CN103937748B - The unicellular from the strain of suspension growth mdck cell and its construction method and application of expression people's TMPRSS2 albumen can be stablized - Google Patents

Abstract

Stablize the unicellular mdck cell strain from suspension growth for expressing people's TMPRSS2 albumen, construction method and its application in avian influenza virus are cultivated the present invention relates to a kind of.The invention belongs to biological technical field.MDCK Sus TMPRSS2 are named as from the strain of suspension growth mdck cell the invention discloses a kind of the unicellular of stably expression people's TMPRSS2 albumen, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved in, the preserving number of its cell line is：CGMCC No.8851.Simultaneously the invention provides the construction method of this cell line, and its application mode in avian influenza virus Multiplying culture.It is achieved thereby that the trypsase of TPCK treatment need not be added in avian influenza virus Multiplying culture, so as to efficiently solve irreversible influence of the exogenous protease on host cell growth, the propagation efficiency of avian influenza virus is significantly improved.

Description

The unicellular from the strain of suspension growth mdck cell of expression people's TMPRSS2 albumen can be stablized And its construction method and application

Technical field

Unicellular from the strain of suspension growth mdck cell and its application the present invention relates to one kind, more particularly to one kind can stablize table The unicellular mdck cell strain from suspension growth of intelligent's TMPRSS2 albumen, construction method and its in culture avian influenza virus In application.The invention belongs to biological technical field.

Background technology

Substitute SPF chicken embryos using continuous cell line at present carries out the extensive of avian influenza virus in bioreactor Propagation preparation is the study hotspot that R＆D institution or live vaccine manufacturing enterprise competitively carry out.

The prerequisite that the HA albumen of avian influenza virus particle surface is combined with virus receptor on host cell is HA albumen In its basic amino acid（Lys or Arg）Site is effectively cracked, and HA1 albumen and HA2 albumen is formed, by two between both Sulfide linkage is connected.HA1 albumen is responsible for recognizing the sugar chain acceptor on host cell membrane that the N-terminal of HA2 albumen has one section of fusogenic peptide to be responsible for disease Malicious cyst membrane is merged with host cell membrane, in H⁺Ion channel is formed by the M2 albumen of virus under attack and helps viral genetic Discharged to intracellular, the subsequent processes such as reverse transcription, duplication, accurate translation are completed subsequently into nucleus.Avian influenza virus can be The major reason bred in SPF chicken embryos be exactly containing substantial amounts of protease in chick embryo allantoic liquid, can be to virion sublist The HA albumen in face is cracked.And breed one in the zooblast that influenza virus is cultivated in vitro, such as mdck cell, Vero cells As be required for being added in virus propagation process the trypsase processed through TPCK.This protease weakens chymotrypsin Activity, and enhance to basic amino acid（Such as Arg, lys）The cracking ability in site such that it is able to ensure avian influenza virus HA The correct cracking of albumen.

But the addition of exogenous protease is irreversible to the influence that the growth conditions of host cell are caused, and is particularly planted Protease accumulation after malicious continuous passage is extremely serious on the influence of host cell physiological status, and then influences the increasing of avian influenza virus Grow efficiency.

Goal of the invention

The invention discloses it is a kind of stablize expression people's TMPRSS2 albumen it is unicellular from suspension growth mdck cell strain and Its construction method, and application of this cell line in avian influenza virus Multiplying culture.

The invention particularly discloses following technical scheme：

Stablize the unicellular from the strain of suspension growth mdck cell of expression people's TMPRSS2 albumen the invention discloses a kind of, MDCK-Sus-TMPRSS2 is named as, Classification And Nomenclature is restructuring MDCK-Sus-TMPRSS2 cells, is preserved in China Microbiological bacterium Preservation administration committee common micro-organisms center is planted, address exists, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences Institute of microbiology.The preserving number of its cell line is：CGMCC No.8851.Preservation date is on March 6th, 2014.

Here TMPRSS2 refers to II type transmembrane serine protease（typeⅡ transmembrane protease serines, TMPRSS2）It is a newly discovered class Special Proteins enzyme, the albumen is successively intracellular domain from N-terminal toward C-terminal （cyto.D）, membrane spaning domain (TM), trunk region（Low-density lipoprotein A receptoroids area LDLRA, cleaning the street rich in cysteine Husband receptor area SRCR, prodomain Pro）, digestion domain (catalytic domain).Wherein transmembrane region is incorporated into cell membrane On, trunk region and digestion domain then expression and localization in the outside of cell membrane, the function with proteopepsis enzymolysis.Its albumen Gene order is as shown in SEQ ID NO.1.

Meanwhile, it is the cell for being adapted to full suspension growth that the present invention is further disclosed the MDCK-Sus-TMPRSS2 Strain.This technical characteristic is mainly the mdck cell of the adherent growth for being different from traditional.

Meanwhile, in this application, we further disclose the unicellular structure side from the strain of suspension growth mdck cell Method, it is characterised in that comprise the following steps：

1）Single-cell suspension grows the suspension transfection process of mdck cell

Inoculation initial cell density is 5 × 10⁵The MDCK-Sus cells of cells/ml are lain in 50mlTPP culture tubes, are hanged It is used for plasmid transfection after floating overnight incubation, suspension culture culture medium is replaced by into Opti-MEM culture mediums before transfection is incubated 10 points Clock；

It is a transducing units with 10ml cell suspensions, 100ulPEI reagents is added into the Opti-MEM of 1ml mixing, 10~200ug pIRES-TMPRSS2 plasmid vectors are added into the Opti-MEM of 1ml mixing, 5 are incubated at room temperature respectively Minute；

Above-mentioned two mixed liquor is mixed by the way of being added dropwise over after the completion of incubation, is incubated 15 minutes at room temperature, Form PEI-DNA transfection composites；

PEI-DNA transfection composites are added to 10 times of MDCK-Sus cell suspensions of volume by the way of being added dropwise over In, mixing is vibrated when instilling, it is incubated 15 minutes in room temperature, mixed once at interval of 3 minutes；

After incubation terminates, the mixture after incubation is positioned on shaking table and is cultivated, 55~120rpm of rotating speed；

After 8 hours, it is centrifuged 10 minutes with 1000rpm, by the culture supernatant removal containing remaining transfection composite；

The normal suspension culture medium for adding MDCK-Sus cell lines continues to vibrate the culture that suspends；

2）The positive suspension cell clone's for being transfected screens

The MDCK-Sus cell lines of 18~24 hours are reclaimed according to 1 through centrifugation after transfection:2~1:12 ratio is disperseed again Cultivated into the fresh suspending nutrient solutions of 15ml, 37 DEG C, 5%CO₂, rotating speed is 100~200rpm, is screened by G418, is obtained The restructuring MDCK-Sus-TMPRSS2 cell clones of sustained suspension growth can be stablized in G418 screening resistances.

Specifically, this screening process is that the MDCK-Sus cell lines of 18~24 hours are pressed through recovery is centrifuged after transfection According to 1:2~1:12 ratio is dispersed in the fresh suspending nutrient solutions of 15ml and cultivates again, 37 DEG C, 5% CO2, and rotating speed is 100~ 200rpm, the working concentration of G418 is 400ug/ml, cell initial density substantially 3~5 × 105cells/ in suspending nutrient solution ml.Frequency according to a not good liquor is changed for 5 days carries out G418 and persistently screens, first round screening time substantially 4~6 weeks.It is selected to The restructuring MDCK-Sus-TMPRSS2 cell clones continued to multiply in the case where there is G418 screening pressures continue suspension subculture.The biography Generation operation is realized so that removal culture supernatant and resuspended cell precipitation is centrifuged.G418 screening operation concentration is carried in second wheel screening Up to 600ug/ml continues screening of pressurizeing.It is final obtain can in G418 screening resistances stabilization sustained suspension growth restructuring MDCK-Sus-TMPRSS2 cell clones, and it is preservation to build.

Meanwhile, the present invention further discloses a kind of cell culture, the cell culture contains the MDCK- of restructuring Sus-TMPRSS2 cells.

Further, we also disclosed the unicellular MDCK- from the strain of suspension growth mdck cell or containing restructuring Purposes of the cell culture of Sus-TMPRSS2 cells in avian influenza virus Multiplying culture.

Meanwhile, the cell culture that we further disclose disclosed in the present invention does not contain serum.

Further, during we also disclosed the avian influenza virus Multiplying culture, be free of in cell culture There is the trypsase that TPCK is processed.

Based on foregoing public technology scheme, the present invention further disclose it is unicellular from suspension growth mdck cell strain enter The method that row avian influenza virus bioreactor is bred on a large scale, comprises the following steps：

1）Restructuring MDCK-Sus-TMPRSS2 cells are carried out being suspended entirely with feed supplement association type in batches in bioreactor Culture, obtains MDCK-Sus-TMPRSS2 cell culture fluids,

2）When the cell density in MDCK-Sus-TMPRSS2 cell culture fluids reaches 4 × 10⁶During cells/ml, according to MOI is 0.0001~1 ratio access avian influenza virus and virus multiplication maintaining liquid；

3）PH6.8~7.2, DO are set in virus propagation process（Dissolved oxygen）15~35%, 28~37 DEG C of temperature is cultivated 3 days Progeny influenza virus can be harvested afterwards.

Further, the virus multiplication maintaining liquid component is we disclosed as follows：

The first aspect of the present invention, establishing first can stablize expression people TMPRSS2 albumen and can certainly be hanged with unicellular The MDCK recombinant cell strains of length of growing floating on water.The recombinant cell strain is MDCK-Sus-TMPRSS2, and preserving number is：CGMCC No.8851.Carrier for expression of eukaryon using stable transfection TMPRSS2 containing someone protein expression box elements enters MDCK-Sus cells In strain, and by obtaining the recombinant cell clone that stabilization expresses the albumen after resistance screening repeatedly.

The second aspect of the present invention, there is provided the method for building MDCK-Sus-TMPRSS2 recombinant cell strains.In embodiment party In case, by the open reading frame of the TMPRSS2 albumen of people（ORF）Sequence is connected into carrier for expression of eukaryon, by the eukaryotic expression Carrier is transfected into MDCK-Sus cells.The transfection process is the transfection process of suspension cell, is different from traditional attached cell Transfection procedures.After expression vector is imported in MDCK-Sus cells, using the neomycin resistance on the expression vector（neo^r）Through G418 resistance screenings and pressurization screening, obtain the restructuring mdck cell clone that can stablize expression people's TMPRSS2 albumen.Through RT- PCR detects its mRNA level in-site, and western blot detect that its protein expression level all shows that recombinant cell clone stabilization is obtained The encoding gene of people's TMPRSS2 albumen is simultaneously smoothly expressed, and using indirect immunofluorescence method detects the recombinant cell clone energy The expression and localization of enough stabilization expression people's TMPRSS2 albumen, and the albumen is in cell membrane.

The third aspect of the invention, using the restructuring MDCK-Sus-TMPRSS2 cell lines of invention biological anti- Answer and carried out many plants of propagation of H9 subtype avian influenza virus in device, the recombinant cell strain culture that suspends is included among these and is used Nutrient solution, cultivate program, the virus multiplication maintaining liquid that H9 subtype avian influenza virus propagation is used connects toxic agent amount, receives malicious program Etc. content.

Brief description of the drawings

The preserving number of cell line is：CGMCC No.8851.Preservation date is on March 6th, 2014, is preserved in Chinese micro- life Thing culture presevation administration committee common micro-organisms center.

Fig. 1 is the RT-PCR qualification result figures of MDCK-Sus-TMPRSS2 recombinant cell strains and female parent MDCK-Sus cells；

Wherein：M：DL2000 marker

1：MDCK-Sus cell controls

2：MDCK-Sus-TMPRSS2 recombinant cell strains.

Fig. 2 is that MDCK-Sus-TMPRSS2 recombinant cell strains identify knot with female parent MDCK-Sus cell western blot Fruit is schemed；

Wherein M：Albumen marker

1：MDCK-Sus cell controls

2：MDCK-Sus-TMPRSS2 recombinant cell strains.

Fig. 3 A MDCK-Sus-TMPRSS2 recombinant cell strain indirect immunofluorescene assay results

Can detect that recombinant cell strain has specificity fluorescent；

Fig. 3 B female parent MDCK-Sus cellmediated immunity fluoroscopic examination results

Without specificity fluorescent detection

Fig. 4 is MDCK-Sus-TMPRSS2 recombinant cell strains and female parent MDCK-Sus cell suspension growth curve maps；

Wherein：

■ represents MDCK-Sus-TMPRSS2 recombinant cell strains, ◆ represent MDCK-Sus cell lines.

Fig. 5 cultivates growth curve chart for MDCK-Sus-TMPRSS2 recombinant cell strains suspend in 3L reactors.

Specific embodiment

The structure of the pIRES-TMPRSS2 expression vectors of embodiment 1

1.1 reagents

The plasmid vector of the albumen of TMPRSS2 containing someone ORF is purchased from Sino Biological Inc.（Article No.： HG13070-G）.Trizol, MMLV Transcription System, pMD19-T carriers, oligo dT, various restriction enzymes, ExTaq Enzyme, T4DNA ligase equimolecular biologic materials are purchased from TAKARA companies, and pIRES plasmids are by national veterinary biologicses work Journey Technical Research Center is preserved.Glue reclaim kit, plasmid are small to refer to that big extraction reagent kit is purchased from Qiagen companies.

1.2 primers synthesize：Primer for expanding people's TMPRSS2 albumen ORF sequences is synthesized by Shanghai life work.

p1：GCCGCTAGCTAATACGACTCACTATAGGG（It is the restriction enzyme sites of Nhe I at line）

p2：GCCTCTAGATAGAAGGCACAGTCGAGG（It is the restriction enzyme sites of Xba I at line）

The structure of 1.3 pIRES-TMPRSS2 expression vectors and carry greatly

Using above-mentioned primer, with Yi Qiao Divine Land, Beijing Bioisystech Co., Ltd plasmid vector（Article No.：HG13070-G）For Template, the complete ORF fragments of people TMPRSS2 are amplified through PCR reactions, and PCR reaction conditions are as shown in the table.By the purpose fragment It is connected on pMD19-T carriers, sequencing result carries out sequence alignment, amplified fragments and people's TMPRSS2 albumen on genebank Coded sequence it is consistent;

PCR reactive components：	Volume（ul）
		Plasmid masterplate	1
P1	1
		P2	1
10×Buffer	5
		dNTP	2
Ex-Taq	1
			39
Response procedures：
		95℃	5 minutes
30 circulate 95 DEG C	30 seconds
		56℃	30 seconds
72℃	90 seconds
		72℃	10 minutes

.The purpose fragment and pIRES plasmids expanded using the double digestions of I/Xba of Nhe I treatment PCR, by purpose fragment and The skeleton fragment of pIRES plasmids carries out glue reclaim, and nucleic acid attended operation is carried out after quantifying, and in conversion to DH5 α, selects positive bacteria Fall and largely expand, digestion verification recombinant eukaryon expression vector pIRES-TMPRSS2 after plasmid extraction.Through Qiagen companies plasmid Big extraction reagent kit extracting obtains quality transfection plasmid higher.

1.4 results

Be connected into purpose fragment in carrier for expression of eukaryon using two restriction enzyme sites by this experiment, obtains pIRES- TMPRSS2 carrier for expression of eukaryon is used for follow-up transfection assay.Identified using the double digestion pIRES-TMPRSS2 carriers of I/Xba of Nhe I Result display purpose fragment connection is correct.Carried greatly through plasmid and obtain quality plasmid higher, the value of OD260/OD280 is 1.81, For the experiment of follow-up plasmid transfection.

The structure screening of the restructuring MDCK-Sus-TMPRSS2 recombinant cell clones of embodiment 2

2.1 materials：

Reagent：DMEM、G418、Opti-MEM、PEI（25kD is linear）Reagent is purchased from Invitrogen companies.Rabbit-anti people The primary antibody of TMPRSS2 albumen is purchased from Santa Cruz biotech companies, the goat anti-rabbit igg of FITC marks, the goat-anti of HRP marks Rabbit igg is purchased from Hangzhou Lian Ke biotech companies.Trizol, MMLV Transcription System, the life of oligo dT, ExTaq enzymes equimolecular Thing reagent is purchased from TAKARA companies.

Biomaterial：MDCK-Sus cell lines are voluntarily tamed amplification by National Research Center of Veterinary Biologicals Engineering and Technology Build and be, the unicellular culture medium from suspension growth of the cell line by National Research Center of Veterinary Biologicals Engineering and Technology from Row exploitation is prepared, with DMEM/F12（1:1）Culture medium based on culture medium, adds some nutritional ingredients, is filtered through 0.22um, puts Kept in dark place in 4 DEG C.Nutritional ingredient is as shown in the table：

Each composition is purchased from sigma companies above, is prepared by description of product requirement.Filtered through 0.22um, be placed in 4 DEG C of lucifuges and protect Deposit.

The screening of 2.2 G418 activities

MDCK-Sus cells are inoculated in 50mlTPP culture tubes, and wherein volume of culture is 10ml, and initial cell density is 5 ×10⁵cells/ml.In different culture tubes add various concentrations G148, concentration be 100,200,300,400,500, 600th, 700,800ug/ml, is placed in 37 DEG C, 5%CO₂, rotating speed is that suspension culture is carried out on the culture shaking table of 100~250rpm, often Day sampling is observed and determines cell survival rate, is cultivated 6-12 days, records cell death situation, determines the effective work of minimum of G418 Concentration.

The suspension cell transfection of 2.3 cationic polymers mediation

Inoculation initial cell density is 5 × 10⁵The MDCK-Sus cell lines of cells/ml hang in 50mlTPP culture tubes It is used for plasmid transfection after floating overnight incubation, suspension culture culture medium is replaced by into Opti-MEM culture mediums before transfection is incubated 10 points Clock.It is a transducing units with 10ml cell suspensions, 100ulPEI reagents is added into the Opti-MEM of 1ml mixing, by 10 ~200ug pIRES-TMPRSS2 plasmid vectors add into the Opti-MEM of 1ml mixing, are incubated 5 minutes at room temperature respectively. Above-mentioned two mixed liquor is mixed by the way of being added dropwise over after the completion of incubation, is incubated 15 minutes at room temperature, form PEI- DNA transfection composites.The MDCK-Sus cells that above-mentioned 1ml transfection composites are added to 10ml are hanged by the way of being added dropwise over In liquid, mixing is vibrated when instilling, be incubated 15 minutes in room temperature, mixed once at interval of 3 minutes.Incubation will transfection after terminating The cell for finishing is positioned on the shaking table of the normal culture that suspends, and with normal culture, only change culture rotating speed is remaining condition of culture 55~120rpm.8 hours after transfection, it is centrifuged 10 minutes with 1000rpm, the culture supernatant containing remaining transfection composite is gone Remove, the normal suspension culture medium for being replaced with MDCK-Sus cell lines continues to vibrate the culture that suspends.

The screening of 2.4 MDCK-Sus-TMPRSS2 cell clones：

The MDCK-Sus cell lines of 18~24 hours are reclaimed according to 1 through centrifugation after transfection:2~1:12 ratio is disperseed again Cultivated into the fresh suspending nutrient solutions of 15ml, 37 DEG C, 5%CO₂, rotating speed is 100~200rpm, the work of G418 in suspending nutrient solution Make concentration for 400ug/ml, cell initial density substantially 3~5 × 10⁵cells/ml.Frequency according to a not good liquor is changed in 5 days is entered Row G418 is persistently screened, first round screening time substantially 4~6 weeks.It is selected to be continued to multiply in the case where there are G418 screening pressures Restructuring MDCK-Sus-TMPRSS2 cell clones continue suspension subculture.Passage operation removes culture supernatant simultaneously to be centrifuged Re-suspended cell precipitates to realize.G418 screening operation concentration is improved to 600ug/ml in second wheel screening continues screening of pressurizeing. It is final obtain can in G418 screening resistances stabilization sustained suspension growth restructuring MDCK-Sus-TMPRSS2 cell clones.

2.5 RT-PCR, western blot, indirect immunofluorescence identification：

The MDCK-Sus-TMPRSS2 recombinant cell clones that will be screened are through being collected by centrifugation, after PBS 3 times by Trizol methods extracted total RNA from cell, it is specific as follows：By every 1~2 × 10⁷Individual cell adds the ratio of 0.5mlTrizol liquid Treatment recombinant cell clone, adds 0.1ml chloroforms after being stored at room temperature 5 minutes, acutely vibrate repeatedly 15 seconds, 4 DEG C of 12000g centrifugations 10 minutes.Careful upper strata aqueous phase of drawing adds 0.25ml isopropanols in new centrifuge tube, and room temperature is placed 10 minutes, 4 DEG C of 12000g Centrifugation 10 minutes.Supernatant discarded after centrifugation, adds 1ml75% ethanol, and gentle resuspended precipitation, 4 DEG C of 12000g are centrifuged 10 minutes, protect Precipitation is stayed, with the RNA that DEPC water dissolves are extracted.Vessel, pipette tips, water used by above-mentioned experiment etc. are processed simultaneously using DEPC High pressure, whole process is worn gloves and is operated.Process of reverse-transcription is as follows：The RNA 20ul of extracting, oligo dT 3ul, dNTP mix 4ul is mixed and acted on 10 minutes after 65 DEG C, is immediately placed on ice and adds following reagent：MMLV 1ul、5×buffer 8ul、 RNase inhibitor 1ul, DTT 3ul.37 DEG C effect 2~3 hours after for follow-up PCR operate.It is with the cDNA after reverse transcription Template, enters performing PCR amplification, and design parameter is operated with embodiment one.

The MDCK-Sus-TMPRSS2 recombinant cell clones that will be screened are through being collected by centrifugation, be used for after PBS 3 times Western blot are detected.SDS-PAGE protein electrophoresises are carried out with 5% concentration glue and 8% separation gel, using half dry type transferring film Be transferred to albumen on glue on pvdf membrane by operation, after being closed through BSA, is marked using the rabbit source primary antibody and HRP of people's TMPRSS2 albumen Goat-anti rabbit secondary antibody be incubated turn purposeful albumen pvdf membrane, using chromogenic reagent identify destination protein expression.

The MDCK-Sus-TMPRSS2 recombinant cell clones that will be screened are through being collected by centrifugation, PBS and using 75% cold Acetone is fixed in 96 orifice plates, adds the rabbit source primary antibody 100ul of people's TMPRSS2 albumen（1:200 dilutions）, 30 points are incubated in 37 DEG C Primary antibody is removed after clock.After PBS 3 times, the goat-anti rabbit secondary antibody 100ul of FITC marks is added（1:500 dilutions）, in 37 DEG C Remove secondary antibody after being incubated 30 minutes, with after PBS 3 times in fluorescence microscopy Microscopic observation recombined human TMPRSS2 albumen in MDCK Expression on cell.

Above-mentioned qualification test is identified with MDCK-Sus cells as blank according to same operation step.

2.6 results

2.6.1 the screening of G418 working concentrations determines

It is G418 screening operation concentration with the concentration that whole MDCK-Sus cells were killed in 5 days.According to cell survival rate Measurement result, determine 400ug/ml be normally screening when working concentration, pressurize screening in select 600ug/ml.

2.6.2 the clone of recombinant cell, screening and identification

, by the screening of 6 weeks in the G418 screening environment of 400ug/ml, obtaining normally to hold for cell after transfection The recombinant cell strain of continuous growth.The cell line of survival is dispersed to the G418 screening TPP trainings containing 600ug/ml and 800ug/ml Continuation pressurization screening in pipe is supported, it is final to obtain the MDCK-Sus-TMPRSS2 recombinant clones for being capable of continued propagation.

With reference to Fig. 1, through RT-PCR detections find restructuring mdck cell strain it is amplifiable go out 1492bp band, sequence number 2 in Fig. 1 Indicated, it is in the same size with purpose band, and female parent MDCK-Sus cells, 1 amplification for being indicated is feminine gender in Fig. 1.

With reference to Fig. 2, western blot detections find that restructuring mdck cell strain expresses the destination protein of 70kDa, in Fig. 2 Sequence number 2 is indicated, and female parent MDCK-Sus cells are expressed without destination protein, and 1 is indicated in Fig. 2.

Indirect immunofluorescene assay goes out people TMPRSS2 protein expressions in the surface of cell membrane of recombinant cell, it is seen that specificity Green fluorescence, as shown in Figure 3A, wherein being green fluorescence at white bright spot.And female parent MDCK-Sus cells have no special after testing Property green fluorescence, as shown in Figure 3 B.

Embodiment 3 recombinates the suspension culture performance measurement of MDCK-Sus-TMPRSS2 cell lines

3.1 materials and methods

It is to preserve that maternal MDCK-Sus cells are built by National Research Center of Veterinary Biologicals Engineering and Technology's domestication.

Restructuring MDCK-Sus-TMPRSS2 cell lines screen acquisition as previously described, and build and be.

Suspension growth culture medium is as previously described.

MDCK-Sus cells and restructuring MDCK-Sus-TMPRSS2 cells are with 3 × 10⁵The initial density inoculation of cells/ml In the culture shaking flask of 250ml, 37 DEG C, 5%CO are positioned over₂, rotating speed for 180rpm culture shaking table in carry out suspension culture.Often Cell density and survival rate is measured by sampling within 24 hours, draws growth curve, calculate specific cell growth rate.

In 3L bioreactors（Dutch Applikon Products）In, MDCK-Sus cells and restructuring MDCK-Sus- TMPRSS2 cells are inoculated in 1.5L initial incubation volumes with the initial density of 3~5 × 105cells/ml, set culture rotating speed 60~180rpm, 37 DEG C of cultivation temperature, DO is 50%, pH for 7.1 carry out initial batch experiments, every 24 hours sampling and measuring cells Density, calculates specific cell growth rate.3~5 days backsight cell growth degree of culture carry out feed-batch culture, and feed supplement liquid component is as follows Shown in table：

Raw material components	Component content in the medium
		DMEM / F12（1:1）	28g/L
Yeast is from hydrolysate	0.01~1g/L
		Soybean protein hydrolyate	0.01~0.5g/L
Reduced glutathione	0.01~0.5mg/L
		Monoethanolamine	0.01~0.5ml/L
Pluronic F-68	0.2~1g/L

Above reagent is purchased from Sigma companies, is prepared according to the description of product, is filtered through 0.22um, is placed in 4 DEG C of lucifuges Preserve.

3.2 results

The growth curve for recombinating MDCK-Sus-TMPRSS2 cells is as shown in Figure 4.Compare with maternal MDCK- Sus cells, The growth rate for recombinating MDCK-Sus-TMPRSS2 cells is substantially unchanged, and cultured cells obtains maximum in 250ml shaking flasks Cell density is 2.7 × 10⁶Cells/ml, average specific growth rate is 0.438d^-1, there is no difference with parental cells substantially（It is female This cell maximum cell density 2.65 × 10⁶Cells/ml, average specific growth rate 0.453d^-1）, illustrate to turn by foreign gene The recombinant cell growth performance obtained after dye and screening operation does not change.

In order to verify the ability whether restructuring MDCK-Sus-TMPRSS2 cells possess industrialization propagation avian influenza virus, The recombinant cell carried out in 3L bioreactors in batches with feed supplement formula Combined culture, maximum cell density can reach 6.83 ×10⁶cells/ml（As shown in Figure 5）, illustrate that the recombinant cell strain can carry out the extensive culture that suspends, be conducive to bird flu A large amount of propagation of the virus in bioreactor.

The ability of the restructuring MDCK-Sus-TMPRSS2 cell line propagation avian influenza virus of embodiment 4 compares

4.1 test materials

It is to preserve that MDCK-Sus cells are built by National Research Center of Veterinary Biologicals Engineering and Technology's domestication.

Restructuring MDCK-Sus-TMPRSS2 cell lines screen acquisition as previously described, and build and be.

Suspension growth culture medium, suspension culture method are as previously described.

Bird flu H9N2 viruses：A/chicken/Jiangsu/02/09（Abbreviation JS02）、A/chicken/Jiangsu/ 03/11（Abbreviation JS03）、A/chicken/JiaXing/02/11（Abbreviation JX02）、A/chicken/XiGang/04/01（Referred to as XG04）、A/chicken/HangZhou/03/01（Abbreviation HZ03）、A/chicken/HeNan/03/01（Abbreviation HN03）By state Family's veterinary biologicses Engineering Technical Research Centre is separated, identified, preservation.

4.2 test methods

It is base to recombinate MDCK-Sus-TMPRSS2 cells, female parent MDCK-Sus cells in 50mlTPP cell culture tubes The above-mentioned some avian influenza virus of matter continuous passage, every kind of virus is continuous to pass for 15 generations, was determined in the 1st, the 5th, the 10th, the 15th generation respectively The HA potency of virus of proliferation, judges continuous passage ability of two kinds of propagation host cells to avian influenza virus.Used by poison propagation Maintaining liquid component is as shown in the table：

Above reagent is purchased from Sigma companies, is prepared according to the description of product, is filtered through 0.22um, is placed in 4 DEG C of lucifuges Preserve.

It is added without in above-mentioned virus multiplication maintaining liquid during restructuring MDCK-Sus-TMPRSS2 cell propagation avian influenza virus The trypsase of TPCK treatment, and added eventually in virus multiplication maintaining liquid during the MDCK-Sus cells above-mentioned avian influenza virus of propagation Concentration is the trypsase of the TPCK treatment of 0.1~10ug/ml.Remaining virus multiplication condition is consistent.

MDCK-Sus cells and restructuring MDCK-Sus-TMPRSS2 cells are respectively in 1L bioreactors（Dutch Applikon Products）In carry out the propagation of avian influenza virus.With the method for cell suspension cultures in embodiment 3 by cell culture to 4 × 10⁶Virus inoculation operation can be carried out during cells/ml.Aperture is used to be gone for the stainless steel revolving filter of 12um first Except cells and supernatant, the operation such as PBS cleaning.After having cleaned cell fowl is accessed according to the ratio of MOI 0.0001~1 Influenza virus XG04 plants（A/chicken/XiGang/04/01）, virus multiplication maintaining liquid is as it was previously stated, in virus propagation process Setting pH6.8~7.2, DO 15~35%, 28~37 DEG C of temperature.Sampling in every 24 hours repeatedly after 3 freeze thawing with 10000rmp × HA potency in 5min centrifugation detection supernatants, unit is log2/25ul.MDCK-Sus cells and restructuring MDCK-Sus-TMPRSS2 The condition that cell breeds XG04 plants is only the TPCK treatment of 0.1~10ug/ml of addition in MDCK-Sus cells propagation maintaining liquid Trypsase, remaining proliferation conditions is consistent.

4.3 result of the tests

Result is as shown in the table, under conditions of the trypsase for not using TPCK to process, restructuring of the present invention MDCK-Sus-TMPRSS2 cells with normal proliferative avian influenza virus, and can carry out avian influenza virus passage using the recombinant cell When can keep virus multiplication efficiency higher, the hemagglutinative titer of progeny virus still keeps water higher after the generation of continuous blind passage 10 It is flat.Its maternal MDCK-Sus cell continuous the propagation generation of avian influenza virus 5 and recombinant cell strain in the presence of TPCK trypsase Proliferation results difference less, but after 10 generations of continuous propagation, the hemagglutinative titer of its progeny virus is gradually reduced, it is impossible to obtain preferable Cultivation effect.

The full culture breeding ratio that suspends has been carried out compared with as a result as shown in the table to bird flu XG04 plants in bioreactor. Restructuring MDCK-Sus-TMPRSS2 cells can obtain highest 2¹⁰The hemagglutinative titer of/25ul, hence it is evident that higher than using female parent MDCK- Sus cells breed the viral effect.Avian influenza virus are bred 3~4 days can receive poison, and generation time is short, is grasped in reactor tank Make convenient and simple, with preferable actual application prospect.

SEQUENCE LISTING

<110>Jiangsu Province Agriculture Science Institute

<120>The unicellular from the strain of suspension growth mdck cell and its structure side of expression people's TMPRSS2 albumen can be stablized Method

With application

<130> 20140402

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1479

<212> DNA

<213>People

<400> 1

atggctttga actcagggtc accaccagct attggacctt actatgaaaa ccatggatac 60

caaccggaaa acccctatcc cgcacagccc actgtggtcc ccactgtcta cgaggtgcat 120

ccggctcagt actacccgtc ccccgtgccc cagtacgccc cgagggtcct gacgcaggct 180

tccaaccccg tcgtctgcac gcagcccaaa tccccatccg ggacagtgtg cacctcaaag 240

actaagaaag cactgtgcat caccttgacc ctggggacct tcctcgtggg agctgcgctg 300

gccgctggcc tactctggaa gttcatgggc agcaagtgct ccaactctgg gatagagtgc 360

gactcctcag gtacctgcat caacccctct aactggtgtg atggcgtgtc acactgcccc 420

ggcggggagg acgagaatcg gtgtgttcgc ctctacggac caaacttcat ccttcaggtg 480

tactcatctc agaggaagtc ctggcaccct gtgtgccaag acgactggaa cgagaactac 540

gggcgggcgg cctgcaggga catgggctat aagaataatt tttactctag ccaaggaata 600

gtggatgaca gcggatccac cagctttatg aaactgaaca caagtgccgg caatgtcgat 660

atctataaaa aactgtacca cagtgatgcc tgttcttcaa aagcagtggt ttctttacgc 720

tgtatagcct gcggggtcaa cttgaactca agccgccaga gcaggattgt gggcggcgag 780

agcgcgctcc cgggggcctg gccctggcag gtcagcctgc acgtccagaa cgtccacgtg 840

tgcggaggct ccatcatcac ccccgagtgg atcgtgacag ccgcccactg cgtggaaaaa 900

cctcttaaca atccatggca ttggacggca tttgcgggga ttttgagaca atctttcatg 960

ttctatggag ccggatacca agtagaaaaa gtgatttctc atccaaatta tgactccaag 1020

accaagaaca atgacattgc gctgatgaag ctgcagaagc ctctgacttt caacgaccta 1080

gtgaaaccag tgtgtctgcc caacccaggc atgatgctgc agccagaaca gctctgctgg 1140

atttccgggt ggggggccac cgaggagaaa gggaagacct cagaagtgct gaacgctgcc 1200

aaggtgcttc tcattgagac acagagatgc aacagcagat atgtctatga caacctgatc 1260

acaccagcca tgatctgtgc cggcttcctg caggggaacg tcgattcttg ccagggtgac 1320

agtggagggc ctctggtcac ttcgaagaac aatatctggt ggctgatagg ggatacaagc 1380

tggggttctg gctgtgccaa agcttacaga ccaggagtgt acgggaatgt gatggtattc 1440

acggactgga tttatcgaca aatgagggca gacggctaa 1479

Claims (7)

1. it is a kind of to stablize the unicellular from the strain of suspension growth mdck cell of expression people's TMPRSS2 albumen, it is named as MDCK-Sus- TMPRSS2, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and the preserving number of its cell line is： CGMCCNo.8851。

2. as claimed in claim 1 unicellular from the strain of suspension growth mdck cell, it is characterised in that：The MDCK-Sus- TMPRSS2 is the cell line for being adapted to full suspension growth.

3. a kind of cell culture, it is characterized in that, the cell culture contains in claim 1 ~ 2 described in any one MDCK-Sus-TMPRSS2 cells.

4. the unicellular cell training from described in the strain of suspension growth mdck cell or claim 3 described in claim 1 or 2 Support purposes of the thing in avian influenza virus Multiplying culture.

5. purposes as claimed in claim 4, it is characterized in that, do not contain serum in the cell culture.

6. the purposes as described in claim 4 or 5, it is characterized in that, during avian influenza virus Multiplying culture, in cell culture The trypsase of TPCK treatment is not contained.

7. it is a kind of using described in claim 1 or 2 it is unicellular from suspension growth mdck cell strain carry out avian influenza virus life The method that thing reactor is bred on a large scale, it is characterized in that comprising the following steps：1）Will be unicellular described in claim 1 or 2 The culture that suspended entirely with feed supplement association type in batches is carried out in bioreactor from the strain of suspension growth mdck cell, MDCK- is obtained Sus-TMPRSS2 cell culture fluids, 2）4 are reached when the cell density in MDCK-Sus-TMPRSS2 cell culture fluids × 10⁶During cells/ml, avian influenza virus and virus multiplication maintaining liquid are accessed according to the ratio that MOI is 0.0001~1；3）Virus PH6.8~7.2, DO are set in breeding（Dissolved oxygen）15~35%, 28~37 DEG C of temperature, culture can harvest filial generation stream after 3 days Influenza Virus, the virus multiplication maintaining liquid component is as follows,

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