CN1556205A - Method of purifying active virus - Google Patents
Method of purifying active virus Download PDFInfo
- Publication number
- CN1556205A CN1556205A CNA2004100126059A CN200410012605A CN1556205A CN 1556205 A CN1556205 A CN 1556205A CN A2004100126059 A CNA2004100126059 A CN A2004100126059A CN 200410012605 A CN200410012605 A CN 200410012605A CN 1556205 A CN1556205 A CN 1556205A
- Authority
- CN
- China
- Prior art keywords
- antibody
- purifying
- virus
- host cell
- centrifugal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
A process for purifying living virus includes preparing the antibody of host cell, purifying it with agarose carrying staphylococcal protein A, mixing it with the cell breaking system of reproductive virus, removing deposit, mixing its suspension with said agarose, and removing deposit.
Description
Technical field
The present invention relates to a kind of method of the live virus of purifying, specifically, the present invention relates to a kind of from the host cell system of virus of proliferation the method for purifying live virus.
Background technology
Method for purifying virus commonly used both at home and abroad at present mainly contains: methods such as density gradient centrifugation, differential centrifugation, affinity chromatography and direct immunization precipitation technology.Below be illustrated with regard to the feature of these technology.
1. density gradient centrifugation
No matter selecting cesium chloride or sucrose for use is medium, all has following relative merits.
Advantage:
All relate to the operation of virus all carries out under cold condition, helps keeping of virus activity.
Shortcoming:
1) it is long that virus is left the culture time, centrifugally often needs several hrs, even tens hours, so be unfavorable for keeping of virus activity.
2) differentiate not exclusively.This is because the virus infected cell complicated component certainly exists the material close even identical with viral density, is difficult to the result who separates with virus.
3) damage of virion is bigger in ultracentrifugal process.
4) owing to the centrifuge tube finite volume, applied sample amount only is the long-pending 5%-10% of gradient liquid, so can't realize the high-throughput preparation, therefore can't realize industrialization.
5) ultracentrifugation is to the requirement height of equipment, wasteful, therefore cost height.
2. differential centrifugation technology
This technology and density gradient centrifugation are compared, and want simple in operation, and following technical characterstic is arranged.
Advantage:
1) do not relate to the preparation of gradient liquid, simple to operate, centrifugal under cold condition.
2) sample size of primary treatment is bigger, and is favourable to producing.
Shortcoming:
The resolving power of present technique method is far away from density gradient centrifugation, can be used for the separate and subside coefficient and differs and reach
10 times particle can not be realized and the sizable separate impurities of virion is removed, thereby can only be as a kind of method of easy thick purified virus, and this can't reach application demand.
3. affinity chromatography technology
This technology is used for genetic engineered product-proteinic separation and purification, the i.e. aftertreatment of genetic engineered product now more.Also have many people to use it for the preparation purifying of recombinant virus and virus vaccines, this method also has its relative merits.
Advantage:
1) Zhi Bei viral purity height.
2) be easy to amplify production.
Shortcoming:
1) in the process of crossing post, several times the wash-out of damping fluid experiences peracidity or overbasic condition, and is very big to the activity influence of virus.
2) in crossing the post process, virus is gone through wash-out again as the antibody coupling on antigenic substance and the base for post matter, and the structural integrity of virus easily goes to pot, and also can the biologic activity of virus be impacted.
4. direct immunization precipitation technology
This technology is used for the detection of viral protein more, less see its be used for virus separation and purification.And this technology is identical with affinity chromatography technology ultimate principle, also needs the damping fluid of extreme condition to carry out the process of wash-out, isolated viral, so, have the identical shortcoming of affinity chromatography technology.These are this not mediocre giving unnecessary details.
Therefore existing these extract and the method for purified virus all has common characteristics, and promptly they are all operated at virus particle itself, as: centrifugal is for sedimentation virus itself; Affinity chromatography is that antiviral antibody is coupled on the base for post matter, by the antigen-antibody combination virus and impurity is separated again, and last wash-out again descends the virus particle with antibodies.Undoubtedly, these methods are bigger to the damage of virus particle, the cost costliness, and majority can not enter industrialization.
Traditional method for purifying virus, on the one hand bigger as differential centrifugation or density gradient centrifugation to the virus damage, those comparatively fragile envelope virus particularly, Shou Huo virus always has some impurity more or less on the other hand; Affinity chromatography and some also are that virus is had bigger damage at the purification process of virion directly for another example.
Utilization of the present invention be the immunology theory and technology, but, it is a kind of reverse immune isolation technique thinking, its operation is opposite with traditional immune separation system, all operations are not all at virus particle itself, there be not combining between virus and the antibody to take place, so be referred to as " oppositely co-immunoprecipitation technology " (ReverseCoimmunoprecipita) with dissociation process.Clearly, this reverse co-immunoprecipitation technology helps protecting the integrity and the biologic activity of virus, and simple to operate, with low cost, be suitable for amplifying and produce.
Summary of the invention
The object of the present invention is to provide a kind of method of the live virus of purifying, this method separated product purity height, infectious strong, easy and simple to handle, low production cost.
To achieve these goals, the present invention has mainly adopted following technical measures, and the method for purification live virus comprises the following steps:
A), prepare the antiserum(antisera) of anti-host cell with the broken composition immune animal of the host cell of the uninfecting virus of cultivating;
B) in antiserum(antisera), extract the total antibody that contains foreign protein with the saturated ammonium sulphate method;
C) with the sepharose 4B extraction, the total antibody of purifying that have SP;
Total antibody that D) will extract is broken into the branch system with the host cell that virus multiplication infects to be mixed, centrifugal, discards host cell fragment and immunocomplex; Collecting supernatant, obtain to contain the suspension of virus and a certain amount of assorted antibody, is the viral suspension of purifying for part;
E) viral suspension that will partly purify mixes with the sepharose 4B that has SP, and is centrifugal to adsorb assorted antibody, collects supernatant, is the virus of purifying.
The present invention has enumerated some kinds of viruses and proliferative cell system thereof; clearly; its protection authority should not be only limited to these viruses and cell, and should comprise use that this technology is separated with method and all wild viruses of purifying, gene recombined virus, recombinant viral vector ... or the like.
In a preferred version of the present invention, steps A) sero-fast preparation, mix with the equivalent incomplete Freund's adjuvant with the broken composition antigen of the host cell of cultivating, add the 9-11mg bacille Calmette-Guerin vaccine by per 1 milliliter of said mixture again, make milky suspension as antigen, immune animal prepares the antiserum(antisera) of anti-host cell.In another preferred version of the present invention, step B) the first purifying of antibody extracts the first antibody purification that contains other antibody and proteic anti-host cell with the saturated ammonium sulphate method, and uses 0.1MNaHCO in antiserum(antisera)
3/ 0.5MNaCl solution is diluted to first antibody purification the solution of 4-6mg/ml; Step C) preparation of the total antibody of high purity is hatched 900-1100rpm/min for 37 ℃ altogether with first antibody purification and the sepharose 4B that has SP, centrifugal 10-12min, after the washing, again the immunoglobulin IgG in the coupling is eluted and collects gently, realize total purifying antibody.In another preferred version of the present invention, step D) Bing Du first purifying is broken into branch system 11000-14000rpm/min, 4 ℃ of centrifugal 10--15min with the host cell of virus multiplication, get supernatant mixed with anti-host cell antibody in 1: 2 by volume, 4 ℃, 20-25 rev/min of shaken overnight, with mixture with 10000-13000rpm/min, 4 ℃ of centrifugal 13-16min, abandoning cellular immunization mixture precipitation, collect supernatant liquor, is to be first purified virus.In another preferred version of the present invention, step e) purifying of virus, with supernatant liquor and the sepharose 4B that has SP in 4 ℃ of overnight incubation, absorption antibody, 900-1100rpm/min, centrifugal 10-12min asks in collections, is purified virus.
The virus of indication and host cell are in the present invention: blue tongue rims and VERO host cell, or herpes simplex virus I-type and VERO host cell, or herpes simplex virus I I type and VERO host cell, or human rotavirus and MA
104Host cell, or adenovirus and VERO host cell, or human cytomegalic inclusion disease virus and HEL host cell, or Coxsackie B virus and VERO host cell ... or the like.
Related virus and cell are except that blue tongue rims HbC strain among the present invention, and all the other viruses and cell all are to have announced also general virus and cell.Blue tongue rims HbC strain is from Hubei Medical Univ.'s journal, and 1999,20 (4): 265; CBA, 1999,13 (11): 59).
Related virus all is human or animal's common disease substance among the present invention, or the zoonosis pathogenic agent, is that important health virus is or/and economic virus.So related cell all is very valuable cell, especially VERO cell among the present invention, be the specified cell that can be used for producing of the Chinese government.
The virus and the host cell of indication are in a specific embodiment of the present invention, blue tongue rims and host cell VERO.
The method of purifying live virus provided by the invention is compared with traditional method, has following advantage:
(1) the viral purity of method purifying provided by the invention is very high.Because impurity by corresponding antibody in conjunction with and sedimentation is gone down; Excessive anticellular antibody and assorted antibody can combine with the sepharose 4B that has albumin A simultaneously, separates from viral suspension.
(2) the virus particle structure behind the purifying is very complete.Because this method all operations is not all at virion itself, neither exist antiviral antibody combine with virus with dissociation process in because of acid-base function and high ion concentration break virus ionic contradiction, do not exist poisons ultracentrifugation due to illness or column chromatography to cause the problem of viral ion damaged yet.
(3) kept viral biologic activity.Advantages such as because the advantage that this purification system had both had above-mentioned (2), it is simple also to have the operating process and the step that relate to virus in experiment, and all can finish at 4 ℃, and stripped (the leaving culturing cell) time of virus is short.
(4) with low cost.Do not relate to ultracentrifugation and affinity column technology because prepare purified virus, and the crosslinked sepharose 4B of SP can use repeatedly with this system.
(5) high flux property.This purification system very easily obtains amplifying, and has high-throughout feature, and with low cost, can be used for large-scale industrial production.
(6) have guiding significantly.This purification process can be widely used in other any virus or proteinic extraction, both can be used for suitability for industrialized production, can be used for the preparation of laboratory high purity virus again.
Description of drawings
Fig. 1. be the viral purity graphic representation after the SephadexG-200 gel-filtration analysis purification
Through behind the reverse immunoprecipitation, draw Sephadex in the A280 place
As seen G-200 chromatography curve only has a precipitous ultraviolet absorption peak.
Fig. 2. be phospho-wolframic acid negative staining electromicroscopic photograph before and after the reverse immunoprecipitation purifying
Fig. 2-A. purifying provirus suspension, background impurities is a lot, but complete virus particle is high-visible.×30,000
Fig. 2-B. is clean through reverse immunoprecipitation purifying rear backdrop, inclusion-free; Virus particle is complete, clear in structure.Viral suspension is through concentrating.×60,000
Fig. 3. for country allows to produce the infectious figure of proliferative cell research virus particle that makes BTV-HbC3 with the Vero cell
The Vero cell of Fig. 3-A. uninfecting virus except that virus inoculation not, all on all four control cells of all the other conditions (* 200)
Fig. 3-Vero cytopathy the image (* 200) of B. inoculation after BTV-HbC336 hour
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Be to be understood that, these embodiment only are used to the present invention is described and are not used in restriction the scope of protection of present invention, unreceipted concrete experiment condition and method in the following example, usually according to normal condition as chief editors such as J. Sa nurse Brookers, Science Press, 1992, molecular cloning experiment guide (second edition); D.L. Spector etc., Science Press, 2001, or connect according to manufacturer and execution such as advise.
The sero-fast preparation of embodiment 1 various anti-host cells
The sero-fast preparation of 1 anti-blue tongue rims host cell VERO
Select for use country to allow to produce with Vero cell proliferation blue tongue rims; Meanwhile, treat that the Cultivation of Vero of virus inoculation does not prepare cell antigen by the viral same operation step of results after converging into individual layer.The sensitive cells of results is by multigelation three times, with fragmentation; 5000rpm/min10 ℃ of centrifugal 12min, supernatant prepares anti-Vero cell antiserum(antisera) as Vero cell antigen immune animal.
The Vero cell antigen are mixed with the equivalent incomplete Freund's adjuvant, add the 9-11mg bacille Calmette-Guerin vaccine by every milliliter again, make milky suspension, be injected in after emulsification fully that to reach the back in the experimental group rabbit four-footed lift subcutaneous, each site 0.2ml; Inject 1 time with method after 2 weeks.In the 4th week beginning booster immunization, the Vero cell antigen that will not add adjuvant in promptly continuous three days are injected tame rabbit ear vein, and dosage increases progressively every day, is respectively 0.1ml, 0.2ml, 0.3ml.Last injection 1 week of back, heart blood sampling, separation of serum.
The sero-fast preparation of host cell Vero of 2 anti-herpes simplex virus I types and herpes simplex virus I I type sensitivity
Select for use country to allow to produce with Vero cell proliferation I herpes simplex virus type type and II hsv; Meanwhile, treat that the Cultivation of Vero of virus inoculation does not prepare cell antigen by the viral same operation step of results after converging into individual layer.The sensitive cells of results is by multigelation three times, with fragmentation; 5000rpm/min10 ℃ of centrifugal 12min, supernatant prepares anti-Vero cell antiserum(antisera) as Vero cell antigen immune animal.
The Vero cell antigen are mixed with the equivalent incomplete Freund's adjuvant, add the 9-11mg bacille Calmette-Guerin vaccine by every milliliter again, make milky suspension, be injected in after emulsification fully that to reach the back in the experimental group rabbit four-footed lift subcutaneous, each site 0.2ml; Inject 1 time with method after 2 weeks.In the 4th week beginning booster immunization, the Vero cell antigen that will not add adjuvant in promptly continuous three days are injected tame rabbit ear vein, and dosage increases progressively every day, is respectively 0.1ml, 0.2ml, 0.3ml.Last injection 1 week of back, heart blood sampling, separation of serum.
The sero-fast preparation of 3 anti-adenovirus host cell Vero
Select Vero cell proliferation adenovirus for use; Meanwhile, treat that the Cultivation of Vero of virus inoculation does not prepare cell antigen by the viral same operation step of results after converging into individual layer.The sensitive cells of results is by multigelation three times, with fragmentation; 5000rpm/min10 ℃ of centrifugal 12min, supernatant prepares anti-Vero cell antiserum(antisera) as Vero cell antigen immune animal.
The Vero cell antigen are mixed with the equivalent incomplete Freund's adjuvant, add the 9-11mg bacille Calmette-Guerin vaccine by every milliliter again, make milky suspension, be injected in after emulsification fully that to reach the back in the experimental group rabbit four-footed lift subcutaneous, each site 0.2ml; Inject 1 time with method after 2 weeks.In the 4th week beginning booster immunization, the Vero cell antigen that will not add adjuvant in promptly continuous three days are injected tame rabbit ear vein, and dosage increases progressively every day, is respectively 0.1ml, 0.2ml, 0.3ml.Last injection 1 week of back, heart blood sampling, separation of serum.
4 anti-human rotavirus's host cell MA
104Sero-fast preparation
Select MA for use
104The cell proliferation human rotavirus; Meanwhile, treat the not cultivation MA of virus inoculation
104Cell prepares cell antigen by the viral same operation step of results after converging into individual layer.The sensitive cells of results is by multigelation three times, with fragmentation; 5000rpm/min10 ℃ of centrifugal 12min, supernatant is as MA
104The cell antigen immune animal prepares anti-MA
104The cell antiserum(antisera).
With MA
104Cell antigen mix with the equivalent incomplete Freund's adjuvant, add the 9-11mg bacille Calmette-Guerin vaccine by every milliliter again, make milky suspension, are injected in after emulsification fully that to reach the back in the experimental group rabbit four-footed lift subcutaneous, each site 0.2ml; Inject 1 time with method after 2 weeks.In the 4th week beginning booster immunization, will not add the MA of adjuvant in promptly continuous three days
104Cell antigen are injected tame rabbit ear vein, and dosage increases progressively every day, is respectively 0.1ml, 0.2ml, 0.3ml.Last injection 1 week of back, heart blood sampling, separation of serum.
The sero-fast preparation of 5 anti-human cytomegalic inclusion disease virus host cell HEL
Select hel cell proliferation of human cytomegalovirus for use; Meanwhile, treat that the cultivation hel cell of virus inoculation does not prepare cell antigen by the viral same operation step of results after converging into individual layer.The hel cell of results is by multigelation three times, with fragmentation; 5000rpm/min10 ℃ of centrifugal 12min, supernatant prepares anti-hel cell antiserum(antisera) as the hel cell antigen-immunized animal.
Hel cell antigen is mixed with the equivalent incomplete Freund's adjuvant, add the 9-11mg bacille Calmette-Guerin vaccine by every milliliter again, make milky suspension, be injected in after emulsification fully that to reach the back in the experimental group rabbit four-footed lift subcutaneous, each site 0.2ml; Inject 1 time with method after 2 weeks.In the 4th week beginning booster immunization, the hel cell antigen that will not add adjuvant in promptly continuous three days is injected tame rabbit ear vein, and dosage increases progressively every day, is respectively 0.1ml, 0.2ml, 0.3ml.Last injection 1 week of back, heart blood sampling, separation of serum.
The sero-fast preparation of 6 anti-Coxsackie B virus host cell Vero
Select Vero cell proliferation Coxsackie B virus for use; Meanwhile, treat that the Cultivation of Vero of virus inoculation does not prepare cell antigen by the viral same operation step of results after converging into individual layer.The sensitive cells of results is by multigelation three times, with fragmentation; 5000rpm/min10 ℃ of centrifugal 12min, supernatant prepares anti-Vero cell antiserum(antisera) as Vero cell antigen immune animal.
The Vero cell antigen are mixed with the equivalent incomplete Freund's adjuvant, add the 9-11mg bacille Calmette-Guerin vaccine by every milliliter again, make milky suspension, be injected in after emulsification fully that to reach the back in the experimental group rabbit four-footed lift subcutaneous, each site 0.2ml; Inject 1 time with method after 2 weeks.In the 4th week beginning booster immunization, the Vero cell antigen that will not add adjuvant in promptly continuous three days are injected tame rabbit ear vein, and dosage increases progressively every day, is respectively 0.1ml, 0.2ml, 0.3ml.Last injection 1 week of back, heart blood sampling, separation of serum.
Embodiment 2 contains the preparation of total antibody of anti-host cell antibody
1 contains the preparation of total antibody of anti-blue tongue rims host cell VERO antibody
Extract in antiserum(antisera) and the first anti-Vero cell antibody of purifying with the saturated ammonium sulphate method, ultraviolet spectrophotometer is surveyed its A
280Value, thus draw the concentration of IgG, use 0.1M NaHCO again
3/ 0.5M NaCl solution is diluted to 5mg/ml with it, and-20 ℃ of preservations are standby.
To hatch 2h or 4 ℃ altogether through the antibody of preliminary purification and 37 ℃ of the sepharose 4Bs that has SP according to condition given on the product description spends the night, next day 1000rpm/min, centrifugal 10min, gently after the washing for several times, again the immunoglobulin IgG in the coupling is eluted and collects, realize total purifying antibody.
2 contain the preparation of total antibody of anti-herpes simplex I C-type virus C or II C-type virus C host cell Vero antibody
Extract in antiserum(antisera) and the first anti-Vero cell antibody of purifying with the saturated ammonium sulphate method, ultraviolet spectrophotometer is surveyed its A
280Value, thus draw the concentration of IgG, use 0.1M NaHCO again
3/ 0.5M NaCl solution is diluted to 5mg/ml with it, and-20 ℃ of preservations are standby.
To hatch 2h or 4 ℃ altogether through the antibody of preliminary purification and 37 ℃ of the sepharose 4Bs that has SP according to condition given on the product description spends the night, next day 1000rpm/min, centrifugal 10min, gently after the washing for several times, again the immunoglobulin IgG in the coupling is eluted and collects, realize total purifying antibody.
3 contain the preparation of total antibody of anti-adenovirus host cell Vero antibody
Extract in antiserum(antisera) and the first anti-Vero cell antibody of purifying with the saturated ammonium sulphate method, ultraviolet spectrophotometer is surveyed its A
280Value, thus draw the concentration of IgG, use 0.1M NaHCO again
3/ 0.5M NaCl solution is diluted to 5mg/ml with it, and-20 ℃ of preservations are standby.
To hatch 2h or 4 ℃ altogether through the antibody of preliminary purification and 37 ℃ of the sepharose 4Bs that has SP according to condition given on the product description spends the night, next day 1000rpm/min, centrifugal 10min, gently after the washing for several times, again the immunoglobulin IgG in the coupling is eluted and collects, realize total purifying antibody.
4 contain anti-human rotavirus's host cell MA
104The preparation of total antibody of antibody
In antiserum(antisera), extract and the first anti-MA of purifying with the saturated ammonium sulphate method
101Cell antibody, ultraviolet spectrophotometer are surveyed its A
280Value, thus draw the concentration of IgG, use 0.1M NaHCO again
3/ 0.5M NaCl solution is diluted to 5mg/ml with it, and-20 ℃ of preservations are standby.
To hatch 2h or 4 ℃ altogether through the antibody of preliminary purification and 37 ℃ of the sepharose 4Bs that has SP according to condition given on the product description spends the night, next day 1000rpm/min, centrifugal 10min, gently after the washing for several times, again the immunoglobulin IgG in the coupling is eluted and collects, realize total purifying antibody.
5 contain the preparation of total antibody of anti-human cytomegalic inclusion disease virus host cell HEL antibody
Extract in antiserum(antisera) and the first anti-hel cell antibody of purifying with the saturated ammonium sulphate method, ultraviolet spectrophotometer is surveyed its A
280Value, thus draw the concentration of IgG, use 0.1M NaHCO again
3/ 0.5M NaCl solution is diluted to 5mg/ml with it, and-20 ℃ of preservations are standby.
To hatch 2h or 4 ℃ altogether through the antibody of preliminary purification and 37 ℃ of the sepharose 4Bs that has SP according to condition given on the product description spends the night, next day 1000rpm/min, centrifugal 10min, gently after the washing for several times, again the immunoglobulin IgG in the coupling is eluted and collects, realize total purifying antibody.
6 contain the preparation of total antibody of anti-Coxsackie B virus host cell Vero antibody
Extract in antiserum(antisera) and the first anti-Vero cell antibody of purifying with the saturated ammonium sulphate method, ultraviolet spectrophotometer is surveyed its A
280Value, thus draw the concentration of IgG, use 0.1MNaHCO again
3/ 0.5M NaCl solution is diluted to 5mg/ml with it, and-20 ℃ of preservations are standby.
To hatch 2h or 4 ℃ altogether through the antibody of preliminary purification and 37 ℃ of the sepharose 4Bs that has SP according to condition given on the product description spends the night, next day 1000rpm/min, centrifugal 10min, gently after the washing for several times, again the immunoglobulin IgG in the coupling is eluted and collects, realize total purifying antibody.
The first purification of embodiment 3 live viruses
The first purification of 1 blue tongue rims
The virus of breeding on the results Vero cell, smudge cells, 11000rpm/min, 4 ℃ of centrifugal 10min; Get supernatant, mix by 1: 2 volume ratio with antibody purified, 4 ℃, 20 rev/mins of shaken overnight fully combine antigen and antibody and form Vero cellular immunization mixture; Next day, with 12000rpm/min, 4 ℃ of centrifugal 15min discarded the immunocomplex precipitation with mixture, and supernatant is the blue tongue rims particle suspension of just purifying from the Vero cell culture.
The first purification of 2I herpes simplex virus type and II herpes simplex virus type
The virus of breeding on the results Vero cell, smudge cells, 11000rpm/min, 4 ℃ of centrifugal 10min; Get supernatant, mix by 1: 2 volume ratio with antibody purified, 4 ℃ of 20 rev/mins of shaken overnight fully combine antigen and antibody and form Vero cellular immunization mixture; Next day, with 12000rpm/min, 4 ℃ of centrifugal 15min discarded the immunocomplex precipitation with mixture, and supernatant is the I herpes simplex virus type particle suspension or the II herpes simplex virus type particle suspension of just purifying from the Vero cell culture.
The first purification of 3 adenovirus
The virus of breeding on the results Vero cell, smudge cells, 11000rpm/min, 4 ℃ of centrifugal 10min; Get supernatant, mix by 1: 2 volume ratio with antibody purified, 4 ℃ of 20 rev/mins of shaken overnight fully combine antigen and antibody and form Vero cellular immunization mixture; Next day, with 12000rpm/min, 4 ℃ of centrifugal 15min discarded the immunocomplex precipitation with mixture, and supernatant is the adenovirion suspension of just purifying from the Vero cell culture.
4 human rotaviruss' first purification
Results MA
104The virus of breeding on the cell, smudge cells, 11000rpm/min, 4 ℃ of centrifugal 10min; Get supernatant, mix by 1: 2 volume ratio with antibody purified, 4 ℃ of 20 rev/mins of shaken overnight fully combine antigen and antibody and form MA
104The cellular immunization mixture; Next day, with 12000rpm/min, 4 ℃ of centrifugal 15min discarded the immunocomplex precipitation with mixture, and supernatant is from MA
101Human rotavirus's particle suspension of just purifying in the cell culture.
The first purification of 5 human cytomegalic inclusion disease viruss
The virus of breeding on the results hel cell, smudge cells, 11000rpm/min, 4 ℃ of centrifugal 10min get supernatant; Mix by 1: 2 volume ratio with antibody purified, 4 ℃ of 20 rev/mins of shaken overnight fully combine antigen and antibody and form the hel cell immunocomplex; Next day, with 12000rpm/min, 4 ℃ of centrifugal 15min discarded the immunocomplex precipitation with mixture, and supernatant is the human cytomegalic inclusion disease virus particle suspension of just purifying from the hel cell culture.
The first purification of 6 Coxsackie B virus
The virus of breeding on the results Vero cell, smudge cells, 11000rpm/min, 4 ℃ of centrifugal 10min get supernatant, mix by 1: 2 volume ratio with antibody purified, 4 ℃ of 20 rev/mins of shaken overnight fully combine antigen and antibody and form Vero cellular immunization mixture; Next day, with 12000rpm/min, 4 ℃ of centrifugal 15min discarded the immunocomplex precipitation with mixture, and supernatant is the Coxsackie B virus particle suspension of just purifying from the Vero cell culture.
The high purifying of embodiment 4 live viruses
The high purifying of 1 blue tongue rims
With the blue tongue rims supernatant liquor after just purifying and the sepharose 4B that has SP in 4 ℃ of overnight incubation, next day 1000rpm/min, centrifugal 10min collects supernatant, is the blue tongue rims of purifying.
The high purifying of 2I herpes simplex virus type and II herpes simplex virus type
With the I herpes simplex virus type after just purifying or II herpes simplex virus type supernatant liquor with have the sepharose 4B of SP in 4 ℃ of overnight incubation, next day 1000rpm/min, centrifugal 10min collects supernatant, is the I herpes simplex virus type or the II herpes simplex virus type of purifying.
The high purifying of 3 adenovirus
With the adenovirus supernatant liquor after just purifying and the sepharose 4B that has SP in 4 ℃ of overnight incubation, next day 1000rpm/min, centrifugal 10min collects supernatant, is the adenovirus of purifying.
4 human rotaviruss' high purifying
With the human rotavirus's supernatant liquor after just purifying and the sepharose 4B that has SP in 4 ℃ of overnight incubation, next day 1000rpm/min, centrifugal 10min collects supernatant, is the human rotavirus of purifying.
The high purifying of 5 human cytomegalic inclusion disease viruss
With the human cytomegalic inclusion disease virus supernatant liquor after just purifying and the sepharose 4B that has SP in 4 ℃ of overnight incubation, next day 1000rpm/min, centrifugal 10min collects supernatant, is the human cytomegalic inclusion disease virus of purifying.
The high purifying of 6 Coxsackie B virus
With Coxsackie B virus supernatant liquor of just purifying and the sepharose 4B that has SP in 4 ℃ of overnight incubation, next day 1000rpm/min, centrifugal 10min collects supernatant, is the Coxsackie B virus of purifying.
The characteristic of virus behind embodiment 5 purifying
1) the viral purity after purifying is analyzed in the SephadexG-200 gel-filtration
Collect elutriant, survey its A
280Value is made ultraviolet absorption curve figure (Fig. 1), discloses: a chromatographic peak only appears in the viral suspension behind the purifying.This result has proved through reverse co-immunoprecipitation method and has handled that impurity component is removed fully from virus particle, and it is highly purified that virus is obtained.
2) viral purity and integrity after the transmission electron microscope observing analysis is purified
To make negative staining, transmission electron microscope observing and take the photograph sheet (Fig. 2) through the viral suspension before and after reverse co-immunoprecipitation is purified.The result proves: the viral suspension background impurities before the purifying is a lot, and complete virus particle is high-visible; By contrast, the viral suspension background behind the purifying is clean, inclusion-free, complete, the clear in structure of virus particle.
3) TCID
50The biologic activity of virus behind the analysis of experiments purifying
Blue tongue rims after purified are to the TCID of Vero cell
50For: 10
-5.7/ ml; Blue tongue rims are to the TCID of Vero cell before the purifying
50For: 10
-6.6/ ml (Fig. 3).
Claims (1)
1, a kind of method of the live virus of purifying, this method comprises the following steps:
A) sero-fast preparation: mixes with the equivalent incomplete Freund's adjuvant as antigen with the broken composition of the host cell of cultivating, press per 1 milliliter of said mixture adding 9-11mg bacille Calmette-Guerin vaccine again, make milky suspension, immune animal prepares the antiserum(antisera) of anti-host cell;
B) the first purifying of antibody: extract in antiserum(antisera) with the saturated ammonium sulphate method and to contain other antibody and proteic antibody purification just, and use 0.1M NaHCO
3/ 0.5M NaCl solution is diluted to first antibody purification the solution of 4-6mg/ml;
C) preparation of the total antibody of high purity: first antibody purification and the sepharose 4B that has SP are hatched for 37 ℃ altogether, 900-1100rpm/min, centrifugal 10-12min is after the washing, again the immunoglobulin IgG in the coupling is eluted and collects, realize total purifying antibody;
D) Bing Du first purifying: with the broken composition system of the host cell 11000-14000rpm/min of propagation, 4 ℃ of centrifugal 10-15min get supernatant and mixed in by volume 1: 2 with anti-host cell antibody, and 4 ℃, 20-25 rev/min of shaken overnight; With 10000-13000rpm/min, 4 ℃ of centrifugal 13-16min abandon cellular immunization mixture precipitation, collect supernatant liquor with mixture;
E) Bing Du purifying: with supernatant liquor and the sepharose 4B that has SP in 4 ℃ of overnight incubation, absorption antibody, 900-1100rpm/min, centrifugal 10-12min asks in collections, is the virus of purifying.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410012605 CN1260354C (en) | 2004-01-02 | 2004-01-02 | Method of purifying active virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410012605 CN1260354C (en) | 2004-01-02 | 2004-01-02 | Method of purifying active virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1556205A true CN1556205A (en) | 2004-12-22 |
| CN1260354C CN1260354C (en) | 2006-06-21 |
Family
ID=34350979
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410012605 Expired - Fee Related CN1260354C (en) | 2004-01-02 | 2004-01-02 | Method of purifying active virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1260354C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102350000A (en) * | 2011-10-24 | 2012-02-15 | 陈冬娥 | Application of heterogeneous bluetongue virus dsRNA (double-strand ribonucleic acid) in preparation of endogenic interferon inducer |
| CN102350001A (en) * | 2011-10-24 | 2012-02-15 | 武汉大学 | Preparation method of heterologous bluetongue virus double-strand RNA (ribonucleic acid) endogenous interferon inducer |
-
2004
- 2004-01-02 CN CN 200410012605 patent/CN1260354C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102350000A (en) * | 2011-10-24 | 2012-02-15 | 陈冬娥 | Application of heterogeneous bluetongue virus dsRNA (double-strand ribonucleic acid) in preparation of endogenic interferon inducer |
| CN102350001A (en) * | 2011-10-24 | 2012-02-15 | 武汉大学 | Preparation method of heterologous bluetongue virus double-strand RNA (ribonucleic acid) endogenous interferon inducer |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1260354C (en) | 2006-06-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1313152C (en) | Vaccines | |
| CN1065997A (en) | Vaccine | |
| CN1579553A (en) | Method for preparing II-type pig's ring-virus nucleic vaccine and the use thereof | |
| CN1816619A (en) | Method for purifying virus | |
| CN1468256A (en) | Purification of hbv antigens for use in vaccines | |
| CN1260354C (en) | Method of purifying active virus | |
| CN1759177A (en) | Adenoviral serotype 34 carriers, nucleic acid and consequent virus thereof | |
| CN1498963A (en) | Method for producing virus like granules of human papilloma virus | |
| CN1095951A (en) | Poly I: C compound immunologic adjuvant and contain the vaccine of this adjuvant | |
| CN1943789A (en) | The DNA vaccine of an anti-infection of hepatitis C virus | |
| CN1645145A (en) | Method and reagent box for inspecting mycelian protein antibody of white candida | |
| CN86100441A (en) | In vitro differential diagnosis method for EBV virus related diseases | |
| CN1301749C (en) | Polyepitope DNA vaccine of anti-simple herpes virus-2 infection and its preparing method | |
| CN1162182C (en) | Method of protein production using mitochondrial translation system | |
| CN1059927C (en) | Reconstituted hepatitis B vaccine with corboxy end having anterior surface antigen 1 determinant | |
| CN86106762A (en) | Novel dna and polypeptide | |
| CN1556216A (en) | Detecting method of blue tongue virus and reagent box | |
| CN1238500C (en) | Immunology epitope of hog cholera virus feature and application thereof | |
| CN1736482A (en) | Recombined chicken pox virus vaccine rFPV-12LSH5H9, its construction method and use | |
| CN1922201A (en) | A process for the preparation and purification of recombinant proteins | |
| CN1598581A (en) | Method for testing SARS virus antigen | |
| CN112725370B (en) | PRRSV ORF5 fusion gene DNA vaccine and preparation method thereof | |
| CN1192038C (en) | Monoclonal antibodies against HIV-1 and vaccines made thereof | |
| CN1583171A (en) | Preparation of immuno-stimulation composition for hemolycin in monad | |
| CN1566144A (en) | Functional protein in SARS coronavirus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |