CN102526722A - Rabies vaccine for human beings - Google Patents
Rabies vaccine for human beings Download PDFInfo
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- CN102526722A CN102526722A CN2010106077352A CN201010607735A CN102526722A CN 102526722 A CN102526722 A CN 102526722A CN 2010106077352 A CN2010106077352 A CN 2010106077352A CN 201010607735 A CN201010607735 A CN 201010607735A CN 102526722 A CN102526722 A CN 102526722A
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Abstract
The invention provides a rabies vaccine for human beings, which contains an outer membrane fragment of a split rabies virus particle and tetanus toxoid; the outer membrane fragment comprises furcella and matrix protein, wherein each dosage of rabies vaccine for human beings contains 10-150 micrograms of virus protein; and the dosage of the tetanus toxoid is 3-10 Lf/dosage. Compared with the existing virus purification stock solution, the rabies vaccine for human beings contains less other proteins, has higher immunogenicity and effect, and is more secure for use.
Description
Technical field
The present invention relates to the medical prevention field, specifically, relate to a kind of rabies vaccine of human.
Background technology
Rabies are by the acute infectious disease due to the rabies virus infection, are a kind of infecting both domestic animals and human acute infectious disease.Have that to surpass 50,000 people dead because of rabies an every year, 99% dead the time occur in Asia, Africa and South America.
Rabies virus (RV) is the height neurotropic virus, is the not segmentation minus-stranded rna virus in the Rhabdoviridae Rhabdovirus, and profile is the bullet shape, and nucleocapsid is symmetry in the shape of a spiral, and the surface has peplos, contains single stranded RNA, is to cause rabic pathogen.Rabies virus has two kinds of major antigens: a kind of is glycoprotein antigen on the outer virionic membrane, can combine to make virus to have neurotoxicity with acetylcholinergic receptor, and makes and produce neutralizing antibody and hemagglutination inhibition antibody in the body, and neutralizing antibody has protective effect; Another kind is the nucleoprotein antigen of internal layer, can make and produce complement fixation antibody and precipitin, unprotect effect in the body.
The peplos component of RV is made up of transmembrane glycoprotein G and substrate M albumen.Glycoprotein forms about 400 trimerization furcellas, and these furcellas are closely aligned on virus surface.
Rabies vaccine is to prevent rabies that unique effective medicine takes place at present.The rabies vaccine that uses at present mainly contains three types: the fire extinguishing vaccine of cultivating through animal brain, through the inactivated vaccine of duck embryo culture and the inactivated vaccine cultivated through cell (human diploid cell, chick-embryo cell, Ren Mus primary cell and Vero passage cell).
The rabies vaccine major part is the immunity after being used to expose, and therefore requires vaccine in human body, to produce immune response faster.But no matter existing rabies vaccine is to adopt which kind of Virus culture system, all is to process the vaccine that contains whole virus particles finally.The composition that it contains and antigen is irrelevant, like the residual protein of the histone of viral foreign protein, viral nucleic acid and lipid and cultivation virus, when increasing the antigenic content of virus, these foreign proteins also increase thereupon, so the side reaction of vaccine has just strengthened.
Summary of the invention
Technical problem to be solved by this invention is, in order to overcome defective of the prior art, provide a kind of and can in human body, produce sooner, higher immune response, and side reaction few the human novel vaccine.
A kind of human rabies vaccine is characterized in that, described vaccine contains adventitia fragment and the tetanus toxoid of rabies whole virus particles after cracking.Described cracking is that the rabies whole virus particles is through the chemical method cracking.Described adventitia fragment comprises furcella and the stromatin that glycoprotein forms.Tetanus toxoid has the effect of quick enhance immunity to rabies virus VLP; Can obtain the effect of senior middle school and antibody in a short time; And neutralizing antibody holds time longlyer, bites and patient that incubation period is short for mad dog poisons, and a kind of safer effective Antirabic Vaccine is provided.
Described human rabies vaccine is to be prepared by the CTN-1V of rabies virus fixed virus, aGV strain or other optional strains in cell adapted rabies virus fixed virus.
Among the present invention, the every dosage of human rabies vaccine is for containing virus protein 10-150 μ g, and the consumption of tetanus toxoid is 3Lf/ agent-10Lf/ agent.
Described rabies vaccine comprises rabies virus adventitia, stromatin and supraspinal glycoprotein composition.
A kind of method for preparing human rabies vaccine, the step of this method comprises:
(1) prepares particulate adventitia fragment of cracked mad dog lytic virus and tetanus toxoid respectively;
(2) these two kinds of compositions are mixed according to a certain percentage;
(3) with mixed vaccine according to liquid dosage form or dosage form packing of freeze-dried formulation vaccine or lyophilizing;
(4) with packing after the calibrating of the process of the product after packing finished product, promptly obtain required Antirabic Vaccine.
Described two kinds of compositions according to the mixed proportion of antigenic content are: virus protein 10-150 μ g/ agent; The consumption of tetanus toxoid is 3Lf/ agent-10Lf/ agent.
The particulate adventitia fragment of the cracked mad dog lytic virus of the preparation of described step (1) comprises the steps:
The rabies totivirus liquid of A, preparation purification;
B, Triton-100 is added lytic virus granule in the viral liquid behind the purification according to the 0.2-1.0% final concentration;
Viral liquid after C, the cracking carries out secondarily purified, collects outer virionic membrane fragment part, purifying antigen once more with 10-60% SDGC or column chromatography method;
D, through 30KD-100KD filter membrane ultrafiltration and concentration, dialysis is reclaimed employing virus cracking liquid after the desaccharide.
The present invention carries out cracking through the rabies virus granule that cell culture, purification obtain with chemical method with rabies virus fixed virus; Extraction and purification contain the outer virionic membrane fragment of furcella and stromatin; A certain amount of tetanus toxoid of remix is configured to a kind of rabies virus split vaccine.The present invention also provides the method for preparing this split vaccine.This virolysis vaccine is used to prevent the rabies of people because of the rabies virus infection initiation.
Existing rabies whole virus vaccine has been carried out transformation to mad dog split vaccine of the present invention and upgrading makes it to reach better immune effect and safety.The present invention adds decomposition agent with the rabies totivirus after purified, and extraction and purification contain the outer virionic membrane fragment of furcella and stromatin, and a certain amount of tetanus toxoid of remix is through being configured as the rabies virus split vaccine.Make in this vaccine, except that containing immunogenic envelope antigen exists, greatly reduced the viral endogenous cytotoxic material that other might property causes untoward reaction, like viral nucleic acid and lipid.Thereby can overcome existing totivirus commercially available vaccine and have immune limitation and postvaccinal untoward reaction.
The used rabies virus fixed virus of the present invention is: CTN-1V, aGV strain.Nat'l Pharmaceutical & Biological Products Control Institute is responsible for preparation and provides above-mentioned viral seed culture of viruses.Certainly, vaccine of the present invention also can adopt other through cell adapted rabies virus fixed virus.
The method of rabies vaccine of the present invention configuration comprises: according to the virus concentration of purified virus liquid, Triton-100 is added in the viral liquid behind the purification, make fully thoroughly cracking of whole virus particles.Viral liquid after the cracking carries out secondarily purified, with SDGC or column chromatography method, collects the fragment that contains peplos and furcella.Through the filter membrane ultrafiltration and concentration, reclaim employing virus cracking liquid after the dialysis desaccharide.With the buffer dilution, add 1% thimerosal, vaccinogen liquid is prepared in aseptic filtration through secondary, and behind the antigen stabilizing agent that adding suits, behind assay approvals such as sterility test, protein concentration and purity testing, potency test, packing is vaccine.
The invention provides a kind of human rabies vaccine and preparation method thereof, compare with existing viral purification stock solution, its foreign protein that contains significantly reduces and has reduced side effect, and has higher immunogenicity and effectiveness, uses safer.
The specific embodiment
In order to make technological means of the present invention, creation characteristic, to reach purpose and effect and be easy to understand and understand, the present invention is done further elaboration below in conjunction with embodiment.
Embodiment 1 rabies whole virus particles cracking and extraction purification
With the Vero cell in 37 ℃ of cultivations, and through passing continuously for 4 generations.After the 4th generation cell culture 3-5 days, use phosphate buffer drip washing, the virus inoculation titre is for being not less than 7.5LgLD
50The rabies virus fixed virus CTN-1V strain of/ml.And add cell maintenance medium in 35 ℃ of cultivations 24 hours.Change to fresh cell maintenance medium, continue to cultivate 2-4 days according to above-mentioned condition, CPE reaches when cytopathy ++～++ in+time, gather in the crops, merge viral liquid.According to 1: 4000 adding beta-propiolactone, place 22 ℃, 8 days inactivation of viruses.After the inactivation test checking is qualified, with 100KD filter membrane ultrafiltration and concentration.Add the protamine sulfate of 10mg/ml in the viral liquid after concentrating, making its final concentration is 0.1mg/ml, after 8000rpm is centrifugal, and the supernatant aseptic filtration.Again through the Sepharose4FF gel filtration chromatography, collect totivirus peak liquid, according to the virus protein concentration of the totivirus liquid of purification, with Triton-100 slowly add in the totivirus liquid behind the purification according to 0.2% final concentration and gentle agitation even.In 2-8 ℃ of gentle vibration 60 minutes, make fully thoroughly cracking of virion.Viral liquid after the cracking carries out secondarily purified, uses earlier the 10-60% SDGC, collects the segmental liquid phase of outer virionic membrane that is rich in furcella and stromatin.Purification with the PBS dilution, is transferred PH7.2 once more.Through 100KD filter membrane ultrafiltration and concentration, reclaim the outer virionic membrane fragment that contains furcella and stromatin after the dialysis desaccharide.With the phosphate buffer dilution, add 1% thimerosal, prepare vaccinogen liquid through 0.45 μ g and 0.22 μ g secondary aseptic filtration.After behind the assay approvals such as sterility test, protein concentration and purity testing, potency test, add antigen stabilizing agent human albumin, transfer pH7.0, contain virus protein 10-150 μ g/0.5ml/ agent according to every dose, packing is subsequent use.
Embodiment 2
Get human rabies virion crack protein (mainly containing the particulate adventitia fragment of lytic virus), the tetanus toxoid stock solution (Wuhan Biological Products Inst.) of above-mentioned preparation, according to the mixed proportion of antigenic content be: virus protein 10-150 μ g/ agent; The consumption of tetanus toxoid is 3Lf/ agent-10Lf/ agent; Aqueous vaccine is processed in combination respectively, encapsulation then, calibrating packing at last.
Embodiment 3
Get human rabies purification stock solution (diploid cell) (Nat'l Pharmaceutical & Biological Products Control Institute), rabies virus granule crack protein and tetanus toxoid stock solution (Wuhan Biological Products Inst.) make up respectively according to the ratio of table 1 and process aqueous vaccine.If the Antirabic Vaccine is a matched group, wherein only in matched group, adding 1% human albumin is protective agent, carries out the specificity experiment of four kinds of combination-vaccines respectively according to the method for three ones of Chinese Pharmacopoeias.Experimental result is all qualified, explains that the vaccine of the rabies virus granule crack protein that adds tetanus toxoid is safely and effectively.
Embodiment 4
With the vaccine of 5 kinds of combinations of the foregoing description 3 immune mouse respectively, each immune 0.5ml, blood sampling after 14 days is observed in immunity twice respectively in 0 day, 7 days, detects the serum neutralizing antibody and tires.
Table 1
| The vaccine kind | (IU) tires/agent |
| CTN-IV strain 10ug/ agent | 3.16 |
| CTN-IV strain 75ug/ agent | 5.56 |
| CTN-IV strain 75ug/ agent+3Lf/ agent | 6.34 |
| CTN-IV strain 75ug/ agent+7Lf/ agent | 7.50 |
| Human rabies purification stock solution (Vero cell) | 2.65 |
Therefore, explain that tetanus toxoid can obviously increase the effect of tiring of rabies vaccine, play the effect of enhance immunity, in the preparation of vaccine, can significantly reduce human rabies purification stock solution antigen amount, have excellent protection and render a service.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technical staff of the industry should understand; The present invention is not restricted to the described embodiments; That describes in the foregoing description and the description just explains principle of the present invention; The present invention also has various changes and modifications under the prerequisite that does not break away from spirit and scope of the invention, and these variations and improvement all fall in the scope of the invention that requires protection.
Claims (6)
1. a human rabies vaccine is characterized in that, described vaccine contains adventitia fragment and the tetanus toxoid of rabies whole virus particles after cracking; Described adventitia fragment comprises furcella and stromatin.
2. human rabies vaccine according to claim 1 is characterized in that, the every dosage of described human rabies vaccine is for containing virus protein 10-150 μ g, and the consumption of tetanus toxoid is 3Lf/ agent-10Lf/ agent.
3. human rabies vaccine according to claim 1 is characterized in that, described human rabies vaccine is to be prepared by the CTN-1V of rabies virus fixed virus, aGV strain or other optional strains in cell adapted rabies virus fixed virus.
4. the method for preparing of a human rabies vaccine, the step of this method comprises:
(1). prepare particulate adventitia fragment of cracked mad dog lytic virus and tetanus toxoid respectively;
(2). these two kinds of compositions are mixed according to a certain percentage;
(3). tightly is packing or lyophilizing with mixed vaccine according to liquid dosage form or freeze-dried formulation vaccine;
(4). with packing after the calibrating of the process of the product after packing finished product, promptly obtain required Antirabic Vaccine.
5. method for preparing according to claim 4 is characterized in that, two kinds of composition mixed proportions are in the step (2): virus protein 10-150 μ g/ agent; The consumption of tetanus toxoid is 3Lf/ agent-10Lf/ agent.
6. method for preparing according to claim 4 is characterized in that, the particulate adventitia fragment of the cracked mad dog lytic virus of the preparation of described step (1) comprises the steps:
The rabies totivirus liquid of A, preparation purification;
B, Triton-100 is added lytic virus granule in the viral liquid behind the purification according to the 0.2-1.0% final concentration;
Viral liquid after C, the cracking carries out secondarily purified, collects outer virionic membrane fragment part, purifying antigen once more with 10-60% SDGC or column chromatography method;
D, through 30KD-100KD filter membrane ultrafiltration and concentration, dialysis is reclaimed employing virus cracking liquid after the desaccharide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010106077352A CN102526722A (en) | 2010-12-23 | 2010-12-23 | Rabies vaccine for human beings |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010106077352A CN102526722A (en) | 2010-12-23 | 2010-12-23 | Rabies vaccine for human beings |
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| CN102526722A true CN102526722A (en) | 2012-07-04 |
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| CN2010106077352A Pending CN102526722A (en) | 2010-12-23 | 2010-12-23 | Rabies vaccine for human beings |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434570A (en) * | 2016-10-09 | 2017-02-22 | 泰安京泰生物技术有限公司 | RV (rabies virus) splitting solution and application thereof |
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2010
- 2010-12-23 CN CN2010106077352A patent/CN102526722A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434570A (en) * | 2016-10-09 | 2017-02-22 | 泰安京泰生物技术有限公司 | RV (rabies virus) splitting solution and application thereof |
| CN106434570B (en) * | 2016-10-09 | 2019-05-28 | 泰安京泰生物技术有限公司 | A kind of hydrophobin lysate and its application |
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Application publication date: 20120704 |
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