CN102559599A - Vero-SIAT1 cell line and application thereof - Google Patents
Vero-SIAT1 cell line and application thereof Download PDFInfo
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Abstract
The invention relates to a Vero-SIAT1 cell line, a method for reproduction of influenza viruses in the Vero-SIAT1 cell line and a novel influenza inactivated vaccine prepared by the method, and especially discloses the method for reproduction of influenza viruses in the Vero-SIAT1 cell line. The method for reproduction of influenza viruses in the Vero-SIAT1 cell line comprises the following steps of inoculating Vero-SIAT1 cells with influenza viruses, carrying out culture, collecting a virus solution, carrying out inactivation, and carrying out purification to obtain a monovalent stock solution. The invention belongs to the technical field of vaccine preparation technologies.
Description
Technical field
The present invention relates to make up a kind of Vero-SIAT1 clone, and in this clone the method for breeding influenza virus, and the novel influenza inactivated vaccine of method preparation thus.Particularly disclose a kind of method of in the Vero-SIAT1 cell, breeding influenza virus, influenza virus has been inoculated the Vero-SIAT1 cell, cultivated, gathered in the crops viral liquid, prepared unit price stoste through deactivation, purifying.The invention belongs to vaccine production Technology field.
Background technology
Influenza (abbreviation influenza) is the acute respiratory transmissible disease that is caused by influenza virus (be called for short influenza virus), and clinical manifestation is heating, headache, myalgia, weak, rhinitis, pharyngalgia and cough, and gastrointestinal upset can be arranged.Influenza can increase the weight of potential disease (cardiopulmonary disease) or cause secondary bacterial pneumonia or former influenza virus property pneumonia, the elderly and suffer from various chronic diseases or the person of having a delicate constitution severe complication occurs after suffering from influenza easily.Transmission of influenza virus is rapid, popular extensively, and antigen is prone to variation, and crowd's specific immunity situation is unstable.
Influenza Virus orthomyxovirus section, according to its separately nucleocapsid endoantigen difference be divided into first type, B-mode and three kinds of viruses of third type.Wherein first type and second type influenza virus are bigger to threat of human.Influenza pandemic has certain seasonality, and north China popular generally all occurs in winter, and the south four seasons all have case to take place, and onset peak is in summer and winter.Influenza A virus all has discovery in the animal (comprising pig, horse and various bird) and the mankind.Though few exceptions occurs, human nonspecific infection H1, H2 or H3 and N1 or N2 subtype virus.The influenza virus of animal hypotype directly causes people's parainfluenza once in a while.Though the human probability of this type of virus infection is not high, case fatality rate is very high.Though Influenza B virus is mostly relevant with light-duty influenza with low sickness rate, also can cause the same with influenza A virus serious popular once in a while.Influenza B virus only infects the mankind, the pathogenic agent the Childhood of mainly being.The antigenic variation degree of Influenza B virus is not as influenza A virus.Influenza virus is propagated to Susceptible population by respiratory secretions, and the main factor is to sneeze, cough or even the small-particle aerosol of talk generation by the infected.
Influenza is the highly infective disease that is caused by a kind of very easily virus of sudden change.Estimate that according to WHO when burst was popular, it can the bamboo telegraph in the whole world, influences 10% to 20% of total population.Even in the non-explosive popular time, influenza also causes 3,000,000 to 5,000,000 serious cases and 250,000 to 500,000 examples dead every year.Unique feasible influenza preventive measures are vaccine inoculation at present.The history in existing more than 50 year since influenza vaccines are used, what generally use both at home and abroad is vaccinum influenzae inactivatum, is that influenza virus is cultivated in the chicken embryo after formaldehyde or the deactivation of β propiolactone are processed.Current vaccine has 3 types: 1. whole virus vaccine, form by complete influenza virus particles, and contain surface antigen and internal antigens, have good immunogenicity, but reactionogenicity is also stronger.Lipoids in the peplos belongs to pyrogeneous substance, often causes exothermic reaction.2. split-virus vaccine is that influenza virus is used the cracking agent cracking, processes behind the purifying.This vaccine has been removed viral nucleic acid and high molecular weight protein, but keeps HA and NA albumen and part matrix M albumen and nucleoprotein (NP), compares with whole virus vaccine, and immunogenicity and security are all better, at home and abroad is widely used at present.3. subunit vaccine, influenza virus prepares through cracking with after being further purified only contains HA and the proteic vaccine of NA, has safe and effective and the few characteristics of untoward reaction.The two kinds of vaccines in back can be used for children.
Adopt chick embryo technique to produce influenza vaccines safety, effective; And the virus safe property problem that relates to is few; But also exist certain limitation, like the problem of bacterial contamination in the production process (intracellular toxin), remain to be developed the new bird exogenous factor pollution problem of high growth strain, residual ovalbumin problem (inapplicable), potential and industrial scale is little, the PT long, under the situation of flu outbreak, be difficult to purchase at short notice and produce millions of required eggs of novel vaccine etc. to the egg allergy sufferers.Therefore; Many production of vaccine commercial city is at the research cell culture method; The cell cultures vaccine can or be very popular people to the prevailing disease that takes place any time in 1 year and all makes rapid answer, and the danger of the chicken embryo (possibly influence biological load and vaccine endotoxin content) that can avoid polluting.And the influenza virus that in mammalian cell, grows more approaches the virus in the clinical sample than the isolating virus of chicken embryo culture, therefore possibly produce more effective vaccine.
First type and Influenza B virus are through the sialyloligosaccharide on HA protein binding host cell surface glycolipid or the gp.Human influenza virus preferentially combines α 2, the 6-sialyl, but bird flu virus mainly combines α 2, the 3-sialyl.Human tracheal epithelial cell mainly comprises α 2, the 6-sialyl, however the duck intestinal epithelial cells mainly has α 2, the 3-sialyl.Therefore increase α 2,6 and connect receptor level and will increase the interaction of stream of people influenza virus and cell surface, and then influenza virus A1, A3 blood subgroup and Type B are bred have higher virus titer in cell.
The Vero cell is the cell strain that WHO allows to be used for production of vaccine, and it is responsive to multiple virus, and is responsive too to influenza virus.Though there is α 2 in the Vero cell, the 6-sialyl, the amount of its content nobody tracheal epithelial cell is high, causes the breeding titre of human influenza virus in the Vero cell not high.In our invention, stably transfection Vero cell is with the eDNA of SIAT1, and then increases the acceptor of human influenza virus.Vero-SIAT1 clone not only helps breeding human influenza virus, and helps the research of influenza vaccines.
Summary of the invention
The present invention makes up a kind of Vero-SIAT1 clone, and a kind of preparation method of novel influenza vaccine is provided, and is problems such as the culture medium security of making influenza vaccines, culture medium source to solve what exist at present with the chicken embryo.
Make up Vero-SIAT1 clone: stably transfection Vero cell is with the eDNA of SIAT1, the galactose alpha 2 on its product catalysis gp, 6-sialylation, and then the acceptor of increase human influenza virus.The Vero cell is had the cDNA of SIAT1 and the plasmid transfection of neomycin resistance gene, and is screened by G418 vitriol.Behind the stable transfection, prove, in the Vero cell, cross expression SIAT1 gene through Western blot and immunofluorescence microscopy.Show that through SNA and MAA dyeing process compare with parent's vero cell, the vero cell of stable transfection is expressed the α 2 of 7 times of a large amounts respectively, the α 2 of 6-sialyl and 3 times of low amounts, 3-sialyl.In addition, influenza virus A1, A3 blood subgroup and Type B are bred in the Vero-SIAT1 cell and are had higher virus titer.
Influenza virus A1, A3 blood subgroup and Type B were pressed 1: 10 respectively
-1～1: 10
-7Be inoculated in to contain the Vero-SIAT1 cell that grows to individual layer of following composition virus-culturing fluid: the MEM nutrient solution; 0.2～2% bovine serum albumin; The trypsinase that 2 μ g/ml～20 μ g/ml trypsinase or TPCK handle; Use HEPES or 5%～8% sodium hydrogen carbonate solution adjustment pH value of solution value to 7.0～8.0; The Xin Meisu of 20 μ g/ml～500 μ g/ml.Under 33 ℃～35 ℃ conditions, being cultured to cytopathy (CPE) reaches ++ +～++ ++ in+time, results contain the supernatant of influenza virus; Adopt as above with this virus-culturing fluid that method goes down to posterity, set up the production of novel influenza inactivated vaccine with original species word bank, main seed bank and work seed bank.
Infect the Vero-SIAT1 cell with work seed bank virus liquid with as above method, gather in the crops viral liquid with 1: 100～1: 10000 formaldehyde solution or beta-propiolactone deactivation; Adopt DEAE Streamline and/or DEAESepharose FF and/or Sepharose 4FF chromatography method to carry out purifying; (chromatography condition: the basic damping fluid of DEAE Streamline and/or DEAE Sepharose FF: pH6.8-8.0,0.001-0.2Mol/l PBS damping fluid or Tris damping fluid, wash-out use sodium chloride concentration to be 0.01-1Mol/l to obtain virus stock solution used; Sepharose 4FF elution buffer is pH6.8-8.0,0.001-0.2Mol/l PBS damping fluid, contains 0.1-0.15Mol/l sodium-chlor); Detect virus antigen content (standard antigen and standard antiserum(antisera) are provided by NIBSC) with the SRID method, regulate the antigenic content of stoste, preparation influenza virus A1, A3 blood subgroup and Type B unit price or multivalence are mixed work in-process; Work in-process are carried out packing, be finished product---Influenza Vaccine behind the assay approval.
Advantage of the present invention is: the present invention obtains the Vero-SIAT1 clone of stably express SIAT1 through the method that makes up for the first time, and the producing and manufacturing technique that utilizes Vero-SIAT1 cells produce Influenza Vaccine is provided.
Description of drawings
Fig. 1 is a pcDNA3.1-SIAT1-cmyc vector construction of the present invention.We obtain the pBluescript-SIAT1 carrier through gene synthetic method, behind the SIAT1 gene, add the cmyc label then, and purpose is for easy to detect, are connected to screening and expression goal gene SIAT1 on pcDNA3.1 (-) carrier at last.A. be template with the pBluescript-SIAT1 carrier, PCR obtains the SIAT1-cmyc fragment; B. the SIAT1-cmyc fragment is connected on pcDNA3.1 (-) carrier and obtains pcDNA3.1-SIAT1-cmyc; C. identify pcDNA3.1-SIAT1-cmyc with Not I and BamHI double digestion, the result is correct.
Fig. 2 detects SIAT1 protein expression in the Vero cell for Western.Mock is normal vero cell; PcDNA3.1 is that the empty carrier of 48h transient transfection vero cell is as negative control; SIAT1-cmyc be 48h transient transfection vero cell have purpose fragment carrier as positive control, Vero-SIAT1-cmyc is the vero clone that has the stable transfection of SIAT1-cmyc.From figure, can see; Compare with the pcDNA3.1 negative control with Mock; The obvious high expression level SIAT1 of Vero-SIAT1-cmyc experimental group purpose fragment; And it is also high than positive controls expression amount, explains that the stable transfection expression SIAT1 clone that we screen can high expression level SIAT1 goal gene.
Fig. 3 is that the method for immunocytochemical stain detects SIAT1 protein expression in the Vero cell.Compare with the pcDNA3.1 negative control with Mock; The obvious high expression level SIAT1 of Vero-SIAT1-cmyc experimental group purpose fragment; And it is also obviously high than positive controls expression amount, explains that the stable transfection expression SIAT1 clone that we screen can high expression level SIAT1 goal gene.
Fig. 4 is for utilizing the western method, and SNA detects α-2,6 and connects glycosidic link, and MAA detects α-2,3 and connects glycosidic link.Compare with the pcDNA3.1 negative control with Mock, the α of Vero-SIAT1-cmyc experimental group-2,6 connects glycosidic link obviously to be increased; And α-2,3 connects glycosidic link and obviously reduces, explain that vero clone is crossed expression SIAT1 gene after; Can catalysis α-2; 6 connect the formation of glycosidic link, and emulative minimizing α-2,3 connects glycosidic link.Positive controls changes not obvious, possibly be because the time ratio of 48h is shorter, though the SIAT1 gene is expressed, also be not clearly for catalysis, and the SIAT1 gene expression amount of positive controls is starkly lower than steady commentaries on classics experimental group.
Fig. 5 surely changes the virus titer in the cell for the plaque experiment detects the H1N1 influenza virus at Vero and Vero.Compare with Mock, behind the H1N1 influenza virus that infects same amount, the titre of Vero-SIAT1-cmyc experimental group cell obviously improves, and improves 4.9 times.
Fig. 6 is the duplicating efficiency that in Vero and Vero-SIAT1-cmyc cell, compares human influenza virus.Compare with the Vero cell, behind the H3N2 and Type B influenza virus that infect same amount, the titre of Vero-SIAT1-cmyc experimental group cell obviously improves, and improves 4.1 and 2.8 times respectively.
Preferred implementation
The influenza virus A1 blood subgroup was pressed 1: 10
-4Be inoculated in the Vero-SIAT1 cell that grows to individual layer of the MEM virus-culturing fluid that uses HEPES Xin Meisu adjust pH to 7.0, that contain 1% bovine serum albumin, 5 μ g/ml trypsinase, 200 μ g/ml; Under 35 ℃ of conditions, being cultured to cytopathy (CPE) reaches ++ +～++ ++ in+time, results contain the supernatant of influenza virus.
Embodiment 2
Influenza virus A1 industry type was pressed 1: 10
-7Be inoculated in the Vero-SIAT1 cell that grows to individual layer of the MEM virus-culturing fluid that uses 5%～8% sodium hydrogen carbonate solution adjustment pH value of solution Xin Meisu value to 7.5, that contain 0.2% bovine serum albumin, 20 μ g/ml trypsinase, 20 μ g/ml; Under 35 ℃ of conditions, being cultured to cytopathy (CPE) reaches ++ +～++ ++ in+time, results contain the supernatant of influenza virus.
Embodiment 3
The influenza virus A1 blood subgroup was pressed 1: 10
-1Be inoculated in the Vero-SIAT1 cell that grows to individual layer of the MEM virus-culturing fluid that uses 5%～8% sodium hydrogen carbonate solution adjustment pH value of solution Xin Meisu value to 8.0, that contain 2% bovine serum albumin, 2 μ g/ml trypsinase, 500 μ g/ml; Under 35 ℃ of conditions, being cultured to cytopathy (CPE) reaches ++ +～++ ++ in+time, results contain the supernatant of influenza virus.
Embodiment 4
Adopt arbitrary method results influenza virus A3 blood subgroup and Type B virus liquid among the embodiment 1-3.
Embodiment 5
Arbitrary method is set up novel influenza virus of A 1, A3 blood subgroup and Type B production with original species word bank, main seed bank and work seed bank among the employing embodiment 1-3.
Embodiment 6
Infect the Vero-SIAT1 cell with influenza virus A1 blood subgroup work seed bank virus liquid with arbitrary method among the embodiment 1-3, gather in the crops viral liquid with formaldehyde solution deactivation in 1: 100; Adopt DEAE Streamlin chromatography method to carry out purifying, chromatography condition: DEAE Streamline basis damping fluid: pH6.8,0.001Mol/lPBS damping fluid, wash-out uses sodium chloride concentration to be 0.01Mol/l; Obtain virus stock solution used; Detect virus antigen content with the SRID method.
Embodiment 7
Infect the Vero-SIAT1 cell with influenza virus A1 blood subgroup work seed bank virus liquid with arbitrary method among the embodiment 1-3, gather in the crops viral liquid with beta-propiolactone deactivation in 1: 1000; Adopt DEAE Sepharose FF chromatography method to carry out purifying, chromatography condition: the basic damping fluid of DEAE Sepharose FF: pH8.0,0.2Mol/l Tris damping fluid, wash-out uses sodium chloride concentration to be 1Mol/l; Obtain virus stock solution used; Detect virus antigen content with the SRID method
Embodiment 8
Infect the Vero-SIAT1 cell with influenza virus A1 blood subgroup work seed bank virus liquid with arbitrary method among the embodiment 1-3; Gather in the crops viral liquid with formaldehyde solution deactivation in 1: 10000; Adopt Sepharose 4FF chromatography method to carry out purifying, Sepharose 4FF elution buffer is pH7.5,0.1Mol/l PBS damping fluid, contains 0.1Mol/l sodium-chlor; Obtain virus stock solution used; Detect virus antigen content with the SRID method.
Embodiment 9
Adopt embodiment 7-9 method to obtain influenza virus A3 blood subgroup and Type B virus stock solution used.
Embodiment 10
Regulate the antigenic content of influenza virus A1, A3 blood subgroup and Type B virus stock solution used respectively, preparation influenza virus A1, A3 blood subgroup and Type B unit price inactivated vaccine work in-process.
Embodiment 11
Regulate the antigenic content of stoste, mix influenza virus of A 1, A3 blood subgroup and Type B virus stock solution used, preparation trivalent influenza inactivated vaccine work in-process.
Embodiment 12
Work in-process are carried out packing, are finished product behind the assay approval---the influenza inactivated vaccine.
Claims (7)
1. make up a kind of Vero-SIAT1 clone, this clone can be used as the matrix of preparation novel influenza vaccine.It is characterized in that:
Stably transfection Vero cell is with human α 2, the cDNA of 6-sialytransferase (SIAT1), and the galactose alpha 2 on its product catalysis gp, the 6-sialylation, and then increase the acceptor of human influenza virus.The Vero cell is had the cDNA of SIAT1 and the plasmid transfection of neomycin resistance gene, and is screened by G418 vitriol.Behind the stable transfection, prove, in the Vero cell, cross expression SIAT1 gene through Western blot and immunofluorescence microscopy.Show that through SNA and MAA dyeing process compare with parent's vero cell, the vero cell of stable transfection is expressed the α 2 of 7 times of a large amounts respectively, the α 2 of 6-sialyl and 3 times of low amounts, 3-sialyl.In addition, influenza virus A1, A3 blood subgroup and Type B are bred in the Vero-SIAT1 cell and are had higher virus titer.Vero-SIAT1 clone not only helps breeding human influenza virus, and helps the research of influenza vaccines.
2. utilize Vero-SIAT1 clone to prepare the method for novel influenza inactivated vaccine, it is characterized in that:
1) respectively with influenza virus A1, A3 blood subgroup and Type B after virus-culturing fluid dilution with 1: 10
-1～1: 10
-7The Vero-SIAT1 cell of individual layer has been grown in inoculation, under 33 ℃～35 ℃ conditions, is cultured to cytopathy (CPE) and reaches ++ +～++ ++ in+time, results contain the supernatant of influenza virus respectively;
2) supernatant of selecting step 1) to obtain is set up the production of novel influenza inactivated vaccine respectively with original species word bank, main seed bank and work seed bank;
3) use work seed bank virus liquid to adopt the method for step 1) to infect the Vero-SIAT1 cell respectively, the viral liquid of obtain is carried out deactivation;
4) virus antigen of deactivation adopts chromatography method to distinguish purifying, obtains each virus stock solution used;
5) detect the antigenic content of each virus stock solution used,
6) regulate the antigenic content of influenza virus A1, A3 blood subgroup and Type B virus stock solution used respectively, preparation influenza virus A1, A3 blood subgroup and Type B unit price inactivated vaccine work in-process; Or regulate the antigenic content of each stoste, mix influenza virus of A 1, A3 blood subgroup and Type B virus stock solution used, preparation trivalent influenza inactivated vaccine work in-process.
3. the said method of claim 2, wherein influenza virus infects the condition of Vero-SIAT1 cell:
The composition of virus-culturing fluid:
The MEM nutrient solution;
0.2～2% bovine serum albumin;
The trypsinase that 2 μ g/ml～20 μ g/ml trypsinase or TPCK handle;
Use HEPES or 5%～8% sodium hydrogen carbonate solution adjustment pH value of solution value to 7.0～8.0;
The Xin Meisu of 20 μ g/ml～500 μ g/ml.
4. the said method of claim 2, wherein the inactivation of viruses agents useful for same comprises 1: 100～1: 10000 formaldehyde solution and beta-propiolactone.
5. the said method of claim 2, wherein purified virus comprises DEAEStreamline with chromatography media, DEAE Sepharose FF, Sepharose 4FF.
6. the said method of claim 2; Chromatography condition is: the basic damping fluid of DEAE Streamline and/or DEAESepharose FF: pH6.8-8.0,0.001-0.2Mol/l PBS damping fluid or Tris damping fluid, wash-out use sodium chloride concentration to be 0.01-1Mol/l; Sepharose 4FF elution buffer is pH6.8-8.0,0.001-0.2Mol/l PBS damping fluid, contains 0.1-0.15Mol/l sodium-chlor.
7. the said product of claim 1 is the unit price of influenza virus A1, A3 blood subgroup and the Type B of preparation or the multivalence mixture of each hypotype.
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Cited By (1)
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| CN106754722A (en) * | 2016-11-10 | 2017-05-31 | 中国食品药品检定研究院 | Vero cell line of stabilization expression bovine trypsinogen and application thereof |
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Application publication date: 20120711 |