CN103898066A - Vero (Rabies Purified Vaccine for Human Use) cell cold-adapted strain of influenza A virus and application thereof - Google Patents
Vero (Rabies Purified Vaccine for Human Use) cell cold-adapted strain of influenza A virus and application thereof Download PDFInfo
- Publication number
- CN103898066A CN103898066A CN201410039468.1A CN201410039468A CN103898066A CN 103898066 A CN103898066 A CN 103898066A CN 201410039468 A CN201410039468 A CN 201410039468A CN 103898066 A CN103898066 A CN 103898066A
- Authority
- CN
- China
- Prior art keywords
- virus
- influenza
- vero cell
- strain
- cell
- 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
The invention provides a Vero cell cold-adapted strain of influenza A virus and an application thereof. The Vero cell cold-adapted strain is named as A/Yunnan/1/2005Vca (H3N2), the microbial preservation number is CCTCC NO. V201253. The strain can be subjected to a continuous passage culture on a Vero cell, the hemagglutination titer is maintained at 1:512-1:1024, and the infectious titer is maintained at 7.0-8.5log10TCID50/ml, and has phenotypes of Ca and Ts and characteristics of Att by identification. The strain as a female parent strain and an epidemic strain is subjected to a genetic reassortment, or six internal genes of the strain and two surface protein genes of the epidemic strain are subjected to a gene reassortment by a reverse genetics technology to obtain the Vero cell cold-adapted strain containing surface antigens of the epidemic strain. The Vero cell cold-adapted strain can be used in the preparation of Vero cell live-attenuated influenza vaccines or live-attenuated influenza vaccines, and can be also used for the production of inactivated influenza vaccines.
Description
Technical field
The present invention relates to a kind of influenza A virus Vero cell acclimatization to cold strain, and relate to using this adapted strain as maternal strain and other epidemic isolates carry out reprovision, thereby obtain the Vero cell acclimatization to cold strain that contains epidemic strain surface antigen, using the seed culture of viruses as preparation Vero cell Gripovax or Gripovax and vaccinum influenzae inactivatum, belong to biological product technical field.
Background technology
Influenza virus belongs to orthomyxoviridae family, and its virion is spherical in shape or thread, and nucleocapsid is helical symmetry, and viral genome is the segmented RNA of sub-thread minus strand, has coating.According to the antigenicity of virus nucleoprotein (NP) and stromatin (MP), influenza virus can be divided into first (A), second (B), third (C) three types, wherein the gene of A type and Influenza B virus contains 8 RNA fragments, and the gene of influenza virus C contains 7 fragments; Influenza A virus again can be different with neuraminidase (NA) antigenicity according to influenza virus hemagglutinin (HA), are further divided into some hypotypes.Influenza A virus is often with popular form appearance, and second type influenza virus is local outburst, and influenza C is mainly that the form of distributing occurs.Wherein A type and Influenza B virus and the mankind have close ties, and influenza A virus, except infecting people, also can infect the animals such as birds, pig, horse.And people contacts frequently with these animals in life, for producing variation, influenza virus provides favourable condition, and the virus after variation can cause influenza pandemic, and degree of variation greatly causes being very popular.
The variation of influenza virus surface antigen is the major cause of influenza pandemic, and the variation of its surface antigen exists two kinds of mechanism: one is called as antigenic drift, and another kind of variation is called as the large variation of antigen.Antigenic drift all can occur on influenza A virus and Influenza B virus, mainly by the point mutation on HA and NA gene and multiple amino acids selective mutation, escape from the immunization of body and infected in the past the antibody obtaining after virus to viral restraining effect, antigenic drift can produce new epidemic strain, also be that influenza virus can infect the mankind simultaneously, and continue the reason of evolving.The large variation of antigen occurs over just on influenza A virus, and when host cell is during simultaneously by the influenza a virus infection of two kinds of different subtypes, newborn progeny virus can obtain the gene segment from two parental viruss, becomes gene resortment virus.Gene resortment produces influenza A virus antigen mutant strain, if this mutant strain is propagated in the Susceptible population of immunoprophylaxis vaccine not, will cause flu outbreak.
In crowd, pandemic influenza A virus has H
1n
1, H
2n
2and H
3n
2three hypotypes.16 kinds of HA of influenza A virus and the hypotype of 9 kinds of NA compositions are also extensively present in some wildlifes, in aquatic bird.If virus is not passed through genetic resortment, bird flu hypotype general tree people produces effective infection, but the virus of this rapid diffusion is because regular gene breaks up again, the PI mankind.For example, since Hong Kong outburst bird flu in 1997, H
5n
1hypotype, through genetic modification, except causing large-scale outbreak in bird, has also produced the event that many cases people infects., there is infecting people's new variant in the variation of finding this virus strain in 2003.According to the data of Agence France-Presse, H
5n
1avian influenza virus amounts to lethal 300 people that exceed in the whole world between 2003 to 2011, cause about 400,000,000 poultry to be slaughtered, and causes about 20,000,000,000 dollars of losses.
Inoculation influenza vaccines are prevention and the effective means that reduces influenza severity, comprise inactivated vaccine and attenuated live vaccine.Nineteen forty-one, the vaccinum influenzae inactivatum prepared of chicken embryo got the Green Light first in the U.S., was used widely in the whole world.What use at present is mainly deactivation influenza three split vaccines of preparing with chicken embryo, comprises influenza A virus H
1n
1strain, H
3n
2strain and Influenza B virus strain.The component of vaccine strain is to be recommended according to the Changing Pattern of annual global influenza virus by WHO, determines with the surface antigen of influenza virus epidemic strain for production of vaccine next year.Influenza A virus association laboratory utilizes reverse Genetics Technique, adopt six internal gene of chicken embryo high yield strain and the HA of wild epidemic strain and NA gene resortment (6+2 reprovision) to obtain, the novel vaccine strain of the HA that contains influenza virus epidemic strain and NA surface glycoprotein, has the characteristic of high yield on chicken embryo.
Gripovax, because possessing collunarium approach inoculation, provides mucosal immunity and cellular immunization, and the feature such as immunoprotection is more lasting and be better than inactivated vaccine.Only have at present the U.S. and Russia to produce Gripovax, its vaccine seed is criticized the influenza virus acclimatization to cold strain that all derives from chicken embryo high yield, is A/Ann Arbor/6/60 (H in the U.S.
2n
2) and B/Ann Arbor/1/66, be A/Leningrad/134/17/57 (H in Russia
2n
2) and B/USSR/60/69.But, have the following disadvantages as culture medium using chicken embryo: (1) may cause chicken transmissible disease because of vaccination wave dispersion because of potentially contaminated; (2) often morph because of continuous passage, cause vaccine not mate with epidemic isolates in crowd, vaccine potency is reduced; (3) long because of culture cycle, be difficult to scale operation and go out to meet the sudden demand of flu outbreak; (4) purge process complexity; (5) easily cause allergic reaction.Especially high pathogenic avian influenza popular in worldwide since 2003, making to adopt chicken embryo to produce influenza virus vaccine risk increases.Therefore, work out vaccine safely and effectively, become influenza vaccines important research direction, be not subject to the restriction of chicken embryo source especially will tackle flu outbreak time, World Health Organization's suggestion substitutes chicken embryo with mammaliancellculture influenza virus and prepares influenza vaccines.
At present, be mainly Madin-Darby canine kidney(cell line) (MDCK) (MDCK) and African green monkey kidney cell (Vero) for the preparation of the mammalian cell of influenza vaccines.Although mdck cell is to cultivate and the good mammal cell line of separated flow Influenza Virus, there is potential tumorigenicity in it, makes it be applied to production of vaccine and have no end of misgivings.African green monkey kidney cell (Vero) is the continuous cell line that vaccine for man is produced that can be used for of World Health Organization's approval, its genetic background is comparatively clear, without potential tumorigenicity, safe and reliable, and can utilize bio-reactor to carry out large scale culturing, feed rate is large in a short time.Vero cell has been widely used in the production of the vaccines such as mad dog, encephalitis, poliomyelitis, hemorrhagic fever with renal syndrome at present.But Vero cell is not the responsive culture medium of influenza virus, the lower and most virus of output can not be on Vero cell propagation continuously.We study Vero cell influenza for many years, now successfully filter out a strain influenza A virus Vero cell high yield adapted strain, can make body produce relatively strong humoral immunization effect and cytotoxic T lymphocyte reaction.On this basis, how further to select influenza virus Vero cell acclimatization to cold strain, can 25 DEG C also can massive duplication and high yield continuously, become a difficult problem for Vero cell Gripovax research and development.
Reverse genetics, for classical genetics, to obtain on the basis of organism genome full sequence, by target gene is carried out to necessary processing and modification, as rite-directed mutagenesis, gene insertion/deletion and gene substitution etc., the modifying factor group containing the essential element of organism by composition sequential build again, allow it assemble out the individuality with vital activity, the genomic structure of postgraduate's object and function, and these modifications may have on the phenotype of organism, proterties the content of the aspects such as which kind of impact.Reverse genetics has been applied to influenza virus across kind mechanism of transmission, virus virulence and Study on Pathogenicity thereof and vaccine research and development at present.Be applied to vaccine research and development, i.e. 6+2 reprovision, for example, by addition reprovision of the HA of six internal gene (PB2, PB1, PA, NP, M, NS) of chicken embryo high yield strain and current popular strain and NA, the chicken embryo high yield strain of acquisition epidemic strain, in order to produce vaccine.Consuming time loaded down with trivial details with respect to conventional flow influenza vaccine method of production, and require enough chicken embryo supplies, reverse genetics can make lead time shortening, the work simplification of vaccine.Therefore reverse genetics is an important means of reply flu outbreak.
In sum, influenza is still the Pandemic infection disease that the mankind still can not effectively control, and inoculation influenza vaccines are Main Means of flu-prevention, and in influenza vaccines, attenuated live vaccine is better than again inactivated vaccine.Use at present the matrix of chicken embryo as preparation influenza vaccines, have a lot of weak points, become the important directions of influenza vaccines development and use African green monkey kidney cell (Vero cell) to prepare influenza vaccines.How to select influenza virus Vero cell acclimatization to cold strain, make its not only 33 DEG C and also 25 DEG C also can massive duplication and high yield continuously, become a difficult problem for Vero cell Gripovax research and development.Because under normal circumstances, 25 DEG C is the minimum temperature that Vero cell maintains normal growth, and at this temperature, the metabolism of cell is all tending towards slowly, therefore can affect influenza virus and copy intracellular, and copying of influenza virus needs cell to provide support.
At present influenza vaccines used to produce seed culture of viruses be all to join adapted strain with chicken embryo high yield strain reprovision or natural chicken, it is generally insensitive on Vero cell.Home and overseas has influenza vaccines that producer directly provides WHO to produce seed culture of viruses inoculation Vero cell to produce that poison amount is low or output is unstable, restricted and utilized Vero cell cultures influenza virus to prepare the application of influenza vaccines.
Summary of the invention
One of object of the present invention is to provide a kind of new influenza A virus Vero cell acclimatization to cold strain, and on Vero cell 25 DEG C can continuous passage and keep stablizing high yield, this influenza A virus Vero cell acclimatization to cold strain has
ca, Tsphenotype and
attcharacteristic.
Influenza A virus Vero cell acclimatization to cold strain provided by the invention, called after A/Yunnan/1/2005Vca (H
3n
2), its preserving number is: CCTCC NO. V201253, and depositary institution is: Chinese Typical Representative culture collection center, preservation address is: Wuhan City, Wuhan University; The preservation time is: on December 20th, 2012.
Two of object of the present invention is to provide described influenza A virus Vero cell acclimatization to cold strain A/Yunnan/1/2005Vca (H
3n
2) preparation method.
Preserving number provided by the invention is the influenza A virus Vero cell acclimatization to cold strain A/Yunnan/1/2005Vca (H of CCTCC NO. V201253
3n
2) obtain by following method:
A. on the Vero cell that grows up to fine and close individual layer for 24 hours, the blood clotting titre of inoculation 1MOI reaches the influenza A virus A/kunming/1/2005Va(H3N2 of 1:1024);
B. be MEM or DMEM/F12 maintaining mother liquor composition, penicillin-Streptomycin sulphate 20,000 U/ml, glutamine solution 0.3-0.6mg/ml, bovine serum albumin 1mg/ml, TPCK-pancreas enzyme concentration 0.5-1.5ug/ml, pH7.0-7.2, under 30 ± 1 DEG C of conditions, the influenza A virus of culturing step A, after 72 hours, is gathered in the crops virus liquid;
C. the virus liquid multigelation of step B being gathered in the crops 3 times, inoculate on the Vero cell that grows up to fine and close individual layer for 24 hours, then repeating step B and C, so on Vero cell, passed continuously for 20 generations, and the blood clotting titre that maintains kind of poison cell harvest liquid is at 1:1024, influenza A virus must go down to posterity;
D. on the Vero cell that grows up to fine and close individual layer for 24 hours, the influenza A virus that goes down to posterity of inoculation 1MOI step C;
E. be MEM or DMEM/F12 maintaining mother liquor composition, penicillin-Streptomycin sulphate 20,000 U/ml, glutamine solution 0.3-0.6mg/ml, bovine serum albumin 1mg/ml, TPCK-pancreas enzyme concentration 0.5-1.5ug/ml, pH7.0-7.2, under 25 ± 1 DEG C of conditions, culturing step D's went down to posterity after influenza A virus 120-168 hour, results virus liquid;
F. the virus liquid multigelation of step e being gathered in the crops 3 times, inoculate on the Vero cell that grows up to fine and close individual layer for 24 hours, repeating step E and F again, so on Vero cell, passed continuously for 72 generations, and the blood clotting titre that maintains kind of poison cell harvest liquid is at 1:1024, obtains influenza A virus Vero cell acclimatization to cold strain.
The influenza A virus A/kunming/1/2005Va(H3N2 of described steps A) be existing influenza A virus Vero cell high yield adapted strain, its preserving number is: CCTCC NO.V200514, the patent No. is ZL 2,009 1 0094223.8.
Preserving number provided by the invention is the influenza A virus Vero cell acclimatization to cold strain A/Yunnan/1/2005Vca (H of CCTCC NO. V201253
3n
2) essential characteristic: (a) transmission electron microscope observing is about 80-120nm to the influenza virus diameter of gained, has the comparatively typical influenza virus form of cyst membrane; (b) gene sequencing shows in continuous passage culturing process, is difficult for producing influenza virus gene variation; (c) influenza virus continuous passage on 25 DEG C of Vero cells is cultivated, and blood clotting titre can remain on 1:1024; (d) HI experiment shows that viral HA antigen can be by goat-anti or the anti-H3 subtype influenza virus of other animal hyper-immune serum specific recognition; (e) A/Yunnan/1/2005Va(H3N2) live virus is after natural way infects SPF chicken, and infected chicken is without morbidity and dead.
Three of object of the present invention is to provide the influenza A virus Vero cell acclimatization to cold strain A/Yunnan/1/2005Vca (H that preserving number is CCTCC NO. V201253
3n
2) application, using influenza A virus Vero cell acclimatization to cold strain as maternal strain and other epidemic isolates carry out reprovision, thereby obtain the Vero cell acclimatization to cold strain of epidemic strain, specifically by following two kinds of methods realization:
First method: obtain the Vero cell acclimatization to cold strain of influenza virus epidemic strain by conventional reprovision technology:
A1. with the volume ratio of 1:100-100:1, be the influenza A virus Vero cell acclimatization to cold strain A/Yunnan/1/2005Vca (H of CCTCC NO. V201253 by preserving number
3n
2) mix with other subtype influenza viruses, obtaining hybrid virus, concrete ratio is determined by viral hemoagglutination titre and different strain growth property;
A2. on 9-10 age in days SPF chicken embryo, by the amount of every embryonic breeding kind 0.1-0.3ml, the hybrid virus of inoculation step A1, hatches 12-48 hour in 33 ± 1 DEG C, and the 2-8 DEG C of cold embryo that spends the night, collects chick embryo allantoic liquid, obtains virus liquid;
Or on fresh Vero cell, by 0.004-0.008ml/cm
2the amount of Vero cell, the hybrid virus of inoculation step A1, is placed in 33 ± 1 DEG C and cultivates 48-72 hour, obtains virus liquid;
Or on fresh mdck cell, by 0.004-0.008ml/cm
2the amount of mdck cell, the hybrid virus of inoculation step A1, is placed in 33 ± 1 DEG C and cultivates 48-72 hour, obtains virus liquid;
A3. anti-A/Yunnan/1/2005Vca (H will be added in the virus liquid of steps A 2
3n
2) antiserum(antisera) to antiserum(antisera) concentration be 1-80%(volume ratio), concrete concentration is 1:10240-1:2560 depending on antibody titer, and 25 DEG C of screenings of going down to posterity on Vero cell, thereby selects the Vero cell acclimatization to cold strain of epidemic strain;
Second method: obtain the Vero cell acclimatization to cold strain of influenza virus epidemic strain with existing reverse Genetics Technique reprovision:
B1. be the influenza A virus Vero cell acclimatization to cold strain A/Yunnan/1/2005Vca (H of CCTCC NO. V201253 by preserving number
3n
2) 6 internal gene, i.e. PB2, PB1, PA, NP, M, NS, is cloned in respectively on upper or other bidirectional transcription vector of bidirectional transcription vector pHW2000 6 internal gene plasmids of Vero cell acclimatization to cold strain of DCRP; By 2 surface protein gene HA of epidemic strain and NA, be also cloned on this bidirectional transcription vector, obtain 2 surface protein gene plasmids of epidemic strain;
B2. after 6 internal gene plasmids of Vero cell acclimatization to cold strain of step B1 institute DCRP being mixed with 2 surface protein gene plasmids of epidemic strain, on cotransfection mammalian cell 297T, Cos1, Vero or mdck cell, cultivate 48-72 hour, harvested cell liquid for 37 ± 1 DEG C;
B3. on 9-10 age in days SPF chicken embryo, by the amount of every embryonic breeding kind 0.1-0.2ml, the harvested cell liquid of inoculation step B2, cultivates 96-120 hour for 25 ± 1 DEG C, and results virus liquid, obtains the Vero cell acclimatization to cold strain of epidemic strain;
Or on the mdck cell that grows up to fine and close individual layer for 24 hours, by 0.04-0.08ml/cm
2the amount of mdck cell, the harvested cell liquid of inoculation step B2, is placed in 25 ± 1 DEG C and cultivates 120-168 hour, and results virus liquid, obtains the Vero cell acclimatization to cold strain of epidemic strain;
Or on the Vero cell that grows up to fine and close individual layer for 24 hours, by 0.04-0.08ml/cm
2the amount of Vero cell, the harvested cell liquid of inoculation step B2, is placed in 25 ± 1 DEG C and cultivates 120-168 hour, and results virus liquid, obtains the Vero cell acclimatization to cold strain of epidemic strain.
difficult point of the present invention is:
(1) influenza A virus Vero cell acclimatization to cold strain also can massive duplication and continuous high yield in the time cultivating for 25 DEG C.
(2) to influenza A virus A/Yunnan/1/2005 Vca (H
3n
2) the malicious culture condition of kind on Vero cell carried out the optimization of system.
(3) influenza A virus Vero cell acclimatization to cold strain also can massive duplication in the time that 25 DEG C of chicken embryos are cultivated and high yield continuously, has attenuation feature simultaneously.
(4) how to apply influenza A virus A/Yunnan/1/2005 Vca (H
3n
2) the hereditary and reverse genetics reprovision with other virus strain, and then acquisition Vero cell acclimatization to cold strain is studied.
the present invention compared with prior art has following advantages and effect:
(1) influenza A virus Vero cell acclimatization to cold strain provided by the invention, can adopt microcarrier fermentor tank to carry out large scale culturing Vero cell and influenza virus, to prepare Gripovax and Pandemic influenza vaccine.More existing influenza vaccines of producing as culture medium using chicken embryo have the following advantages:
can adopt microcarrier fermentor tank large scale culturing Vero cell and influenza virus, production technique is easy to stdn;
adopt standardized Vero cell, do not pollute containing exogenetic biotic factor;
not containing being easy to cause that human allergy's chicken embryo is white;
having avoided influenza virus viral hemagglutinin antigen after chicken embryo culture easily to change reduces vaccine potency;
in the time that flu outbreak is sudden, be not subject to the impact in chicken embryo source, can produce enough vaccines and satisfy the demands.
(2) the present invention is the influenza A virus Vero cell high yield adapted strain A/kunming/1/2005Va(H3N2 of CCTCC NO.V200514 at preserving number) on basis, adopt continuous gradient falling temperature method, passed continuously for 72 generations, successfully selecting preserving number is the influenza A virus Vero cell acclimatization to cold strain A/Yunnan/1/2005Vca (H of CCTCC NO. V201253
3n
2), it can cultivate on Vero cell in continuous passage, and blood clotting titre remains on 1:512-1:1024, and infection titer remains on 7.0-8.5log
10tCID50/ml, has through qualification
ca, Tsphenotype and
attcharacteristic.Set it as maternal plant and epidemic strain influenza virus reprovision, can obtain Vero cell influenza A virus vaccine production seed culture of viruses, greatly promoted exploitation and the production of Vero cell Gripovax and Vero cell Pandemic influenza vaccine.
Brief description of the drawings
Fig. 1 is A/Yunnan/1/2005Va (H
3n
2) at the continuous passage hemagglutinative titer figure of 30 DEG C.
Fig. 2 is A/Yunnan/1/2005Vca (H
3n
2) at 25 DEG C of continuous passage hemagglutinative titer figure.
Embodiment
Further describe by the following examples the present invention, it should be understood that these embodiment, only for the object of illustration, never limit the scope of the invention.
Preserving number is the influenza A virus Vero cell acclimatization to cold strain A/Yunnan/1/2005Vca (H of CCTCC NO. V201253
3n
2) preparation method:
1. on the Vero cell that grows up to fine and close individual layer for 24 hours, the preserving number that the blood clotting titre of inoculation 1MOI reaches 1:1024 is the influenza A virus A/kunming/1/2005Va(H3N2 of CCTCC NO.V200514);
2. be MEM maintaining mother liquor composition, penicillin-Streptomycin sulphate 20,000 U/ml, glutamine solution 0.6mg/ml, bovine serum albumin 1mg/ml, TPCK-pancreas enzyme concentration 1.0ug/ml, pH7.2, under 30 ± 1 DEG C of conditions, the influenza A virus of culturing step 1, after 72 hours, is gathered in the crops virus liquid;
3. the virus liquid multigelation of step 2 being gathered in the crops 3 times, inoculate on the Vero cell that grows up to fine and close individual layer for 24 hours, then repeating step 2 and 3, so on Vero cell, passed continuously for 20 generations, and the blood clotting titre that maintains kind of poison cell harvest liquid is at 1:1024, influenza A virus must go down to posterity;
4. on the Vero cell that grows up to fine and close individual layer for 24 hours, the influenza A virus that goes down to posterity of inoculation 1MOI step 3;
5. be MEM maintaining mother liquor composition, penicillin-Streptomycin sulphate 20,000 U/ml, glutamine solution 0.6mg/ml, bovine serum albumin 1mg/ml, TPCK-pancreas enzyme concentration 1.0ug/ml, pH7.2, under 25 ± 1 DEG C of conditions, culturing step 4 go down to posterity influenza A virus after 72 hours, results virus liquid;
6. the virus liquid multigelation of step 5 being gathered in the crops 3 times, inoculate on the Vero cell that grows up to fine and close individual layer for 24 hours, repeating step 5 and 6 again, so on Vero cell, passed continuously for 72 generations, and the blood clotting titre that maintains kind of poison cell harvest liquid is at 1:1024, obtaining preserving number is the influenza A virus Vero cell acclimatization to cold strain of CCTCC NO. V201253, called after A/Yunnan/1/2005Vca (H
3n
2).
This influenza A virus A/Yunnan/1/2005Vca (H
3n
2) correlation test data see Fig. 1-Fig. 2 and Biao 1-table 2, prove that this strain has on Vero cell the characteristic of 25 DEG C of propagation of efficient stable while cultivating.
Table 1, the influenza virus pathology time in temperature reduction process
Blood clotting has suppressed experimental verification through a large amount of A/Yunnan/1/2005Vca (H repeatedly going down to posterity at 25 DEG C
3n
2) there is not crossed contamination, on serotype, be the influenza virus of a strain H3N2 hypotype.
Table 2, the A/Yunnan/1/2005Vca (H in 25 DEG C of sources
3n
2) blood clotting suppress experimental result
The influenza A virus to 24 hour of the embodiment 1 of inoculation 1MOI grows up on the Vero cell of fine and close individual layer, maintaining mother liquor composition is MEM, penicillin-Streptomycin sulphate 20,000 U/ml, glutamine solution 0.3mg/ml, bovine serum albumin 1mg/ml, TPCK-pancreas enzyme concentration 1.0ug/ml, pH7.2, being placed in 25 ± 1 DEG C cultivates after 168h, results virus liquid, cell harvesting liquid multigelation 3 times, the virus liquid of results is inoculated on Vero cell again, method is the same, on 25 DEG C of Vero cells, so continuous passage is gone down, the blood clotting titre of planting poison cell harvest liquid can maintain 1:512-1:1024.
Influenza A virus Vero cell acclimatization to cold strain A/Yunnan/1/2005Vca (H
3n
2), have
ca, Tsphenotype and
attcharacteristic.
ca(cold adaptation) phenotype: acclimatization to cold phenotype, refers to that strain can effectively grow at 25 DEG C; 25 DEG C and 33 DEG C of cultivations, the difference of the two TCID50 is less than 100 times.
ts(temperature sensitive) phenotype: temperature sensitive phenotype, refers to that strain is at 39 DEG C of limiting growths; TCID50 under 39 DEG C of cultivations is lower than at least 100 times of the TCID50 of 33 DEG C.
att(attenuation) characteristic: attenuation characteristic, experimentation on animals, ferret is the most responsive animal.Strain copies limited and not pathogenic at ferret respiratory tract, have attenuation.Correlation test data, in Table 3-table 4, prove that this strain has
ca, Tsphenotype and
attcharacteristic.
Table 3, A/Yunnan/1/2005Vca (H
3n
2) have
tswith
caphenotype
Table 4, A/Yunnan/1/2005Vca (H
3n
2) ferret experimental result
embodiment 3
The influenza A virus to 24 hour of the embodiment 1 of inoculation 1MOI grows up to fine and close individual layer, on the Vero cell of serum-free culture, maintaining mother liquor composition is SFM(Hyclone), penicillin-Streptomycin sulphate 20,000 U/ml, glutamine solution 0.6mg/ml, bovine serum albumin 1mg/ml, TPCK-pancreas enzyme concentration 0.5ug/ml, pH7.0-7.2, being placed in 25 ± 1 DEG C cultivates after 120-168h, results virus liquid, cell harvesting liquid multigelation 3 times, the virus liquid of results is inoculated on Vero cell again, method is the same, on Vero cell, so continuous passage is gone down, the blood clotting titre of planting poison cell harvest liquid can maintain 1:512.
By the influenza A virus liquid of the embodiment of 1MOI 1, dilution is 10 respectively
-3with 10
-4be inoculated into 9-11 day instar chicken embryo, every embryonic breeding kind 0.2ml, be placed in 25 ± 1 DEG C and cultivate after 96-120h, results virus liquid, cell harvesting liquid multigelation 3 times, the virus liquid of results is inoculated on 9-11 day instar chicken embryo again, method is the same, and on chicken embryo, so continuous passage is gone down, and the blood clotting titre of planting poison cell harvest liquid can maintain 1:1024 ~ 1:2048.
embodiment 5
The influenza A virus to 24 hour of the embodiment 1 of inoculation 1MOI grows up on the mdck cell of fine and close individual layer, be DMEM/F12 maintaining mother liquor composition, penicillin-Streptomycin sulphate 20,000 U/ml, glutamine solution 0.3mg/ml, bovine serum albumin 1mg/ml, TPCK-pancreas enzyme concentration 1.0ug/ml, pH7.0-7.2, being placed in 25 ± 1 DEG C cultivates after 120-168h, results virus liquid, cell harvesting liquid multigelation 3 times, the virus liquid of results is inoculated on mdck cell again, method is the same, on mdck cell, so continuous passage is gone down, the blood clotting titre of planting poison cell harvest liquid can maintain 1:512 ~ 1:1024.
adopt genetic resortment technology to obtain H
1
n
1
influenza virus Vero cell acclimatization to cold strain
1. influenza virus genetic resortment
By non-influenza virus Vero cell adapted strain A/Caledonia/20/99 (H
1n
1) with the A/Yunnan/1/2005Vca (H of embodiment 1
3n
2) virus strain, mix in 9:1 ratio, inoculate respectively two pieces of 9-11 age in days SPF chicken embryos, every embryonic breeding kind 0.2ml, hatches 24h for 33 ± 1 DEG C, and 2 DEG C of cold embryos that spend the night are collected chick embryo allantoic liquid, obtain virus harvest liquid;
2. influenza virus H after reprovision
1n
1the seed selection of Vero cell acclimatization to cold strain
The H that is 1:640 by influenza virus harvest liquid 1. of step and antibody titer
3n
2antiserum(antisera), mixes by the volume ratio of 2:1, in 37 DEG C and after 2 hours, is inoculated on the Vero cell of cultivating 24h, after results virus liquid, then uses same antibody, processes and goes down to posterity once on Vero cell by same procedure; Do not add afterwards antiserum(antisera) and on Vero cell, passed continuously again for 10 generations, results virus liquid;
3. serological identification virus type
The virus liquid blood clotting titre that 2. determination step is gathered in the crops, and be configured to 4 HAU antigens, use H
3n
2positive serum, H
1n
1positive serum, Type B positive serum and virus liquid, carry out hemagglutination-inhibition test (H I) by the quick method of false add, and blood clotting titre is 1:512, through being accredited as H
1n
1influenza virus.
adopt genetic resortment technology to obtain H
5
n
1
influenza virus Vero cell acclimatization to cold strain
1. influenza virus genetic resortment
By non-influenza virus Vero cell adapted strain A/AnHui/1/2005 (H
5n
1) with the A/Yunnan/1/2005Vca (H of embodiment 1
3n
2) virus strain, mix in 1:1 ratio, inoculate fresh Vero cell, initial inoculum size is 0.15ml, maintenance medium mother liquor composition is MEM, TPCK-pancreas enzyme concentration 1ug/ml, pH7.2, is placed in 25 DEG C and cultivates 120-168h, results virus liquid;
2. influenza virus H after reprovision
5n
1the seed selection of Vero cell acclimatization to cold strain
The H that is 1:128 by influenza virus harvest liquid 1. of step and antibody titer
3n
2antiserum(antisera), mixes by 5:8 volume ratio, in 37 DEG C and after 2 hours, is inoculated on the Vero cell of cultivating 24h, cultivates 120-168h for 25 DEG C, results virus liquid.Again above influenza virus Vero cell kind poison harvest liquid is not added to antiserum(antisera) and be inoculated on the Vero cell of cultivating 24h, according to said method on Vero cell, go down to posterity altogether 4 times.Again 4～8 DEG C of 10000rpm of the 5th generation Vero cell kind poison harvest liquid are got to supernatant for centrifugal 20 minutes,, on Vero cell, go down to posterity again 4 times successively in going down to posterity after following ratio 4:1,20:1, the mixed neutralization of 1:40,1:80 with antiserum(antisera), results virus liquid;
3. serological identification virus type
The virus liquid blood clotting titre that 2. determination step is gathered in the crops, and be configured to 4 HAU antigens, use H
3n
2positive serum, H
1n
1positive serum, Type B positive serum and virus liquid carry out hemagglutination-inhibition test (H I) with the quick method of false add, and viral hemoagglutination titre 1:512, through being accredited as influenza virus H
5n
1.
adopt genetic resortment technology to obtain H
3
n
2
influenza virus Vero cell acclimatization to cold strain
1. genetic resortment obtains H
1n
1influenza virus Vero cell acclimatization to cold strain, method is shown in embodiment 6.
2. genetic resortment obtains H
3n
2influenza virus Vero cell acclimatization to cold strain.
A. influenza virus genetic resortment
By non-influenza virus Vero cell adapted strain A/Fujian/196/2009(H
3n
2) with the reprovision H of embodiment 6
1n
1vero cell adapted strain, mixes by the volume ratio of 8:2, inoculates respectively two pieces of 9-11 age in days SPF chicken embryos, and every embryonic breeding kind 0.2ml, hatches 24h for 33 ± 1 DEG C, and 2 DEG C of cold embryos that spend the night are collected chick embryo allantoic liquid, obtain virus harvest liquid;
B. influenza virus H after reprovision
3n
2the seed selection of Vero cell acclimatization to cold strain
By influenza virus harvest liquid and the H of step a
1n
1antiserum(antisera) (antibody titer 1:640), mixes by the volume ratio of 2:1, in 37 DEG C and after 2 hours, is inoculated on the Vero cell of cultivating 24h, and results virus liquid goes down to posterity once on Vero cell by antibody same treatment again; Do not add afterwards antiserum(antisera) and on Vero cell, passed continuously again for 10 generations, results virus liquid;
C. serological identification virus type
The virus liquid blood clotting titre of determination step b results, and be configured to 4 HAU antigens, use H
3n
2positive serum, H
1n
1positive serum, Type B positive serum and virus liquid carry out hemagglutination-inhibition test (H I) with the quick method of false add, and blood clotting titre 1:512, through being accredited as H
3n
2influenza virus.
reverse genetics reprovision obtains H
3
n
2
influenza virus Vero cell acclimatization to cold strain
1. by influenza virus A/Yunnan/1/2005Vca (H
3n
2) 6 internal gene be cloned on bidirectional transcription vector pHW2000;
2. by non-influenza virus Vero cell adapted strain A/Wisconsin/67/2005 (H
3n
2) 2 surface protein genes be cloned in step two-way expression vector pHW2000 1.;
3. by step influenza virus A/Yunnan/1/2005Vca (H 1.
3n
2) 6 internal gene plasmids and step A/Wisconsin/67/2005 (H 2.
3n
2) 2 surface protein gene plasmids, totally 8 plasmids, totally 2 μ g, add transfection reagent Lipofectamine 2000, cotransfection growth 12-24 hour, cover with the 293T cell of 80-90%, cultivate 72h, results virus liquid, multigelation three times, surveying its blood clotting titre is 1:32, shows that virus is saved successfully;
4. by the continuous passage on Vero cell of step virus liquid 3., obtain and there is A/Wisconsin/67/2005 (H
3n
2) the influenza virus Vero cell acclimatization to cold strain of HA/NA;
5. serological identification virus type
reverse genetics reprovision obtains H
5
n
1
influenza virus Vero cell acclimatization to cold strain
1. by influenza virus A/Yunnan/1/2005Vca (H of embodiment 1
3n
2) 6 internal gene be cloned in bidirectional transcription vector pHW2000;
2. by non-influenza virus Vero cell adapted strain A/Anhui/1/2005 (H
5n
1) (H
5n
1△) 2 of influenza vaccines strain surface protein genes, are cloned in step two-way expression vector pHW2000 1..
3. by step influenza virus A/Yunnan/1/2005Vca (H 1.
3n
2) 6 internal gene plasmids and step A/Wisconsin/67/2005 (H 2.
3n
2) 2 surface protein gene plasmids, totally 8 plasmids, totally 2 μ g, add transfection reagent Lipofectamine 2000, cotransfection growth 12-24 hour, cover with the 293T cell of 80-90%, cultivate 48h, results virus liquid, multigelation three times, surveying its blood clotting titre is 1:16, shows that virus is saved successfully;
4. by the continuous passage on Vero cell of step virus liquid 3., obtain and there is A/Anhui/1/2005 (H
5n
1) (H
5n
1the influenza virus Vero cell acclimatization to cold strain of HA/NA △);
5. serological identification virus type
Claims (5)
1. an influenza A virus Vero cell acclimatization to cold strain, called after A/Yunnan/1/2005Vca (H
3n
2), the microbial preservation of this strain number is: CCTCC NO. V201253.
2. the application of influenza A virus Vero cell acclimatization to cold strain in the Vero cell adapted strain of preparing epidemic strain as claimed in claim 1.
3. the application of influenza A virus Vero cell acclimatization to cold strain in preparation influenza vaccines as claimed in claim 1.
4. a preparation method for influenza A virus Vero cell acclimatization to cold strain, is characterized in that through following steps:
A. on the Vero cell that grows up to fine and close individual layer for 24 hours, the blood clotting titre of inoculation 1MOI reaches the influenza A virus A/kunming/1/2005Va(H3N2 of 1:1024);
B. be MEM or DMEM/F12 maintaining mother liquor composition, penicillin-Streptomycin sulphate 20,000 U/ml, glutamine solution 0.3-0.6mg/ml, bovine serum albumin 1mg/ml, TPCK-pancreas enzyme concentration 0.5-1.5ug/ml, pH7.0-7.2, under 30 ± 1 DEG C of conditions, the influenza A virus of culturing step A, after 72 hours, is gathered in the crops virus liquid;
C. the virus liquid multigelation of step B being gathered in the crops 3 times, inoculate on the Vero cell that grows up to fine and close individual layer for 24 hours, then repeating step B and C, so on Vero cell, passed continuously for 20 generations, and the blood clotting titre that maintains kind of poison cell harvest liquid is at 1:1024, influenza A virus must go down to posterity;
D. on the Vero cell that grows up to fine and close individual layer for 24 hours, the influenza A virus that goes down to posterity of inoculation 1MOI step C;
E. be MEM or DMEM/F12 maintaining mother liquor composition, penicillin-Streptomycin sulphate 20,000 U/ml, glutamine solution 0.3-0.6mg/ml, bovine serum albumin 1mg/ml, TPCK-pancreas enzyme concentration 0.5-1.5ug/ml, pH7.0-7.2, under 25 ± 1 DEG C of conditions, culturing step D's went down to posterity after influenza A virus 120-168 hour, results virus liquid;
F. the virus liquid multigelation of step e being gathered in the crops 3 times, be inoculated on the Vero cell that grows up to fine and close individual layer for 24 hours, repeating step E and F again, so on Vero cell, passed continuously for 72 generations, and the blood clotting titre that maintains kind of poison cell harvest liquid is at 1:1024, obtains influenza A virus Vero cell acclimatization to cold strain.
5. method as claimed in claim 4, is characterized in that the influenza A virus A/kunming/1/2005Va(H3N2 of described steps A) be existing influenza A virus Vero cell high yield adapted strain, its preserving number is: CCTCC NO.V200514.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410039468.1A CN103898066B (en) | 2014-01-27 | 2014-01-27 | A kind of influenza A virus Vero cell acclimatization to cold strain and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410039468.1A CN103898066B (en) | 2014-01-27 | 2014-01-27 | A kind of influenza A virus Vero cell acclimatization to cold strain and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103898066A true CN103898066A (en) | 2014-07-02 |
| CN103898066B CN103898066B (en) | 2016-02-17 |
Family
ID=50989640
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410039468.1A Active CN103898066B (en) | 2014-01-27 | 2014-01-27 | A kind of influenza A virus Vero cell acclimatization to cold strain and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103898066B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039270A (en) * | 2015-08-11 | 2015-11-11 | 中国农业大学 | H9N2 subtype bird flu cold adaption attenuated strain and application thereof |
| CN116751818A (en) * | 2023-08-10 | 2023-09-15 | 天津中逸安健生物科技有限公司 | Preparation method of recombinant influenza virus vector therapeutic hypertension vaccine |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101560503A (en) * | 2009-03-13 | 2009-10-21 | 中国医学科学院医学生物学研究所 | Influenza A virus Vero cell adapted strain and application thereof |
-
2014
- 2014-01-27 CN CN201410039468.1A patent/CN103898066B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101560503A (en) * | 2009-03-13 | 2009-10-21 | 中国医学科学院医学生物学研究所 | Influenza A virus Vero cell adapted strain and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 丁士磊: "流感病毒H_3N_2亚型毒株在Vero细胞上冷适应的研究", 《中国优秀硕士学位论文全文数据库 农业科技辑（月刊）》 * |
| 丁士磊等: "流感冷适应减毒活疫苗的研究进展", 《中国生物制品学杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039270A (en) * | 2015-08-11 | 2015-11-11 | 中国农业大学 | H9N2 subtype bird flu cold adaption attenuated strain and application thereof |
| CN105039270B (en) * | 2015-08-11 | 2018-01-02 | 中国农业大学 | H9N2 subtype avian influenza acclimatization to cold attenuated strains and its application |
| CN116751818A (en) * | 2023-08-10 | 2023-09-15 | 天津中逸安健生物科技有限公司 | Preparation method of recombinant influenza virus vector therapeutic hypertension vaccine |
| CN116751818B (en) * | 2023-08-10 | 2024-01-26 | 天津中逸安健生物科技有限公司 | Preparation method of recombinant influenza virus vector therapeutic hypertension vaccine |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103898066B (en) | 2016-02-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102428099B (en) | The epiornitic seedling of delivery Avian pneumo-encephalitis virus | |
| Horimoto et al. | The development and characterization of H5 influenza virus vaccines derived from a 2003 human isolate | |
| CN104232594B (en) | Recombination classes fowl type H1N1 inactivated influenza virus vaccines strain (JS40/PR8) and its preparation method and application | |
| CN108018300A (en) | Distinguish immune and infection animal H7 subtype avian influenza vaccine strains and its preparation method and application | |
| CN1298845C (en) | Recombinant Newcastle disease LaSota low virulent vaccine strain expressing bird flu virus H5 subtype HA protein | |
| CN108728419A (en) | Express aviadenovirus penton Protein reconstitutions newcastle disease vaccine Candidate Strain rAI4-penton and construction method | |
| CN100487119C (en) | Recombination newcastle disease LaSota weak virus vaccine for expressing poultry influenza virus H5 sub type HA protein | |
| CN104195116B (en) | A kind of recombinant Newcastle disease virus and its construction method for expressing goose parvovirus VP3 genes | |
| CN103468651A (en) | Recombination Newcastle vaccine strain rAI4-S1 for expressing infectious bronchitis virus S1 protein and generating method thereof | |
| CN104073513A (en) | Acquisition method and adaptive sites of influenza A virus vaccine mammalian cell adaptive strain | |
| CN101560503B (en) | Influenza A virus Vero cell adapted strain and application thereof | |
| CN103328629A (en) | Novel vaccines against the a/H1N1 pandemic flu virus | |
| Shi et al. | Generation of an attenuated H5N1 avian influenza virus vaccine with all eight genes from avian viruses | |
| CN101619306B (en) | Influenza B virus Vero cell productive adaptive strain and preparation and application thereof | |
| CN105671002B (en) | The building of H9N2 subtype avian influenza virus cell high yield vaccine strain and application | |
| CN104611299B (en) | H9N2 avian flus strain, preparation method, vaccine combination and its application of a kind of artificial recombination | |
| CN103898066B (en) | A kind of influenza A virus Vero cell acclimatization to cold strain and application thereof | |
| RU2736786C1 (en) | Strain "a/chicken/primorsk/419/2018 h9n2" of avian influenza virus of genus alphainfluenzavirus type influenza a virus subtype h9 for controlling antigenic and immunogenic activity of vaccines for avian influenza and for making biopreparations for diagnosis and specific prevention of avian influenza type a subtype h9 | |
| CN103740654B (en) | A kind of Influenza B virus Vero cell acclimatization to cold strain and application thereof | |
| CN103118704A (en) | Modified viral strains, and method for improving the production of vaccine seeds of the influenza virus | |
| Lekcharoensuk et al. | Cloned cDNA of A/swine/Iowa/15/1930 internal genes as a candidate backbone for reverse genetics vaccine against influenza A viruses | |
| CN104073470A (en) | Spinner-flask culture method for H9N2 subtype of avian influenza virus | |
| CN108300702B (en) | Chicken-derived H9N2 avian influenza virus cold-adapted strain screening method and application thereof | |
| CN102223895B (en) | Method for production of pH stable enveloped viruses | |
| CN103333916A (en) | Construction method and application of BHK cell line suitable for newcastle disease virus proliferation |
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 |