously shearing for 2min to form multiple emulsion;
(5) and (4) homogenizing the multiple emulsion formed in the step (4) at high pressure of 900bar for 2min, and reducing the particle size to obtain the compound emulsion.
4. Use of the MF 59-based entrapped antigen nanoemulsion of claim 1 in the production of vaccines.
5. The use of claim 4, wherein the antigen is selected from the group consisting of proteins and polypeptides associated with influenza, measles, chicken pox, rubella, epidemic encephalitis, mumps, tuberculosis, respiratory infectious disease.
6. The use of claim 4, wherein the antigen is selected from the group consisting of proteins and polypeptides associated with AIDS, hepatitis B, syphilis, gonorrhea, trichomonas vaginitis, bacterial vaginitis, amoebic disease, genital herpes infections.
7. A vaccine, characterized in that the vaccine is prepared from the MF 59-based entrapped antigen nanoemulsion of claim 1.
8. The vaccine of claim 7, wherein the antigen is selected from the group consisting of proteins and polypeptides associated with influenza, measles, chicken pox, rubella, epidemic encephalitis, mumps, tuberculosis, and respiratory infectious disease.
9. The vaccine of claim 7, wherein said antigen is selected from the group consisting of proteins and polypeptides associated with AIDS, hepatitis B, syphilis, gonorrhea, trichomonas vaginitis, bacterial vaginitis, amoebic disease, and genital herpes infections.
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| CN102600468A (en) * | 2012-03-01 | 2012-07-25 | 浙江天元生物药业有限公司 | Preparation process of avian influenza virus split vaccine containing MF59 adjuvant |
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