CN101560503A - Influenza A virus Vero cell adapted strain and application thereof - Google Patents
Influenza A virus Vero cell adapted strain and application thereof Download PDFInfo
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Abstract
The invention discloses an influenza A virus Vero cell adapted strain and application thereof. The influenza A virus Vero cell adapted strain is named as A/Yunnan/1/2005Va(H3N2), and the preserving number of the strain is CCTCC NO:V200514. The strain comprises an H3 subgroup hemagglutinin gene, an N2 subgroup neuraminidase gene and 6 internal genes for high yield of characteristic influenza viruses on Vero cells. The blood coagulation titer can be maintained at more than 1,024 by continuous subculturing of the strain on the Vero cells. The strain can be taken as a donor strain and subjected to genetic reassortment with an epidemic strain, or reverse genetics technology is adopted to perform genetic reassortment on the 6 internal genes and 2 surface protein genes of the epidemic strain to obtain the Vero cell adapted strain of the epidemic strain, and finally the adapted strain is used for preparing a Vero cell influenza vaccine or a Vero cell pandemic influenza vaccine.
Description
Technical field
The present invention relates to a kind of influenza A virus Vero cell adapted strain, also relate to this adapted strain, obtain the Vero cell adapted strain of epidemic strain,, belong to biological technical field with preparation Vero stream of cells influenza vaccine as donor strain and epidemic strain reprovision.
Background technology
Influenza (influenza) is a kind of acute respiratory infection that is caused by influenza virus, has hyperinfection, mainly through the air droplet transmission, therefore it can take place suddenly in a short time, rapid spread cause in various degree popular, even is very popular in the world, sickness rate is high and with certain mortality ratio, is the human at present Pandemic infection disease that still can not effectively control.
Influenza virus belongs to orthomyxoviridae family, 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.Influenza A virus often occurs with popular form, and second type influenza virus is local outburst, and influenza C mainly is to occur to distribute form.Wherein first type and Influenza B virus and the mankind have close ties, and influenza A virus also can infect animals such as birds, pig, horse except that infected person, and its hypotype H5 or H7 easily cause high pathogenic avian influenza (HPAI).Therefore the people contacts frequent place with these animals in life, provides good environment for influenza virus produces variation, and the virus after the variation can cause influenza pandemic, and degree of variation greatly then causes being very popular.
The variation of influenza virus surface antigen is the major cause of influenza pandemic, there are two kinds of mechanism in the variation of its surface antigen: a kind of antigenic drift that is called as, comprise influenza A virus and Influenza B virus, mainly be by point mutation on HA and NA gene and multiple amino acids selective mutation, escape from the immunization of body and the antibody that obtained behind the infective virus in the past to the restraining effect of virus.Antigenic drift can produce new epidemic strain, and it also is that influenza virus can infect the mankind simultaneously, and continues the weapon of evolution; Another kind of variation is called as the big variation of antigen, occur over just on the influenza A virus, when host cell 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 can produce the strain of influenza A virus antigenic shift, will cause that influenza is very popular in the world if this mutant strain can be propagated in the Susceptible population of immunization not.
Pandemic influenza A virus has H1N1 in the crowd, three hypotypes of H2N2 and H3N2.The hypotype that 16 kinds of HA in the influenza virus A type and 9 kinds of NA form extensively be present in some wildlifes as: in the aquatic bird.If virus is not passed through genetic resortment, the bird flu hypotype does not generally produce effective infection to the people.But the virus of this rapid diffusion is broken up owing to regular gene again, thereby it is human that it is infected, for example, and since Hong Kong outburst bird flu in 1997, the H5N1 type breaks out in bird except causing on a large scale through genetic modification, and has produced the incident that many cases people infects.The variation of finding this virus strain in 2003, occurred can infected person new variant, may cause new flu outbreak, WHO prediction new flu outbreak after nineteen sixty-eight approaches.
The inoculation influenza vaccines are main means of flu-prevention and complication thereof.The vaccinum influenzae inactivatum of nineteen forty-one chicken embryo preparation gets the Green Light first in the U.S., is used widely in the whole world.The influenza vaccines of using mainly are with influenza three split vaccines of the deactivation of chicken embryo preparation at present, comprise influenza A virus H1N1 strain, H3N2 strain and Influenza B virus strain.The component of vaccine strain be by WHO according to every year global influenza virus the Changing Pattern production of vaccine that is used for next year of recommending determine with the surface antigen of influenza virus epidemic strain.Influenza A virus association laboratory utilizes the genetic resortment technology that chicken embryo high yield strain and wild epidemic strain are carried out reprovision, and the HA that contains the influenza virus epidemic strain that obtains and the novel vaccine strain of NA surface glycoprotein have the characteristic of high yield on the chicken embryo.But the chicken embryo is produced influenza vaccines as culture medium needs complicated purge process, to reduce people to the white transformation reactions that produces of chicken embryo; And go down to posterity in the chicken embryo hemagglutinin antigen of back virus of influenza virus easily changes vaccine potency is reduced; The chicken embryo is difficult to scale operation to satisfy the sudden demand of flu outbreak in addition; Especially high pathogenic avian influenza popular in the worldwide since 2003 makes and adopts the chicken embryo to produce the influenza virus vaccine risk to increase.Work out safely and effectively that vaccine has become influenza vaccines important research direction, especially will when the reply flu outbreak, not be subjected to the restriction of chicken embryo source, the World Health Organization to urge the new influenza vaccines production matrix of countries in the world research.
The mammalian cell that is used to prepare influenza vaccines at present mainly is mdck cell and Vero cell, though mdck cell is to cultivate and the mammal cell line preferably that separates influenza virus, but there is potential tumorigenicity in it, makes it be applied to production of vaccine and has no end of misgivings.African green monkey kidney cell (Vero) is the production of vaccine clone of world health organisation recommendations, adopt now the Vero cells produce vaccine approved listing Poliomyelitis Vaccine, Rabies Vaccine, Vaccinum Encephalitis B etc. are arranged.Beginning in 1998, people such as Kistnar are the influenza all-virus inactivated vaccine with Vero cell development purifying in Austria, excites after the immune BALB/c mouse to have produced higher hemagglutination inhibition antibody, and the emission levels of T cellullar immunologic response and cytokine is also high.But the Vero cell is not the responsive culture medium of influenza virus, the lower and most viruses of output can not be on the Vero cell propagation continuously, make to be difficult to Vero stream of cells influenza vaccine cost height on market, use.Therefore how quickly breeding influenza virus Vero cell high yield adapted strain becomes the difficult problem of Vero stream of cells influenza vaccine research and development.The human cell is that PER.C6 can make influenza virus effectively growth therein, be one of optimum cell of influenza virus cultivation, but it belongs to Dutch antibody and vaccine Crucell house journal owns.
Reverse genetics, for classical genetics, be on the basis that obtains organism genome full sequence, by target gene being carried out necessary processing and modification, as rite-directed mutagenesis, gene insertion/disappearance, gene substitution etc., again by forming the modifying factor group that sequential build contains the essential element of organism, allow it assemble out individuality with vital activity, genomic structure of postgraduate's object and function, and these modifications may have the content of aspects such as which kind of influence to phenotype, the proterties of organism.Reverse genetics has been applied to influenza virus and has striden the research of kind mechanism of transmission, virus virulence and Study on Pathogenicity thereof, directions such as vaccine research and development at present.The way of vaccine research and development is exactly that the chicken embryo high yield strain of acquisition epidemic strain is in order to the production vaccine the A/PR/8/34 of laboratory strain (H1N1) of chicken embryo high yield and current popular strain reprovision in addition.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 that the lead time of vaccine is shortened, work simplification, so reverse genetics is an important means of reply flu outbreak.
In sum, influenza is still the human Pandemic infection disease that still can not effectively control, and the inoculation influenza vaccines are the main means of flu-prevention.Use at present the matrix of chicken embryo, have a lot of weak points as the preparation influenza vaccines, as be difficult to large-scale production, complex process, also have that exogenous factor pollutes may wait shortcoming.Therefore, use passage cell such as Vero cell preparation influenza vaccines to become the important directions that influenza vaccines are developed, and how quickly breeding influenza virus Vero cell high yield adapted strain is the difficult problem of Vero stream of cells influenza vaccine and the research and development of pandemic influenza vaccine.
Summary of the invention
One of the object of the invention is to overcome the deficiencies in the prior art, and a kind of influenza A virus Vero cell high yield adapted strain is provided, and this strain is uploaded at the Vero cell and is commissioned to train fosterly, and the blood clotting titre can remain on more than 1024.
Technical problem to be solved by this invention realizes by following technological approaches:
A kind of influenza virus Vero cell adapted strain, called after A/Yunnan/1/2005Va (H3N2), the preserving number of this strain are CCTCC NO:V200514, depositary institution: Chinese typical culture collection center; Preservation address: Wuhan City, Wuhan University; Preservation date: on November 30th, 2005.
It is the method for the influenza virus Vero cell adapted strain of CCTCC NO:V200514 that two of the object of the invention provides the preparation preserving number.
Preserving number provided by the invention is the influenza virus Vero cell adapted strain of CCTCC NO:V200514, be that tens of kinds of wild influenza virus strains continuous passage on the Vero cell that will collect is cultivated, cultivate and obtain after 25 generations, it has H3 hypotype hemagglutinin gene, N2 hypotype Neuraminidase Gene and has 6 internal gene fragments of high-yield character on the Vero cell.
Preserving number provided by the invention is the essential characteristic of the influenza virus Vero cell adapted strain of CCTCC NO:V200514: (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 the continuous passage culturing process, is difficult for producing the influenza virus gene variation; (c) influenza virus continuous passage on the Vero cell is cultivated, and the blood clotting titre can remain on more than 1024; (d) the HI experiment shows that the HA antigen of virus 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 the SPF chicken, and infected chicken does not have morbidity and dead.
Preserving number of the present invention is the influenza virus Vero cell adapted strain of CCTCC NO:V200514, obtains through following method:
A, the street strain that separation in the clinical sample is obtained are inoculated on the Vero cell that grows up to fine and close individual layer, with DMEM/F12 or MEM as basic medium, wherein adding TPCK-pancreatin to concentration again is that 0.2-1.5ug/ml, bovine serum albumin to concentration are 0.1-2ug/ml, with NaHCO
3The adjusting medium pH value is 7-8; Behind 33 ± 2 ℃ of cultivation 48-96h, gather in the crops viral liquid, finish the 1st cultivation of going down to posterity;
B, withhold the viral liquid that obtains with the above-mentioned the 1st and be inoculated into once more on the Vero cell, after the method for pressing the A step is cultivated, gather in the crops viral liquid; So continuous passage to 25 is gathered in the crops viral liquid during generation, and promptly getting preserving number is the influenza virus Vero cell adapted strain of CCTCC NO:V200514.
Three of the object of the invention provides the application of influenza virus Vero cell adapted strain in the Vero cell adapted strain of preparation epidemic strain.
Four of the object of the invention provides the application of influenza virus Vero cell adapted strain in the preparation influenza vaccines.
Five of the object of the invention provides a kind of method for preparing influenza virus Vero cell adapted strain, prepares Vero stream of cells influenza vaccine to use this adapted strain.The influenza virus Vero cell adapted strain that promptly with the preserving number is CCTCC NO:V200514 is as the donor strain, behind the epidemic strain reprovision, obtain the Vero cell adapted strain of epidemic strain, finally use this adapted strain to prepare Vero stream of cells influenza vaccine or Vero cell pandemic influenza vaccine.
Influenza virus Vero cell adapted strain that guarantor's minus sign provided by the invention is CCTCC NO:V200514 and epidemic strain prepare the method for influenza virus Vero cell adapted strain with the traditional method reprovision, may further comprise the steps:
1) be that the influenza virus Vero cell adapted strain of CCTCC NO:V200514 mixes in proportion with popular subtype influenza virus with preserving number, concrete ratio is determined with different strain growth characteristics by the viral hemoagglutination titre;
2) said mixture is inoculated on 9～10 age in days SPF chicken embryos or Vero cell or the mdck cell, wherein: during inoculation SPF chicken embryo, every embryonic breeding kind 0.1-0.3ml is hatched 12-48h in 33 ± 2 ℃, the 2-8 ℃ of cold embryo that spends the night collected chick embryo allantoic liquid and is virus results liquid; During inoculation Vero cell, each little square vase inoculum size is 0.05-0.2ml, places 33 ± 2 ℃ to cultivate 48-96h, gathers in the crops viral liquid; During the inoculation mdck cell, each little square vase inoculum size is 0.05-0.2ml, places 33 ± 2 ℃ to cultivate 48-96h, gathers in the crops viral liquid;
3) with 2) antiserum(antisera) to the concentration that adds anti-A/Yunnan/1/2005Va (H3N2) in the viral liquid of results is 1%-80%, concrete concentration is suppressed to tire by the antibody blood clotting and the blood clotting titre of virus decide, is seeded in the Vero cell and uploads and be commissioned to train foster 1 time; Perhaps with 2) the viral liquid of results do not add the antiserum(antisera) direct inoculation and uploads to be commissioned to train at the Vero cell and support 1 time;
4) repeat 3 repeatedly) step, make passage, and carry out antibody screening, select the Vero cell adapted strain of epidemic strain, so that use this adapted strain to prepare Vero stream of cells influenza vaccine or Vero cell pandemic influenza vaccine.
To be the influenza virus Vero cell adapted strain of CCTCC NO:V200514 and epidemic strain prepare the method for influenza virus Vero cell adapted strain with the reverse genetic reprovision that learns a skill to preserving number provided by the invention, may further comprise the steps:
1) is that 6 internal gene of the influenza virus Vero cell adapted strain of CCTCC NO:V200514 are cloned respectively in bidirectional transcription vector with preserving number, obtains the recombinant plasmid of 6 internal gene respectively;
2) 2 surface protein genes of epidemic strain are cloned equally in bidirectional transcription vector, obtain the recombinant plasmid of 2 surface protein genes respectively;
3) with above-mentioned 1) and 2) 8 plasmids mix; cotransfection is on mammalian cell 293T or COS or MDCK; perhaps transfection is on the cell mixing of MDCK and 293T or on the cell mixing of MDCK and COS; cultivate 18-24h; get cell culture supernatant; promptly obtain the Vero cell adapted strain of epidemic strain, so that use this adapted strain to prepare Vero stream of cells influenza vaccine or Vero cell pandemic influenza vaccine.
Described bidirectional transcription vector is influenza virus gene bidirectional transcription vector pHW2000.
The present invention compared with prior art has following advantage and effect:
(1) after employing preserving number provided by the invention is the influenza virus Vero cell adapted strain of CCTCC NO:V200514, producing influenza vaccines with the chicken embryo as culture medium and have the following advantages than prior art: 1. can adopt microcarrier fermentor tank large scale culturing Vero cell and influenza virus, production technique is easy to stdn; 2. adopt standardized Vero cell, do not contain exogenetic biotic factor and pollute; 3. do not contain and be easy to cause that human allergy's chicken embryo is white; 4. having avoided influenza virus viral hemagglutinin antigen after the chicken embryo culture easily to change reduces vaccine potency; 5. when flu outbreak is sudden, be not subjected to the influence in chicken embryo source, can produce enough vaccines of meeting the need of market.
(2) at present used influenza vaccines to produce with seed culture of viruses all be to join adapted strain with chicken embryo high yield strain reprovision or natural chicken, it is generally insensitive on the Vero cell.The influenza vaccines that the producer that has directly provides WHO produce seed culture of viruses and are inoculated on the Vero cell, but product poison amount is low or output is unstable, have restricted the application that utilizes Vero cell cultures influenza virus, preparation influenza vaccines.We will separate the influenza virus that obtains in the clinical sample, be seeded in and carry out the continuous passage cultivation on the Vero cell, cultivating preserving number is the influenza virus Vero cell adapted strain of CCTCC NO:V200514, and with its as donor strain and popular strains of influenza viruses reprovision after, obtain Vero cell vaccine production strain, promoted Vero stream of cells influenza vaccine and Vero cell pandemic influenza developing vaccines and application.
Embodiment
Further describe the inventive method by the following examples, it should be understood that these embodiment only are used for the purpose of illustration, never limit the scope of the invention.
Embodiment 1
Preserving number is the preparation method of the influenza virus Vero cell adapted strain of CCTCC NO:V200514:
1) be inoculated on the Vero cell that grows up to fine and close individual layer separating the influenza virus that obtains in the clinical sample, as basic medium, wherein adding TPCK-pancreatin to concentration again is 1ug/ml with DMEM/F12, and bovine serum albumin to concentration is 1ug/ml, with NaHCO
3Regulating the medium pH value is 7.5; Behind 33 ± 1 ℃ of cultivation 72h, gather in the crops viral liquid, finish the 1st cultivation of going down to posterity;
2) the viral liquid of cultivating results that goes down to posterity for the 1st time is inoculated on the Vero cell once more, uses and 1) after the identical method of step and condition cultivate, gather in the crops viral liquid; So continuous passage on the Vero cell, when being passaged to for 25 generations, gather in the crops viral liquid and promptly get influenza virus Vero cell adapted strain, called after A/Yunnan/1/2005Va (H3N2), preserving number is CCTCC NO:V200514, with this strain as the donor strain, with the Vero cell adapted strain that obtains epidemic strain behind other epidemic strain reprovision, to be used for Vero stream of cells influenza vaccine and Vero cell pandemic influenza developing vaccines and application.
Embodiment 2
Influenza virus Vero cell adapted strain A/Yunnan/1/2005Va (H3N2) continuous passage on serum-free Vero cell is cultivated and is kept stablizing high yield.
1) the influenza virus Vero cell adapted strain A/Yunnan/1/2005Va (H3N2) with embodiment 1 gained is inoculated on the Vero cell that grows up to fine and close individual layer of serum-free culture, inoculum size is every little square vase 0.2ml, keeping mother liquor composition is: SFM (Hyclone), TPCK-pancreatin 0.5ug/ml, bovine serum albumin 1.5ug/ml uses NaHCO
3Adjust pH value to 7.2, place 33 ± 1 ℃ of cultivation 72h, gather in the crops viral liquid, promptly finish the 1st be commissioned to train foster;
2) with above-mentioned 1) the 1st the withholding on the Vero cell that the viral liquid that obtains is inoculated into serum-free culture once more of gained, adopt and 1) after identical method and condition cultivate, gather in the crops viral liquid; So plant continuously poison and go down to posterity on the Vero cell, whenever withhold and survey its blood clotting titre after obtaining liquid multigelation three times, the blood clotting titre maintains more than 1024, shows that strain A/Yunnan/1/2005Va (H3N2) has Vero cell high and stable yields characteristic.
Embodiment 3
The influenza virus Vero cell adapted strain A/Yunnan/1/2005Va (H3N2) of embodiment 1 gained and A/Caledonia/20/99 (H1N1) reprovision obtain the method for H1N1 influenza virus Vero cell adapted strain:
1. influenza virus reprovision
The non-adapted strain A/Caledonia/20/99 of Vero cell (H1N1) is mixed by 9: 1 volume ratios with influenza virus Vero cell adapted strain A/Yunnan/1/2005Va (H3N2), inoculation 9～10 age in days SPF chicken embryos, every embryonic breeding kind 0.2ml, hatch 24h for 33 ± 2 ℃, 2 ℃ of cold embryos that spend the night are collected chick embryo allantoic liquid and are virus results liquid;
2. the seed selection of influenza virus H1N1 Vero cell adapted strain behind the reprovision
1. it is that the antiserum(antisera) of 1: 640 anti-A/Yunnan/1/2005Va (H3N2) mixes by 2: 1 volume ratio that viral liquid and the antibody blood clotting of results are suppressed to tire, and with after 2 hours, is inoculated on the Vero cell and cultivates 48h in 37 ℃, gathers in the crops viral liquid; The viral liquid that to gather in the crops again adds identical antiserum(antisera) in proportion and mixes, and in 37 ℃ and after 2 hours, is inoculated on the Vero cell and cultivates 48h, gathers in the crops viral liquid; The viral liquid that to gather in the crops does not again add antiserum(antisera), passes for 10 generations on the Vero cell more continuously, finally obtains the malicious results of the 12nd generation cell kind liquid;
3. serological identification virus type
Get the 12nd and withhold the viral liquid that obtains, survey its blood clotting titre and be configured to 4 HAU antigens, carry out hemagglutination-inhibition test (HI) with H3N2 positive serum, H1N1 positive serum, Type B positive serum and viral liquid with the quick method of false add, blood clotting titre 512, through being accredited as the H1N1 influenza virus, can be used for the preparation of Vero stream of cells influenza vaccine with this strain, perhaps with this strain once more as donor strain and other epidemic strain reprovisions, thereby obtain the Vero cell adapted strain of epidemic strain.
Embodiment 4
The influenza virus Vero cell adapted strain H1N1 of embodiment 3 gained and A/Wisconsin/67/2005 (H3N2) reprovision obtain the method for H3N2 influenza virus Vero cell adapted strain:
1. influenza virus reprovision
The non-adapted strain A/Wisconsin/67/2005 of Vero cell (H3N2) is mixed by 1: 1 volume ratio with embodiment 3 gained influenza virus Vero cell adapted strain H1N1, inoculation has grown up to the mdck cell of fine and close individual layer, every little square vase inoculation 0.1ml cultivates 72h, gathers in the crops viral liquid for 33 ± 2 ℃;
2. the seed selection of influenza virus H3N2 Vero cell adapted strain behind the reprovision
1. the viral liquid of gathering in the crops is inoculated on the Vero cell, and every little square vase inoculation 0.05ml virus liquid was cultivated 72 hours, and was gathered in the crops viral liquid for 33 ℃.It is that the antiserum(antisera) of 1: 3200 anti-H1N1 mixes by 5: 1 volume ratio that the viral liquid that will gather in the crops again and antibody blood clotting suppress to tire, and with after 2 hours, is inoculated on the Vero cell and cultivates 72h in 37 ℃, gathers in the crops viral liquid; The viral liquid that to gather in the crops once more adds identical antiserum(antisera) in proportion and mixes, and in 37 ℃ and after 2 hours, is inoculated on the Vero cell and cultivates 72h, gathers in the crops viral liquid; The viral liquid that to gather in the crops does not again add antiserum(antisera), passes for 5 generations on the Vero cell more continuously, finally obtains the malicious results of the 8th generation cell kind liquid;
3. serological identification virus type
Get the 8th and withhold the viral liquid that obtains, survey its blood clotting titre and be configured to 4 HAU antigens, carry out hemagglutination-inhibition test (HI) with H3N2 positive serum, H1N1 positive serum, Type B positive serum and viral liquid with the quick method of false add, blood clotting titre 512, through being accredited as the H3N2 influenza virus, this strain can be used for the preparation of Vero stream of cells influenza vaccine, thereby perhaps obtains the Vero cell adapted strain of epidemic strain once more as donor strain and other epidemic strain reprovisions with this strain.
Embodiment 5
The influenza virus Vero cell adapted strain A/Yunnan/1/2005Va (H3N2) of embodiment 1 gained and influenza virus H 5 N 1 reprovision obtain the method for H5N1 influenza virus Vero cell adapted strain
1. influenza virus reprovision
Non-influenza virus Vero cell adapted strain H5N1 is mixed by 1: 1 volume ratio with influenza virus Vero cell adapted strain A/Yunnan/1/2005Va (H3N2), be inoculated on the fresh Vero cell, inoculum size is every little square vase 0.15ml, keeping mother liquor composition is: MEM, TPCK-pancreatin 1.2ug/ml, bovine serum albumin 1.7ug/ml uses NaHCO
3Adjust pH value to 7.4, place 33 ± 2 ℃ to cultivate 72h, gather in the crops viral liquid;
2. the seed selection of influenza virus H 5 N 1 Vero cell adapted strain behind the reprovision
With 1. viral liquid and the antibody blood clotting of results suppress the to tire antiserum(antisera) of the anti-A/Yunnan/1/2005Va (H3N2) that is 1: 128, by 5: 8 volume ratio mixing, in 37 ℃ with after 2 hours, be inoculated on the Vero cell, cultivate 96h for 33 ℃, gather in the crops viral liquid, finish the 1st cultivation of going down to posterity; The viral liquid that to gather in the crops does not again add antiserum(antisera), is inoculated into 34 ± 1 ℃ of cultivation 96h on the Vero cell, gathers in the crops viral liquid, so passes for 4 generations on the Vero cell continuously; Again the 5th is gone down to posterity and cultivate the viral liquid of results,, get supernatant in 4～8 ℃ of 10000rpm centrifugations 20 minutes; Getting supernatant by 4: 1 volume ratio and the antiserum(antisera) mixing of anti-A/Yunnan/1/2005Va (H3N2), with 2 hours, is inoculated on the Vero cell in 37 ℃,, gathers in the crops viral liquid, finish the 6th cultivation of going down to posterity in 33 ℃ of cultivation 96h; Successively by 20: 1,1: 40,1: 80 volume ratio with the antiserum(antisera) mixing of anti-A/Yunnan/1/2005Va (H3N2), with 2 hours, was inoculated on the Vero cell in 37 ℃ so again, and in 33 ℃ of cultivation 96h, going down to posterity was cultured to for the 9th generation, gathered in the crops viral liquid;
3. serological identification virus type
Get the 9th and withhold the viral liquid that obtains, survey its blood clotting titre and be configured to 4 HAU antigens, carry out hemagglutination-inhibition test (HI) with H3N2 positive serum, H1N1 positive serum, Type B positive serum and viral liquid with the quick method of false add, viral hemoagglutination titre 512, through being accredited as influenza virus H 5 N 1, this recombined strain is used for the preparation of Vero stream of cells influenza vaccine or Vero cell pandemic influenza vaccine.
Embodiment 6
The influenza virus Vero cell adapted strain A/Yunnan/1/2005Va (H3N2) of embodiment 1 gained obtains the method for A/Wisconsin/67/2005 (H3N2) influenza virus Vero cell adapted strain with the reverse genetic reprovision that learns a skill:
1) 6 internal gene of the influenza virus Vero cell adapted strain A/Yunnan/1/2005Va (H3N2) of embodiment 1 gained is cloned in bidirectional transcription vector PHW2000;
2) with 2 surface protein gene clones of non-influenza virus Vero cell adapted strain A/Wisconsin/67/2005 (H3N2) in two-way expression vector PHW2000;
3) with 2 surface protein gene plasmids of 6 internal gene plasmids of influenza virus A/Yunnan/1/2005Va (H3N2) and A/Wisconsin/67/2005 (H3N2) totally 8 mix, the total 2 μ g of plasmid, behind liposome 8ul mixing, cotransfection growth 18～24 hours, in the COS of fine and close individual layer cell, cultivate 24h, results culture supernatant, surveying its blood clotting titre is 64, acquisition has the influenza virus Vero cell adapted strain of the HA/NA of A/Wisconsin/67/2005 (H3N2), and this strain is used for the preparation of Vero stream of cells influenza vaccine and the preparation of pandemic influenza vaccine.
Claims (8)
1, a kind of influenza virus Vero cell adapted strain, called after A/Yunnan/1/2005Va (H3N2), the microbial preservation of this strain number is CCTCC NO:V200514.
2, influenza virus Vero cell adapted strain as claimed in claim 1 is characterized in that it has H3 hypotype hemagglutinin gene, N2 hypotype Neuraminidase Gene and has 6 internal gene fragments of high-yield character on the Vero cell.
3, a kind of method for preparing influenza virus Vero cell adapted strain as claimed in claim 1 is characterized in that through the following step:
A, the street strain that separation in the clinical sample is obtained are inoculated on the Vero cell that grows up to fine and close individual layer, with DMEM/F12 or MEM as basic medium, wherein adding TPCK-pancreatin to concentration again is that 0.2-1.5ug/ml, bovine serum albumin to concentration are 0.1-2ug/ml, with NaHCO
3The adjusting medium pH value is 7-8; Behind 33 ± 2 ℃ of cultivation 48-96h, gather in the crops viral liquid, finish the 1st cultivation of going down to posterity;
B, withhold the viral liquid that obtains with the above-mentioned the 1st and be inoculated into once more on the Vero cell, after the method for pressing the A step is cultivated, gather in the crops viral liquid; So continuous passage to 25 is gathered in the crops viral liquid during generation, and promptly getting preserving number is the influenza virus Vero cell adapted strain of CCTCC NO:V200514.
4, the application of influenza virus Vero cell adapted strain in the Vero cell adapted strain of preparation epidemic strain according to claim 1.
5, the application of influenza virus Vero cell adapted strain in the preparation influenza vaccines according to claim 1.
6, a kind of method for preparing the Vero cell adapted strain of epidemic strain is characterized in that through following steps:
1) be that the influenza virus Vero cell adapted strain of CCTCC NO:V200514 mixes in proportion with popular subtype influenza virus with preserving number, concrete ratio is determined with different strain growth characteristics by the viral hemoagglutination titre;
2) said mixture is inoculated on 9～10 age in days SPF chicken embryos or Vero cell or the mdck cell, wherein: during inoculation SPF chicken embryo, every embryonic breeding kind 0.1-0.3ml is hatched 12-48h in 33 ± 2 ℃, the 2-8 ℃ of cold embryo that spends the night collected chick embryo allantoic liquid and is virus results liquid; During inoculation Vero cell, each little square vase inoculum size is 0.05-0.2ml, places 33 ± 2 ℃ to cultivate 48-96h, gathers in the crops viral liquid; During the inoculation mdck cell, each little square vase inoculum size is 0.05-0.2ml, places 33 ± 2 ℃ to cultivate 48-96h, gathers in the crops viral liquid;
3) with 2) antiserum(antisera) to the concentration that adds anti-A/Yunnan/1/2005Va (H3N2) in the viral liquid of results is 1%-80%, concrete concentration is suppressed to tire by the antibody blood clotting and the blood clotting titre of virus decide, is seeded in the Vero cell and uploads and be commissioned to train foster 1 time; Perhaps with 2) the viral liquid of results do not add the antiserum(antisera) direct inoculation and uploads to be commissioned to train at the Vero cell and support 1 time;
4) repeat 3 repeatedly) step, make passage, and carry out antibody screening, select the Vero cell adapted strain of epidemic strain, so that use this adapted strain to prepare Vero stream of cells influenza vaccine or Vero cell pandemic influenza vaccine.
7, a kind of method for preparing the Vero cell adapted strain of epidemic strain is characterized in that through following steps:
1) is that 6 internal gene of the influenza virus Vero cell adapted strain of CCTCC NO:V200514 are cloned respectively in bidirectional transcription vector with preserving number, obtains the recombinant plasmid of 6 internal gene respectively;
2) 2 surface protein genes of epidemic strain are cloned equally in bidirectional transcription vector, obtain the recombinant plasmid of 2 surface protein genes respectively;
3) with above-mentioned 1) and 2) 8 plasmids mix; cotransfection is on mammalian cell 293T or COS or MDCK; perhaps transfection is on the cell mixing of MDCK and 293T or on the cell mixing of MDCK and COS; cultivate 18-24h; get cell culture supernatant; promptly obtain the Vero cell adapted strain of epidemic strain, so that use this adapted strain to prepare Vero stream of cells influenza vaccine or Vero cell pandemic influenza vaccine.
8, the method for the Vero cell adapted strain of preparation epidemic strain according to claim 7 is characterized in that described bidirectional transcription vector is influenza virus gene bidirectional transcription vector pHW2000.
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| CN101979517A (en) * | 2010-10-15 | 2011-02-23 | 洛阳普莱柯生物工程有限公司 | Method for producing influenza viruses in large scale by using bioreactor |
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| CN111032861A (en) * | 2017-08-28 | 2020-04-17 | 一般财团法人阪大微生物病研究会 | Staged preparation method of reassortant influenza virus |
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