CN110129272A

CN110129272A - Stablize the PK-15 cell strain for expressing MAP3K8 albumen and its building and application

Info

Publication number: CN110129272A
Application number: CN201910249994.3A
Authority: CN
Inventors: 郑海学; 张克山; 郝军红; �田宏; 茹毅; 李丹; 朱紫祥; 杨帆; 刘湘涛
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2019-03-29
Filing date: 2019-03-29
Publication date: 2019-08-16
Anticipated expiration: 2039-03-29
Also published as: CN110129272B

Abstract

The present invention relates to the PK-15 cell strain for stablizing expression MAP3K8 albumen and its buildings and application.The cell strain is named as PK-15/MAP3K8, and deposit number is CCTCC NO:C2018262.The preparation of the cell strain the following steps are included: target gene MAP3K8 synthesis, the building of recombined lentivirus vector Lv-MAP3K8, the slow virus of packaging expression MAP3K8, slow-virus infection PK-15 cell, screening and culturing and identification obtain the PK-15 cell strain for stablizing expression MAP3K8 albumen.It compares with wild type PK-15 cell, the duplication of FMDV or SVV can be significantly inhibited using PK-15 cell great expression MAP3K8.The molecular mechanism of MAP3K8 gene function and its behind in research pig source cell that is established as of the cell strain provides biomaterial.

Description

Stablize the PK-15 cell strain for expressing MAP3K8 albumen and its building and application

Technical field

The invention belongs to field of biotechnology, and in particular to stablize the PK-15 cell strain and its structure of expression MAP3K8 albumen It builds and applies.

Background technique

Stable cell lines (Stable Cell Line), after referring on plasmid integration to chromosome, with corresponding Plasmid DNA In resistance marker screen the cell line, just obtained the cell line of stable expression.Stable expression of exogenous DNA can be integrated into On host cell chromosome, or it is equivalent to the additional sustainable presence of son, host cell long-term expression target gene can be made.Slow disease Poison is one kind of retrovirus, the immunodeficiency virus including a variety of mammals.It can be by gene after slow virus infected cell It is integrated on host cell chromosome, and expression can be stablized.Therefore, it is often transformed into the tool of transgenosis.In disease treatment, Transgenic animals, animal disease model etc. play a significant role.Slow virus carrier can accommodate biggish DNA fragmentation, and May be infected is a variety of to be in division stage and nondividing phase cell, which effectively can be transferred to brain, liver, flesh for target gene Meat, retina etc. do not cause toxicity or immune response.Target gene can be inserted into slow virus carrier in cell construction, Target gene is integrated on target cell by slow-virus infection target cell, using more in cell line building.With animal Transgenic technology is constantly progressive and develops, transgenic animals animal improvement, medical and health, in terms of all have Wide application prospect.Target gene can be integrated by slow virus carrier as a kind of carrier for deriving from retrovirus In host cell chromosome, expression alien gene steady in a long-term, while it has that transfection efficiency is high, can infect division stage and overstepping one's bounds It splits phase cell, accommodate the advantages that allogenic gene segment is big, be a kind of efficient tool of prepare transgenosis animal.

PK-15 cell (Porcine Kidney Epithelial cells) is also referred to as PK15 or PK(15), derive from pig Kidney, the entitled pig renal epithelial cell of Chinese, normal cellular morphology are Epithelial, adherent growth.The cell compares a variety of viruses Sensitivity, such as pig circular ring virus (PCV), pig parvoviral (PPV), swine fever virus (CSFV), can be applied to pig circular ring virus epidemic disease The preparation of seedling, swine parvovirus vaccine, classical swine fever virus vaccine etc..Ren sus domestica cell PK15 proliferative capacity depend on cell materials, The composite factors such as culture technique, condition of culture.PK-15 cell be widely used in swine fever virus, porcine pseudorabies virus and In the production of the separation of pig parvoviral etc., in vitro culture and related vaccines.Foot and mouth disease virus can be limited by having no at present (Foot-and-mouth disease virus, FMDV) and Senecan are ancient viral (Seneca valley virus, SVV) PK-15 cell line relevant report.

MAP3K8 is also known as TPL2, and extensive expression is suffered from different tissues.The albumen is a kind of serine-Soviet Union Histidine kinase.TPL2 signal transduction rises emphatically in congenital and adaptive immunity and in cancer as potential proto-oncogene It acts on.Effect of the TPL2 in congenital immunity is only limitted to bacterial infection model, the correlative study carried out using virus, helminth It is fewer.TPL2/MAP3K8 has been considered as the key regulator of I type (IFN-α/β) and II type (IFN-γ) IFN, is more The important component of kind virus infection approach adjusts IFN-α/β and IFN- λ with cell-type-specific manner difference and lures It leads.The duplication that research is reported in vesicular stomatitis virus (VSV) in the mouse embryonic fibroblasts of TPL2 defect increases (Schmid, S.; Sachs, D. et al. Mitogen-activated protein kinase-mediated licensing of interferon regulatory factor 3/7 reinforces the cell response to virus. J Biol Chem 2014,289(1), 299-311.).

Summary of the invention

It is an object of the present invention to provide a kind of PK-15 cell strain of stable expression MAP3K8 albumen and its buildings and application.This Invention obtains the cell strain PK-15/MAP3K8 for stablizing expression MAP3K8 albumen by slow virus expression system.With FMDV or SVV The cell strain of building is infected, which can inhibit the duplication of FMDV, SVV.The cell strain be established as virus separation and grind The biological activity for studying carefully MAP3K8 is laid a good foundation.

The PK-15 cell strain of stable expression MAP3K8 albumen provided by the invention, is named as PK-15/MAP3K8, preservation Number is CCTCC NO:C2018261.

The present invention also provides PK-15 cell strains to inhibit the application in FMDV or SVV duplication.

The present invention also provides PK-15 cell strains in pig source cellMAP3K8Gene function and its molecular mechanism grind Application in studying carefully.

The present invention also provides it is a kind of it is stable expression MAP3K8 albumen PK-15 cell strain construction method, specifically include with Lower step:

Step 1: MAP3K8 gene the building of recombined lentivirus vector Lv-MAP3K8: being inserted into slow virus carrier Lv-pCDH's The area MCS；

Step 2: the slow virus of packaging expression MAP3K8: packing Lv-MAP3K8 virus liquid with 293FT cell；It is collected after 48 hours Virus liquid is filtered, is saved backup；

Step 3: slow-virus infection PK-15 cell: the slow-virus infection PK-15 cell that step 2 is obtained terminates after 8 hours；

Step 4: screening and culturing: after infection 48h, carrying out drug screening, culture medium is replaced after 48h~72h and is passed on, training is maintained It supports, identification, obtains the PK-15 cell strain PK-15/MAP3K8 for stablizing expression MAP3K8 albumen.

The present invention is also verified the cell strain PK-15/MAP3K8 that above step screens using qRT-PCR, as a result table Bright MAP3K8 being capable of efficient, stable expression in the cell strain.

PK-15/MAP3K8 cell strain is met FMDV, SVV by the present invention respectively, and qRT-PCR is the results show that with wild type PK- 15 compare, and cell strain PK-15/MAP3K8 inhibits the duplication of FMDV or SVV.

The invention has the following advantages:

1, the present invention constructs the PK-15 cell strain for stablizing expression MAP3K8 albumen using slow virus system, which builds It stands and provides reliable biomaterial for MAP3K8 gene function and its molecular mechanism in research PK-15 cell.

2, the PK-15 cell strain for stablizing expression MAP3K8 albumen that the present invention constructs, utilizes PK-15 cell great expression MAP3K8 can inhibit the duplication of FMDV or SVV, by PK-15/MAP3K8, wild type PK-15 cell connect respectively FMDV and FMDV relative expression quantity is higher than the relative expression quantity of FMDV in PK-15/MAP3K8 cell in SVV, wild type PK-15, and difference is aobvious It writes；SVV relative expression quantity is higher than the relative expression quantity of SVV in PK-15/MAP3K8 cell, difference pole in wild type PK-15 cell Significantly.In short, the PK-15/MAP3K8 cell strain of building has apparent antivirus action, answering for FMDV, SVV can be significantly inhibited System.

Detailed description of the invention

Fig. 1 is the expression quantity comparison diagram of MAP3K8 in cell strain pCDH- PK-15, PK-15/MAP3K8.

Fig. 2 is the qRT-PCR product electrophoresis of MAP3K8 the and β-actin of cell strain pCDH- PK-15, PK-15/MAP3K8 Detection figure.

Fig. 3 is MAP3K8 dose-dependent inhibition FMDV copy pattern.MYC-TPL2 indicates that MYC label and MAP3K8 merge table Up to product.

Fig. 4 is the expression comparison diagram of MAP3K8 albumen in wild type PK-15 and PK-15/MAP3K8 cell after meeting FMDV, * * indicates that p < 0.01, * * * indicate p < 0.001 in figure.

Fig. 5 is FMDV/GAPDH mrna expression amount comparison diagram in PK-15/MAP3K8 cell and wild type PK-15, * in figure Indicate that p < 0.05, * * indicate p < 0.01.

Fig. 6 is that the indirect immunofluorescence of PK-15/MAP3K8 cell and wild type PK-15 after meeting malicious FMDV 16h is observed Photo.A: the PK-15 of infection FMDV 16h；B: the PK-15/MAP3K8 of infection FMDV 16h.

Fig. 7 is SVV/GAPDH mrna expression amount comparison diagram in PK-15/MAP3K8 cell and wild type PK-15, * * in figure Indicate that p < 0.01, * * * indicate p < 0.001.

Fig. 8 is that the indirect immunofluorescence of PK-15/MAP3K8 cell and wild type PK-15 after meeting malicious SVV 16h observes photograph Piece.A: the PK-15 of infection SVV 16h；B: the PK-15/MAP3K8 of SVV16h is infected.

MAP3K8-PK15 or MAP3K8 (Pig)-PK15 indicates PK-15/MAP3K8 in attached drawing.

Preservation information:

The preservation time: on December 19th, 2018；

Depositary institution's title: China typical culture collection center；

Deposit number: CCTCC NO:2018262；

Depositary institution address: wuchang, wuhan area, Hubei China province Luo Jia Shan street 16；

Classification naming: pig source nephrocyte PK-15/MAP3K8.

Specific embodiment

The present invention is further illustrated below in conjunction with the drawings and specific embodiments are illustrated, but embodiment is not the present invention Any type of restriction.Unless specifically indicated, the present invention uses reagent, method and apparatus is routinely try for the art Agent, method and apparatus.

Material therefor source in embodiment: the ancient virus of mouth disease virus strain A/GDMM/CHA/2013, Senecan (Seneca valley virus, SVV) strain and porcine kidney cell (PK-15) are by Lanzhou veterinary institute aftosa epidemiology Team saves；Anti- β-Actin the monoclonal antibody of mouse is purchased from Thermo Scientific company.Opti-MEM,Lipo 2000,0.25% EDTA pancreatin and newborn bovine serum (FBS) are purchased from Gibco company；DMEM cell culture fluid and PBS solution are purchased from Hyclone Company；ECL color developing agent is purchased from Thermo Scientific company；NP-40 lysate and PMSF are purchased from green skies company；Reversion Record kit praises biotech firm's production by Novi.Slow virus Lv-pCDH is purchased from plasmid vector cell protein antibody cell gene Collection (NTCC).Recombined lentivirus vector Lv-MAP3K8 by PPL (Public Protein/Plasmid Library, China) company constructs.The VP3 albumen rabbit primary antibody of FMDV, SVV rabbit primary antibody by Lanzhou veterinary institute aftosa epidemiology Team's preparation, preparation method is with reference to (the preparation and identification [D] Shandong agriculture of Chen Fei foot and mouth disease virus 3C protein polyclone antibody Sparetime university is learned, and 2018.) and (such as Hu Tao teaching prepares rabbit polyclonal antibody interpretation of result with purifying blister stomatitis virus antigen [J], medical evacuation ship, 2019(6): 112-114).Immunofluorescence rabbit secondary antibody is bought in Cell Signaling Technology(CST) company.Rabbit secondary antibody is purchase in Proteintech Group company.

The building process of embodiment 1PK-15/MAP3K8 stable cell line

The building and virus packaging of 1.1 slow virus carriers

Specifically includes the following steps:

Step 1: the building of recombined lentivirus vector Lv-MAP3K8 and Lv-pCDH: chemical synthesis Susscrofa (pig) The CDS sequence of MAP3K8 gene, CDS sequence are shown in XM_021064737.1 on NCBI；The sequence 5 ' end and 3 ' ends, which introduce, digestion Reconnection after site Xba I and BamH I, double digestion target gene and slow virus carrier Lv-pCDH carrier, obtains recombinant slow virus Carrier Lv-MAP3K8.

Step 2: packaging slow virus:

(1) slow virus Lv-pCDH and Lv-MAP3K8 virus liquid is packed respectively with 293FT cell；

1. the digestive inoculation of 293T cell: being prepared by growth conditions 0.25% trypsin digestion of good 293T cell slender Born of the same parents' suspension and adjust concentration be 4 × 10⁵/ ml takes 10 ml to be inoculated in 10 cm culture dishes.In 37 C, 5% CO₂In incubator Overnight incubation, second day cell confluency degree reach 80%, spare.

2. virus packaging:

Virus packaging: by 7.5 μ g slow virus carrier Lv-pCDH and packaging plasmid (3 μ g pMD2G and 6 μ g psPAX), 7.5 μ g recombined lentivirus vector Lv-MAP3K8(hamster) and packaging plasmid (3 μ g pMD2G and 6 μ g psPAX) be added separately to It is mixed in 150 μ l OPTI-MEM, obtains two groups of slow virus carrier mixtures；25 μ lLipo 2000 are taken to be added to 500 μ l It is mixed in OPTI-MEM, is stored at room temperature 5 min, obtains 2000 mixture of Lipo；Two groups of slow virus carrier mixture difference are slow It is added in 2000 mixture of Lipo, 15 min is stored at room temperature after mixing.Then be added dropwise to respectively 1. obtained in convergence degree Up in 80% 293T cell, mix well.The DMEM fresh medium containing 10%FBS is changed to after 6 h.

Slow virus carrier requirement: concentration is 1 μ g/ μ l, and purity: the value of OD260/OD280 is in 1.8~2.0 ranges.

(2) two groups of culture solutions (containing slow virus) is collected after cultivating 48 h, 10 min is centrifuged under the conditions of 1500g, 4 C and goes Cell fragment with 0.45 μm of filter filtering, saves backup then with 0.45 μm of membrane filtration vial supernatant.

1.2 stablize the screening and identification of the PK-15 cell strain PK-15/MAP3K8 of expression MAP3K8

Specifically include the following contents:

1.2.1 slow-virus infection PK-15 cell:

It is dense that 5 × pEGit virus is added according to the ratio of 4:1 in Lv-pCDH, Lv-MAP3K8 virus filtration liquid that 1.1 are obtained Contracting liquid.After 4 C are stood overnight, 20 min are centrifuged under the conditions of 3200g, 4 C.It discards supernatant, is cultivated with the DMEM containing 5% FBS Viral agglomerate is resuspended in liquid, and with the packing of 200 μ l/ pipes, -80 C are saved.

Then slow virus Lv-pCDH, Lv-MAP3K8 are infected into PK-15 cell respectively: viral 200 μ l of volume, viral load About 2 × 10⁷, 2ml is complemented to complete medium, 2 μ l transfection reinforcing agent polybrene (polybrene) is added, is terminated after 8h.Two Group cell is denoted as pCDH-PK-15 and PK-15/MAP3K8 respectively.

1.2.2 the screening of pCDH-PK-15, PK-15/MAP3K8 cell strain:

After infecting 48h, puromycin is added and carries out drug screening, replace culture medium after screening 48h~72h and pass on, maintains training It supports, obtains positive cell strain pCDH-PK-15, PK-15/MAP3K8.By screening obtained positive cell strain PK-15/MAP3K8 The PK-15 cell strain of as stable expression MAP3K8 albumen.

1.2.3qRT-PCR the expression quantity of MAP3K8 is detected:

(1) the qRT-PCR primer of MAP3K8 (pig) and the qRT-PCR primer of β-actin gene, primer sequence such as table 1 are designed (NO:1~4 SEQ ID).

(2) Trizol extracts the RNA of positive pCDH-PK-15, PK-15/MAP3K8 cell strain, and measures the concentration of RNA.

(3) reverse transcription is carried out at the standard of 10 μ l system cDNA by 500ng RNA reverse transcription, by the successful cDNA of reverse transcription As template, qRT-PCR detection is carried out using the primer that step 1 synthesizes as amplimer.Reaction system are as follows: AceQ 10 μ l of qPCRSYBR Green Master Mix, sterilize ddH₂O 7.2 μ l, Primer1(10 μM) 0.4 μ l, Primer2(10 μM) 0.4 μ l, 2 μ l of template cDNA.Amplification program are as follows: 95 DEG C of 5min；95 DEG C of 10s, 60 DEG C of 30s, 40 circulations；95℃ 15s, 60 DEG C of 60s, 95 DEG C of 15s.QRT-PCR table of the MAP3K8 in mRNA level in-site in PK-15/MAP3K8 cell strain as the result is shown About 250 times are improved compared with cellular control unit strain pCDH-PK-15 up to amount.

(4) it after qRT-PCR experiment, is detected using product of the nucleic acid electrophoresis technology to qRT-PCR, is as a result shown Show: for MAP3K8 gene, the band and pCDH-PK-15 cell strain of the qRT-PCR amplified production of PK-15/MAP3K8 cell strain Compare, significant difference；For β-actin gene, the band of the qRT-PCR amplified production of two cell strains is substantially suitable.

QRT-PCR detection and electrophoresis result illustrate: the cell strain of stable expression MAP3K8 has been screened by above step PK-15, and MAP3K8 being capable of efficient, stable expression in the cell strain.

1 real-time quantitative PCR primer of table

Primer	Sequence (5'-3')	Sequence number
			MAP3K8-pig-F	TCGACCAAAGCCGACATCTA	SEQ ID NO:1
MAP3K8-pig-R	TATCAGCTCCCTCATGCCTG	SEQ ID NO:2
			β-actin-pig-F	TCCCTGGAGAAGAGCTACGA	SEQ ID NO:3
β-actin-pig-R	CGCACTTCATGATCGAGTTG	SEQ ID NO:4
			GAPDH-F	ACATGGCCTCCAAGGAGTAAGA	SEQ ID NO:5
GAPDH-R	GATCGAGTTGGGGCTGTGACT	SEQ ID NO:6
			FMDV-F	CACTGGTGACAGGCTAAGG	SEQ ID NO:7
FMDV-R	CCCTTCTCAGATTCCGAGT	SEQ ID NO:8
			SVV-F	AGAATTTGGAAGCCATGCTCT	SEQ ID NO:9
SVV-R	GAGCCAACATAGAAACAGATTGC	SEQ ID NO:10

Embodiment 2

2.1MAP3K8 dose-dependent inhibition FMDV duplication

(1) pCDNA3.1- MAP3K8 (Pig) is prepared:

PCDNA3.1- MAP3K8 (Pig) is synthesized by Shanghai Jin Kairui company, specifically: it is terminated in MAP3K8 (Pig) sequence 5 ' There are BamHI restriction enzyme site and MYC label, 3 ' to be connected to XhoI restriction enzyme site, then by the sequence and pCDNA3.1 (+) carrier point Not Yong BamHI and XhoI double digestion, connection, the pCDNA3.1-MAP3K8 (Pig) recombinated.

MAP3K8 (Pig) sequence information: the CDS sequence with reference to shown in NCBI sequence number XM_013980209.2.

(2) PK-15 cell dosage dependence transfection pCDNA3.1- MAP3K8 (Pig):

0 μ g, 1 μ g and 2 μ gpCDNA3.1- MAP3K8 (Pig) are transfected into 3 groups of identical PK-15 cell (cell confluency degree respectively Reach 80%), meet FMDV respectively afterwards for 24 hours, connects toxic dose by 1:200.Sample is received after meeting malicious 6h.

(3) preparation of PK-15 cell protein sample:

Culture solution is discarded, is rinsed cell 2 times with 1 × PBS, cell fragment is removed；Suitable NP-40 lysate is added, and makes With PMSF is added in first several minutes, make 1 molL of ultimate density of PMSF^-1；Sufficiently after cracking, 12000 rmin^-1From 10 min of the heart, Sample supernatants is collected in the EP pipe newly prepared, is added 5 × SDS Loading Buffer(and has been added β-mercapto Base ethyl alcohol), 100 DEG C of metal baths boil 10~15 min of sample.

(4) SDS-PAGE proteins gel electrophoresis and transfer:

Prepare 10% polyacrylamide protein adhesive；Suitable protein sample loading is taken, while plus albumen pre-dyed Marker as big Small instruction；Electrophoretic voltage first adjust 80 V run 30 min, after protein sample run out of concentration glue enter separation gel after, voltage is adjusted to 120 V, until electrophoresis terminates；200 mA after electrophoresis, 2 h of transferring film；After transferring film, closed using 5% skimmed milk power room temperature 2 h；After closing, TBST washes 2 times, every time 5 min；It is incubated for primary antibody, 4 DEG C overnight (primary antibody can recycle)；TBST expresses 3 It is secondary, 10 min every time；It is incubated for secondary antibody, is incubated at room temperature 2 h；TBST expresses 3 times, every time 10 min；It is acquired with full resolution pricture and is System carries out ECL development and saves result.The albumen primary antibody of FMDV be VP3 albumen rabbit primary antibody, MYC label primary antibody purchase in Sigma company, β-actin primary antibody, rabbit secondary antibody, mouse secondary antibody are purchases in Proteintech Group company.

As shown in figure 3, the protein band of FMDV is gradually with the increase of pCDNA3.1- MAP3K8 (Pig) transfection dosage It is thin out, illustrate the dose-dependent duplication for inhibiting FMDV of MAP3K8, MAP3K8 has the energy for making host anti-virus, the innate immunity Power.

2.2qRT-PCR verifies PK-15/MAP3K8 cell and inhibits FMDV duplication

The following steps are included:

(1) FMDV venom is diluted to the concentration of 1:500 with the DMEM of serum-free.

(2) it will be laid in cell plates after PK-15/MAP3K8 cell dissociation, be placed in 37 DEG C, 5%CO₂In incubator, to cell It is long to 70%~90% when, cell is cleaned twice with the DMEM of serum-free, to remove remaining serum in cell.

(3) venom diluted is added in cell to (cell culture medium of 2ml adds 4ul's according to volume appropriate FMDV), it is placed in 37 DEG C, 5%CO₂Incubator is incubated for after 1 h, is discarded venom, is changed 2%FBS DMEM maintaining liquid into, continue to cultivate. 0,8h, 16h collect sample after connecing poison.

(4) sample RNA is extracted, then reverse transcription utilizes the primer pair MAP3K8-pig-F/R of amplification MAP3K8 gene NO:7~8 primer pair FMDV-F/R(SEQ ID of the 3D albumen of (NO:1~2 SEQ ID), amplification FMDV) and amplification internal reference NO:5~6 primer pair GAPDH-F/R(SEQ ID of GAPDH gene) qRT-PCR, primer are carried out to the cDNA of reverse transcription respectively To sequence, see Table 1 for details.

As shown in figure 4, MAP3K8 is in PK-15/MAP3K8 cell in the sample collected in 0h, 8h, 16h after connecing poison Expression quantity be above control wild type PK-15 cell (difference is extremely significant).This explanation, PK-15/MAP3K8 cell is in FMDV Stimulate lower overexpression MAP3K8.

As shown in figure 5, in the sample collected in 8h, 16h after connecing poison, FMDV/GAPDH in PK-15/MAP3K8 cell Mrna expression amount is lower than FMDV/GAPDH mrna expression amount (significant difference) in wild type PK-15 cell.That is PK-15/ 8h, 16h can inhibit the duplication of FMDV to MAP3K8 cell after connecing poison compared with wild type PK-15.This is because PK-15/ MAP3K8 cell overexpression MAP3K8 under FMDV stimulation, and then inhibit the duplication of FMDV.This shows PK-15/MAP3K8 Cell can be used to overexpression MAP3K8 and then inhibit the duplication of FMDV.

2.3 indirect immunofluorescences verify PK-15/MAP3K8 cell and inhibit FMDV duplication

(1) PK-15 cell is laid in the glass capsule of 20 mm, when cell it is long to 40%~50% when, connect poison；

(2) 16h collects cell after connecing poison, is cleaned 1 time with 1 × PBS；

(3) with 4% paraformaldehyde (every 1 mL of ware), room temperature, which is protected from light, fixes 1 h；

(4) 3 times are cleaned with 1 × PBS, 5 min/ times (light liquid feeding body, prevent cell from being rushed)；

(5) with 0.2% Triton X-100(100 mL PBS+20 μ LTriton100) the penetrating 1h of room temperature；

(6) 3 times are washed with 1 × PBS, 5 min/ times；

(7) with 5% BSA(1 × PBST:50 mL 1 × PBS+50 μ LTween20,5% BSA:10 mL+0.5g BSA) 37 DEG C closing 1 h；

(8) confining liquid is drawn, addition is stayed overnight with the diluted primary antibody of 5 %BSA (the VP3 albumen rabbit primary antibody of FMDV), 4 DEG C；

(9) it is cleaned three times, 10 min/ time, is added with the diluted fluorescein label secondary antibody (immunofluorescence of 1 × PBST with 1 × PBST Rabbit secondary antibody) (being protected from light), 37 DEG C of 1 h of incubation；

(10) 1 × PBST are cleaned three times, and 10 min/ time, every ware adds 100 μ L to subtract mountant mounting (containing DAPI)；

(11) laser co-focusing observation of use instrument fluorescence is used, and saves picture.

As a result as shown in fig. 6, in the sample collected in 16h after connecing poison, the lesion degree of PK-15/MAP3K8 cell is equal Far below the lesion degree of wild type PK-15 cell.That is PK-15/MAP3K8 cell compared with wild type PK-15 The duplication of 16h inhibition FMDV.This is because PK-15/MAP3K8 cell overexpression MAP3K8 under FMDV stimulation, and then inhibit The duplication of FMDV.This shows that PK-15/MAP3K8 cell can be used to overexpression MAP3K8 and then inhibit the duplication of FMDV.

2.4FMDV TCID in cell strain PK-15/MAP3K8₅₀Measurement

FMDV is carried out 10^-1~10^-8Times gradient dilution, single layer PK-15 cell and PK-15/MAP3K8 cell are covered in inoculation respectively 96 porocyte culture plates, each dilution is inoculated with 8 holes, 0.1 hole mL/, and 37 DEG C of 5~7 d of culture in incubator are seen day by day Record cytopathy (CPE) situation is examined, separation poison TCID is calculated according to Reed-Munch method₅₀.It is measured by Reed-Muench method The TCID of FMDV₅₀, the TCID of PK-15 cell₅₀It is 10^-5.8The TCID of/mL, PK-15/MAP3K8 cell strain₅₀Measurement result is 10^-4.4/mL.This shows that PK-15/MAP3K8 cell inhibits the duplication of FMDV compared with wild type PK-15.

Embodiment 3

3.1qRT-PCR verifies PK-15/MAP3K8 cell and inhibits SVV duplication

The following steps are included:

(1) SVV venom is diluted to the concentration of 1:500 with the DMEM of serum-free.

(4) sample RNA is extracted, then reverse transcription carries out qRT-PCR at cDNA, expands drawing for the 3D albumen conserved region of SVV See Table 1 for details for primer pair (SEQ ID NO:5~6) of the object to (NO:9~10 SEQ ID) and amplification internal reference GAPDH gene.

As a result as shown in fig. 7, in the sample collected in 8h, 16h after connecing poison, SVV/ in PK-15/MAP3K8 cell GAPDH mrna expression amount is lower than SVV/GAPDH mrna expression amount in wild type PK-15 cell (difference is extremely significant).Namely Say that 8h, 16h can inhibit the duplication of SVV to PK-15/MAP3K8 cell after connecing poison compared with wild type PK-15.This is because PK-15/MAP3K8 cell overexpression MAP3K8 under SVV stimulation, and then inhibit the duplication of SVV.This shows PK-15/ MAP3K8 cell can be used to overexpression MAP3K8 and then inhibit the duplication of SVV.

3.2 indirect immunofluorescences verify PK-15/MAP3K8 cell and inhibit SVV duplication

(2) 16 h collect cell after connecing poison, are cleaned 1 time with 1 × PBS；

(4) 3 times are cleaned with 1 × PBS, 5 min/ times (light plus, prevent cell from being rushed)；

(6) 3 times are washed with 1 × PBS, 5 min/ times；

(8) confining liquid is drawn, addition is stayed overnight with the diluted primary antibody of 5 %BSA (the rabbit primary antibody of SVV), 4 DEG C；

As a result as shown in figure 8, in the sample collected in 16h after connecing poison, the lesion degree of PK-15/MAP3K8 cell is equal Far below the lesion degree of wild type PK-15 cell.Illustrate PK-15/MAP3K8 cell compared with wild type PK-15 in 16h energy Inhibit the duplication of SVV.This is because PK-15/MAP3K8 cell overexpression MAP3K8 under SVV stimulation, and then inhibit SVV Duplication.This shows that PK-15/MAP3K8 cell can be used to overexpression MAP3K8 and then inhibit the duplication of SVV.

3.3SVV TCID in cell strain PK-15/MAP3K8₅₀Measurement

SVV is carried out 10^-1~10^-8Times gradient dilution, single layer PK-15 cell and PK-15/MAP3K8 cell are covered in inoculation respectively 96 porocyte culture plates, each dilution is inoculated with 8 holes, 0.1 hole mL/, and 37 DEG C of 5~7 d of culture in incubator are seen day by day Record cytopathy (CPE) situation is examined, separation poison TCID is calculated according to Reed-Munch method₅₀.It is measured by Reed-Muench method The TCID of SVV₅₀, the TCID of PK-15 cell₅₀It is 10^-8.5/mL.The TCID of PK-15/MAP3K8 cell strain₅₀Measurement result is 10^-5.5/mL.This shows that PK-15/MAP3K8 cell inhibits the duplication of SVV compared with wild type PK-15.

The embodiment of the above, only presently preferred embodiments of the present invention, is only used to explain the present invention, not limit The scope of the present invention processed to those of ordinary skill in the art certainly can be according to skill disclosed in this specification Art content, makes other embodiments easily by way of replacing or changing, therefore all in the principle of the present invention and technique item The changes and improvements etc. that part is done, should be included in scope of the present invention patent.

SEQUENCE LISTING

<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences

<120>stablize the PK-15 cell strain for expressing MAP3K8 albumen and its building and application

<130>nothing

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

tcgaccaaag ccgacatcta 20

<210> 2

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

tatcagctcc ctcatgcctg 20

<210> 3

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

tccctggaga agagctacga 20

<210> 4

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

cgcacttcat gatcgagttg 20

<210> 5

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

acatggcctc caaggagtaa ga 22

<210> 6

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

gatcgagttg gggctgtgac t 21

<210> 7

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

cactggtgac aggctaagg 19

<210> 8

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

cccttctcag attccgagt 19

<210> 9

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

agaatttgga agccatgctc t 21

<210> 10

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

gagccaacat agaaacagat tgc 23

Claims

1. stablizing the PK-15 cell strain of expression MAP3K8 albumen, which is characterized in that the cell strain is named as PK-15/MAP3K8, Deposit number is CCTCC NO:C2018262.

2. PK-15 cell strain described in claim 1 is inhibiting the application in FMDV or SVV duplication.

3. PK-15 cell strain described in claim 1 is in pig source cellMAP3K8Gene function and its molecular mechanism grind Application in studying carefully.

4. stablizing the construction method of the PK-15 cell strain of expression MAP3K8 albumen, which is characterized in that specifically includes the following steps: