CN103877570A - Rabies live vaccine and method for preparing rabies live vaccine for oral administration - Google Patents
Rabies live vaccine and method for preparing rabies live vaccine for oral administration Download PDFInfo
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- CN103877570A CN103877570A CN201410056975.6A CN201410056975A CN103877570A CN 103877570 A CN103877570 A CN 103877570A CN 201410056975 A CN201410056975 A CN 201410056975A CN 103877570 A CN103877570 A CN 103877570A
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Abstract
The invention discloses a rabies live vaccine and a method for preparing the rabies live vaccine for oral administration. The rabies live vaccine comprises a rabies virus ERAg3m. A strain of the rabies virus ERAg3m is obtained by using a reverse genetic method for transforming a rabies virus strain ERA and has high safety. Therefore, the immune efficacy of the rabies live vaccine containing the rabies ERAg3m virus can be further improved.
Description
Technical field
The present invention relates to biological technical field, particularly, the present invention relates to rabies live vaccine and the method for preparing the oral live vaccine of rabies.
Background technology
Rabies (Rabies) be again mad dog disease or hydrophobia, is the Zoonosis acute infectious disease being caused by rabies virus (Rabies virus).Rabies major effect central nervous system, once people morbidity does not still have effective clinical treatment method at present, be so far the unique case fatality rate of the mankind up to 100% acute infectious disease.The whole world approximately has 55000 people to die from rabies every year, China from 2003 afterwards annual rabies death toll all exceed 2000 people, exceed 10,000,000,000 although China drops into the rabic expense of prevention and control every year, Meteorological occupies the first in the world, prevention effect is but that the whole world is second from the bottom, is only second to India.The propagation of rabies virus has badly influenced the public health security of China, has become a very outstanding social problem.
The existing rabies vaccine of China is to adopt Flury strain rolling bottle is produced on BHK21 cell attenuated live vaccines and inactivated vaccine.Flury attenuated live vaccines, because exist possible virulence to return strong and brain inner infection lethal, now stops producing substantially.Inactivated vaccine is mainly to adopt rolling bottle or microcarrier to produce virus antigen, and then concentrated, purification, adds adjuvant to make, and owing to producing, viral titre is low, reaches the multiple that immune efficacy requirement need to be concentrated high, as spinner culture need to concentrate 30-50 doubly; Meanwhile, during due to cell and Virus culture, add 2～10% Ox blood serum, cause downstream purification difficulty to strengthen, prepared inactivated vaccine production cost is high, the selling price of existing vaccine all more than part, causes governments at all levels' purchase cost high at 20 yuan/, is difficult to large-scale promotion.The external existing rabies vaccine for dog cat is the inactivated vaccine that adopts intramuscular injection, and quality is better, but expensive.And employing oral vaccine is with the obvious advantage: the one, change traditional vaccine preparation way, adopt orally, be easier to expand immune coverage, realize the immunity to pet dog and cat on a large scale; The 2nd, can avoid some house pet, the injury that particularly may cause people when large-scale dog and strong dog injecting immune; The 3rd, cost is low, is easy to promote; The 4th, adopt slow release method, immune period is longer compared with conventional vaccine.
The raising number of China dog cat is huge, may exceed according to estimates 200,000,000, and this is comprising a large amount of wandering dogs and rural area dog.Adopt intramuscular injection immunity by dog Baoding, for large-scale dog and strong dog, especially easily to hurt the medical worker who carries out inoculation, add that wandering dog is difficult to catch, therefore adopt intramuscular injection some time be difficult to carry out.And oral vaccine can address this problem well, only vaccine need be thrown to be sprinkling upon in feedstuff for large-scale dog, strong dog and wandering dog and feed and just can play the effect of immunoprophylaxis, be easy to promote.
It is to carry out wild animal oral hydrophobia vaccine that successful foreign is eliminated rabic experience, reason is that the propagation of most American-European countries rabies virus is mainly as the propagation of the natural reservoir (of bird flu viruses) such as fox, racoon by wild animal, and the main route of transmission of China's rabies virus be by the biting of pet dog, lick lick and the intimate contact of people and pet dog as kissed etc., therefore the rabic primary measure of China's prevention and control is exactly to cut off the propagation of pet dog to human rabies poison, dog is realized to more than 70% immunity, comprise wandering dog and rural area dog.
Summary of the invention
The present invention is intended to one of solve the problems of the technologies described above at least to a certain extent.For this reason, a kind of method that one object of the present invention is to propose rabies live vaccine and prepares the oral live vaccine of rabies, this vaccine can be effective to wild animal, as the immunoprophylaxis of the animals such as wandering dog.
The following research of the present invention based on inventor completes: first inventor utilizes reverse genetics method that Rabies Virus ERA strain is transformed, obtain the high gene engineered strain of a strain safety, utilize this strain to develop a kind of oral vaccine in conjunction with Microspheres Technique and bait technology, this vaccine can be used for wild animal, as the immunoprophylaxis of the animals such as wandering dog.According to the embodiment of the present invention, rabies virus ERAg3m strain is to obtain by reverse genetics method, can be used as oral live vaccine production strain, and safe, immune efficacy is good.
For this reason in one aspect of the invention, the present invention proposes a kind of rabies live vaccine, comprise: rabies ERAg3m virus.Can further improve thus the immunogenicity of rabies live vaccine.
According to embodiments of the invention, in described rabies live vaccine, the concentration of rabies ERAg3m virus is 1 × 10
9.0tCID
50/ ml.Can further improve thus the immunogenicity of rabies live vaccine.
In another aspect of this invention, the present invention proposes a kind of method of preparing the oral live vaccine of rabies, the method comprises the steps:
1) utilize BHK21 cell to carry out full suspension culture to rabies ERAg3m virus, to obtain rabies ERAg3m virus solution;
2) described rabies ERAg3m virus solution is mixed with chitosan solution and sodium tripolyphosphate solution, to prepare chitosan nano microsphere; And
3) described chitosan nano microsphere is carried out to bait parcel, to prepare the oral live vaccine of described rabies.
Can effectively prepare thus the oral live vaccine of rabies, the oral live vaccine instant of these rabies, immunogenicity is strong, the form that oral rabies live vaccine is prepared into microsphere can effectively keep the immunogenicity of rabies ERAg3m virus, to further improve the oral live vaccine immunity of rabies.
In addition, the method for preparing the oral live vaccine of rabies according to the above embodiment of the present invention can also have following additional technical characterictic:
According to embodiments of the invention, in step 2) in, described rabies ERAg3m virus solution is joined in chitosan-acetic acid solution; Chitosan and rabies ERAg3m virus mixed solution are joined in tripolyphosphate sodium water solution, mix homogeneously centrifugal treating, to obtain the Nano microsphere that contains vaccine antigen again.Can further improve thus and prepare chitosan nano microsphere effect.
According to embodiments of the invention, described full suspension culture is cultivated and is completed for 36 hours at the temperature of 37 degrees Celsius.Can further improve thus rabies ERAg3m virus breeding efficiency, to further improve the efficiency of the oral live vaccine of preparation rabies.
According to embodiments of the invention, the concentration of described rabies ERAg3m virus solution is higher than 1 × 10
8.0tCID
50/ ml.Can further improve thus the immunogenicity of the oral live vaccine of rabies.
According to embodiments of the invention, in described chitosan solution, the concentration of chitosan is 0.1%-10%w/v, and the solvent of described chitosan solution is that concentration is 1% acetic acid; The concentration of described sodium tripolyphosphate solution is 0.1%-5%w/v, can further improve thus efficiency and the quality of preparing chitosan nano microsphere.
According to embodiments of the invention, described centrifugal treating completes under 4 degrees Celsius, the condition of 15000～18000rpm for centrifugal 30 minutes.Can further improve thus efficiency and the quality of preparing chitosan nano microsphere.
According to embodiments of the invention, before described chitosan nano microsphere is carried out to bait parcel, further comprise: in described chitosan nano microsphere, adding MEM solution, is 1 × 10 to make the concentration of rabies ERAg3m virus described in described chitosan nano microsphere
9.0tCID
50/ ml.Can further improve thus the concentration of the rabies ERAg3m virus in chitosan nano microsphere, to further improve the immunogenicity of the oral live vaccine of rabies.
According to embodiments of the invention, described bait parcel further comprises: described chitosan nano microsphere is joined in the wax of thawing and carry out the first bait parcel and dry, to obtain the first chitosan nano microsphere wrappage; The chitosan nano microsphere of described sealing wax is joined in the mixing material of Adeps Sus domestica and wax and carry out the second bait parcel and dry, to obtain the second chitosan nano microsphere wrappage; And described the second chitosan nano microsphere wrappage is joined and in Carnis Sus domestica polymer, mixes and shape, to prepare the oral live vaccine of described rabies.Can further improve thus accessibility and the utilization rate of the oral live vaccine of rabies.
Additional aspect of the present invention and advantage in the following description part provide, and part will become obviously from the following description, or recognize by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage accompanying drawing below combination is understood becoming the description of embodiment obviously and easily, wherein:
Fig. 1 is the cloning process schematic diagram of preparing rabies ERAg3m virus in the method for preparing rabies oral vaccine according to another embodiment of the invention.
Fig. 2 is the flow chart of preparing according to one embodiment of present invention the method for the oral live vaccine of rabies.
The specific embodiment
Describe embodiments of the invention below in detail, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has the element of identical or similar functions from start to finish.Be exemplary below by the embodiment being described with reference to the drawings, be intended to for explaining the present invention, and can not be interpreted as limitation of the present invention.
According to an aspect of the present invention, the present invention proposes a kind of rabies live vaccine, according to embodiments of the invention, this mad dog live vaccine comprises: rabies ERAg3m virus.According to a particular embodiment of the invention, rabies virus ERAg3m strain is transformed acquisition by reverse genetics method by Rabies Virus ERA strain, safe, contain thus these rabies ERAg3m virus and can further improve the immune efficacy of rabies live vaccine.
According to a particular embodiment of the invention, the content of rabies ERAg3m virus being not particularly limited in above-mentioned rabies live vaccine, according to concrete example of the present invention, in rabies live vaccine, the concentration of rabies ERAg3m virus can be 1 × 10
9.0tCID
50/ ml.Can further improve thus the immune efficacy of rabies live vaccine.
According to a particular embodiment of the invention, above-mentioned rabies live vaccine is expressed the glycoprotein of coding rabies ERAg3m virus, and rabies ERAg3m viral glycoprotein has following sudden change: the aminoacid deletion that signal peptide is the 2nd; The aminoacid deletion of the 3rd of signal peptide; The 333rd amino acids sports glutamic acid by arginine; Disappearance psi pseudogene district; Before the G gene of described glycoprotein inserts M gene.
According to a particular embodiment of the invention, above-mentioned rabies ERAg3m virus mutation glycoprotein has the aminoacid sequence shown in SEQ ID NO:1.SEQ ID NO:1 aminoacid sequence is:
MQALLFVPLLVFPLCFGKFPIYTIPDKLGPWSPIDIHHLSCP
NNLVVEDEGCTNLSGFSYMELKVGYILAIKMNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYRWLRTVKTTKESLVIISPSVADLDPYDRSLHSRVFPSGKCSGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPDQL
VNLHDFRSDEIEHLVVEELVRKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVETWNEILPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLESSVIPLVHPLADPSTVFKDGDEAEDFVEVHLPDVHNQVSGVDLGLPNWGKYVLLSAGALTALMLIIFLMTCCRRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESHKSGGETRL。
According to a particular embodiment of the invention, in the genome of said mutation strain, comprise nucleotide sequence shown in SEQ ID NO:2.SEQ ID NO:2 nucleotides sequence is classified as:
According to a particular embodiment of the invention, prepare the method for the rabies ERAg3m virus comprising in rabies live vaccine noted earlier, comprising:
(1) utilize expression vector to carry out transfection to host cell;
(2) host cell through transfection obtaining in step (1) is cultivated;
(3) culture obtaining in step (2) is filtered, to obtain rabies ERAg3m virus.
According to a particular embodiment of the invention, the method for preparing rabies ERAg3m virus specifically comprises the following steps: first to utilize expression vector to carry out transfection to host cell.According to concrete example of the present invention, wherein, expression vector carries the first nucleic acid molecules, the first nucleic acid molecule encoding sudden change glycoprotein, and sudden change glycoprotein has following sudden change: the aminoacid deletion that signal peptide is the 2nd; The aminoacid deletion of the 3rd of signal peptide; The 333rd amino acids sports glutamic acid by arginine; Disappearance psi pseudogene district; Before the G gene of glycoprotein inserts M gene.
According to a particular embodiment of the invention, the sudden change glycoprotein of the first nucleic acid molecule encoding has the aminoacid sequence shown in SEQ ID NO:1 above.In some embodiments of the invention, the first nucleic acid molecules has the nucleotide sequence shown in SEQ ID NO:2 above.
According to another embodiment of the invention, 5 ' end of the first nucleic acid molecules is connected with the nucleic acid molecules of coding hammerhead ribozyme, and 3 ' end of the first nucleic acid molecules is connected with the nucleic acid molecules of coding hepatitis D ribozyme.Can promote thus the recovery of total length viral RNA.
In some embodiments of the invention, utilizing expression vector to carry out in transfection host cell, expression vector and assistant carrier can be carried out to cotransfection to host cell.According to a particular embodiment of the invention, assistant carrier can comprise: the first carrier, Second support, the 3rd carrier and the 4th carrier.Wherein, the first carrier carries N gene order; Second support carries P gene order; The 3rd carrier carries L gene order; And the 4th carrier carry coding t7 rna polymerase nucleic acid molecules.
In some embodiments of the invention, expression vector and first to fourth carrier are pBlue plasmid.
In some embodiments of the invention, can be BHK-21 cell for the host cell of transfection.
In some embodiments of the invention, can specifically comprise for the preparation of the method for rabies virus ERA attenuated vaccine mutant: while being cultured to 6 orifice plate 90% degree of converging in the MEM complete medium of the new-born calf serum by BHK-21 cell in 10%; Described BHK-21 cell is carried out to cotransfection by expression vector and the first carrier, Second support, the 3rd carrier and the 4th carrier liposome 2000 methods in 4ug/ hole; BHK-21 cell after described cotransfection is placed in to 5% CO
2in incubator and at the temperature of 37 ℃, cultivate after 4-6 hour and discard supernatant; Add the MEM complete medium of 5% described new-born calf serum, and be placed in 5% CO
2in incubator, cultivate 72 hours at the temperature of 37 ℃, by cell freeze thawing 3 times, centrifugal collection supernatant also adopts the filter membrane of 0.22um to filter, to obtain described rabies virus ERA attenuated vaccine mutant.According to a particular embodiment of the invention, above-mentioned expression vector is pBlueERA; The first carrier is pBlueN (2ug/ hole), and Second support is pBlue P (1ug/ hole), and the 3rd carrier is pBlueL (1ug/ hole), and the 4th carrier is pT7 (2ug/ hole).
According to a particular embodiment of the invention, rabies ERAg3m virus obtains from ATCC.ERAg3m infects BHK-21 cell, in the MEM culture medium of supplementing 5% new-born calf serum, grows.The centrifugal 20000g of supernatant, 1 hour.Collecting precipitation extracts ERA genome, detected through gel electrophoresis genomic integrity with RNA viruses nucleic acid extraction kit.Reverse transcription test kit generates ERAg3m genome cDNA.According to ERAg3m sequence in Genebank, design primer, divides 4 sections to synthesize full length sequence.
1F：GTCACT
GGTAACTGG
TGTTAAGCGTCTGATGAGTCCGTGAGGACG AAACTATAGGAAAGGAATTCCTATAGTCACGCTTAACAACCAGATCAAAG（KpnⅠ/Ham）（SEQ?ID?NO：3）
1R：
CTGCAGGTTTTTTTCATG（PstⅠ）（SEQ?ID?NO：4）
2F：
CCCGGGAGGCAACACC（SamⅠ）（SEQ?ID?NO：5）
2R：
ACTAGTTAATAGTTTTTTTCAC（SpeⅠ）（SEQ?ID?NO：6）
3F：
CTGCAGAACATCCCTCAAAAGACTCAAGGAAAGATGCAGGCTCTC（PstⅠ）（SEQ?ID?NO：7）
3R：
CCCGGGGTTTTTTTTCAAAAAGAACCCCCC（SamⅠ）（SEQ?ID?NO：8）
4F：
ACTAGTCATTAGATCAGAAG（SpeⅠ）（SEQ?ID?NO：9）
4R:GCGGCCGCGCCCTCCCTTAGCCATCCGAGTGGACGTGCGTCCTCCTTCGGATGCCCAGGTCGGACCGCGAGGAGGTGGAGATGCCATGCCGACCCACGCTTAACAAATAAACAACA(NotⅠ/HdvRz)（SEQ?ID?NO：10）
G protein mutation primer
5F：GCTCATACAAGTC
GAAACTTGGAATGAGATCCT（SEQ?ID?NO：11）
5R：AGGATCTCATTCCAAGT
TTCGACTGACTTGTAGTGAGC（SEQ?ID?NO：12）
Utilize above-mentioned primer, react by a series of PCR, 4 fragments of amplification covering gene group, utilize the intersegmental overlapping restriction enzyme site of sheet, in carrier pBluescript II (+), be linked and packed into the ERAg3mcDNA clone of sudden change, connect respectively hammerhead ribozyme (Ham) and hepatitis D ribozyme (HdvRz) at cDNA two ends, the correct plasmid that checks order is pBlueERA.Concrete clone's process See Figure 1.
According to a particular embodiment of the invention, take pBlueERAg3m as template, design primer, increase respectively N, P, L gene order, and be cloned into respectively pBluescript II (+) carrier T7 promoter downstream, after order-checking is correct, obtain pBlueN, pBlue P, pBlueL.
Extract e. coli bl21 (DE3) genome, pcr amplification t7 rna polymerase gene is also cloned in pcDNA3.1 (+) plasmid CMV/T7 promoter downstream, after order-checking is correct, obtains pT7.
According to a particular embodiment of the invention, the above-mentioned reverse Genetics Technique that utilizes carries out gene mutation, knocks out and position displacement take rabies virus cDNA as object, the mutant of recovery is more safer than existing vaccine strain, more effectively, more cheap.
According to a further aspect in the invention, the present invention proposes a kind of method of preparing the oral live vaccine of rabies.The method comprises: 1) utilize BHK21 cell to carry out full suspension culture to rabies ERAg3m virus, to obtain rabies ERAg3m virus solution; 2) described rabies ERAg3m virus solution is mixed with chitosan solution and sodium tripolyphosphate solution, to prepare chitosan nano microsphere; And 3) described chitosan nano microsphere is carried out to bait parcel, to prepare the oral live vaccine of described rabies.
Utilize thus said method by rabies ERAg3m virus be prepared into can oral edible microsphere formation, to further improve the immunogenicity of rabies ERAg3m virus.
World Health Organization (WHO) is thought: no matter dog is carried out to oral vaccination, be use separately or combine with injection inoculation, and a kind of new method that dog rate of vaccination is improved greatly is all provided, go around especially everywhere, the dog of no supervision only.Due to dog is carried out to injection inoculation, be one of main bugbear in the rabies control of many different regions, the world, so WHO has carried out the research of dog group and dog immunization rate in some countries in Africa, Latin America and Asia.Recognize that injecting immune mode is in the limitation of eliminating aspect rabies, WHO has strengthened the research to dog oral immunity, and the required safer and more effective vaccine of this kind of vaccination ways and the exploitation of bait.This research, by full virus suspension culture and the oral technology combination of microsphere, has significantly reduced the cost of final oral vaccine, has simultaneously strengthened again immune effect, have concurrently easy to use, safe, be easy to the advantages such as popularization, have market potential.
Describe in detail according to the method for preparing the oral live vaccine of rabies of the embodiment of the present invention below with reference to Fig. 2.
S100: preparation rabies ERAg3m virus solution
In certain embodiments, utilize BHK21 cell to carry out full suspension culture to rabies ERAg3m virus, to obtain rabies ERAg3m virus solution.Can further improve thus proliferative speed and the survival quality of rabies ERAg3m virus.
According to concrete example of the present invention, the cultivation of rabies ERAg3m virus can adopt new-born calf serum, can further improve thus culture efficiency, and the concentration of the new-born calf serum of employing is also not particularly limited, for example, can be 8%.Can cultivate better thus these rabies ERAg3m virus.According to a particular embodiment of the invention, other technological parameters in the cultivation process of virus are also not particularly limited, and the temperature of for example cultivating may be controlled to 37 ℃, but is not limited in 37 degrees Celsius, as long as the optimum temperature of viral growth; Incubation time can be 36 hours, but is not limited to 36 hours, as long as can cultivate the rabies ERAg3m virus that obtains sufficient amount; Cultivating rotating speed can be 100r/m, but is also not limited only to 100r/m, as long as can be effectively by cell suspension.
According to a particular embodiment of the invention, the rabies ERAg3m virus that collection employing said method prepares is also prepared rabies ERAg3m virus solution.According to concrete example of the present invention, the concentration of the rabies ERAg3m virus solution of preparation is also not particularly limited, and the concentration of for example virus liquid can be 1 × 10
8.0tCID
50/ ml.
S200: prepare chitosan nano microsphere
In certain embodiments, rabies ERAg3m virus solution is mixed with chitosan solution and sodium tripolyphosphate solution, to prepare chitosan nano microsphere.
According to a particular embodiment of the invention, the above-mentioned step of preparing chitosan nano microsphere may further include: described rabies ERAg3m virus solution is joined in chitosan-acetic acid solution; Chitosan and rabies ERAg3m virus mixed solution are joined in tripolyphosphate sodium water solution, mix homogeneously centrifugal treating, to obtain the Nano microsphere that contains vaccine antigen again.Can prepare thus the chitosan nano microsphere that comprises rabies ERAg3m virus, to further prepare the oral live vaccine of rabies.Can effectively prepare thus the polysaccharide Nano microsphere that comprises rabies ERAg3m virus.So that further rabies ERAg3m virus is prepared into can be oral live vaccine so that expansion rabies ERAg3m virus purposes.
According to a particular embodiment of the invention, to the concentration of the chitosan-acetic acid solution adding in rabies ERAg3m virus solution and be not particularly limited, according to concrete example of the present invention, in chitosan-acetic acid solution, the concentration of chitosan can be 0.1%-10%w/v.According to another concrete example of the present invention, the solvent of the chitosan-acetic acid solution of employing is that concentration is 1% acetic acid, can farthest increase thus the dissolubility of chitosan.
According to a particular embodiment of the invention, the concentration of the tripolyphosphate sodium water solution adding also and be not particularly limited, for example, can be 0.1%-5%w/v.Because chitosan is with cationic macromolecule polyalcohol, can form nanoscale compound polyelectrolyte with the sodium tripolyphosphate effect with anion, therefore can further improve the ratio of briquetting of preparing microsphere.
According to a particular embodiment of the invention, the above-mentioned mixture by preparation chitosan solution, rabies ERAg3m virus solution and tripolyphosphate sodium water solution carries out the condition of centrifugal treating and is not particularly limited, for example can be under 4 degrees Celsius, the condition of 15000～18000rpm centrifugal 30 minutes, can further virus be concentrated completely thus, be convenient to control the concentration of effective virus antigen.
S300: bait parcel
In certain embodiments, the above-mentioned chitosan nano microsphere preparing is carried out to bait parcel, to prepare the oral live vaccine of rabies.Can improve thus the oral live vaccine of rabies.
According to a particular embodiment of the invention, bait parcel may further include: chitosan nano microsphere is joined in the wax of thawing and carry out the first bait parcel and dry, to obtain the first chitosan nano microsphere wrappage; The chitosan nano microsphere of sealing wax is joined in the mixing material of Adeps Sus domestica and wax and carry out the second bait parcel and dry, to obtain the second chitosan nano microsphere wrappage; And the second chitosan nano microsphere wrappage is joined and in Carnis Sus domestica polymer, mixes and shape, to prepare the oral live vaccine of rabies.Can obtain thus the chitosan nano microsphere through bait parcel, to further improve the attractability of the oral live vaccine of rabies, to attract target animals initiatively to eat the oral live vaccine of these rabies.
According to a particular embodiment of the invention, the attractant adopting in said method is not limited in Adeps Sus domestica, as long as can effectively attract target animals food calling vaccine, and research by experiment, Adeps Sus domestica apparently higher than other attractants, can improve the utilization rate of oral vaccine to the attractability of target animals further.In addition, according to the specific embodiment of the invention, the object of paraffin used is mainly vaccine sizing, for attracting target animals.
According to a particular embodiment of the invention, before chitosan nano microsphere is carried out to bait parcel, can further include: in chitosan nano microsphere, adding MEM solution, is 1 × 10 to make the concentration of the rabies ERAg3m virus in chitosan nano microsphere
9.0tCID
50/ ml.Can improve thus the concentration of rabies ERAg3m virus, to further improve the immunocompetence of the oral live vaccine of rabies.
Below with reference to specific embodiment, present invention is described, it should be noted that, these embodiment are only descriptive, and do not limit the present invention in any way.
The preparation of embodiment 1 rabies virus antigen
(1) BHK-21 cell suspension domestication
Select the well-grown monolayer adherence cell of BHK-21 cell clone, after 0.01% pancreatin one 0.01%EDTA Digestive system digestion, suspending into final concentration is 2 × 10
5/ mL～5 × 10
5the Cell sap of/mL, inoculum concentration is looked the number of tank body size and culture fluid and is determined.Suspension nutrient composition comprises: 88% MEM, and 8% new-born calf serum, 1%L mono-glutamine and antibiotic etc., then use (7.2%) NaHCO
2adjust pH to 7.4 left and right, inoculation 5L bioreactor, 37 ℃, 100rpm stir culture.Contact passed for 60 generations in this way, observation of cell form counting.
(2) antigen preparation
Choose rabies ERAg3m strain 13 generations cell adapted through BHK-21, LD
50be 10
-7.0/ ml, 55-60 is for suspension cell in inoculation, and the floating initial concentration of inoculation rear overhang is 2 × 10
5.0individual/ml, 100 turn/min, 37 ℃ of dissolved oxygen are to cultivate under 45% condition, every sampling in 8 hours, measure cytoactive, within 96-120 hour, batch-type results virus liquid is frozen, measures TCID
50and LD
50.
The preparation of embodiment 2 microspheres
1g chitosan is dissolved in 1% acetum, under the mixing speed of 200r/min, rabies virus antigen is joined in this chitosan solution again, making its rabies virus antigen final concentration is 1 × 10
8.0tCID
50/ ml, then slowly add tripolyphosphate sodium water solution, mix homogeneously, under 4 degrees Celsius, the condition of 15000-18000rpm, carry out centrifugal and collecting precipitation thing, distilled water carries out lyophilization after washing precipitate, to obtain chitosan nano microsphere, wherein, the mass ratio of chitosan and sodium tripolyphosphate is (3:1)～(8:1).
The application of the oral live vaccine of embodiment 3 rabies virus microsphere
Oral rabies virus microsphere live vaccine is inoculated to 60 ages in days without 10 of the wandering dogs of rabies virus antibodies, establish 10 of inactivated vaccine inoculation contrast dogs, 10 of blank dogs simultaneously; After immunity inoculation, after 21 days, adopt street virus fatal dose to attack above-mentioned dog and the blank dog that inoculates respectively the oral live vaccine of rabies virus microsphere and inactivated vaccine.Concrete outcome is in table 1.
Table 1 rabies vaccine counteracting toxic substances protection result
| Group | Immunity number of elements | The dead number of elements of counteracting toxic substances | Counteracting toxic substances protective rate |
| Oral live vaccine inoculation group | 10 | 1 | 90% |
| Inactivated vaccine inoculation group | 10 | 2 | 80% |
| Blank group | 10 | 10 | 0% |
Conclusion: oral live vaccine dog 90% is protected, and inactivated vaccine dog 80% is protected, blank dog is all dead, unprotect.The counteracting toxic substances protective rate of the oral live vaccine of rabies virus microsphere is significantly higher than the counteracting toxic substances protective rate of inactivated vaccine thus.
In the description of this description, the description of reference term " embodiment ", " some embodiment ", " example ", " concrete example " or " some examples " etc. means to be contained at least one embodiment of the present invention or example in conjunction with specific features, structure, material or the feature of this embodiment or example description.In this manual, the schematic statement of above-mentioned term is not necessarily referred to identical embodiment or example.And specific features, structure, material or the feature of description can be with suitable mode combination in any one or more embodiment or example.
Although illustrated and described embodiments of the invention above, be understandable that, above-mentioned enforcement is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art can change above-described embodiment within the scope of the invention in the situation that not departing from principle of the present invention and aim, modification, replacement and modification.
Claims (10)
1. a rabies live vaccine, is characterized in that, comprises: rabies ERAg3m virus.
2. rabies live vaccine according to claim 1, is characterized in that, in described rabies live vaccine, the concentration of rabies ERAg3m virus is 1 × 10
9.0tCID
50/ ml.
3. a method of preparing the oral live vaccine of rabies, is characterized in that, comprising:
1) utilize BHK21 cell to carry out full suspension culture to rabies ERAg3m virus, to obtain rabies ERAg3m virus solution;
2) described rabies ERAg3m virus solution is mixed with chitosan solution and sodium tripolyphosphate solution, to prepare chitosan nano microsphere; And
3) described chitosan nano microsphere is carried out to bait parcel, to prepare the oral live vaccine of described rabies.
4. method according to claim 3, is characterized in that, in step 2) in, described rabies ERAg3m virus solution is joined in chitosan-acetic acid solution;
The mixed solution that contains chitosan and rabies ERAg3m virus is added to tripolyphosphate sodium water solution, and mix homogeneously centrifugal treating, to obtain the Nano microsphere that contains vaccine antigen.
5. method according to claim 3, is characterized in that, described full suspension culture is cultivated and completed for 36 hours at the temperature of 37 degrees Celsius.
6. method according to claim 4, is characterized in that, the concentration of described rabies ERAg3m virus solution is higher than 1 × 10
8.0tCID
50/ ml.
7. method according to claim 4, is characterized in that, in described chitosan-acetic acid solution, the concentration of chitosan is 0.1%-10%w/v, and the solvent of described chitosan-acetic acid solution is that concentration is 1% acetic acid; The concentration of described tripolyphosphate sodium water solution is 0.1%-5%w/v.
8. method according to claim 4, is characterized in that, described centrifugal treating completes under 4 degrees Celsius, the condition of 15000～18000rpm for centrifugal 30 minutes.
9. method according to claim 3, is characterized in that, in step 3) described chitosan nano microsphere is carried out to bait parcel before, further comprise:
In described chitosan nano microsphere, adding MEM solution, is 1 × 10 to make the concentration of rabies ERAg3m virus described in described chitosan nano microsphere
9.0tCID
50/ ml.
10. method according to claim 3, is characterized in that, the described bait parcel in step 3) further comprises:
Described chitosan nano microsphere is joined in the wax of thawing and carry out the first bait parcel and dry, to obtain the first chitosan nano microsphere wrappage;
The chitosan nano microsphere of described sealing wax is joined in the mixing material of Adeps Sus domestica and wax and carry out the second bait parcel and dry, to obtain the second chitosan nano microsphere wrappage; And
Described the second chitosan nano microsphere wrappage is joined and in Carnis Sus domestica polymer, mixes and shape, to prepare the oral live vaccine of described rabies.
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| CN113117066A (en) * | 2021-03-22 | 2021-07-16 | 华南农业大学 | Preparation method of chitosan nanoparticle vaccine for avian influenza |
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