CN104225593A - Water-soluble composite immunologic adjuvant and vaccine composition comprising same - Google Patents
Water-soluble composite immunologic adjuvant and vaccine composition comprising same Download PDFInfo
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Abstract
The invention belongs to the technical field of immunologic adjuvants and discloses a water-soluble composite immunologic adjuvant. The water-soluble composite immunologic adjuvant is prepared by multi-phosphonitrile high-molecular polymer and CpG motif recombinant plasmid presented by SEQ ID No.1 according to a certain weight ratio, wherein the side chain of the multi-phosphonitrile high-molecular polymer is para-sodium propionate phenoxy, and the molecular weight ranges between 0.7 to 1 million units. The invention further discloses a method for preparing the immunologic adjuvant, a vaccine composition containing the composite immunologic adjuvant and a preparation method thereof, as well as the application of the composite immunologic adjuvant in the preparation of the vaccine composition. According to biological and immunological characteristics of the multi-phosphonitrile high-molecular polymer and the CpG recombinant plasmid, two molecules are prepared into the novel composite immunologic adjuvant according to a certain compatibility, an immunological enhancement which is safer and more efficient than a traditional oil adjuvant can be achieved, and the water-soluble composite immunologic adjuvant has a wider market prospect.
Description
Technical field
The present invention relates to immunological adjuvant technical field, be specifically related to have the Water Soluble Compound immunological adjuvant of immunological enhancement and preparing the application in vaccine combination.
Background technology
Foot and mouth disease (FMD) is that the artiodactyl caused by foot and mouth disease virus (FMDV) is acute, hot, high degree in contact sexually transmitted disease, endanger one of the most serious infectious disease of animal husbandry, be classified as first of 18 kinds of category-A infectious disease in International Animal Health code by World Organization for Animal Health (OIE) and FAO (Food and Agriculture Organization of the United Nation) (FAO), the whole world elimination plan and the biological weapons security pact that are in recent years listed in again great transnational animal epidemic organize emphasis check object.Still put prevention first with immunity for the control of foot and mouth disease at present, although the prevention that the development of various new generation vaccine is foot and mouth disease brings new opportunity.But due to the deficiency of various new generation vaccine in immune protection effectiveness, cause it can't replace traditional inactivated vaccine completely.In the world, some countries in South America are obtained " without foot and mouth disease statehood " by inactivated foot-and-mouth disease vaccine immunoprophylaxis.In the foot and mouth disease prevention and control of China, its inactivated vaccine is still main flow immune vaccine, and its market share is still very large, in the foot and mouth disease of China and neighboring countries and regions controls, played important function.
At the beginning of inactivated foot-and-mouth disease vaccine research and development, general is all use water adjuvant to carry out compatibility foot and mouth disease inactivation antigen, but british animal health research in 1997 the new oily adjuvant Mnotnadie ISA25 and 206 of Pirbright use for laboratory two kinds prepare foot and mouth vaccine immune swine and cattle and sheep.Follow-up research finds, oily adjuvant, compared to the vaccine prepared by water adjuvant compatibility inactivation antigen, pig and cattle and sheep can produce stronger antibody response, cause inactivated vaccine afterwards all to adopt oily adjuvant.But the use of oily adjuvant but greatly increases the cost of vaccine, and its side effect is comparatively large, usually can cause the even irritated problem such as dead of the redness of injection site, pain, fever.Therefore, if the suitable water adjuvant of a kind of and oily adjuvant effect can be found to prepare inactivation antigen vaccine, the market prospect of so this safe and efficient vaccine will be more wide.
Many phosphonitriles composition is a kind of novel potential macromolecular compound with immunological adjuvant effect, current research has proved that it has immunological enhancement, after itself and the compatible immune mouse of respiratory system epidemic disease pathogen antigen, prove that many phosphonitriles not only improve the level representing autarcetic IgA antibody, and the IFN γ level representing Th1 type can not only be strengthened in acquired immunity, the secretion level of the IL-4 representing Th2 type can also be strengthened.But, the high molecular polymer that many phosphonitriles molecule is made up of with the side chain having multiple different organic substituent an organic backbone, make it have organic and performance that is inorganic polymer concurrently, introduce different side chains, the Synthesis, Characterization of Polyphosphazenes with difference in functionality can be prepared.At present, many phosphonitriles molecule is used as immunological adjuvant and is also in research and development initial stage, its immunoenhancement result not yet reaches the level suitable with oily adjuvant effect.
In addition, current research both domestic and external has proved that CpG motif is a kind of based on body natural immunity signal pathway, the molecule with immune-stimulating effect, and the recombiant plasmid that the research before inventor also demonstrates containing CpG motif is also a kind of well immunostimulant.But although above-mentioned CpG recombiant plasmid has stronger antigen immune increased activity ability, it also can't reach the effect of Traditional adjuvants completely as single adjuvant component.
Summary of the invention
For the above-mentioned defect existed in prior art, one aspect of the present invention provides a kind of Water Soluble Compound immunological adjuvant, it is formed according to the proportions that weight ratio is 5:2 by many phosphonitriles high molecular polymer and the recombiant plasmid comprising the CpG motif shown in SEQ ID NO:1, the side chain of wherein said many phosphonitriles high molecular polymer is to sodium propionate phenoxy group, and molecular weight is between 70-100 ten thousand unit.
In a preferred embodiment of the present invention, this many phosphonitriles high molecular polymer is prepared by following steps:
1, the synthesis of polydichlorophosphazenes
Ammonium chloride and phosphorus chloride are mixed using sulfamic acid, calcium sulphate dihydrate and trichloro-benzenes as solvent, synthesis polydichlorophosphazenes;
2, to the synthesis of methyl propionate benzenpropanoic acid sodium
Para hydroxybenzene methyl propionate is mixed using oxolane as solvent with sodium hydride, synthesizes methyl propionate benzenpropanoic acid sodium;
3, the synthesis of poly-[two (to methyl propionate phenoxy group)] phosphonitrile
The polydichlorophosphazenes that first two steps are synthesized and to the mixing of methyl propionate benzenpropanoic acid sodium using oxolane as solvent, poly-[two (to methyl propionate the phenoxy group)] phosphonitrile of synthesis;
4, the synthesis of poly-[two (to propanoic acid toloxyl)] phosphonitrile
Potassium hydroxide solution is added dropwise to, poly-[two (to propanoic acid the toloxyl)] phosphonitrile of synthesis in poly-[two (to methyl propionate phenoxy group)] phosphonitrile of synthesis;
5, the synthesis of poly-[two (to propanoic acid phenoxy group)] phosphonitrile
Precipitate with deionized water is dissolved, and then precipitate from deionized water with dehydrated alcohol, again precipitate with deionized water is dissolved, add hydrochloric acid solution titration and regulate final solution pH=6.3, finally by saturated brine precipitation, obtain poly-[two (to propanoic acid phenoxy group)] phosphonitrile;
6, the synthesis of poly-[two (to sodium propionate phenoxy group)] phosphonitrile
Poly-[two (to propanoic acid the phenoxy group)] phosphonitrile obtained is dissolved in deionized water, uses sodium hydrate regulator solution pH=7.4, by dehydrated alcohol precipitation, obtain poly-[two (to sodium propionate phenoxy group)] phosphonitrile;
7, screened by the molecular weight of poly [two (to sodium propionate phenoxy group)] phosphonitrile, acquisition molecular weight is poly [two (to sodium propionate the phenoxy group)] phosphonitrile between 70-100 ten thousand unit.
In another preferred embodiment of the present invention, the recombiant plasmid in this Water Soluble Compound immunological adjuvant is for comprising the puc18 recombiant plasmid of the CpG motif shown in SEQ ID NO:1.
The present invention provides a kind of method preparing Water Soluble Compound immunological adjuvant on the other hand, comprises the steps:
1, prepare many phosphonitriles high molecular polymer, the side chain of described many phosphonitriles high molecular polymer is to sodium propionate phenoxy group, and molecular weight is between 70-100 ten thousand unit,
2, preparation comprises the recombiant plasmid of the CpG motif shown in SEQ ID NO:1,
3, by many phosphonitriles high molecular polymer and the recombiant plasmid that comprises the CpG motif shown in SEQ ID NO:1 according to weight ratio be the ratio mixing of 5:2, being dissolved in pH is in the water-soluble solution of 6-8, and fully mixing is formulated.
In a preferred embodiment of the present invention, prepare many phosphonitriles high molecular polymer to comprise the steps:
1, the synthesis of polydichlorophosphazenes
Ammonium chloride and phosphorus chloride are mixed using sulfamic acid, calcium sulphate dihydrate and trichloro-benzenes as solvent, synthesis polydichlorophosphazenes;
2, to the synthesis of methyl propionate benzenpropanoic acid sodium
Para hydroxybenzene methyl propionate is mixed using oxolane as solvent with sodium hydride, synthesizes methyl propionate benzenpropanoic acid sodium;
3, the synthesis of poly-[two (to methyl propionate phenoxy group)] phosphonitrile
The polydichlorophosphazenes that first two steps are synthesized and to the mixing of methyl propionate benzenpropanoic acid sodium using oxolane as solvent, poly-[two (to methyl propionate the phenoxy group)] phosphonitrile of synthesis;
4, the synthesis of poly-[two (to propanoic acid toloxyl)] phosphonitrile
Potassium hydroxide solution is added dropwise to, poly-[two (to propanoic acid the toloxyl)] phosphonitrile of synthesis in poly-[two (to methyl propionate phenoxy group)] phosphonitrile of synthesis;
5, the synthesis of poly-[two (to propanoic acid phenoxy group)] phosphonitrile
Precipitate with deionized water is dissolved, and then precipitate from deionized water with dehydrated alcohol, again precipitate with deionized water is dissolved, add hydrochloric acid solution titration and regulate final solution pH=6.3, finally by saturated brine precipitation, obtain poly-[two (to propanoic acid phenoxy group)] phosphonitrile;
6, the synthesis of poly-[two (to sodium propionate phenoxy group)] phosphonitrile
Poly-[two (to propanoic acid the phenoxy group)] phosphonitrile obtained is dissolved in deionized water, uses sodium hydrate regulator solution pH=7.4, by dehydrated alcohol precipitation, obtain poly-[two (to sodium propionate phenoxy group)] phosphonitrile;
7, screened by the molecular weight of poly [two (to sodium propionate phenoxy group)] phosphonitrile, acquisition molecular weight is poly [two (to sodium propionate the phenoxy group)] phosphonitrile between 70-100 ten thousand unit.
In another preferred embodiment of the present invention, prepare the recombiant plasmid comprising the CpG motif shown in SEQ ID NO:1 and comprise the steps:
1, the CpG motif shown in SEQ ID NO:1 is synthesized;
2, design the number of base adding EcoRI and SalI restriction enzyme site at the end of described motif respectively, and become double-stranded DNA;
3, by the method for gene recombinaton, described double-stranded DNA is incorporated in the plasmid of EcoRI and SalI enzyme action, obtains the recombiant plasmid comprising the CpG motif shown in SEQ ID NO:1.
In another preferred embodiment of the present invention, it is in the PBS solution of 7.2 that many phosphonitriles high molecular polymer is dissolved in pH with the recombiant plasmid comprising the CpG motif shown in SEQ ID NO:1, stirs fully mixing in 30 minutes.
The present invention provides a kind of vaccine combination on the other hand, and it contains Water Soluble Compound immunological adjuvant of the present invention and the immunogen of effective dose.
In a preferred embodiment of the present invention, immunogen is foot and mouth disease inactivation antigen, is more preferably Asia I type inactivated foot-and-mouth disease vaccine antigen or O type inactivated foot-and-mouth disease vaccine antigen.
The present invention provides the method preparing vaccine combination of the present invention on the other hand, comprises mixing Water Soluble Compound immunological adjuvant of the present invention and immunogenic step.
Further aspect of the present invention additionally provides Water Soluble Compound immunological adjuvant of the present invention and is preparing the application in vaccine combination.
In a preferred embodiment of the present invention, immunogen is foot and mouth disease inactivation antigen, is more preferably Asia I type inactivated foot-and-mouth disease vaccine antigen or O type inactivated foot-and-mouth disease vaccine antigen.
Compared with prior art, the present invention has following characteristics and advantages:
First, CpG recombiant plasmid is as immunological adjuvant, and being proved in cellullar immunologic response is a kind of well Th1 type immunostimulant, but its immune persistence is shorter.Many phosphonitriles high molecular polymer, as immunological adjuvant, have stronger effect, and the persistent period is long in stimulation humoral immunity of organism compared with CpG recombiant plasmid, but it is obviously not enough in inducing cellular immune response.The feature that the present invention utilizes many phosphonitriles high molecular polymer and these two kinds of immunological adjuvants of CpG recombiant plasmid stimulating body to produce mutual supplement with each other's advantages in immunne response, appropriate design has also prepared NEW TYPE OF COMPOSITE immunological adjuvant, reach the effect that can promote that humoral immunoresponse(HI) also can promote cellullar immunologic response, and for different epidemic disease vaccine, all there is the compatibility and specific aim.
Secondly, in animal vaccine production, the NEW TYPE OF COMPOSITE immunological adjuvant prepared due to the present invention also has water miscible feature, has therefore greatly simplified the processing step of vaccine compatibility, thus decreases the production cost of vaccine.
Finally, adopt vaccine prepared by NEW TYPE OF COMPOSITE immunological adjuvant of the present invention, easilier than oily adjuvant when immunity to inoculate, easy absorption, in inoculation position noresidue, reaches safe, efficient and lasting object, there is the advantage of the Traditional adjuvants such as alternative oily adjuvant, there is more wide market prospect.
Accompanying drawing explanation
Fig. 1 is many phosphonitriles adjuvant component synthesis process flow diagram.
Fig. 2 is the Efficacy evaluation figure of different molecular weight many phosphonitriles vaccine.
Fig. 3 is the specific antibody level comparison diagram of different vaccine group induction.
Fig. 4 be different vaccine group respectively immunity 7 days, 14 days and 21 days time serum in the comparison diagram of IL-12 content.
Detailed description of the invention
Below in conjunction with accompanying drawing, the present invention is described in further detail, but not as a limitation of the invention.
Embodiment 1: the synthesis technique of many phosphonitriles adjuvant component
1, the synthesis of polydichlorophosphazenes
Ammonium chloride and phosphorus chloride are mixed using sulfamic acid, calcium sulphate dihydrate and trichloro-benzenes as solvent, under 190-195 DEG C of condition, reacts 4h.Synthesis polydichlorophosphazenes.
2, to the synthesis of methyl propionate benzenpropanoic acid sodium
Para hydroxybenzene methyl propionate is mixed with sodium hydride using oxolane as solvent room temperature reaction 4h, synthesizes methyl propionate benzenpropanoic acid sodium.
3, the synthesis of poly-[two (to methyl propionate phenoxy group)] phosphonitrile
React 58h by the polydichlorophosphazenes of first two steps synthesis with to the mixing of methyl propionate benzenpropanoic acid sodium using oxolane as solvent 65 DEG C, generate poly-[two (to methyl propionate phenoxy group)] phosphonitrile.
4, the synthesis of poly-[two (to propanoic acid toloxyl)] phosphonitrile
In poly-[two (to methyl propionate the phenoxy group)] phosphonitrile generated, be added dropwise to potassium hydroxide solution, at 60 DEG C, react 2h, generate poly-[two (to propanoic acid toloxyl)] phosphonitrile.
5, the synthesis of poly-[two (to propanoic acid phenoxy group)] phosphonitrile
Pour out liquid, precipitate with deionized water is dissolved, and then precipitate from deionized water with dehydrated alcohol, again precipitate with deionized water is dissolved, add hydrochloric acid solution titration and regulate final solution pH=6.3, finally by saturated brine precipitation, gathered [two (to propanoic acid phenoxy group)] phosphonitrile.
6, many phosphonitriles molecule: the synthesis of poly-[two (to sodium propionate phenoxy group)] phosphonitrile
Poly-[two (to propanoic acid the phenoxy group)] phosphonitrile obtained is dissolved in deionized water, use sodium hydrate regulator solution pH=7.4, by dehydrated alcohol precipitation, finally obtain many phosphonitriles molecule: poly-[two (to sodium propionate phenoxy group)] phosphonitrile.
Embodiment 2: according to molecular size range to the segmentation of many phosphonitriles
Molecular weight segmentation is carried out to poly-[two (to sodium propionate the phenoxy group)] phosphonitrile of many phosphonitriles molecule, namely the bag filter of PSPP is adopted, poly-[two (to sodium propionate the phenoxy group)] phosphonitrile of polyphosphazene molecule is dialysed, accurately to control the molecular weight of polyphosphazene.
Use the bag filter of 300,000 and 700,000 to dialyse to poly-[two (to sodium propionate the phenoxy group)] phosphonitrile of polyphosphazene molecule respectively, obtain the polyphosphazene molecule that molecular weight is respectively more than less than 300,000,300,000-70 ten thousand and 700,000.
Embodiment 3: many phosphonitriles molecule of different molecular weight and the immunological adjuvant Effect Evaluation of Asia I type foot-and-mouth disease antigen compatibility immune mouse are tested
By many phosphonitriles molecule of the different molecular weight prepared (dosage be 100ug/ only) and Asia I type foot and mouth disease inactivation antigen according to the rules dose compatibility (Asia I type foot and mouth disease inactivation antigen is produced by middle peasant Witter company, and dosage is standard dose) prepare experimental vaccine.
By many phosphonitriles vaccine of the different molecular weight of compatibility of the present invention, antigen alone vaccine (antigen control, i.e. Asia I type foot and mouth disease inactivation antigen), PBS (blank) respectively immunization trial Mus, detect each group of antibody titer with LPB-ELISA test kit (being produced by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences), evaluate the immunoadjuvant function of the many phosphonitriles of different molecular weight.
Antibody titre results shows, many phosphonitriles vaccine that relative molecular weight is greater than 700,000, and its antibody titer is apparently higher than many phosphonitriles vaccine of other molecular size ranges, and specific experiment result is see Fig. 2.Consider in practical application simultaneously, excessive its dissolubility of many phosphonitriles composition of molecular weight is corresponding to diminish, therefore the present invention finally selected molecular weight be that poly-[two (to sodium propionate phenoxy group)] phosphonitrile of many phosphonitriles molecule between 70-100 ten thousand unit is as immunological adjuvant.
The preparation of embodiment 4:CpG DNA plasmid, a large amount of amplification, Isolation and purification
1, the synthesis of CpG motif sequence and screening
According to steric effect and animal reservoir's specificity of CpG motif, design, synthesize multiple CpG motif, full chain thio-modification is carried out for preventing from degrading very soon, mice, rabbit and pig body carry out the Experiment of Compatibility of important epidemic disease vaccine (foot and mouth disease, swine fever and pork measles), therefrom filter out the best adjuvant core sequence of pig, as described in SEQ ID NO:1.
2, the recombinant clone of CpG motif sequence
Artificial resynthesis contains the non-sulfo-of CpG motif, the best adjuvant core sequence of non-methylated above-mentioned SEQ ID NO:1, design the number of base of EcoRI and SalI restriction enzyme site respectively at its end simultaneously, generate AAT TCT CGT CGT TTG TCG TTT TGT CGT TG and complementary series thereof, adopt temperature control degeneration, renaturation technology becomes double-stranded DNA, by genetic engineering recombinant technique, orientation is cloned into puc18 carrier, transformation of E. coli, use amicillin resistance screening positive clone, extract plasmid order-checking qualification, confirm the SEQ ID NO:1 containing CpG motif sequence to be cloned in plasmid vector.To correctly insert the puc18 recombiant plasmid called after recombinant C pG-DNA plasmid of SEQ ID NO:1.
3, a large amount of amplifications of recombinant C pG-DNA plasmid, Isolation and purification
Select the positive strain of high copy, be inoculated into 1000ml containing in the LB culture medium of ammonia benzyl, cultivate after 12 ~ 14 hours for 37 DEG C in 220rpm shaking table, extract recombinant C pG-DNA plasmid in a large number with SDS alkaline lysis.The plasmid of thick extraction after the LiCl that 5M is ice-cold is separated, its supernatant equal-volume isopropanol precipitating, then precipitate by 70% washing with alcohol, centrifugal removing supernatant; By the TE buffer solution of precipitation containing RNase, room temperature treatment after 30 minutes, then uses phenol: chloroform 2 times, 2 times of dehydrated alcohol pelleting centrifugation; After 1ml aquesterilisa dissolution precipitation, add 0.5ml PEG-MgCl
2solution (30mM MgCl
2in add 40%PEG8000), fully place 15 minutes after mixing, centrifugal 20 minutes of 13000rpm, precipitate the washing with alcohol 2 times with 70%, use TE buffer or sterilized water dissolution precipitation again, measure its content and purity with nucleic acid-protein detector, 4 DEG C save backup.
Embodiment 5: the preparation of vaccine and the preparation of strain
By the CpG DNA plasmid of extraction purification according to the dosage of 200 μ g/ head parts and the many phosphonitriles adjuvant dose compatibility according to 500ug/ head part, being dissolved in pH is in the PBS solution of 7.2, and magnetic agitation 30min, namely abundant mixing makes NEW TYPE OF COMPOSITE adjuvant, for subsequent use.
By NEW TYPE OF COMPOSITE adjuvant of the present invention and O type foot and mouth disease inactivation antigen dose compatibility (O type foot and mouth disease inactivation antigen is produced by middle peasant Witter company, and dosage is standard dose) preparation experiment vaccine according to the rules.By commercially available standard 206 adjuvant and O type foot and mouth disease inactivation antigen compatibility preparation standard control vaccine.Single many phosphonitriles composition 500ug/ head part is prepared control vaccine as adjuvant and O type foot and mouth disease inactivation antigen compatibility.Meanwhile, using the independent immune group of O type foot-and-mouth disease antigen as antigen control group, using PBS immune group as blank group.
Strain O/MYA98/BY/2010 provides for middle peasant Witter company.
Embodiment 6: immunity inoculation experiment and experimental result
Adopt the various vaccines of preparation in embodiment 5 to carry out musculi colli injecting immune to experiment pig respectively, injection consumption is the routine dose in actual production.Each group of antibody titer is detected with LPB-ELISA test kit (being produced by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences); the level of cytokine kit detection cell cytokine secretion; and counteracting toxic substances was carried out to test pig in 90 days in immunity, calculate protective rate.
As can be seen from the result of Fig. 3, the specific antibody level of the new generation vaccine induction of composite adjuvant compatibility prepared by the present invention, all high at the vaccine of whole immunologic surveillance phase internal ratio tradition 206 adjuvant compatibilities, sustainable more than 90 days, the new generation vaccine of composite adjuvant compatibility and single many phosphonitriles molecule as adjuvant compatibility vaccine compared with, antibody horizontal obtains and greatly improves, and illustrates that the compound of two kinds of molecules is very necessary.
In addition, much research proves that IL-12 plays pivotal role in the natural immunity and acquired immunity, is especially a kind of important regulate factors in cellullar immunologic response.IL-12 is independent or the T cell of activation can be stimulated to breed with IL-2 synergism, strengthens CTL activity, is strengthened propagation and the cytotoxic effect of NK cell by the generation of induction IFN-γ.
The results show of the present invention is in cellular immunization, and the vaccine of 206 adjuvant compatibilities that the new generation vaccine of composite adjuvant compatibility prepared by the present invention is more traditional has certain advantage.Detect in experiment pig serum when immune 7 days, 14 days, 21 days and represent the immunoreactive IL-12 content of Th1 type, find that the IL-12 content in the new generation vaccine experimental group porcine blood serum of the composite adjuvant compatibility prepared in the present invention in 14 days, 21 days of immunity is significantly higher than the vaccine group of traditional 206 adjuvant compatibilities.With single many phosphonitriles molecule as adjuvant compatibility vaccine compared with, the IL-12 content that new generation vaccine of the present invention induction produces is also higher, and concrete outcome is see Fig. 4.
Immune 90 days time; according to standard dose (dosage is 1000TcID50/ head), counteracting toxic substances is carried out to experiment pig; observed in the new generation vaccine group calculating the composite adjuvant compatibility adopting the present invention to prepare by continuous 10 days; 4 are had not fall ill in 5 experiment pig; protective efficacy is 80%, suitable with the vaccine group effect of standard 206 adjuvant compatibility, and blank group and antigen control group are then all fallen ill; protective rate is 0, and concrete outcome is in table 1.
Table 1 each experimental group protective rate measurement result
| Experimental group | Protective rate |
| 206 vehicle control groups | 5/5 |
| Many phosphonitrile+CpG composite adjuvant groups | 4/5 |
| Many phosphonitriles adjuvant group | 2/5 |
| Antigen control group | 0/5 |
| Blank group | 0/5 |
Claims (12)
1. a Water Soluble Compound immunological adjuvant, it is formed according to the proportions that weight ratio is 5:2 by many phosphonitriles high molecular polymer and the recombiant plasmid comprising the CpG motif shown in SEQ ID NO:1, the side chain of wherein said many phosphonitriles high molecular polymer is to sodium propionate phenoxy group, and molecular weight is between 70-100 ten thousand unit.
2. Water Soluble Compound immunological adjuvant according to claim 1, many phosphonitriles high molecular polymer is wherein prepared by following steps:
(1) synthesis of polydichlorophosphazenes
Ammonium chloride and phosphorus chloride are mixed using sulfamic acid, calcium sulphate dihydrate and trichloro-benzenes as solvent, synthesis polydichlorophosphazenes;
(2) to the synthesis of methyl propionate benzenpropanoic acid sodium
Para hydroxybenzene methyl propionate is mixed using oxolane as solvent with sodium hydride, synthesizes methyl propionate benzenpropanoic acid sodium;
(3) synthesis of poly-[two (to methyl propionate phenoxy group)] phosphonitrile
The polydichlorophosphazenes that first two steps are synthesized and to the mixing of methyl propionate benzenpropanoic acid sodium using oxolane as solvent, poly-[two (to methyl propionate the phenoxy group)] phosphonitrile of synthesis;
(4) synthesis of poly-[two (to propanoic acid toloxyl)] phosphonitrile
Potassium hydroxide solution is added dropwise to, poly-[two (to propanoic acid the toloxyl)] phosphonitrile of synthesis in poly-[two (to methyl propionate phenoxy group)] phosphonitrile of synthesis;
(5) synthesis of poly-[two (to propanoic acid phenoxy group)] phosphonitrile
Precipitate with deionized water is dissolved, and then precipitate from deionized water with dehydrated alcohol, again precipitate with deionized water is dissolved, add hydrochloric acid solution titration and regulate final solution pH=6.3, finally by saturated brine precipitation, obtain poly-[two (to propanoic acid phenoxy group)] phosphonitrile;
(6) synthesis of poly-[two (to sodium propionate phenoxy group)] phosphonitrile
Poly-[two (to propanoic acid the phenoxy group)] phosphonitrile obtained is dissolved in deionized water, uses sodium hydrate regulator solution pH=7.4, by dehydrated alcohol precipitation, obtain poly-[two (to sodium propionate phenoxy group)] phosphonitrile;
(7) screened by the molecular weight of poly [two (to sodium propionate phenoxy group)] phosphonitrile, acquisition molecular weight is poly [two (to sodium propionate the phenoxy group)] phosphonitrile between 70-100 ten thousand unit.
3. Water Soluble Compound immunological adjuvant according to claim 1, wherein recombiant plasmid is for comprising the puc18 recombiant plasmid of the CpG motif shown in SEQID NO:1.
4. prepare a method for Water Soluble Compound immunological adjuvant, comprise the steps:
(1) prepare many phosphonitriles high molecular polymer, the side chain of described many phosphonitriles high molecular polymer is to sodium propionate phenoxy group, and molecular weight is between 70-100 ten thousand unit,
(2) preparation comprises the recombiant plasmid of the CpG motif shown in SEQ ID NO:1,
(3) many phosphonitriles high molecular polymer and the recombiant plasmid comprising the CpG motif shown in SEQ ID NO:1 are mixed according to the ratio that weight ratio is 5:2, being dissolved in pH is in the water-soluble solution of 6-8, and fully mixing is formulated.
5. method according to claim 4, wherein prepares many phosphonitriles high molecular polymer and comprises the steps:
(1) synthesis of polydichlorophosphazenes
Ammonium chloride and phosphorus chloride are mixed using sulfamic acid, calcium sulphate dihydrate and trichloro-benzenes as solvent, synthesis polydichlorophosphazenes;
(2) to the synthesis of methyl propionate benzenpropanoic acid sodium
Para hydroxybenzene methyl propionate is mixed using oxolane as solvent with sodium hydride, synthesizes methyl propionate benzenpropanoic acid sodium;
(3) synthesis of poly-[two (to methyl propionate phenoxy group)] phosphonitrile
The polydichlorophosphazenes that first two steps are synthesized and to the mixing of methyl propionate benzenpropanoic acid sodium using oxolane as solvent, poly-[two (to methyl propionate the phenoxy group)] phosphonitrile of synthesis;
(4) synthesis of poly-[two (to propanoic acid toloxyl)] phosphonitrile
Potassium hydroxide solution is added dropwise to, poly-[two (to propanoic acid the toloxyl)] phosphonitrile of synthesis in poly-[two (to methyl propionate phenoxy group)] phosphonitrile of synthesis;
(5) synthesis of poly-[two (to propanoic acid phenoxy group)] phosphonitrile
Precipitate with deionized water is dissolved, and then precipitate from deionized water with dehydrated alcohol, again precipitate with deionized water is dissolved, add hydrochloric acid solution titration and regulate final solution pH=6.3, finally by saturated brine precipitation, obtain poly-[two (to propanoic acid phenoxy group)] phosphonitrile;
(6) synthesis of poly-[two (to sodium propionate phenoxy group)] phosphonitrile
Poly-[two (to propanoic acid the phenoxy group)] phosphonitrile obtained is dissolved in deionized water, uses sodium hydrate regulator solution pH=7.4, by dehydrated alcohol precipitation, obtain poly-[two (to sodium propionate phenoxy group)] phosphonitrile;
(7) screened by the molecular weight of poly [two (to sodium propionate phenoxy group)] phosphonitrile, acquisition molecular weight is poly [two (to sodium propionate the phenoxy group)] phosphonitrile between 70-100 ten thousand unit.
6. method according to claim 4, wherein prepares the recombiant plasmid comprising the CpG motif shown in SEQ ID NO:1 and comprises the steps:
(1) the CpG motif shown in SEQ ID NO:1 is synthesized;
(2) design the number of base adding EcoRI and SalI restriction enzyme site at the end of described motif respectively, and become double-stranded DNA;
(3) by the method for gene recombinaton, described double-stranded DNA is incorporated in the plasmid of EcoRI and SalI enzyme action, obtains the recombiant plasmid comprising the CpG motif shown in SEQ ID NO:1.
7. method according to claim 4, wherein many phosphonitriles high molecular polymer being dissolved in pH with the recombiant plasmid comprising the CpG motif shown in SEQ IDNO:1 is in the PBS solution of 7.2, stirs fully mixing in 30 minutes.
8. a vaccine combination, it contains the Water Soluble Compound immunological adjuvant according to any one of claim 1-3 and the immunogen of effective dose.
9. vaccine combination according to claim 8, wherein immunogen is foot and mouth disease inactivation antigen, is preferably Asia I type inactivated foot-and-mouth disease vaccine antigen or O type inactivated foot-and-mouth disease vaccine antigen.
10. prepare a method for the vaccine combination described in claim 8 or 9, comprise the Water Soluble Compound immunological adjuvant according to any one of hybrid right requirement 1-3 and immunogenic step.
The application in vaccine combination prepared by Water Soluble Compound immunological adjuvant according to any one of 11. claim 1-3.
12. application according to claim 11, wherein immunogen is foot and mouth disease inactivation antigen, is preferably Asia I type inactivated foot-and-mouth disease vaccine antigen or O type inactivated foot-and-mouth disease vaccine antigen.
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| CN1814757B (en) * | 2005-02-03 | 2012-09-05 | 中国农业科学院兰州兽医研究所 | Novel CpGDNA adjuvant, its preparing method and vaccine containing same |
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| CN1814757B (en) * | 2005-02-03 | 2012-09-05 | 中国农业科学院兰州兽医研究所 | Novel CpGDNA adjuvant, its preparing method and vaccine containing same |
Non-Patent Citations (3)
| Title |
|---|
| MUTWIRI G ET AL: "Innate immunity and new adjuvants", 《OIE REV SCI TECH》 * |
| MUTWIRI G ET AL: "Poly[di(sodium carboxylatoethylphenoxy) phosphazene] (PCEP) is a potent enhancer of mixed Th1/Th2 immune responses in mice immunized with influenza virus antigens", 《VACCINE》 * |
| 侯保才等: "聚磷腈免疫佐剂的研究应用概况", 《中国现代应用药学》 * |
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