CN102839157A

CN102839157A - MDCK cell system capable of stably coexpressing Hst3GIIV and beta1-4GalT1 and establishing method and application thereof

Info

Publication number: CN102839157A
Application number: CN2012103744921A
Authority: CN
Inventors: 刘秀梵; 孙庆; 张文俊; 王晓泉; 胡顺林
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2012-07-12
Filing date: 2012-09-27
Publication date: 2012-12-26

Abstract

The present invention provides a MDCK-hST3GalIV-β1-4GalT1 cell line and a construction method thereof, in particular to transfecting the eukaryotic expression plasmid encoding the hST3GalIV gene sequence and the eukaryotic expression plasmid encoding the β1-4GalT1 gene sequence into MDCK cells, Finally, the MDCK cell line MDCK-hST3GalIV-β1-4GalT1 capable of stably expressing hST3GalIV and β1-4GalT1 genes was obtained, which can be used for large-scale cell culture and production of avian influenza virus vaccine strains.

Description

Stable coexpression hST3GalIV and β 1-4GalT1 mdck cell system and construction method and application

Technical field

The present invention relates to engineering cell system MDCK-hST3GalIV- β 4GalT1 and its construction method, constructed cell line can be used for the mass cell metaplasia production of avian influenza vaccine strain.

Background technology

The hemagglutinin (HA) of influenza surface is combined so as to initial viral host cells infected by the sialyloligosaccharide acceptor with host cell surface, fowl source and the partially thermophilic combination α 2 of horse source stream Influenza Virus, 3- sialyloligosaccharides acceptor (Neu5Ac α 2,3Gal), and people source and the partially thermophilic combination α 2 of pig source stream Influenza Virus, 6- sialyloligosaccharides acceptor (Neu5Ac α 2,6Gal).Therefore, the type and abundance of cell surface sialic acid receptor, adaptability of the influence influenza virus to different hosts.Different sialic acid receptor infected by influenza, which infects in host cell, replicates and spread, produces important influence.

What currently acquired approval was produced is the inactivated vaccine in chicken embryo source.The vaccine of mammalian cell production shows preferable protective effect, current Madin-Darby dogs renal epithelial cell system in animal model（MDCK）It is considered as to cultivate A types and the most suitable cell line of Type B influenza virus.

Traditional influenza virus vaccine is produced by chicken embryo, and virus is bred, concentrated, inactivating, being further purified by culture, and preparation, sterile filling finally detect and let pass.Except research and development use high yield recombinant strain as the kind poison of A type influenzas, basic production technique almost had no change at past 60 years.Not only cost price is high, and the residuals of chicken embryo are also possible to cause allergic reaction, in addition, when the whole world is very popular caused by Lingao pathogenic influenza virus face to face, traditional chicken embryo production method may be insufficient for the demand of global vaccine marketplaces.Therefore, research institution and vaccine company are developing new production technology cooperatively, to reach the purpose of time-saving and efficiency.In order to obtain the alternative of influenza vaccines production, vaccine company has had attempted to many new approach.The cell line of tissue cultures has caused the attention of people as a kind of supplement or alternative of the chicken embryo mode of production.Cell production systems mainly have the advantage that：1) basic skills (production of such as totivirus, multistep cracking, purity higher influenza antigens or attenuated live vaccine) of clinical verification can be obtained in chicken embryo production system using many, simultaneously, shorten the supply time of chicken embryo production, stable prod supply chain；2), due to the high consistency of Cells for production feature, the risk for introducing exogenous pollution can be reduced using the more advanced malicious production system of controllable kind；3) a kind of production of vaccine mode more efficiently, stably, repeatable is provided, can initial action at any time using a kind of variable format and the bioreactor of closing, it is possible to extend manufacture cycle as needed；4) ability that part avian influenza strain produces vaccine is improved.Except above-mentioned advantage, at least there is a kind of theoretic advantage with mammalian cell production vaccine：The separation and duplication of the influenza virus carried out in chicken embryo would generally produce the selection of particular phenotype, and this selection is often different from correct mankind's separation strains.On the contrary, the separation of the influenza virus carried out in cell, which is replicated, will not produce the Phenotypic Selection of this passage dependence.So as to be conducive to the haemagglutinin antigen for expressing native conformation, optimize the specificity of antibody.Renal tissue of the Madin-Darby dog renal epithelial cells system (MDCK) from an adult sleuth.The clinical test results for the Seasonal Influenza Vaccine originated on mdck cell show that the virus in mdck cell source is by that after continuous passage, can keep the stability of antigen, the security and immunogenicity of such vaccine are suitable with the vaccine that chicken embryo is originated.

People source beta galactose glycosides α 2, sialic acid (Neu5Ac) can be transferred to 2 type sugar chains (Gal β 1-4GlcNAc) of the end of glycoprotein by 3- sialyltransferases IV (hST3GalIV), or 1 galactolipin (Gal) on type sugar chain (Gal β 1-3GlcNAc), it is α 2-3 that key is connected between Neu5Ac and the Gal of end.HST3GalIV acts preferentially on 2 type sugar chains (Gal β 1-4GlcNAc), and next is only 1 type sugar chain (Gal β 1-3GlcNAc).

(the UDP- galactolipins of people source β 1-4 galactosyltransferases 1：2-Acetamido-2-deoxy-D-glucose base β 1-4 galactosyltransferases, referred to as β 1-4GalT1, EC2.4.1.38), it is one group of II type film combination glycoprotein, galactosyl (Gal) can be transferred on the N- acetyl glucosamines (GlcNAc) of sugar chain, company's key between Gal and GlcNAc is β 1-4.

The content of the invention

This research establishes mdck cell system -- the MDCK-hST3GalIV- β 4GalT1 cells for improving avian influenza virus α 2,3- sialic acid abundance first.Pass through stable coexpression hST3GalIV and β 1-4GalT1, β 1-4GalT1 catalysis galactolipins can be strengthened the effect that key is connected is connected with β 1-4 with N- acetyl glucosamines, hST3GalIV catalysis sialic acids connect the effect that key is connected with galactolipin with α 2-3, so as to improve mdck cell surface α 2,3- sialic acid abundance.Stable expression cell line cell surface α 2,3- sialic acids are significantly higher than maternal mdck cell system, compared with maternal mdck cell system, separation strains and the vaccine candidate strain of avian influenza virus form bigger plaque in MDCK-hST3GalIV- β 4GalT1 cell lines, and growth titre is higher.

After stable coexpression hST3GalIV and β 1-4GalT1 mdck cell system sets up, it is more suitable for the growth of avian influenza virus, either virus titer or plaque formation ability will be higher, avian influenza virus is also demonstrated to α 2, the inclined preferendum of 3- sialic acids, it is not only research α 2,3- sialic acid receptors provide convenient instrument, carry out receptor-binding characteristic, the appearance for detecting avian influenza virus variant for monitoring avian influenza virus in real time, find that the avian influenza strain that receptor-binding characteristic changes is with a wide range of applications.In addition, MDCK-hST3GalIV- β 4GalT1 cell lines all show preferable application prospect in terms of Anti-avian influenza virus drugs, vaccine strain screening and the production of Cell Culture Vaccine.

The present invention stable expression people source beta galactose glycosides α 2,3- sialyltransferases IV (hST3GalIV) and β 1-4 galactosyl transferases (β 1-4GalT1) mdck cell system, are MDCK-hST3GalIV- β 4GalT1；Preserving number is：CGMCC No.5967.The gene of gene and coding β 1-4GalT1 of the described MDCK-hST3GalIV- β 4GalT1 cell lines simultaneously containing coding hST3GalIV.The gene order such as SEQ ID No of the coding hST3GalIV:Shown by 1, the amino acid sequence such as SEQ ID No of the hST3GalIV coded by it:Shown by 3；The gene order such as SEQ ID No of the coding β 1-4GalT1:Shown by 2, the amino acid sequence such as SEQ ID No of the β 1-4GalT1 coded by it:Shown by 4.The coding hST3GalIV gene orders and β 1-4GalT1 gene orders come from people.

Another aspect of the present invention includes in the genome of hST3GalIV and β 1-4GalT1 genetic recombination to mdck cell there is provided the method for building MDCK-hST3GalIV- β 4GalT1 cell lines, methods described.

The construction method of the MDCK-hST3GalIV- β 4GalT1 cell lines, it is that, to mdck cell, final obtain can stablize the cell line MDCK-hST3GalIV- β 4GalT1 for expressing hST3GalIV and β 1-4GalT1 genes by the eukaryon expression plasmid with coding hST3GalIV gene orders and the eukaryotic expression plasmids with coding β 1-4GalT1 gene orders.

In another aspect of the present invention, a kind of method for improving avian influenza virus growth titre is provided, this method includes, H5N1 hypotypes or the strain of H9N2 subtype avian influenza virus are inoculated in the MDCK-hST3GalIV- β 4GalT1 cell lines that the present invention is built, condition of culture of the virus in the cell line is 35 DEG C, 5%CO2.Strain grows the raising of titre in MDCK-hST3GalIV- β 4GalT1 cell lines, and advantage is provided for the cellularised production of avian influenza vaccine.

More specifically, in one embodiment of the invention, the carrier for expression of eukaryon of hST3GalIV genes and the eukaryotic expression vector transfection of β 1-4GalT1 genes will be encoded into mdck cell system, build stable coexpression hST3GalIV and β 1-4GalT1 mdck cell system, one plant of expression hST3GalIV and β 1-4GalT1 Madin-Darby MDCKs system MDCK-hST3GalIV- β 4GalT1 cell clones were preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC April 10 in 2012 in the present invention, China, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica), preserving number is CGMCC No.5967.

It our experiments show that, α 2 in the MDCK-hST3GalIV- β 4GalT1 cells, 3- sialic acid receptor contents are significantly higher than maternal mdck cell, the ability that avian influenza virus is bred in MDCK-hST3GalIV- β 4GalT1 cell lines of the present invention is significantly higher than maternal mdck cell and MDCK-hST3GalIV, and the titre that avian influenza virus is determined on MDCK-hST3GalIV- β 4GalT1 cells is above the titre on maternal mdck cell and MDCK-hST3GalIV cells.It can be used for improving avian influenza virus yield using MDCK-hST3GalIV- β 4GalT1 cell lines and avoid the side reaction effect that the residue of chicken embryo in the inactivated vaccine of chicken embryo source is brought, available for α 2, the mass cell culture and production of the virus vaccine strain of 3- sialic acid receptor abilities.

Brief description of the drawings

Fig. 1:Mdck cell survivorship curve under different G418 concentration

Abscissa：Time (number of days)

Ordinate：Cell survival rate (%)

The concentration that cell is all killed in 10 ~ 14 days is selected to be used as G418 screening concentration, i.e. 0.8mg/mL.

Fig. 2:MDCK and MDCK-hST3GalIV- β 4GalT1 cell RT-PCR qualification results

(A) electrophoresis result of ST3GalIV genes, 1：MDCK-hST3GalIV- β 4GalT1 cells, 2：Maternal mdck cell, M：200bp Marker；

(B) electrophoresis result of β 1-4GalT1 genes, 1：MDCK-hST3GalIV- β 4GalT1 cells, 2：Maternal mdck cell, M：200bp Marker.

Fig. 3:The combination of Flow cytometry difference mdck cell surface α 2,3- sialic acid receptors and specific agglutination element MAA.

(A) mdck cell blank control；Mdck cell is combined with MAA；MDCK-ST cells are combined with MAA；MDCK-STGT cells are combined with MAA；

(B) comparison of the 3 detection fruit average value of combination of different mdck cell surface α 2,3- sialic acid receptors and MAA.

(*) represents there is significant difference (p compared with MDCK-MAA groups<0.05)；

(* *) represents to be respectively provided with significant difference (p compared with MDCK MAA and MDCK-ST MAA groups<0.05).

Note：

MDCK-ST represents MDCK-ST3GalV cell lines；

MDCK-STGT represents MDCK-hST3GalIV- β 4GalT1 cell lines.

Fig. 4:Reproduction curve of the avian influenza virus on mdck cell, MDCK-ST3GalV cells and MDCK-hST3GalIV- β 4GalT1 cells.

(A) reproduction curves of the LH5N1 on three kinds of cells；

(B) reproduction curves of the LR38/H5N1 on three kinds of cells；

(C) reproduction curves of the F/H9N2 on three kinds of cells.

Note：

M represents mdck cell system；

ST represents MDCK-ST3GalV cell lines；

STGT represents MDCK-hST3GalIV- β 4GalT1 cell lines.

Fig. 5:Infection titer (TCID50) of the different fowl influenza virus strains on maternal mdck cell, MDCK-ST3GalIV cells and MDCK-hST3GalIV- β 4GalT1 cells.

(*) represents that MDCK-hST3GalIV- β 4GalT1 cells have significant difference (p compared with maternal mdck cell measurement result<0.05)；

(* *) represents that MDCK-hST3GalIV- β 4GalT1 cells are respectively provided with significant difference (p compared with maternal mdck cell and MDCK-ST3GalV cells<0.05).

Note：

MDCK-ST3 represents MDCK-ST3GalIV cells；

MDCK-STGT2 represents MDCK-hST3GalIV- β 4GalT1 cells.

Fig. 6:Avian influenza virus is determined in the PFU on maternal mdck cell, MDCK-ST3GalIV cells and MDCK-hST3GalIV- β 4GalT1 cells.

(* *) represents that MDCK-hST3GalIV- β 4GalT1 cells are respectively provided with significant difference (p compared with maternal mdck cell and MDCK-ST3GalIV cells<0.05).

Note：

MDCK-ST represents MDCK-ST3GalIV cells；

MDCK-STGT represents MDCK-hST3GalIV- β 4GalT1 cells.

Embodiment

The present invention is described in detail hereinafter with reference to embodiment and accompanying drawing, and the implementation is merely intended to illustrate the present invention, and the scope being not intended to limit the present invention, the scope of the present invention is specifically limited by appended claims.

The structure of embodiment 1MDCK-hST3GalIV cell lines and MDCK-hST3GalIV- β 4GalT1 cell lines

1. materials and methods

1.1 reagents and instrument

The eukaryon expression plasmid of ST3GalIV and β the 1-4GalT1 ORFs of source containing people is purchased from Genecopoeia companies (article No.：EX-C0523-M02 and EX-C0234-M02)；DMEM culture mediums and serum-free Opti-MEM are purchased from Invitrogen companies；Hyclone is purchased from Hyclone companies；Small amount plasmid extraction kit Plasmid Miniprep Kit are purchased from Qiagen companies；RNA extracts kits are purchased from TaKaRa companies；

HD transfection reagents, purchased from Roche companies；G418 (40mg/mL) solution is purchased from Shanghai Sheng Gong bioengineering Co., Ltd.

Mdck cell is purchased from the American Type Culture Collection committee of Chinese Academy of Sciences cell bank.

Common inverted microscope and fluorescence inverted microscope are Leica products；Spectrophotometric is calculated as NanoDrop companies ND1000；Gel imaging system is genome company's product.

1.2 design of primers

Primer for expanding people source beta galactose glycosides α 2,3- sialyltransferases IV (hST3GalIV)：

3G4F：5’-ATGGTCAGCAAGTCCCGCTGGAAG-3’（SEQ ID No:5）

3G4R：5’-TCAGAAGGACGTGAGGTTCTTG-3’（SEQ ID No:6）

Primer for expanding people source β 1-4 galactosyl transferases 1 (β 1-4GalT1)：

B4G1F：5’-ATGAGGCTTCGGGAGCCGCTCCTGAG-3’（SEQ ID No:7）

B4G1R：5’-CTAGCTCGGTGTCCCGATGTCCAC-3’（SEQ ID No:8）.

1.3 determine suitable G418 concentration in screening and culturing medium

Mdck cell is inoculated with 48 porocyte culture plates and adds the geneticin containing various concentrations when length is to 80-90% abundance（G418）Culture medium：0th, 0.1,0.4,0.6,0.8,1,1.5 and 2mg/mL, puts 37 DEG C, 5%CO2 incubator cultures, observes daily and record cell growth status.The screening concentration that cell growth curve determines G418 is drawn in culture after 14 days.

1.4HD transfects the screening of mdck cell and resistant cell colonies

Inoculation about 2 × 10^-5Mdck cell is to 6 porocyte plates, and cell density when 80%~90% to can be used for transfecting.Will

HD transfection reagents and plasmid solution equilibrate to room temperature, prepare following mixed liquor in centrifuge tube：By the eukaryon expression plasmid hST3GAL4 of encoding ss-galactosidase glycosides α 2,3- sialyltransferase IV gene orders and the eukaryon expression plasmid β 1-4GalT1 of the gene order of coding β 1,4- galactosyl transferases 1（It is purchased from Genecopoeia companies）Each 1 μ g are added in 100 μ L serum-free Opti-MEM culture mediums, after mixing, then add 5 μ L

HD transfection reagents are into above-mentioned mixed liquor, after fully mixing, and are incubated at room temperature 15 minutes, while being washed twice with serum-free Opti-MEM culture mediums.Above-mentioned compound is covered in cell surface, it is 2mL to add serum-free Opti-MEM culture mediums to cumulative volume, is placed in 37 DEG C, 5%CO₂Culture is removed after being cultivated 24 hours in incubator, the fresh DMEM culture mediums containing 10% hyclone is replaced by and continues to cultivate.Trypsin digestion cell is used after 36 hours, by 1:10 ratio is passaged in 48 porocyte plates, it is screening and culturing medium culture to continue with the 10% hyclone DMEM culture mediums of the G418 containing 0.8mg/mL, per the μ L of hole 200, changes primary screening culture medium within every 3 days, G418 is persistently screened after 10-14 days, it is seen that resistant cell growth.Without the groups of cells of plasmid transfection as negative control, cell is cultivated in the nutrient solution containing G418 to death.Growth selection drug-resistant colonies in good condition, continue on for monoclonal cell screening：Cell density is diluted to 1/100 μ L with the screening and culturing medium of the G418 containing 0.8mg/mL, 50 μ L in 96 porocyte plates are laid on, 50 μ L screening and culturing mediums are added per hole.Selection expression highest clone carries out monoclonal screening by limiting dilution assay again after persistently screening 10 days, to ensure to filter out monoclonal resisting cell.The cell filtered out expands in 96 holes, 24 holes and 6 porocyte culture plates successively to be cultivated and freezes in liquid nitrogen.

1.5RT-PCR identification

The cell line and maternal mdck cell filtered out is respectively in 6 porocyte culture plates, and every plant of cell spreads a hole, and the DMEM culture medium adhere-wall cultures of 10% hyclone are contained with 2.5mL.When cell length to 90-100% degree of converging, cell is collected, PBS is washed twice, 1mL0.25% pancreatin is added, 37 DEG C digest 5 minutes, add 1mL10%DMEM piping and druming, even cell suspension will be blown and be transferred to centrifugation 1000rpm centrifugations 10 minutes, abandoning supernatant in centrifuge tube, PBS is added and blow even, it is transferred in 1.5mL centrifuge tubes, 2000rpm is centrifuged 5 minutes, abandoning supernatant, and blot PBS, 1mL Trizol are added, are mixed, are placed 10 minutes on ice.200 μ L precooling chloroforms are added, are shaken 15 seconds, room temperature is placed 2 minutes；4 DEG C of 12000rpm are centrifuged 10 minutes；Draw upper water and make an appointment 500 μ L to 1.5mL centrifuge tubes, add the 500 pre- cold isopropanols of μ L, mix, -20 DEG C are placed 15 minutes；4 DEG C of 12000rpm are centrifuged 10 minutes；Abandoning supernatant, adds 1mL75% pre-cooled ethanols washing precipitation；4 DEG C of 12000rpm are centrifuged 3 minutes；Abandoning supernatant, adds 1mL precoolings absolute ethyl alcohol and washs precipitation again；4 DEG C of 12000rpm are centrifuged 3 minutes；Liquid is exhausted, room temperature dries precipitation in 10 minutes；Add 50 μ L ddH₂O dissolves RNA.All pipette tips, centrifuge tube, ddH are tested above₂O and 75% pre-cooled ethanol are handled and high pressure with DEPC, and whole process is worn gloves and tested.

The RNA for taking 2 μ L to dissolve, RNA concentration and A260/A280 are detected with spectrophotometer ND1000.The total serum IgE of proposition forms cDNA by following system reverse transcription, and step is as follows：Be firstly added the μ L of total rna solution 22, the Oligo dT3 μ L of extraction, blow it is even after, be placed in 70 DEG C of metal baths and act on 5 minutes rearmounted ice baths, add following reagent：The μ L of RNase inhibitor 2, μ L and dNTP the Mix4 μ L of 5 × buffer solution, 8 μ L, M-MuLV reverse transcriptase 1, total reaction volume are 40 μ L, are placed in 37 DEG C of water-baths and are incubated 2 ~ 3 hours, then start RT-PCR reactions, make its reverse transcription into cDNA.

Using reverse transcription into cDNA as template, pass through PCR reaction amplifications people source beta galactose glycosides α 2,3- sialyltransferases IV (hST3GalIV) gene by primer of 3G4F and 3G4R；B4G1F and B4G1R is (β 1-4GalT1) gene of primer amplification β 1-4 galactosyl transferases 1.PCR reaction cycle parameters：94 DEG C of pre-degenerations 3 minutes；94 DEG C are denatured 45 seconds, and 55 DEG C are annealed 45 seconds, and 72 DEG C extend 90 seconds, totally 30 circulations.1% Ago-Gel is prepared, and is dyed with EB, 10 μ LPCR products are added per hole, electrophoresis, gel imaging system detection electrophoresis result are carried out under 100V voltages.After PCR primer is reclaimed through glue reclaim kit, serve Hai Shenggong bioengineering Co., Ltd and go sequencing.

2. result

The determination of 2.1 culture medium G418 working concentrations

We select all kill the G418 concentration of mdck cell as screening concentration in 10-14 days.According to cell growth curve (Fig. 1), when G418 concentration is between 0.8 ~ 1.0mg/mL, cell is all dead in 10~14 days.Therefore, we select 0.8mg/mL to be the concentration of G418 in pressure screening and culturing medium in this experiment.

2.2RT-PCR qualification result

From RT-PCR results (Fig. 2), the cell line of one plant of screening amplifies 1002bp band, and band is single and clear, in the same size with target gene (hST3GalIV)；The cell line of another plant of screening it is amplifiable go out two band 1002bp band and 1197bp band, it is in the same size with target gene (hST3GalIV and β 1-4GalT1) respectively.And maternal mdck cell has not seen the band of amplification in control group.Illustrate that purpose band has been incorporated into mdck cell by we.This two plants of mdck cells are conventionally expanded and frozen.We, which divide, names the mdck cell for only expanding a gene for MDCK-ST3GalIV cells, and the mdck cell for amplifying two genes is named as MDCK-hST3GalIV- β 4GalT1 cells.

The screening of 2.3 aim cells

After normal mdck cell transfected plasmids, screened using the DMEM culture mediums containing 0.8mg/mL G418 and 10% hyclone, obtain resistant cell clone.In the culture medium containing G418, the cell death of untransfected.The resisting cell of acquisition is mixed after clone carries out limiting dilution, identification selection with G418 and obtains the strain of monoclonal resisting cell.

In the present invention, we carry out RT-PCR identifications to the MDCK-ST3GalIV cells and MDCK-hST3GalIV- β 4GalT1 cells that build, passage cell strain can be continued under without the screening of G418 pressure simultaneously, as a result show that foreign gene can stablize expression at least 10 more than generation.These qualification results show, in resulting MDCK-ST3GalIV cell monoclonal cell lines, and ST3GalIV gene open reading frame sequences cDNA has been incorporated into mdck cell genome, can be passed on and stable constantly transcript and expression with mdck cell system；In MDCK-hST3GalIV- β 4GalT1 cells, people source ST3GalIV and people source β 1-4GalT1 gene open reading frame sequences cDNA have been incorporated into mdck cell genome, can be passed on and stable constantly transcript and expression with mdck cell system.

The present invention successfully builds one plant of stable MDCK-ST3GalIV cell line for expressing people source ST3GalIV genes and one plant of stable expression people source ST3GalIV and people source β 1-4GalT1 MDCK-hST3GalIV- β 4GalT1 cell lines first, to improve the α 2 of cell surface, 3- sialic acid abundance.The foundation of MDCK-hST3GalIV- β 4GalT1 stable cell lines, it is not only avian influenza virus α 2,3- sialic acids tendency Journal of Sex Research provides important research tool, for the monitoring of avian influenza virus receptor binding specificities variant, Anti-avian influenza virus drugs screening, the measure of neutralizing antibody and the production application for mass producing cell culture avian influenza virus vaccine, it is with a wide range of applications.

The expression of the flow cytomery cell surface α 2,3- sialic acids of embodiment 2

1 materials and methods

1.1 reagents and instrument

Digoxigenin labeled sugar identification reagent box (DIG Glycan Differentiation Kit) and Anti-Digoxigenin-Fluorescein (Anti-DIG FITC) are purchased from Roche companies；Flow cytometer FACSAria is U.S. company BD product.

The expression of 1.2 FCM analysis cell surface sialic acids

Maternal MDCK is laid on each 3 holes of 6 porocyte plates respectively with transfection mdck cell.(about 1.5 × 10 during cell length to 90% abundance⁶Individual cells/well), pancreatin digestion adds a small amount of 10%DMEM, collects respectively per hole cell into EP pipes, 1400rpm is centrifuged 8 minutes, collects cell precipitation.It is resuspended and is washed twice with PBS, then with Buffer I (50mM Tris-HCl, 0.15M NaCl, 1mM MgCl₂、1mM MnCl₂、1mM CaCl₂, pH7.5) wash once.With the closing dilution configured【Close the preparation of dilution：10 times of dilution confining liquids are in TBS (0.05M Tris-HCl, 0.15M NaCl, pH7.5)】It is uniform that cell precipitation is resuspended, closed 1 hour on ice.Repeated washing step, then the MAA processing cells marked respectively with an anti-dig as stated above, while setting one group of blank control, that is, is not added with primary antibody afterwards.The method of one process resistant is：Plus 4 μ L MAA uniform in 30 μ L Buffer I cell precipitation, ice bath 1 hour is resuspended.Repeated washing step, adds secondary antibody, i.e. 1 μ L Anti-DIG FITC uniform resuspension cell precipitation, ice bath 1 hour in 30 μ l Buffer I, last PBS, which washs 3 times, is used for flow cytomery.

2. result

In order to detect α 2, content of the 3- sialic acid receptors on maternal mdck cell, MDCK-ST3GalIV cells and MDCK-hST3GalIV- β 4GalT1 cells, we use the α 2 that digoxin (DIG) is marked, 3- sialic acids specific agglutination element, Korea's sophora japonica lectin (MAA) is used as secondary antibody as primary antibody, the anti-dig antibody of FITC marks.Korea's sophora japonica lectin (MAA) can specifically combine SA α -2,3Gal.Testing result is shown, α 2 in MDCK-ST3GalIV cells, the content of 3- sialic acid receptors is significantly higher than maternal mdck cell, and the content of α 2 in MDCK-hST3GalIV- β 4GalT1 cells, 3- sialic acid receptor is all remarkably higher than maternal mdck cell and MDCK-ST3GalIV cells (Fig. 3).Fluorescence intensity Dao Shuo areas peak figure is displayed that, compared with maternal mdck cell, the Dao Shuo areas of MDCK-ST3GalIV cells and MDCK-hST3GalIV- β 4GalT1 cells are offset to the right, and the offset of MDCK-hST3GalIV- β 4GalT1 cells is more (Fig. 3).Test result indicates that, the stable expression in MDCK-hST3GalIV cells of ST3GalV genes, cell surface α 2, the abundance of 3- sialic acid receptors are improved, and people source ST3GalV genes are co-expressed with β 1-4GalT1 genes in MDCK-hST3GalIV- β 4GalT1 cells（Fig. 3）, further increase the α 2 on its surface, 3- sialic acid receptor abundance.

Embodiment 3TCID₅₀Measure and Virus plaque measure

1 materials and methods

1.1 strains and reagent

H5N1:Street strain A/Mallard/Huadong/lk/2005 (L plants) preserves (Zhang W J by the veterinary college Ministry of Agriculture of Yangzhou University livestock and poultry Infectious Diseases Lab, Xue T, Wu X W, Zhang P H, Zhao G, Peng D X, Hu S L, Wang X Q, Liu X W, Liu W B, Liu X F.Increase in viral yield in eggs and MDCK cells of reassortant H5N1vaccine candidate viruses caused by insertion of 38amino acids into the NA stalk.Vaccine, 2011,29:8032-8041.) applicant promises to undertake provides 20 years from the applying date to the public.

The strain of LR38 (H5N1) vaccine candidate:Restructuring low virulent strain (the Zhang W J that reverse Genetics Technique is built are utilized for the veterinary college Ministry of Agriculture of Yangzhou University livestock and poultry Infectious Diseases Lab, Xue T, Wu X W, Zhang P H, Zhao G, Peng D X, Hu S L, Wang X Q, Liu X W, Liu W B, Liu X F.Increase in viral yield in eggs and MDCK cells of reassortant H5N1vaccine candidate viruses caused by insertion of 38amino acids into the NA stalk.Vaccine, 2011,29:8032-8041.), HA and NA gene sources are promised to undertake in street strain A/Mallard/Huadong/lk/2005. applicant and provided 20 years to the public from the applying date.

H9N2:Street strain A/Chicken/Shanghai/F/98 (F plants) is preserved by the veterinary college Ministry of Agriculture of Yangzhou University livestock and poultry Infectious Diseases Lab（Shi Huoying,Liu XiuFan,ZhangXiaorong,et al.Generation of an attenuated H5N1avian influenza virusvaccine with all eight genes from avian viruses,Vaccine,25:7379-7384.）Applicant promises to undertake to be provided 20 years from the applying date to the public.

The trypsase of TPCK modifications is purchased from Sigma companies.

1.2TCID₅₀Measure

Maternal MDCK, MDCK-ST3GalIV and MDCK-hST3GalIV- β 4GalT1 cells are laid on 96 orifice plates respectively, are washed twice with serum-free DMEM when cell density is to 90%, wash away dead cell and the serum of residual, then virus inoculation.Virus liquid is made into 10 times of gradient dilutions with serum-free DMEM in the centrifuge tube of ice bath, from 10^-1~10^-10.The trypsase of TPCK modifications is added in viral dilution liquid simultaneously, makes its final concentration of 1 μ g/mL.Viral dilution liquid is inoculated into 96 orifice plates, each dilution factor is inoculated with 8 holes, and 100 μ L/ holes are subsequently placed in 35 DEG C, 5%CO₂Incubator adsorb 1 hour, just shaken every 20 minutes it is even once.After absorption 1 hour, the viral dilution liquid for abandoning inoculation is inhaled, serum-free DMEM is added, per the μ L of hole 200, while also adding the trypsase of TPCK modifications, makes its final concentration of 1 μ g/mL.H5N1 hypotypes street strain cultivates 48 hours, H5N1 hypotypes recombinate low virulent strain and H9N2 hypotypes street strain cultivates and draws within 72 hours the μ L of supernatant 50 respectively afterwards into 96 hole blood-coagulation-boards, add isometric 1% chicken erythrocyte suspension, the blood clotting result per hole is observed after 30 minutes, the presence or absence of cytopathy is represented with the presence or absence of hemagglutination activity, tissue culture infective dose (50%tissue culture infective dose, TCID are calculated by Reed-Muench methods₅₀) result.

1.3 Virus plaque

Maternal mdck cell, MDCK-ST3GalIV cells and MDCK-hST3GalIV- β 4GalT1 cells are laid in 6 porocyte culture plates respectively, when cell density is to 90%, growth-promoting media is discarded, is washed twice with serum-free DMEM, dead cell and the serum of residual are washed away, then virus inoculation.With serum-free DMEM dilution virus storage liquid (10^-1~10^-8), the trypsase of the μ g/mL TPCK of final concentration 1 modifications is added, each dilution factor is inoculated with 3 holes, gently rocks 6 porocyte culture plates, the viral dilution liquid of addition is uniformly laid on cell surface, be placed in 35 DEG C, 5%CO₂Incubator is adsorbed 1 hour.After absorption 1 hour, the viral dilution liquid for abandoning inoculation is inhaled, is washed twice with serum-free DMEM；Add first layer covering liquid (1.6% agar：2 times of 2%DMEM=1：1) cell surface, is laid on after mixing to be allowed to be completely covered, the trypsase of addition final concentration 1 μ g/mL TPCK modifications in H5N1 hypotypes restructuring low virulent strain and H9N2 hypotypes street strain coagulant liquid, 1.5mL/ hole, after solidification, Tissue Culture Plate is inverted, 35 DEG C, 5%CO is placed in₂Incubator culture.Tissue Culture Plate is taken out after 48 ~ 72 hours, second layer covering liquid (16% agar is added：2 times of 2%DMEM：1 ‰ dimethyl diaminophenazine chloride=9：9：2), per hole 1.5mL, after solidification, Tissue Culture Plate is inverted, 35 DEG C, 5%CO is placed in₂Incubator culture.Tissue Culture Plate is taken out after 24 ~ 48 hours, plaque is counted, the infection titer of virus is represented with plaque forming unit (Plaque forming unit, PFU).

2. result

2.1TCID₅₀Measurement result

Surface α 2 is improved in order to detect, whether the MDCK-hST3GalIV- β 4GalT1 cells of 3- sialic acids are more suitable for the propagation of avian influenza virus, we select to select several plants of measure TCID in H5N1 hypotypes and H9N2 hypotype strains₅₀(Fig. 5).Test result indicates that, the titre that whether H5N1 hypotypes or H9N2 hypotypes strain are determined on MDCK-hST3GalW- β 4GalT1 cells is above the titre (p on maternal mdck cell and MDCK-hST3GalIV cells<0.05) the MDCK-hST3GalIV- β 4GalT1 cell lines of our structures, are tentatively illustrated because the α 2 on surface, 3- sialic acids abundance improves and be more suitable for than maternal mdck cell and MDCK-hST3GalIV cells the propagation of avian influenza virus.

2.2 Virus plaque

Test result indicates that, three plants of avian influenza virus can form plaque on maternal mdck cell, MDCK-ST3GalIV cells and MDCK-hST3GalIV- β 4GalT1 cells, but plaque individual more plastidogenetic than other two is bigger on MDCK-hST3GalIV- β 4GalT1 cells, the hole of identical viral dilution, there is the more of plaque, the time is also earlier.PFU determine result show, the result highest (Fig. 6) that virus of the same race is determined on MDCK-hST3GalIV- β 4GalT1 cells.Virus plaque experimental result further demonstrates that the MDCK-hST3GalIV- β 4GalT1 cell lines that we build are more suitable for the propagation of avian influenza virus.

TCID50 and the result of plaque formation experiment show, the MDCK-hST3GalIV- β 4GalT1 cells of coexpression of anthropogenic ST3GalIV and β 1-4GalT1 genes are significantly higher than maternal mdck cell system and single expression hST3GalIV MDCK-hST3GalIV cell lines to the sensitiveness of avian influenza virus, and MDCK-hST3GalIV- β 4GalT1 cell lines are more suitable for the propagation of avian influenza virus.

SEQUENCE LISTING

<110>Yangzhou University

<120>Stable coexpression hST3GalIV and β 1-4GalT1 mdck cell system and construction method and application

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aactactccc gggatcagcc catcttcctg cggcttgagg attatttctg ggtcaagacg 240

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Claims

1. A MDCK cell line stably expressing hST3GalIV and β1-4GalT1 is MDCK-hST3GalIV-β4GalT1, which contains hST3GalIV and β1-4GalT1 gene sequences; the gene sequence of the hST3GalIV is as shown in SEQ ID No: 1, the The gene sequence of β1-4GalT1 is shown in SEQ ID No: 3; the deposit number of the cell line is CGMCC No. 5967.

2. the construction method of the MDCK cell line that stably expresses hST3GalIV and β1-4GalT1 according to claim 1 is characterized in that, the eukaryotic expression plasmid with the gene sequence encoding hST3GalIV and the gene sequence with the β1-4GalT1 gene sequence The eukaryotic expression plasmid was transfected into MDCK cells, and finally the cell line MDCK-hST3GalIV-β4GalT1 which could stably express hST3GalIV and β1-4GalT1 genes was obtained.

3. The method for constructing the MDCK-hST3GalIV-β4GalT1 cell line according to claim 2, characterized in that it comprises the following specific steps:

1) Transfection of MDCK cells and screening of resistant cell clones

Transfection: MDCK cells were used for transfection when the density of MDCK cells reached 80% to 90% in 6-well plates; each of the eukaryotic expression plasmids encoding the hST3GalIV gene sequence and the eukaryotic expression plasmid encoding the β1-4GalT1 gene sequence were placed in 100 In μL serum-free Opti-MEM medium, add 5 μL FuGENE ^® HD transfection reagent after mixing, mix well and incubate at room temperature for 15 minutes; cover the above complex on the cell surface, add serum-free Opti-MEM medium To a total volume of 2 mL, culture in a 37°C, 5% CO ₂ incubator for 24 hours, remove the culture, and replace it with fresh DMEM medium containing 10% fetal bovine serum to continue the culture;

Screening of resistant cell clones: Digest cells with trypsin 36 hours after transfection, passage to 48-well cell plates at a ratio of 1:10, and continue to use 10% fetal bovine serum DMEM medium containing 0.8 mg/mL G418 For screening medium culture, 200 per well μL, change the selection medium every 3 days. After G418 continuous selection for 10-14 days, the growth of resistant cells can be seen; select resistant cell colonies with good growth status and continue to use for monoclonal cell selection: use a medium containing 0.8 mg/mL G418 selection medium Dilute the cell density to 1 cell/100 μL, spread 50 μL in a 96-well cell plate, add 50 μL selection medium to each well; select the clone with the highest expression level after continuous selection for 10 days and press the limiting dilution again Monoclonal screening was carried out to ensure the selection of monoclonal resistant cells; the selected cells were expanded and cultured in 96-well, 24-well and 6-well cell culture plates in turn and frozen in liquid nitrogen;

2) Identification of MDCK-hST3GalIV-β4GalT1 cell line

Extract the RNA of the above-mentioned resistant cells, and reverse transcribe to form cDNA, use the reverse transcribed cDNA as a template, and use the PCR method to amplify human β-galactoside α2,3-sialic acid by PCR respectively Transferase IV gene and β1-4 galactosyltransferase 1 gene; the amplification results show that the MDCK-hST3GalIV-β4GalT1 cell line has been successfully constructed,

Primers used to amplify human β-galactoside α2,3-sialyltransferase IV (hST3GalIV):

3G4F: 5'- ATGGTCAGCAAGTCCCGCTGGAAG-3'

3G4R: 5'-TCAGAAGGACGTGAGGTTCTTG-3'

Primers used to amplify human β1-4 galactosyltransferase 1 (β1-4GalT1):

B4G1F: 5'- ATGAGGCTTCGGGAGCCGCTCCTGAG-3'

B4G1R: 5'- CTAGCTCGGTGTCCCGATGTCCAC-3'.

4. The MDCK-hST3GalIV-β1-4GalT1 cell line according to claim 1 is used to improve the growth titer and plaque formation ability of avian influenza virus isolates.

5. The application of the MDCK-hST3GalIV-β1-4GalT1 cell line described in claim 1 in the production of vaccines.