CN106755110A - A kind of preparation method that Vero/Slam/V is subcloned for strengthening the sensitive cells of PPRV duplications - Google Patents
A kind of preparation method that Vero/Slam/V is subcloned for strengthening the sensitive cells of PPRV duplications Download PDFInfo
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Abstract
A kind of preparation method that Vero/Slam/V is subcloned for strengthening the sensitive cells of PPRV duplications, comprising goat lymphocytic signals activation factor Slam/V primer sequences, slam/V Lentivirals build, the acquisition of slam/V replication defect type slow virus, slam/V slow-virus infection target cells Vero, screening, the checking of sensitive cells subclone Vero/Slam/V, strengthen checking of PPR virus neurological susceptibility etc..The present invention includes thering is the positive cell subclone for improving PPRV replication capacities, and positive cell subclone has good biological characteristics and genetic stability, and the Slam/V of its stabilization expression has functions that to be remarkably reinforced PPRV duplications.
Description
Technical field
It is more particularly to a kind of for strengthening the present invention relates to the technology of preparing of sensitive cells subclone in field of biology
The sensitive cells that PPRV is replicated is subcloned the preparation method of Vero/Slam/V.
Background technology
PPR (Peste des petits ruminants, PPR) is by PPR virus (Peste des
Petits ruminants virus, PPRV) cause, with hyperpyrexia (more than 40 DEG C), conjunctivitis, eye, nose mucus or purulence point
Secretion, canker sore, cough, the diarrhoea of stench are main clinical characteristics, and pneumonia and hemorrhagic enteritis are main pathological change
Acute, hot, highly contagious disease.The disease belongs to paramyxovirus section (Paramyxo-viridae) Morbillivirus
(Morbillivirus) member, what is belonged to together also has rinderpest virus (Rinderpestvirus, RPV), CDV
(Canine distemper virus, CDV), sea dog pestivirus (Porpoise distemper, PDV), dolphin pestivirus
(Dolphin distemper, DDV) and measles virus (Measles Virus, MV).Virus isolation and Identification is current popular
The basis of PPRV strain biological characteristic researches, but PPRV is in the cell line that Vero, BHK-21, PK-15 etc. are commonly used or is difficult
Adapt to, or do not produce typical cells lesion (CPE), or pass on restrovirus loss several times, separation rate is relatively low, and it is right to influence and limit
The research of the separation, biological characteristics and mechanism of causing a disease of PPRV street strains.Therefore, set up and be easy to the sensitive thin of PPRV culture propagation
Born of the same parents, further investigation and vaccine virus production to carrying out PPRV all have important application value.
The content of the invention
The technical problem to be solved in the present invention overcomes prior art to be difficult to be separated to high titre PPR virus
Deficiency, building one kind of the lentivirus-mediated stable integration of targeting PPR virus specific cells acceptor is used to strengthen
The sensitive cells that PPRV is replicated is subcloned the preparation method of Vero/Slam/V.
To solve the above problems, present invention employs following technical proposals：
A kind of preparation method that Vero/Slam/V is subcloned for strengthening the sensitive cells of PPRV duplications, including goat letter
Number lymphocyte activator molecule Slam/V primer sequences, by building goat Slam/V Lentivirals, obtain Slam/V
Replication defect type slow virus like-particles, Vero target cells, screening stabilization table are infected with the Slam/V slow virus like-particles for obtaining
Vero positive cells up to Slam/V are subcloned, Slam activity, the heredity of checking positive cell subclone Vero/Slam/V expression
Stability and its cultivation effect replicated to PPRV.
The sequence SEQ ID NO:1 to SEQ ID NO:2 include：
SEQ ID NO.1:GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCTAGATCTGCGGAAAGGTGACT,
SEQ ID NO.2:GGGGACAACTTTTGTATACAAAGTTGTGGCATGCTGAGGGCCAAGAGTGAG。
The Slam/V Lentivirals build, including Slam/V and pDONRTM221 donor vehicles carry out B, P site
Restructuring obtains entry vector pDONR/Slam/V, and pDONR/Slam/V and pLenti6.2/V5-DESTTM
Vector expression vectors carry out the restructuring of L, R site and obtain expression cloning pDEST/Slam/V.
The acquisition of the complete slow virus of Slam/V, is by pDEST/Slam/V plasmids and package combination pLP1, pLP2
And VSV-G transfects human embryonic kidney cell line 293-FT jointly, harvesting supernatant is obtained with infective Slam/V replication defectives
Type slow virus like-particles.
The Vero/Slam/V positives sensitive cells subclone, Vero is infected with the Slam/V slow virus like-particles for obtaining
Cell, and resistance screening is carried out using blasticidin S blasticidin, obtain the Vero positive cells of stabilization expression Slam/V
Subclone.
The Vero/Slam/V positive cells subclone improves the verification method of PPR virus print effect, adopts
With skills such as target gene amplification, indirect immunofluorescence, western-blot immune-blotting methods technology and CPEs
Art separately verifies slam/V genome conformities, transcription, and slam/V protein expressions, reactivity and PPRV are in expression slam/V sun
Cultivation effect on property cell subclone.
The preparation of Vero/slam/V functional areas of the present invention sensitive cells subclone and comprising the following steps that for verification method：
A. the primer for Slam/V functional areas is designed and synthesized, the primer is with same with the B sites that carrier P sites match
Source arm；
B. goat PBLC is separated, its total serum IgE is extracted；
C. amplification Slam/V functional areas, and and pDONRTM221 donor vehicles utilize BPⅡEnzyme Mix
Enzyme carries out B, P site homologous recombination, restructuring thing conversion OneMach1TM T1R Chemically Competent
Cells competent cells, select positive colony, and the fidelity of checking sequence is sequenced, and obtain entry vector pDONR/Slam/V；
D. by above-mentioned pDONR/Slam/V and pLenti6.2/V5-DESTTM Vector expression vectors pass through
LRII plus Enzyme Mix enzymes carry out L, R site homologous recombination, obtain expression skeleton pDEST/Slam/V；
E. the expression skeleton pDEST/Slam/V plasmids for obtaining and Packaging Mix pLP1, pLP2 and VSV-G corotation
Dye 293-FT cells, obtain Slam/V replication defect type slow virus like-particles；
F. with the Slam/V replication defect type slow virus like-particles vero cells infections of above-mentioned acquisition；
G.blasticidin resistance screenings, obtain the Vero positive cells subclone of expression Slam/V functional areas；
H. it is thin with target gene amplification, indirect immunofluorescence, western-blot immune-blotting methods technology and cause respectively
The technologies such as born of the same parents' lesion effect separately verify slam/V genome conformities, transcription, slam/V protein expressions, reactivity and PPRV
Cultivation effect on expression slam/V positive cell subclones.
Lymphocytic signals activation factor (Signaling lymphocyte activation molecule, SLAM, and
Claim CD150) be PPRV infection cell receptor, two characteristic domains with immunoglobulin superfamily：The V knots of N-terminal
Structure domain and the C2 domains of C-terminal, wherein V structure domain are the functional areas combined with virus protein, comprising specific with virus protein
With reference to key amino acid site.Therefore, the acceptor of expression Slam/V functional areas can improve the quick of cell separation virus really
Perception, but the country is currently without the sensitive cells that can improve PPR separation.Therefore select Slam/V domain conducts
Target sets up PPRV sensitive cells.
In consideration of it, the present invention is by slow virus expression system, using Gateway technologies by goat Slam/V function area genes
Stable integration obtains permanent table on Vero cytogene group chromosomes by blasticidin S blasticidin resistance screenings
Also it is PPRV for PPR virus separation, identification provide reliable tools up to the stable cell subclone of Slam/V functional areas
The research such as course of infection, mechanism of causing a disease lays the foundation, and the further investigation and vaccine virus production to carrying out PPRV all have important
Application value.Embody as follows：
Firstth, the Gateway technologies that are constituted using BP and LR two reactions are quick, accurate, efficiently build pDEST/Slam/
V expression vectors.The step of technology significantly simplify gene cloning and subclone, it is ensured that be correctly oriented and reading frame,
It is easy to analysis and the protein expression of functional gene.Target DNA fragmentation being binned between carrier by site-specific
Transfer.
Secondth, Vero/Slam/V sensitive cells subclone is existed Slam gene stable integrations using slow virus expression system
On Vero cytogene group chromosomes, good genetic stability is made it have, that is, pass on and do not lose, although transient transfection also has
There is certain effect, but only reside within Qualify Phase, the real positive cell that there is stabilization enhancing PPRV to replicate is not set up
Subclone.
3rd, the Vero/Slam/V sensitive cells for building is subcloned compared with normal Vero cells, is slightly changed in form
Become, verified through a series of experiments such as cell growth curve, cytotoxicity experiments, its biological characteristics is no abnormal, so no matter
Scientific research still produces vaccine, and Vero/Slam/V sensitive cells subclone can be completely used for small lot or extensive raw
Product is used.
4th, compared with normal Vero cells, the Vero/Slam/V sensitive cells subclone of structure can be significantly improved
PPRV separation rates, one PPRV causes the lesion time of Vero/Slam/V sensitive cells subclone than causing normal Vero cytopathies
Time substantially shortens, secondly the copy number of Vero/Slam/V sensitive cells subclone isolated viral is improved than normal Vero cells
More than 100 times.
No matter the Vero/slam/V sensitive cells subclone that the present invention is prepared using slow virus expression system is from gene water
When flat, transcriptional level or protein level are verified, good activity is shown.Vero/slam/V grows with normal Vero
Without significant difference, illustrate the protein on cells of expression does not have overt toxicity to characteristic.Vero/slam/V and normal Vero cells phase
Than the cytopathy time substantially shortens after inoculation PPRV, it is seen then that the slam/V of Vero/slam/V sensitive cells subclone expression
The separation rate of PPRV can be significantly improved.
Brief description of the drawings
Fig. 1 is slam/V gene integrations and its heredity in the sensitive cells of genome amplification checking Vero/slam/V functional areas
Stability nucleic acid electrophoresis figure；
Fig. 2 is indirect immunofluorescence slam gene tables from the sensitive cells of protein level checking Vero/slam/V functional areas
Up to fluorogram；
Fig. 3 is the western- of activity analysis behind Vero/slam/V functional areas sensitive cells expression slam/V functional areas
Blot result protein electrophoresis figures；
Fig. 4 is neurological susceptibility cytological maps of the PPRV to Vero/slam/V functional areas sensitive cells；
Fig. 5 is cultivation effects of the real-time RT-PCR checkings PPRV on the sensitive cells of vero/slam/V functional areas
Block diagram.
A is amplification slam/V genes after the cell screening Amplification Culture of Vero/slam/V functional areas in Fig. 1；B is Vero/
Slam/V functional areas cell expands slam/V genes after passing for 30 generations；M is DL2000 molecular weight Marker；
A is normal vero cells in Fig. 2；B is vero/slam/V functional areas cell；C is Immunofluorescence test slam/V bases
Because of the expression in vero cells；D is table of the Immunofluorescence test slam/V genes in the cell of vero/slam/V functional areas
Reach；
A is that PPRV causes vero cytopathic effects in Fig. 4；B is that PPRV causes vero/slam/V functional areas cytopathy effect
Should.
Specific embodiment
Below in conjunction with the accompanying drawings and embodiment is described in further detail to the present invention.
Embodiment 1
1st, design and synthesize the primer of the signal lymphocyte activator molecule Slam/V functional areas for goat, the primer with
The matching of carrier P sites can carry out homologous recombination, i.e., with B sites homology arm, sequence is as follows：
SEQ ID NO.1:GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCTAGATCTGCGGAAAGGTGACT,
SEQ ID NO.2:GGGGACAACTTTTGTATACAAAGTTGTGGCATGCTGAGGGCCAAGAGTGAG。
2nd, goat signal lymphocyte activator molecule RNA templates are prepared
Gather and separating health goat PBLC, extract its total serum IgE.
3rd, the DNA of Slam/V functional areas is prepared
Amplification Slam/V functional areas, set up 50ul amplification systems, as follows：
Above-mentioned click is mixed, and carries out RT-PCR amplifications, and reaction condition is：
4th, Slam/V entry vectors build
Gel is reclaimed and purifies above-mentioned PCR primer, and and pDONRTM221 donor vehicles utilize BPⅡ
Enzyme Mix enzymes carry out B, P homologous recombination, and BP recombining reaction systems are：
Click on and mix, 25 DEG C carry out recombining reaction, proteinase k are added after 2h, in system, after 37 DEG C of reaction 10min
Product converts OneMach1TM T1RChemically Competent Cells competent cells, select positive gram
It is grand, the fidelity of checking sequence is sequenced, entry vector is obtained, it is named as pDONR/slam/V.
5th, the structure of pDONOR/slam/V expression vectors
By above-mentioned pDONR/slam/V functional areas and pLenti6.2/V5-DESTTM Vector expression vectors
By LRII plus Enzyme Mix enzymes carry out L, R site homologous recombination, and LR recombining reaction systems are：
Click on and mix, 25 DEG C carry out recombining reaction, proteinase k, 37 DEG C of reaction 10min are added after 16h, in system
Product enters Ones of the 50uL in thawed on ice afterwardsMach1TM T1RChemically Competent Cells competence
Mixed in cell, ice bath 30min, 42 DEG C of thermal shock 60s (being sure not vibration), 2~3min of ice bath add the SOC cultures of 450uL preheatings
Base, sealed membrane sealing, is mixed, 37 DEG C of horizontal rotating speed 225rpm, vibrates 1h, 4000rpm centrifugation 2min, and about 350uL is abandoned in suction, remaining
100uL bacterium solutions coat the LB flat boards of ammonia benzyl resistance, and after upright flat board absorbs liquid 10min, 37 DEG C are inverted culture 14h to single bacterium
Drop out existing.Random picking is grown on the several single bacterium colonies on LB (ammonia benzyl resistance), plasmid is extracted after Zengjing Granule in a small amount, through PCR
Sequence verification its fidelity of the positive is accredited as, the expression skeleton that checking is suitably from obtained is named as pDEST/slam/V.
6th, the acquisition of slam/V replication defect types slow virus like-particles
PDEST/slam/V functional areas and optimized ViraPowerTMPackaging Mix (pLP1, pLP2 and
VSV-G) cotransfection HEKC 293-FT (transfection of the method for liposome 3000), it is sick slowly that 48h harvests the slam/V without replication capacity
Malicious supernatant.4 DEG C of 3500rpm, centrifugation 5min collect supernatant to remove cell fragment, and as slam/V replication defect types are sick slowly
Malicious like-particles, -70 DEG C save backup.
7th, the screening of slam/V replication defect types slow virus like-particles vero cells infection and positive cell subclone
Vero cell culture fluids are abandoned in suction, add 3ml slam/V replication defect type slow virus liquid, 37 DEG C of incubation 2h to add
7ml normal cell nutrient solutions, change the fresh DMEM nutrient solutions containing blasticidin after 2d, carry out resistance screening.Work as appearance
During the cell clone of blasticidin resistances, pancreatin digestion goes to new blake bottle, and resistance screening is continued after cell is covered with.
Until when cell is no longer dead, carrying out limiting dilution, cell subclone island, and Amplification Culture, continuous passage are looked for, carried out a series of
Subsequent authentication, analysis.
8th, by genome amplification from gene level verify Vero/slam/V cell subclones in slam/V gene integrations and
Its genetic stability
One bottle of Amplification Culture and the continuous Vero/slam/V functional areas cell passed after 30 generations are taken, nutrient solution, pancreatin are abandoned in suction
After digestion, 500ul PBS are resuspended, and its genomic DNA is extracted using genome DNA extracting reagent kit, with SEQ IDNO.1, SEQ
IDNO.2 is primer, is expanded, and amplification system and amplification condition are as described above.
9th, result
As shown in figure 1, equal Successful amplification is to slam/V functional areas base in Vero/slam/V functional areas and its 30 generation cells
Cause, clip size is consistent with expection, is 429bp, and sequence verification is suitable, illustrates that slam/V functional areas have successfully been incorporated into Vero thin
In born of the same parents' genome chromosome, in 30 generations of continuous biography, do not lose, it is seen that with good genetic stability.
Embodiment 2
1-7 steps are with embodiment 1.
8th, slam/V gene expressions in the cell of Vero/slam/V functional areas are verified from protein level by indirect immunofluorescence
Situation
The positive vero/slam/V functional areas positive cell subclone of step 7 screening in case study on implementation 1 is inoculated in and adds in advance
Enter 6 porocyte culture plates of film flying, after culture 48h, take out film flying, cell face is rinsed 2 times with PBS (pH7.6), drain
80% acetone of precooling is added after liquid, -20 DEG C of refrigerators is put into and is fixed 25min, acetone is abandoned in suction, with PBST buffer solutions (pH7.6)
Washing cell monolayer 3 times.At room temperature 45min is closed with the PBS confining liquids containing 3~5%BSA.Confining liquid is discarded, is washed with PBS
Cell 3 times.PBS 1 is added per hole:The anti-Slam primary antibodies of the goat (Santa Cruz Bioisystech Co., Ltd) of 200 dilutions, 37 DEG C
1h is acted in wet box, primary antibody is abandoned, cell monolayer is washed with PBST buffer solutions 3 times.1 is added per hole:The different sulphur cyanogen of FITC of 500 dilutions
Fluorescence secondary antibody is abandoned in the fluorescein-labeled donkey anti goat igg of acid, 37 DEG C of incubation 1h in wet box, suction, and cell is rinsed 3 times with PBST, is inhaled
2 are added dropwise after dry flushing liquor and drip 50% glycerite, be placed in fluorescence microscopy Microscopic observation coloration result and take a picture.
9th, result
Found by Immunofluorescence test, slam/V genes are expressed under the mediation of pseudotype virus, and expression
Albumen can be recognized that and slam expression of receptor amounts are very low in normal vero cells by slam FLAs.Further
The vero/slam/V positive cells subclone for demonstrating structure from protein level can improve the expression quantity of slam/V acceptors.Such as
Fig. 2.
Embodiment 3
1-7 steps are with embodiment 1.
8th, the activity analysis after Vero/slam/V positive cells subclone expression slam/V
Digested with pancreatin, PBS collects vero/slam/V positive cells subclone, through 12% after cell pyrolysis liquid cracking
PVDF transfer membranes are transferred to after SDS-PAGE electrophoresis, PBS washes film 3 times, each 5min, the PBS that 5% skimmed milk power is added dropwise was closed
Night.PBST washes film 3 times, each 5min, adds 1:The anti-slam primary antibodies of goat of 200 dilutions, 37 DEG C combine 1.5h.Film 3 is washed with PBS
Secondary, each 10min adds the anti-goat ELIAS secondary antibody of donkey of 1: 500 dilution, and jog is incubated 1h at room temperature, and PBST washes film 3 times, often
Secondary 5min, through four chloro- 1- how phenol substrate colour developing, amino black staining analysis result, with check expression product whether have specificity
Immunocompetence.
9th, result
After SDS-PAGE electrophoresis, there is positive band in 14kDa or so in the foreign protein of expression, and blank control group is not
There is specific band, illustrate the active structure of foreign protein of expression, it is anti-with goat respectively after being transferred to PVDF transfer membranes
Slam primary antibodies and donkey anti goat igg/HRP effects, as a result show to occur specific color band on purpose band position (as schemed
3), there is not band in normal vero compared with control cells, and illustration purpose albumen slam/V functional areas can recognize have by slam antibody
Immunologic competence.
Embodiment 4
1-7 steps are with embodiment 1.
8th, inoculation PPRV viruses, the neurological susceptibility that checking PPRV is subcloned to vero/slam/V positive cells
Each two bottles of vero/slam/V and vero normal cells are taken simultaneously, while setting blank, nutrient solution is abandoned, is inoculated with
PPRV virus liquids 3ml, 37 DEG C of incubation 2h, add maintaining liquid 7ml, put 37 DEG C, observe cytopathogenic effect situation.
9th, result
After inoculation PPRV, compared with normal vero cells (A in such as Fig. 2), PPRV causes sub- gram of vero/slam/V positive cells
Grand (B in such as Fig. 2) the lesion time substantially shortens, and such as Fig. 4 shows, the slam/V of vero/slam/V positive cells subclone expression
With raising PPRV neurological susceptibility effects.This result is provided compared with good tool for breeding PPRV.
Embodiment 5
1-7 steps are with embodiment 1.
8th, inoculation PPRV viruses, real-time RT-PCR are expressed from transcriptional level checking transgene positive cells subclone
Goat slam/V to the cultivation effect of PPRV
PPRV is inoculated with vero/slam/V sensitive cells subclone and normal vero cells respectively, respectively at 48h, 72h and
96h collects virus, after -70 ° of refrigerator multigelations, respectively takes 400ul, and Trizol methods extract its RNA respectively, and it is dense that ND2000 determines its
Degree, dilution makes all RNA be maintained at a concentration (100ng/uL), and each sample 3ul is drawn using the good PPRV of laboratory proofing
Thing, probe primer and β-actin house-keeping gene primers are diluted to 10umol/L respectively, according to One StepPrimer ScriptTM
RT-PCR Kit (Perfect Real time) specification is matched, synchronous amplification PPRV and β-actin genes, each sample weight
It is multiple 3 times.Then, with the Relative copy number for comparing PPRV in the different samples of threshold method analysis.
9th, result
As shown in Figure 5, no matter which of 48h, 72h or 96h at time point, PPRV is in transgenosis vero/slam/V sensitive cells
On subclone breed Relative copy number than normal Vero cells will be high, illustrate vero/slam/V sensitive cells subclone
The slam of stabilization expression has the function of being remarkably reinforced PPRV breedings.
Organization Applicant
----------------------
Street :Chengguan District, Lanzhou City, Gansu Province saltern Bao Xujia level grounds 1
City :Lanzhou
State :Gansu
Country :China
PostalCode : 730046
PhoneNumber : 0931-8343385
FaxNumber : 0931-8340977
EmailAddress : jingningcaixiong@163.com
<110> OrganizationName :Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
Application Project
-------------------
<120> Title :A kind of preparation method that Vero-Slam-V is subcloned for strengthening the sensitive cells of PPRV duplications
<130> AppFileReference : V Domain of Human SLAM(CDw150) Is Essential for
Its Function as a Measles Virus Receptor
<140> CurrentAppNumber :
<141> CurrentFilingDate : ____-__-__
Sequence
--------
<213> OrganismName : Ovis aries
<400> PreSequenceString :
ggggacaagt ttgtacaaaa aagcaggctt aatgctagat ctgcggaaag gtgact 56
<212> Type : DNA
<211> Length : 56
SequenceName : SEQ ID NO.1
SequenceDescription :
Sequence
--------
<213> OrganismName : Ovis aries
<400> PreSequenceString :
ggggacaact tttgtataca aagttgtggc atgctgaggg ccaagagtga g 51
<212> Type : DNA
<211> Length : 51
SequenceName : SEQ ID NO.2
SequenceDescription :
Claims (7)
1. it is a kind of for strengthen PPRV duplication sensitive cells be subcloned Vero/Slam/V preparation method, it is characterised in that bag
Goat signal lymphocyte activator molecule slam/V primer sequences are included, by building goat slam/V Lentivirals, is obtained
Slam/V replication defect type slow virus like-particles are obtained, with the slam/V slow-virus infection Vero target cells for obtaining, screening stabilization
Express the Vero positive cells subclone of slam/V, the work of the Slam/V of checking positive cell subclone Vero/Slam/V expression
Property, genetic stability and its to PPRV replicate cultivation effect.
2. it is according to claim 1 it is a kind of for strengthen PPRV duplication sensitive cells be subcloned Vero/Slam/V system
Preparation Method, it is characterised in that the slam/V primers carry the B sites homology arm with Carrier-matching, its sequence is：
SEQ ID NO.1:GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCTAGATCTGCGGAAAGGTGACT,
SEQ ID NO.2:GGGGACAACTTTTGTATACAAAGTTGTGGCATGCTGAGGGCCAAGAGTGAG。
3. it is according to claim 1 it is a kind of for strengthen PPRV duplication sensitive cells be subcloned Vero/Slam/V system
Preparation Method, it is characterised in that the structure of the Slam/V expression vectors, including Slam/V and pDONRTM221 donor vehicles carry out B,
P sites recombinate, and obtain entry vector pDONR/Slam/V, pDONR/Slam/V and pLenti6.2/V5-DESTTM
Vector expression vectors carry out the restructuring of L, R site and obtain expression cloning pDEST/Slam/V.
4. it is according to claim 1 it is a kind of for strengthen PPRV duplication sensitive cells be subcloned Vero/Slam/V system
Preparation Method, it is characterised in that the acquisition of the Slam/V replication defect types slow virus like-particles, is by pDEST/Slam/V plasmids
Human embryonic kidney cell line 293-FT is transfected jointly with package combination pLP1, pLP2 and VSV-G, and harvesting supernatant is had
Infective Slam/V replication defect types slow virus like-particles.
5. it is according to claim 1 it is a kind of for strengthen PPRV duplication sensitive cells be subcloned Vero/Slam/V system
Preparation Method, it is characterised in that the Vero positive cells subclone of the screening stabilization expression slam/V, is to use blasticidin S
Blasticidin carries out resistance screening, obtains the Vero positive cells subclone of stabilization expression Slam/V.
6. a kind of preparation that Vero/Slam/V is subcloned for strengthening the sensitive cells of PPRV duplications as claimed in claim 5
Method, it is characterised in that the Vero cells are 3.5ug/ml to the tolerable concentration of blasticidin blasticidin Ss.
7. a kind of preparation that Vero/Slam/V is subcloned for strengthening the sensitive cells of PPRV duplications as claimed in claim 1
Method, it is characterised in that the sensitive cells is subcloned the checking of Vero/Slam/V, using target gene amplification method, indirectly
Immunofluorescence method, western-blot immune blotting detection methods and CPE separately verify slam/V genomes
Integrate, transcribe, the propagation of slam/V protein expressions, reactivity and PPRV on the positive cell subclone of expression slam/V
Effect.
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