CN1843507B - Mumps virus ingredient vaccine for human, and its preparation method and uses - Google Patents
Mumps virus ingredient vaccine for human, and its preparation method and uses Download PDFInfo
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- CN1843507B CN1843507B CN2006100106849A CN200610010684A CN1843507B CN 1843507 B CN1843507 B CN 1843507B CN 2006100106849 A CN2006100106849 A CN 2006100106849A CN 200610010684 A CN200610010684 A CN 200610010684A CN 1843507 B CN1843507 B CN 1843507B
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Abstract
The invention relates to a mumps virus ingredient vaccine for human, and its preparation method and uses, wherein the preparing process comprises isolating a parotiditis virus from human body infectedby parotiditis virus through conventional methods, adapting the virus to Vero cell culture to prepare single-layer static or rotary Vero cell culture, carrying out virus deactivation, decomposition and purification, predicating the two antigenic components of HN and NP by means of molecular weights, using the antigen composition as the semi-finished vaccine after protein content measurement according to the semi-finished vaccine protein concentration, diluting to 100mul/ml with physiological saline, formulating Al(OH)3 with a concentration of 10.87mg/ml simultaneously, finally mixing the twoformulations.
Description
Technical field
The present invention relates to biological technical field, more particularly, the present invention relates to a kind of mumps virus ingredient vaccine for human.Simultaneously, the invention still further relates to the preparation method and the application of described vaccine.
Background technology
Mumps Vaccine is one of viral vaccine of the conventional inoculation of global child.At present, the type of service of this vaccine all adopts attenuated live vaccine in various countries, because its application, successfully at majority state, especially the parotitic sickness rate of child has been controlled in many areas of developed country.But along with the extensive use of this attenuated live vaccine in the crowd and the reduction of corresponding sickness rate, it might be the same with other similar viral lived vaccine, exists virulence changes and form a specific hypotype in the crowd probability to occur.In addition, between the different strains of this class vaccine also performance the diversity of immunology effect of degree varies is arranged, the inductive crowd's of institute immunology effect still is not the best.Therefore,, on the basis of existing attenuated mumps vaccine, live, carry out the research and development of corresponding vaccine, can promote the effectiveness and the safety of this class vaccine from the development angle of vaccine.From the angle of vaccine biotechnology, exploration has better immunogenic ingredient vaccine or subunit vaccine, will be the optimised form of next step vaccine development and use.
At present, use the viral vaccine of subunit form only to be the hepatitis B virus surface antigen vaccine, and, only have the cracking influenza virus vaccine to use as a kind of preliminary ingredient vaccine form as viral ingredient vaccine.Therefore, further study the component or the subunit form vaccine of multiple viral disease, have the important techniques meaning.And utilize mumps virus to come at first object as this work, because therefore this viral construction features and biological characteristics have comparatively feasible technical meaning.
Mumps virus belongs to paramyxovirus, its genome is the minus strand single-stranded RNA, form by 15500 nucleotide, its 7 albumen of encoding, they are respectively NP, P, M, F, SH, HN and L albumen, and HN wherein and NP albumen are significant aspect the specific immunity originality of virus, and the two all can induce the humoral immunization and the cell immune response of body, especially HN can induce body to produce neutrality antibody, and blood clotting suppresses neutralizing antibody.Wherein NP is the 70KD size, and HN is the 77KD size, and has hemagglutination activity.In addition, because mumps virus can grow in multiple tissue culture cells,, thereby help the large-scale culture preparation as the Vero cell.
Summary of the invention
The objective of the invention is to utilize the biotechnology means, a kind of mumps virus ingredient vaccine for human is provided at the pressing for of the deficiencies in the prior art and realistic situation.Simultaneously, the present invention also provides a kind of preparation method of described ingredient vaccine.The cultivation of Vero cell tissue, virolysis and component separation method that this method is set up can be used for preparing the ingredient vaccine of several paramyxovirus.
Purpose of the present invention is achieved by following technical proposals.
The invention provides a kind of mumps virus ingredient vaccine for human, this vaccine is to adopt following method to prepare: adopt conventional method to be separated to a strain mumps virus in the popular human body of parotitis; This virus can be grown at this cell inner stablity, and reach 10 after being adapted to the Vero cell culture
5.0CCID
50/ ml output, the blood clotting titre can reach 1: 8, and the pathological changes cycle is 5～6 days; Prepare the static or rotation Vero cell culture of monolayer then; Deactivation and cracking and purifies and separates through virus obtain corresponding virus protein component, and according to its molecular weight size, decidable is HN and two antigenic components of NP; This antigen component after measuring protein content as the semi-finished product vaccine; According to semi-finished product vaccine protein concentration, be diluted to 100 μ l/ml with normal saline, the Al of cofabrication 10.87mg/ml concentration (OH)
3, the two is to mix times (V/V), and make experimental ingredient vaccine, and it is detected: its semi-finished product protein content＞2000 μ g/ml, finished product protein content are 100 μ g/ml, and sterility test is (-), formaldehyde residual volume＜0.5mg/ml; This vaccine is required mumps virus ingredient vaccine.
The present invention provides the preparation method of described mumps virus ingredient vaccine for human simultaneously, and this method adopts the step of following order:
(1) separation of mumps virus
In the human body that infects mumps virus, be separated to a strain mumps virus, the patient's who fell ill 1～4 day mouthwashes changes in the little plate of sterilization, add the Hanks liquid mix homogeneously that is equivalent to mouthwashes amount 50%, low-speed centrifugal 10 minutes, get supernatant, add penicillin and streptomycin, place refrigerator and inoculate the Vero cell that grows up to monolayer after 2 hours, rearmounted 37 ℃ of cultivations.Every day, observation had or not cytopathy, cultivated after 6 days, cytopathy occurred, after hemagglutination test confirms to truly have viral infection; And carrying out neutralization test with commercial mumps virus antibody, the clear and definite strain that is separated to is mumps virus.
To separate mumps virus be SH gene and flanking sequence thereof, according to gene type, determine that it belongs to the F genotype; This virus can be grown at this cell inner stablity, and reach 10 after being adapted to the Vero cell culture
5.0CCID
50/ ml output, the blood clotting titre can reach 1: 8, and the pathological changes cycle is 5～6 days.
(2) the static or rotation Vero cell culture of preparation monolayer
Adopt the Vero cell of 0.25% pancreatin-0.01%EDTA digestion formation monolayer, room temperature leaves standstill 10min, discards pancreatin, puts room temperature 10-15min again, treat cell dissociation form single after, with the DMEM-8%BCS re-suspended cell, be diluted to 10
5.0Cell/ml is by 10
6.0Cell/static the cultivation of bottle graft kind Luo Shi bottle; By 4 * 10
6.0Cell/bottle, inoculation rolling bottle rotating and culturing; Cultivated 2 days for 37 ℃, press the 0.01-0.05moi virus inoculation, change the DMEM-1%BCS culture fluid simultaneously, 37 ℃ are continued to cultivate 5～6 days, treat to gather in the crops when pathological changes appears in cell virus, sampling and measuring titre; Its titre should be 10
5.0CCID
50/ ml.
(3) Bing Du deactivation and cracking and purifies and separates:
Gather in the crops viral liquid after titer determination and aseptic detection, add formalin stock solution by 1: 2000 (V/V), 37 ℃ of deactivations 7 days, every sampling in 2 hours, inactivation of virus curve (seeing figure-1) was measured in beginning sampling every day in second day in first day; After 7 days, inactivation of viruses concentrates after filtration, concentrates 100 times on the original volume basis, presses 2%TritonX-100, and the ratio of 11%SDS adds inactivation of viruses liquid, room temperature effect 2 hours.Handling back inactivation of viruses liquid is filtered by the Sapharose4B post, its virolysis product separating resulting is seen figure-2, see figure-3 through the blood clotting experiment analysis results, determine to get sample separation, through PAGE-SDS (12%) electrophoretic analysis, and the comprehensive blood clotting experimental analysis of Western blot (figure-5), determine that this separation component comprises two major antigen compositions, according to its molecular weight size, decidable is HN and two antigenic components of NP; This antigen component after measuring protein content as the semi-finished product vaccine.
(4) preparation of mumps virus experimental vaccine
According to semi-finished product vaccine protein concentration, be diluted to 100 μ l/ml with normal saline, the Al of cofabrication 10.87mg/ml concentration (OH)
3, the two makes experimental ingredient vaccine with to doubly (V/V) mixing, is required mumps virus ingredient vaccine for human.
The present invention carries out the detection of protein content, aseptic, formaldehyde residual volume to described mumps virus ingredient vaccine for human, and testing result is as follows:
Semi-finished product protein content:＞2000 μ g/ml;
Finished product protein content: 100 μ g/ml
Sterility test: (-)
The formaldehyde residual volume detects:＜0.5mg/ml
Compared with prior art, the present invention has following outstanding advantage:
(1) best in quality: the mumps virus ingredient vaccine that the present invention developed, only contain two strong protein subunits of immunogenicity of HN, NP;
(2) effectiveness height; With mumps virus ingredient vaccine and commercial mumps virus attenuated live vaccine difference immune mouse that the present invention developed, ELISA result detects and shows the antibody titer of antibody titer that this ingredient vaccine produces apparently higher than the commercialization attenuated live vaccine; Prove absolutely this ingredient vaccine owing to only contain the strong protein subunit of immunogenicity, therefore can induce body to produce higher immunne response effect;
(3) safety is good; The mumps virus ingredient vaccine that the present invention developed is for inactivated whole virus, because after virus handled through decomposition agent, relative molecular mass diminished, and has only kept and has antigenic protein subunit; Therefore, side reaction reduces greatly, has better safety.
Description of drawings
Fig. 1 is the inactivation of virus curve; Among the figure: with 0 expression, infection titer is 10 before the inactivation of virus
5.0CCID
50/ ml, 8 hours postoperative infection titres of deactivation reduce to 0, thoroughly deactivation.
Fig. 2 be second peak, 14～23 pipes PAGE-SDS (12%) electrophoresis pattern, the Western blot collection of illustrative plates of 17 pipes.
Fig. 3 is the electrophoresis result of 17,18 pipes, two bands as seen occur, and size is 70KD.
Fig. 4 is the Western blot result of 17 pipes, adopts the mumps virus antiserum anti-as one, two specific bands as seen occur, and the molecular weight size of band position can clearly be HN and NP albumen in comprehensive blood clotting and the electrophoresis.
The specific embodiment
The present invention is further illustrated below in conjunction with specific embodiment, but this is not the qualification to content of the present invention.
Except as otherwise noted, the percent that is adopted among the present invention is percetage by weight.
Embodiment 1
---deactivation and the cracking and the purifies and separates of virus
Gather in the crops viral liquid after titer determination and aseptic detection, add formalin stock solution by 1: 2000 (V/V), 37 ℃ of deactivations 7 days, every sampling in 2 hours, beginning sampling every day in second day was measured the inactivation of virus curve, as shown in Figure 1 in first day.After 7 days, inactivation of viruses concentrates after filtration, concentrates 100 times on the original volume basis, presses TritonX-100 2%, and the ratio of SDS1% adds inactivation of viruses liquid, room temperature effect 2 hours.Handling back inactivation of viruses liquid is filtered by the Sapharose4B post, its virolysis product separating resulting sees Table 1, see Table 2 through the blood clotting experiment analysis results, determine to get sample separation, through PAGE-SDS (12%) electrophoretic analysis, and Western blot (sees the comprehensive blood clotting experimental analysis of Fig. 2～Fig. 4), determines that this separation component comprises two major antigen compositions, according to its molecular weight size, decidable is HN and two antigenic components of NP; This antigen component after measuring protein content as the semi-finished product vaccine.
Table 1. virolysis product separating resulting
After processing as shown in table 1 back inactivation of viruses liquid filters via the Sapharose4B post, adopt test tube to collect two peaks altogether.
---the preparation of mumps virus experimental vaccine
According to semi-finished product vaccine protein concentration, be diluted to 100 μ l/ml with normal saline, the Al of cofabrication 10.87mg/ml concentration (OH)
3, the two makes experimental ingredient vaccine so that doubly (V/V) mixed, and made is the mumps virus ingredient vaccine that the present invention develops.
Test example 1
---to the immunogenicity analysis of described ingredient vaccine
Get some of PSF mices, with 20 be one group, set 50 a μ g/ dosage group respectively, 100 a μ g/ dosage group, a 150 μ g/ dosage group, right limb subcutaneous injection is got blood after 4 weeks behind mice, separation of serum, the titre of detection antiviral antigen-antibody, it the results are shown in Table 2.Testing result shows that the sun commentaries on classics of material for anti parotitis antiviral antibody has appearred in 100 μ g/ dosage group 100% ground.Use the neutralization test of these antibody to show, the inductive antibody of this mumps virus experimental vaccine has neutralization to mumps virus and renders a service.Adopt the ELISA test kit to detect the titre of antiviral antigen-antibody, the sun that anti-corresponding virus antigen-antibody has appearred in 100 μ g/ dosage group 100% ground, result visible immunity back changes.
Table 2. immunity back antiviral antibody sun changes the result
Test example 2
---the safety analysis of ingredient vaccine
The mice of using this ingredient vaccine is carried out comprehensive and systematic pathological analysis show, this experimental vaccine can not cause that any organ produces visible pathology and changes.
Mice pathological replacement result: the heart, liver, spleen, lung, kidney, the parotid gland, each internal organs of brain there is no unusually.
Test example 3
---the blood clotting experiment analysis results
Table 3.
Table 3 be depicted as each the pipe sample blood clotting result, the blood clotting degree with ++ ++, +++, ++ ,+, ± ,-expression.The result is that blood clotting all appears in the sample that number is all managed at second peak ++ ++, and first peak and plateau respectively manage the blood clotting result be-.But preliminary judgement second peak is the sample separation component thus, and contains HA albumen.Again sample is respectively managed at second peak and make doubling dilution.
Table 4. hemagglutinative titer result
Table 4 is with+criterion as a result of, and respectively manages hemagglutinative titer and all be higher than provirus hemagglutinative titer (1: 8), determines further that therefore second peak is the sample separation component.
Claims (2)
1. a mumps virus ingredient vaccine for human is characterized in that, this vaccine is to adopt following method to prepare:
(1) separation of mumps virus: the mouthwashes that will infect 1～4 day patient of mumps virus morbidity changes in the little plate of sterilization, add the Hanks liquid mix homogeneously that is equivalent to mouthwashes amount 50%, low-speed centrifugal 10 minutes, get supernatant, add penicillin and streptomycin, place refrigerator and inoculate the Vreo cell that grows up to monolayer after 2 hours, rearmounted 37 ℃ of cultivations; Every day, observation had or not cytopathy, cultivated after 6 days, cytopathy occurred, after the blood clotting experiment confirm truly has viral infection; And carrying out neutralization test with business-like mumps virus antibody, the clear and definite strain that is separated to is mumps virus;
(2) the static or rotation Vero cell culture of preparation monolayer: adopt the Vero cell of 0.25% pancreatin 0.01%EDTA digestion formation monolayer, room temperature leaves standstill 10min, discards pancreatin, put room temperature 10～15min again, treat cell dissociation form single after, with the DMEM-8%BCS re-suspended cell, be diluted to 10
5.0Cell/ml is by 10
6.0Cell/static the cultivation of bottle graft kind Luo Shi bottle; By 4 * 10
6.0Cell/bottle, inoculation rolling bottle rotating and culturing; Cultivated 2 days for 37 ℃, by 0.01～0.05moi virus inoculation, change the DMEM-1%BCS culture fluid simultaneously, 37 ℃ are continued to cultivate 5～6 days, treat to gather in the crops when pathological changes appears in cell virus, sampling and measuring titre; Its titre is 10
5.0CCID
50/ ml;
(3) Bing Du deactivation and cracking and purifies and separates: gather in the crops viral liquid after titer determination and aseptic detection, add formalin stock solution by 1: 2000 (V/V), 37 ℃ of deactivations 7 days, in first day every sampling in 2 hours, the inactivation of virus curve is measured in beginning sampling every day in second day; After 7 days, inactivation of viruses concentrates after filtration, concentrates 100 times on the original volume basis, presses TritonX-1002%, and the ratio of SDS1% adds inactivation of viruses liquid, room temperature effect 2 hours; After inactivation of viruses liquid filters via the Sapharrose4B post after handling, adopt test tube to collect two peaks altogether, second peak is the sample separation component; Its virolysis product separates, through the blood clotting experimental analysis, determine to get sample separation, through the 12%PAGE-SDS electrophoretic analysis, and Western blot, comprehensive blood clotting experimental analysis determines that this separation component comprises two major antigen compositions, be judged to be HN and two antigenic components of NP according to its molecular weight size, this antigen component after measuring protein content as the semi-finished product vaccine;
(4) preparation of mumps virus experimental vaccine: according to semi-finished product vaccine protein concentration, be diluted to 100 μ l/ml, the AL of cofabrication 10.87mg/ml concentration (OH) with normal saline
3, the two to be to mix doubly (V/V), and make experimental ingredient vaccine and it is detected, its semi-finished product protein content＞2000 μ g/ml, sterility test is (-), formaldehyde residual volume＜0.5mg/ml; This vaccine is required mumps virus ingredient vaccine.
2. the preparation method of a mumps virus ingredient vaccine for human is characterized in that, this method adopts the step of following order:
(1) separation of mumps virus: the mouthwashes that will infect 1～4 day patient of mumps virus morbidity changes in the little plate of sterilization, add the Hanks liquid mix homogeneously that is equivalent to mouthwashes amount 50%, low-speed centrifugal 10 minutes, get supernatant, add penicillin and streptomycin, place refrigerator and inoculate the Vreo cell that grows up to monolayer after 2 hours, rearmounted 37 ℃ of cultivations; Every day, observation had or not cytopathy, cultivated after 6 days, cytopathy occurred, after the blood clotting experiment confirm truly has viral infection; And carrying out neutralization test with business-like mumps virus antibody, the clear and definite strain that is separated to is mumps virus;
(2) the static or rotation Vero cell culture of preparation monolayer: adopt the Vero cell of 0.25% pancreatin 0.01%EDTA digestion formation monolayer, room temperature leaves standstill 10min, discards pancreatin, put room temperature 10～15min again, treat cell dissociation form single after, with the DMEM-8%BCS re-suspended cell, be diluted to 10
5.0Cell/ml is by 10
6.0Cell/static the cultivation of bottle graft kind Luo Shi bottle; By 4 * 10
6.0Cell/bottle, inoculation rolling bottle rotating and culturing; Cultivated 2 days for 37 ℃, by 0.01～0.05moi virus inoculation, change the DMEM-1%BCS culture fluid simultaneously, 37 ℃ are continued to cultivate 5～6 days, treat to gather in the crops when pathological changes appears in cell virus, sampling and measuring titre; Its titre is 10
5.0CCID
50/ ml;
(3) Bing Du deactivation and cracking and purifies and separates: gather in the crops viral liquid after titer determination and aseptic detection, add formalin stock solution by 1: 2000 (V/V), 37 ℃ of deactivations 7 days, in first day every sampling in 2 hours, the inactivation of virus curve is measured in beginning sampling every day in second day; After 7 days, inactivation of viruses concentrates after filtration, concentrates 100 times on the original volume basis, presses TritonX-1002%, and the ratio of SDS1% adds inactivation of viruses liquid, room temperature effect 2 hours; After inactivation of viruses liquid filters via the Sapharrose4B post after handling, adopt test tube to collect two peaks altogether, second peak is the sample separation component; Its virolysis product separates, through the blood clotting experimental analysis, determine to get sample separation, through the 12%PAGE-SDS electrophoretic analysis, and Western blot, comprehensive blood clotting experimental analysis determines that this separation component comprises two major antigen compositions, be judged to be HN and two antigenic components of NP according to its molecular weight size, this antigen component after measuring protein content as the semi-finished product vaccine;
(4) preparation of mumps virus experimental vaccine: according to semi-finished product vaccine protein concentration, be diluted to 100 μ l/ml, the AL of cofabrication 10.87mg/ml concentration (OH) with normal saline
3, the two to be to mix doubly (V/V), and make experimental ingredient vaccine and it is detected, its semi-finished product protein content＞2000 μ g/ml, sterility test is (-), formaldehyde residual volume＜0.5mg/ml; This vaccine is required mumps virus ingredient vaccine.
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101570566B (en) * | 2008-04-30 | 2013-02-20 | 上海泽润生物科技有限公司 | Vero cell cracked protein, preparation method and application thereof |
| RU2526131C2 (en) * | 2008-04-30 | 2014-08-20 | Шанхай Зерун Биотек Ко., Лтд. | Vero cell lysis proteins, method for production thereof and kit for determining host cell proteins for vero cells containing lysis proteins |
| CN102018956A (en) * | 2010-11-16 | 2011-04-20 | 中国医学科学院医学生物学研究所 | F-gene type attenuated live mumps vaccine and preparation method and application thereof |
| CN106290862B (en) * | 2016-08-05 | 2017-10-31 | 北京市华信行生物科技有限公司 | Mumps virus HN antigens and its purposes in detection material for anti parotitis antiviral antibody |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994025600A1 (en) * | 1993-04-30 | 1994-11-10 | Smithkline Beecham Biologicals S.A. | Recombinant antigens from mumps virus and their use in vaccines |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994025600A1 (en) * | 1993-04-30 | 1994-11-10 | Smithkline Beecham Biologicals S.A. | Recombinant antigens from mumps virus and their use in vaccines |
| CN1126492A (en) * | 1993-04-30 | 1996-07-10 | 史密斯克莱·比奇曼生物公司 | Recombinant antigens from mumps virus and their use in vaccines |
Non-Patent Citations (4)
| Title |
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| .腮腺炎病毒及疫苗研究进展.国外医学.预防.诊断.治疗用生物制品分册21 4.1998,21(4),148-151. * |
| 任桂杰 王志玉.副粘病毒包膜糖蛋白分子生物学研究进展.病毒学报,21 1.2005,21(1),72-75. * |
| 徐海明.腮腺炎病毒及疫苗研究进展.国外医学.预防.诊断.治疗用生物制品分册21 4.1998,21(4),148-151. * |
| 陈志慧.流行性腮腺炎病毒及其疫苗.中国计划免疫10 2.2004,10(2),120-124. * |
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