CN104178457A - MDCK (Madin-Darby Canine Kidney) cell line for stably coexpressing human-derived Siat7e and ST3GalIV and application thereof - Google Patents
MDCK (Madin-Darby Canine Kidney) cell line for stably coexpressing human-derived Siat7e and ST3GalIV and application thereof Download PDFInfo
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- CN104178457A CN104178457A CN201410411768.8A CN201410411768A CN104178457A CN 104178457 A CN104178457 A CN 104178457A CN 201410411768 A CN201410411768 A CN 201410411768A CN 104178457 A CN104178457 A CN 104178457A
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Abstract
The invention belongs to the technical field of biology, and relates to an engineering cell line clone MDCK-hSiat7e-ST3GalIV and application thereof, particularly an engineering cell line clone strain MDCK-hSiat7e-ST3GalIV of which the collection number is CGMCC No:9330. The invention also relates to application of the cell line in lowering MDCK cell adherence performance and enhancing growth titer of avian influenza virus isolates, and application in anti-avian influenza virus drug screening and neutralizing antibody determination.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the clone MDCK-hSiat7e-ST3GalIV of engineering cell system and construction process thereof, relate more specifically to a clone MDCK-hSiat7e-ST3GalIV of strain engineering cell system, and described clone reducing mdck cell adherent performance and improving the growth titre of avian influenza virus strain isolated, thereby for the mensuration of the screening of Anti-avian influenza virus drugs, neutralizing antibody and for the full suspension culture scale operation of the serum-free Cell Culture Vaccine of MDCK.
Background technology
Avian influenza virus (AIV) belongs to the A of orthomyxoviridae family type Influenza Virus, surface glycoprotein hemagglutinin (HA) and neuraminidase (NA) have respectively 16 kinds and 9 kinds of [Webster RG, Bean WJ, Gorman OT, Chambers TM, & Kawaoka Y (1992) Evolution and ecology of influenza A viruses.Microbiological reviews56 (1): 152-179.].Wherein, HPAI (High Pathogenic AI) (H5 and H7 hypotype) has worldwide caused huge financial loss to aviculture, brings serious threat to human health simultaneously.In Mammals, all influenza viruses all derive from aquatic bird, but influenza virus seldom occurs across kind of a propagation, and this is to be determined by the interaction of influenza virus and its acceptor.Influenza virus is infected host cell by its surperficial hemagglutinin (HA) and cell surface sialyloligosaccharide receptors bind.The structure of cell receptor and viral HA receptor binding site is the principal element that determines influenza virus host specificity.Common influenza virus acceptor has two kinds, and one is sialic acid α 2,3 semi-lactosis (SA α 2,3-GAL), and another kind is sialic acid α 2,3 semi-lactosis (SA α 2,6GAL) semi-lactosis.Although it is sialic oligosaccharides that all influenza viruses all identify end, but various flows Influenza Virus HA bind receptor characteristic difference, most of avian influenza virus are preferentially in conjunction with SA α 2,3-GAL acceptor, human influenza virus is preferentially in conjunction with SA α 2,6GAL acceptor [Rogers G, et al. (1983) Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity.Nature304 (5921): 76-78.].
Mdck cell is to carry out influenza to study the most frequently used cell, is also one of clone of producing at present influenza virus vaccine, but its surperficial SA α 2,3Gal acceptor abundance is lower, causes virus titer low.Therefore, improve mdck cell surface SA α 2,3Gal acceptor abundance is likely improved the breeding titre of avian influenza vaccine strain.α 2,3-sialytransferase can be transferred to Neu5Ac the end Gal of glycoprotein and glycolipid, between Neu5Ac and Gal, connecting key is α 2,3[Ito T & Kawaoka Y (2000) Host-range barrier of influenza A viruses.Veterinary microbiology74 (1): 71-75.].Sialic acid conventionally with sugar chain N-terminal second-to-last sugar-be mainly that semi-lactosi (galactose, Gal) is with α 2,3 or α 2,6 glycosidic links connections.6 different α 2 from people's cell or tissue, are cloned and have identified, 3-sialytransferase cDNA.Phylogenetic tree analysis shows [Tsuji S (1996) Molecular cloning and functional analysis of sialyltransferases.Journal of Biochemistry120 (1): 1-13.], this family can be divided into two subfamilies, a subfamily is ST3GalI and ST3GalII, another subfamily is ST3GalIII, ST3GalIV, ST3GalV and ST3GalVI.
It is all the method that adopts chicken embryo to produce that traditional influenza vaccines produce, and the inactivated vaccine in chicken embryo source, although taked effective purifying process, still exists many deficiencies.The residue of not only cost costliness, and chicken embryo may cause allergic reaction.Main problem is the variation that the virus in chicken embryo source usually causes HA after the continuous passage of chicken embryo, this variation also may occur in other sections of virus [Rocha EP, et al. (1993) Comparison of10influenza A (H1N1and H3N2) haemagglutinin sequences obtained directly from clinical specimens to those of MDCK cell-and egg-grown viruses.The Journal of general virology74:2513-2518.], result can not be mated completely with the epidemic isolates in crowd the vaccine in chicken embryo source.The method of simultaneously producing with chicken embryo must be longer culture cycle and loaded down with trivial details operation steps and be easy to contaminated [Tree JA, Richardson C, Fooks AR, Clegg JC, & Looby D (2001) Comparison of large-scale mammalian cell culture systems with egg culture for the production of influenza virus A vaccine strains.Vaccine19 (25): 3444-3450.].And the vaccine that application mammalian cell is produced demonstrates reasonable provide protection [Govorkova E at animal model; Murti G; Meignier B; De Taisne C, & Webster R (1996) African green monkey kidney (Vero) cells provide an alternative host cell system for influenza A and B viruses.Journal of virology70 (8): 5519-5524.].Mdck cell is considered to cultivate A type and the most suitable clone of Influenza B virus [Robertson J (1998) An overview of host cell selection.Developments in biological standardization98:7-11; Discussion73-14.].This clone produces rapidly influenza virus after having infection, obtains at short notice the feature of high titre.The virus in MDCK source is after continuous passage, keep stability [the Govorkova E of antigen, Kodihalli S, Alymova I, Fanget B, & Webster R (1998) Growth and immunogenicity of influenza viruses cultivated in Vero or MDCK cells and in embryonated chicken eggs.Developments in biological standardization98:39-51; Discussion73-34.].
Traditional mdck cell is cultivated most employing serum adherent culture, however serum composition complexity, and contain the composition harmful to cell, and increased the difficulty of product separation purifying; Serum source is limited, expensive, and production cost is high; The quality of serum, because animal individual, the serum place of production, product batch number difference produce fluctuation, makes experiment and production be difficult to realize stdn.Mdck cell is the cell of anchorage dependence, only attaches to its ability survival of stromal surface, propagation and expression product.Traditional cultural method adopts two-dimentional monolayer to cultivate [Frisch SM & Francis H (1994) Disruption of epithelial cell-matrix interactions induces apoptosis.The Journal of cell biology124 (4): 619-626.], owing to being subject to substrate surface area restriction, in addition the long and high puzzlement of production cost of digestion process complexity, production time, is difficult to realize mdck cell large scale culturing and influenza vaccines scale operation.And zooblast suspension culture system be except can breaking through cell growth surface restriction, cell culture environment homogeneous more, is beneficial to production-scale amplification and monitoring.Therefore, the cell that exigence can adapt to suspension growth meets the industrial production of magnifying, and the foundation of the full suspension cell line of MDCK has great meaning for the industrialization process of production of vaccine.
The Siat7e gene (ST6GalNac V) that existing bibliographical information proves the mankind is relevant with the adherent performance of cell, by transfection relatively the Hela cell of Siat7e gene and the adherent Hela cell of untransfected, the cell attachment performance of transfection has obviously obtained reduction, and its adherent performance enhancing [Jaluria P that got back while having suppressed the expression of Siat7e gene by SiRNA technology, Betenbaugh M, Konstantopoulos K, Frank B, & Shiloach J (2007) Application of microarrays to identify and characterize genes involved in attachment dependence in HeLa cells.Metabolic engineering9 (3): 241-251.].Siat7e gene claims again ST6GalNac V, belong to the member of α 2-6 sialytransferase family, its effect is mainly sialic acid to be transferred to [Tsuchida A on the N-acetylglactoside that ganglioside props up from donor CMP-Neu5Ac, et al. (2003) Synthesis of disialyl Lewis a (Lea) structure in colon cancer cell lines by a sialyltransferase, ST6GalNAc VI, responsible for the synthesis of α-series gangliosides.Journal of Biological Chemistry278 (25): 22787-22794.].
The mdck cell that the present invention has set up coexpression of anthropogenic Siat7e gene and ST3GalIV is first the method for MDCK-hSiat7e-ST3GalIV cell.By stablizing coexpression hSiat7e and ST3GalIV, can make people source Siat7e gene express in mdck cell, thereby reduce the adherent performance of mdck cell; People source ST3GalIV gene is expressed in mdck cell simultaneously, improves mdck cell surface SA α 2,3Gal acceptor abundance, and then improve the breeding titre of avian influenza vaccine strain.Be not only the application of the full suspension culture of serum-free of research MDCK, lay a good foundation for the application of scale operation Cell Culture Vaccine, improve the breeding titre of avian influenza vaccine strain simultaneously and saved production technique cost.In addition, MDCK-hSiat7e-ST3GalIV clone is all showing good application prospect aspect the production of Anti-avian influenza virus drugs, vaccine strain screening and Cell Culture Vaccine.
Summary of the invention
First object of the present invention has been to provide the method that builds MDCK-hSiat7e-ST3GalIV clone, described method comprises the open reading frame cDNA of the open reading frame cDNA of hSiat7e and ST3GalIV is cloned in mdck cell system, builds up the mdck cell of stablize coexpression hSiat7e and ST3GalIV to be.
A strain hSiat7e of the present invention and the positive Madin-Darby Madin-Darby canine kidney(cell line) of ST3GalIV are that MDCK-hSiat7e-ST3GalIV cell clone is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC on June 26th, 2014, China, Beijing), preserving number is CGMCC No.9330.
In the present invention, utilize the highly effective eukaryon expression plasmid of people source Siat7e and ST3GalIV gene, transfection mdck cell, express positive monoclonal cell strain through G418 resistance and RT-PCR Screening and Identification, we have carried out the detection of qRT-PCR to the 3 strain monoclonal cell strains that obtain, obtain people source Siat7e and the ST3GalIV gene the highest monoclonal cell called after MDCK-G5G4 of expression amount simultaneously, obtained the mdck cell system of stable double expression(DE) Siat7e and ST3GAL4 gene.Detect α-2 in MDCK-hSiat7e-ST3GalIV cell through flow cytometer, 3 and the content of α 2,6 sialic acid acceptors be significantly higher than maternal mdck cell.Show, people source Siat7e and ST3GalV gene obtain stably express in MDCK-hSiat7e-ST3GalIV cell.
Second object of the present invention is that MDCK-hSiat7e-ST3GalIV clone can reduce the adherent performance of mdck cell and improve avian influenza virus growth titre simultaneously.We find transfection the cell of Siat7e gene can lose the ability that forms tight junctions with flanking cell, extensibility is not as maternal cell simultaneously.Therefore reduced the adherent performance of mdck cell, for the application of the full suspension culture of serum-free of research MDCK, laid a good foundation for the application of scale operation Cell Culture Vaccine.HA and TCID simultaneously
50test result all show, no matter be clade2.3.2.1H5N1 hypotype or clade7.2H5N1 hypotype, all to MDCK-hSiat7e-ST3GalIV clone susceptible more, virus titre also all higher than maternal mdck cell.
The invention also discloses the application of described clone in the growth titre that reduces the adherent performance of mdck cell and raising avian influenza virus strain isolated.Described clone is produced the application in Cell Culture Vaccine in the mensuration of the screening of Anti-avian influenza virus drugs, neutralizing antibody and in the full suspension culture of serum-free of MDCK.
In in the present invention by the eukaryotic expression vector transfection of people source Siat7e and ST3GalIV gene to mdck cell being, be built into the mdck cell system that stablizes coexpression hSiat7e and ST3GalIV.In described cell, people source Siat7e gene has obtained expression, reduces the adherent performance of mdck cell; α 2 simultaneously, 3-sialic acid content receptor is significantly higher than maternal mdck cell, and the titre that bird flue virus H 5 N 1 subtype is measured in MDCK-hSiat7e-ST3GalIV clone is higher than the result of measuring on maternal mdck cell.Be not only the application of the full suspension culture of serum-free of research MDCK, lay a good foundation for the application of scale operation Cell Culture Vaccine, improve the breeding titre of avian influenza vaccine strain simultaneously and saved production technique cost.
Brief description of the drawings
Fig. 1: mdck cell survival curve under different G418 concentration.X-coordinate: time (number of days) ordinate zou: cell survival rate (%)
The expression of people source hSiat7e and ST3GalIV gene mRNA in Fig. 2: MDCK and 3 strains transformation clone cell.Note: MDCK-G5G4 represents MDCK-hSiat7e-ST3GalIV cell
Fig. 3: MDCK and MDCK-hSiat7e-ST3GalIV cell RT-PCR qualification result
(A) electrophoresis result of hSiat7e gene, 1:MDCK-hSiat7e-ST3GalIV cell, 2: maternal mdck cell, M:200bp Marker; (B) electrophoresis result of ST3GalIV gene, 1:MDCK-hSiat7e-ST3GalIV cell, 2: maternal mdck cell, M:200bp Marker.
Fig. 4: MDCK and MDCK-hSiat7e-ST3GalIV cellular form change
Note: MDCK-G5G4 represents MDCK-hSiat7e-ST3GalIV cell
Fig. 5: flow cytometer detects mdck cell and the reactivity of MDCK-hSiat7e-ST3GalIV cell to different connecting-type specific agglutination elements
(A) mdck cell contrast; The contrast of MDCK-hSiat7e-ST3GalIV cell; Mdck cell is combined with SNA; MDCK-hSiat7e-ST3GalIV cell is combined with SNA; Mdck cell is combined with MAA; MDCK-hSiat7e-ST3GalIV cell is combined with MAA
(B) comparison of 3 detected result mean values that mdck cell and MDCK-hSiat7e-ST3GalIV cell surface sialic acid acceptor are combined with SNA and MAA
(*) represent to have significant difference (p<0.05) compared with MDCK-SNA;
(* *) represents to have significant difference (p<0.05) compared with MDCK MAA.
Note: MDCK-G5G4 represents MDCK-hSiat7e-ST3GalIV clone.
Fig. 6: the reproduction curve of avian influenza virus on mdck cell and MDCK-hSiat7e-ST3GalIV cell
(A) reproduction curve of YZC3 on two kinds of cells; (B) reproduction curve of rYZC3 on two kinds of cells;
(C) reproduction curve of wt-DT on two kinds of cells; (D) reproduction curve of rH5N1/PR8 on two kinds of cells.
Note: M represents mdck cell system; G5G4 represents MDCK-hSiat7e-ST3GalIV clone.
Fig. 7: the infection titer (TCID of fowl influenza virus strain on maternal mdck cell and MDCK-hSiat7e-ST3GalIV cell
50)
(*) represent that MDCK-hSiat7e-ST3GalIV cell has significant difference (p<0.05) compared with maternal mdck cell measurement result;
Note: MDCK-G5G4 represents MDCK-hSiat7e-ST3GalIV cell.
In the present invention, MDCK-hSiat7e-ST3GalIV cell clone (MDCK-G5G4) was preserved in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 26th, 2014, Institute of Microorganism, Academia Sinica), Classification And Nomenclature is Madin-Darby canine kindey cell, and preserving number is CGMCC No.9330.
Embodiment
The structure of 1MDCK-hSiat7e-ST3GalIV clone
Primer
Contain the eukaryon expression plasmid of people source Siat7e and ST3GalIV open reading frame purchased from Genecopoeia company (article No.: EX-V1581-M03 and EX-C0523-M02); The GenBank accession number of people source Siat7e and ST3GalIV gene is respectively NM_030965.1 and NM_001254757.1.The primer sequence is in table 1.
Table 1.Siat7e and ST3GAL4 gene RT-PCR and qRT-PCR primer Table 1.Primers for detecting Siat7e and ST3GAL4genes by RT-PCR and qRT-PCR
athe GenBank accession number of GAPDH is: NM_001003142.1.
G418 concentration in screening culture medium
We select the G418 concentration that can all kill mdck cell in 10-14 days as screening concentration.According to cell growth curve (Fig. 1), G418 concentration is between 0.8～1.0mg/mL time, and cell is all dead in 10～14 days.Therefore, we to select 0.8mg/mL be the concentration of G418 in pressure screening culture medium in this experiment.
The mono-clonal screening of object cell and the detection of genetic expression
After normal mdck cell transfection plasmid, use the DMEM substratum that contains 0.8mg/mL G418 and 10% foetal calf serum to screen, obtain the cell clone with resistance.In the substratum that contains G418, the necrocytosis of untransfected.The resistant cell of acquisition is mixed to clone G418 to carry out obtaining the strain of monoclonal anti sexual cell after limiting dilution, qualification selection.
In order to select people source Siat7e and the people source ST3GalIV gene the highest monoclonal cell of expression amount simultaneously, we have carried out the detection of qRT-PCR to the 3 strain cells that obtain.Result (seeing Fig. 2) shows that the Siat7e of 3 obtained strain cells and the expression of ST3GalIV gene there are differences, and the mRNA of Siat7e and ST3GalIV gene all can detect, but can't detect in maternal MDCK in 3 strain clone strains.Wherein the double expression(DE) level of MDCK-G5G4-F2 cell Siat7e and ST3GalIV gene is significantly higher than other three strain clones strains (p<0.05), and therefore the double gene expression amount of MDCK-G5G4-F2 is the highest.
RT-PCR qualification result
From RT-PCR result (Fig. 3), the cell strain of screening can amplify the band of two band 680bp and the band of 1002bp, in the same size with goal gene (hSiat7e and ST3GalIV) respectively.And in control group, maternal mdck cell is not seen the band of amplification.Illustrate that we have been incorporated into object band in mdck cell.This two strains mdck cell is frozen according to ordinary method amplification.We will amplify the MDCK-hSiat7e-ST3GalIV clone called after MDCK-G5G4 of two genes.
Cellular form comparison
The cellular form of the MDCK-hSiat7e-ST3GalIV cell of maternal mdck cell and double expression(DE) is as Fig. 4, and the mdck cell that we find to express people source Siat7e affects adhesion between cell and the extensibility of cell.Transfection the cell of Siat7e gene can lose the ability that forms tight junctions with flanking cell, extensibility is not as maternal cell (Fig. 4) simultaneously.Therefore reduce the adherent performance of mdck cell, for the application of the full suspension culture of serum-free of research MDCK, lay a good foundation for the application of scale operation Cell Culture Vaccine.
In the research work of this part, we carry out RT-PCR qualification to the MDCK-hSiat7e-ST3GalIV cell building, and can, under screening without G418 pressure, continue passage cell strain simultaneously, and result shows that foreign gene can be more than at least 10 generations of stably express.These qualification results show, the MDCK-hSiat7e-ST3GalIV cell obtaining, people source Siat7e and ST3GalIV gene open reading frame sequence cDNA have been incorporated in mdck cell genome, can go down to posterity and stable transcript and expression constantly along with mdck cell system.
2 flow cytometers detect cell surface α 2, the sialic expression of 3-
In order to detect α-2,3 and α 2, the content of 6 sialic acid acceptors on MDCK and engineered cells, we use two kinds of lectins of digoxigenin labeled to detect α-2 as primary antibodie, 3 and α-2, the expression amount of 6 connecting-type acceptors: black elderberry lectin (Sambucusnigra agglutinin, SNA) specific recognition 2,6 connecting-type sialic acids, Koryo Chinese scholartree amurensin lectin (Maackia amurensis agglutinin, MAA) specific recognition 2,3 connecting-type sialic acids (Roche); The anti-DIG antibody of FITC mark is anti-as two.Detected result shows, α-2 in MDCK-hSiat7e-ST3GalIV cell, 3 and the content of α 2,6 sialic acid acceptors be significantly higher than maternal mdck cell (Fig. 5 (A)).Fluorescence intensity Dao Shuo district peak figure also shows, compared with maternal mdck cell, and the α 2 of MDCK-hSiat7e-ST3GalIV cell, 6-sialic acid and α 2, the content Dao Shuo district of 3-sialic acid acceptor is skew (Fig. 5 (B)) to the right all.Experimental result shows, people source Siat7e and ST3GalV gene stably express in MDCK-hSiat7e-ST3GalIV cell, has improved cell surface α 2,6-sialic acid acceptor and α 2, the abundance of 3-sialic acid acceptor.
The mensuration of 3HA titre and TCID
50mensuration
Strain: Clade2.3.2.1H5N1: the A/chicken/Yangzhou/1117/2011 of street strain (YZC3 strain); The strain of rYZC3 vaccine candidate is the restructuring low virulent strain that the veterinary college Ministry of Agriculture of Yangzhou University livestock and poultry Infectious Diseases Lab utilizes reverse Genetics Technique to build, HA and NA gene source are in the A/chicken/Yangzhou/1117/2011 of street strain (YZC3 strain) [Zhang Yan, Zhang Wenjun, Lie group brightness, Liu Xiaowen, Liu Wenbo, the structure [J] of Liu Xiu .Clade2.3.2H5N1 subtype avian influenza of ancient India vaccine candidate strain. journal of animal science and veterinary medicine, 2012,06:915-921]
Clade7.2H5N1: the A/Chicken/Huadong/4/2008 of street strain (wt-DT strain), the strain of rH5N1/PR8 vaccine candidate is the restructuring low virulent strain that the veterinary college Ministry of Agriculture of Yangzhou University livestock and poultry Infectious Diseases Lab utilizes reverse Genetics Technique to build, HA and NA gene source are in the A/Chicken/Huadong/4/2008 of street strain (wt-DT strain) [Zhang Wenjun, Xue Tao, Wu little Wei, Zhang Yan, Zhong Lei, Peng great Xin, the structure [J] of Liu Xiu .H5 subtype avian influenza of ancient India vaccine variation Candidate Strain. Chinese poultry resource, 2011, 08:14-18] [Wenjun Zhang, et al. (2011) .Increase in viral yield in eggs and MDCK cells of reassortant H5N1vaccine candidate viruses caused by insertion of38amino acids into the NA stalk Vaccine.29, 8032 – 8041.]
HA titration
For more maternal mdck cell and α-2, the ability of MDCK-hSiat7e-ST3GalIV (MDCK-G5G4) the cell cultures avian influenza virus of 3 sialic acid acceptor overexpressions, we have selected respectively the clade2.3.2.1YZC3 of street strain strain (H5N1) and clade7.2wt-DT strain (H5N1), and the vaccine candidate strain rYZC3 (H5N1) and the rH5N1/PR8 (H5N1) that build cultivate on these two kinds of cells, and the HA that measures different time points tire (Fig. 6).Experimental result shows, virus 60h left and right HA in cell cultivation process tires and reaches peak value, and wherein, the HA peak value of 4 strain virus on MDCK-STGT2 cell tired all higher than maternal mdck cell, and the tire speed that rises of HA is faster.Show, avian influenza virus has better fecundity on the MDCK-hSiat7e-ST3GalIV of coexpression Siat7e and ST3GAL4 gene (MDCK-G5G4) cell.
TCID
50measure
Improve surperficial α 2 in order to detect, whether the sialic MDCK-hSiat7e-ST3GalIV cell of 3-is more suitable for the propagation of avian influenza virus, and we are chosen in H5N1 hypotype strain and select several strains to measure TCID
50(Fig. 7).Experimental result shows, the titre that H5N1 hypotype strain is measured on MDCK-hSiat7e-ST3GalIV cell is higher than the titre on maternal mdck cell (p<0.05), our MDCK-hSiat7e-ST3GalIV clone of building of preliminary explanation is because surperficial α 2, and 3-sialic acid abundance improves and is more suitable for the propagation of avian influenza virus than maternal mdck cell.
Claims (4)
1. coexpression of anthropogenic Siat7e and beta galactose glycosides α 2 are stablized in a strain, and the mdck cell of 3-sialytransferase IV is MDCK-hSiat7e-ST3GalIV, and its preserving number is CGMCC NO:9330.
2. the application of clone claimed in claim 1 in the growth titre that reduces the adherent performance of mdck cell and raising avian influenza virus strain isolated.
3. the application of clone claimed in claim 1 in the mensuration of the screening of Anti-avian influenza virus drugs, neutralizing antibody.
And clone claimed in claim 1 produce the application in Cell Culture Vaccine in the full suspension culture of the serum-free of MDCK.
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| CN116355857A (en) * | 2023-05-10 | 2023-06-30 | 北京华夏兴洋生物科技有限公司 | Suspension-cultured bovine kidney cells, and preparation method and application thereof |
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| CN102839157A (en) * | 2012-07-12 | 2012-12-26 | 扬州大学 | MDCK cell system capable of stably coexpressing Hst3GIIV and beta1-4GalT1 and establishing method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102839157A (en) * | 2012-07-12 | 2012-12-26 | 扬州大学 | MDCK cell system capable of stably coexpressing Hst3GIIV and beta1-4GalT1 and establishing method and application thereof |
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| Title |
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| MONTEERARAT Y 等: "Inhibition of H5N1 highly pathogenic influenza virus by suppressing a specific sialyltransferase", 《ARCH VIROL.》, vol. 155, no. 6, 30 June 2010 (2010-06-30) * |
| 张启龙 等: "共表达Siat7e和ST3GalI基因MDCK细胞株的建立", 《中国兽药杂志》 * |
| 张启龙 等: "共表达人Siat7e和ST3GalI基因MDCK细胞株的建立", 《中国兽药杂志》, vol. 48, no. 3, 31 March 2014 (2014-03-31) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116355857A (en) * | 2023-05-10 | 2023-06-30 | 北京华夏兴洋生物科技有限公司 | Suspension-cultured bovine kidney cells, and preparation method and application thereof |
| CN116355857B (en) * | 2023-05-10 | 2023-09-12 | 北京华夏兴洋生物科技有限公司 | Suspension-cultured bovine kidney cells, and preparation method and application thereof |
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