CN109266615A - A kind of cell BHK/slam/v based on PPR virus receptor - Google Patents
A kind of cell BHK/slam/v based on PPR virus receptor Download PDFInfo
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- CN109266615A CN109266615A CN201810942663.3A CN201810942663A CN109266615A CN 109266615 A CN109266615 A CN 109266615A CN 201810942663 A CN201810942663 A CN 201810942663A CN 109266615 A CN109266615 A CN 109266615A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C12N2740/00—Reverse transcribing RNA viruses
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- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
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Abstract
The present invention provides a kind of cell BHK/slam/v based on PPR virus receptor, its deposit number are as follows: CCTCC NO:C201739, PPR virus receptor-functional areas goat lymphocytic signals activation factor V of cell expression have the function of that PPR virus duplication is remarkably reinforced, the cell shortens the malicious time (shortening to 3-4d from 4-7d) of receipts after cytopathogenic effect by overexpression PPR virus specific cells receptor, improve PPR virus poison valence (ct value is increased to 17.01 from 20.19), same volume viral copy number is also increased considerably (copy number is increased to 4.9X108 from 5.6X106), cost has been saved simultaneously.Good tool is provided for PPR virus separation and vaccine virus production, also provides cell model for PPR virus pathogenic mechanism research.The present invention also provides the preparation of the cell and applications.
Description
Technical field
The present invention relates to the foundation of cell line in field of biology, in particular to PPR virus recipient cell system
Building and application, specifically a kind of cell BHK/slam/v based on PPR virus receptor.The cell line is named as
BHK/Slam/V is preserved in China typical culture collection center (Wuhan, China Wuhan University), deposit number in 2017.7.6:
CCTCC NO:C201739。
Background technique
Peste des petits ruminants (Peste des petits ruminants, PPR) is by PPR virus (Peste des
Petits ruminants virus, PPRV) cause sheep and goat, especially lamb fever, stomatitis, diarrhea, pneumonia etc. is characterized
Acute, hot highly contagious disease.2007, peste des petits ruminants was passed to China Ali, Tibet, Ministry of Agriculture's meeting for the first time
It adopts an effective measure immediately with local government, controls epidemic situation rapidly.Peste des petits ruminants is passed to me again in by the end of November, 2013
State, epidemic situation involve multiple provinces.The disease is OIE statutory report animal epidemic, and the animal epidemic that whole world plan is eradicated, China
It is classified as a kind of animal epidemic.The Ministry of Agriculture prints and distributes " national peste des petits ruminants elimination plan (2016-on December 24th, 2015
The year two thousand twenty) " notice.Various regions animal and veterinary department prompt action, epidemic situation are effectively controlled, utmostly reduce epidemic situation and make
At loss.2016, all provinces in the whole nation were completed peste des petits ruminants overall state in the respective administrative areas and are assessed.Immune province after
Continuous to implement to be immunized, the nonimmune province such as Fujian, Hainan continues to carry out Monitoring and supervision work.By 2018, all immune provinces in the whole nation
Part reaches immune without peste des petits ruminants area (hereinafter referred to as immune without epidemic-stricken area) standard, and exits to all immune provinces developments immune
Risk assessment；According to assessment result, the whole nation is gradually exited immune.Nonimmune province reach nonimmune no peste des petits ruminants area (with
The lower nonimmune no epidemic-stricken area of abbreviation) standard.
To the year two thousand twenty, except the land border county (group field) for adjoining peste des petits ruminants epidemic situation country or along 30 kilometers of models of boundary line
It encloses other than interior immune isolation strip, strives that the whole nation reaches nonimmune no epidemic-stricken area standard.(http://www.moa.gov. cn/
Govpublic/SYJ/201512/t20151225_4966587.htm it) to realize this target, shoulder heavy responsibilities.This disease is studied, point
From basis and the key for virus being targeting research, and finding suitable target cell is the thing stood in the breach.Biology at present
The cell or virus in known field are not easy to adapt to or virocyte poison valence is lower, some cells, and virus adapts to pass not yet
Several generations just can't detect virus, seriously affect and limit the separation to the field PPRV acquisition strain, identification and biology
The research of characteristic and pathogenic mechanism etc..
The study found that entering cell after the cell entry animal upper respiratory tract with cyst membrane and host cell membrane amalgamation mode, opening
Dynamic virus infection.Lymphocytic signals activation factor (Signaling lymphocyte Activation molecule,
SLAM, also known as CD150) be PPR virus infection cell receptor (Pawar R M, et al.Effect of si
RNA mediated suppression of signaling lymphocyte activation molecule on
replication of peste des petits ruminants virus in vitro[J].Virus Res, 2008a,
136 (1-2): 118-123.), this receptor expression quantity in resting cell is very low, but its expression quantity is sharply after cell activation
Rise, promotes the specific binding of virus with cell receptor.Data shows that SLAM has two of immunoglobulin superfamily
Characteristic domain: the V structure domain of N-terminal and the C2 structural domain of C-terminal, wherein V structure domain is the functional areas in conjunction with virus protein,
Key amino acid site comprising 8 with virus protein specific binding, is distributed in H protein and SLAM interacts and to be formed
(Hashiguchi T, et al.Structure of the measles virus hemagglutinin on β-pleated sheet face
bound to its cellular receptor SLAM[J].Nat Struct Mol Biol.2011,18(2):135-
41.Howie D,et al.The role of SAP in murine CD150(SLAM)-mediated T-cell
proliferation and interferon gamma production[J]. Blood,2002,100(8):2899-
2907. cover Beijing research [D] of saussurea involucrata PPR virus hemagglutinin and its receptor: Chinese Academy of Agricultural Sciences's research
Raw institute, 2012.).The genetic affinity of different animals SLAM molecule is far and near directly to determine virus to the infection characterization of host
(Ohishi K,Ando A,Suzuki R,et al.Host virus specificity of morbilliviruses
predicted by structural modeling of the marine mammal SLAM,a receptor[J].Comp
Immunol Microbiol Infect Dis,2010, 33(3):227-241.).Actually mainly pass through hemagglutinin (H) egg
White configuration changes the C2 knot in the V structure domain and nearly film of identification cell surface receptor slam, slam on molecular structure including remote film
Structure domain, wherein V structure domain is necessary to PPRV H albumen identifies and is in combination.
Summary of the invention
The technical problem to be solved by the present invention is to be difficult to be separated to this hardly possible of high titre PPR virus for current
Topic provides a kind of cell that can be used for the separation of PPR virus high titre.
To solve the above problems, present invention employs following technical proposals:
A kind of cell BHK/slam/v based on PPR virus receptor, the PPR virus recipient cell
Sequence after the translation of the functional areas V are as follows: SEQ ID NO.1:LDLRKGDSPRLEDGYEFHLENLSLRILKSRKED EGWYFISL
EENVSVQHFSLQLKLYEQVSTPQIKVLNSTQEDGNCSLMLACVVEKGDHVTYNWSEEAGAPLLSPT
NSSHLLYLTLGPQHA。
The cell BHK/slam/v based on PPR virus receptor is slow by constructing using SEQ ID NO.1
Virus expression carrier obtains replication defect type slow virus, with the slow virus Supernatant infection bhk cell of acquisition, screening expression
The bhk cell of the functional areas slam/v clones and verifies its reinforcing effect to PPR virus duplication.
The cloning vector for constructing the expression skeleton of the PPR virus receptor is pDONRTM221P5-P2 (2-
Fragment), expression vector pLenti4/V5-DESTTM Vector；It is carried with above-mentioned cloning vector and expression
The matched primer sequence of body such as SEQ ID NO.2 and SEQ ID NO.3:
SEQ ID NO.2:GGGGACAACTTTGTATACAAAAGTTGTAATG
CTAGATCTGCGGAAAGGTGACT,
SEQ ID NO.3:GGGGACCACTTTGTACAAGAAAGCTGGGTT
GGCATGCTGAGGGCCAAGAGTGAG。
It is packed using the expression skeleton cotransfection 293-FT cell, 100 square meter cell bottles is selected, in 750ul
Expression skeleton is separately added into opti- MEM, the amount of pLP1, pLP2 and VSV-G plasmid is 12-15ug；Infect target cell
BHK, sense make the time as 1-3h；Screening surely turns cell, makes the blasticidin S-HCl blasticidin S of bhk cell death
Minimum concentration is 3.0ug/ml.
The cell BHK/slam/v is verified using following methods: target gene amplification；Western-Blot is immune
Trace；Induced cytopathic effect and real time fluorescent quantitative calculate Relative copy number.
The present invention uses high-throughput gene clone technology (Gateway Cloning Technology) in insertion first
Two side recombination sequences of attL1 and attL2 are integrated at target DNA fragment both ends, to construct the structure and title of a channel-like
Be " entry clones " (Gateway Entry Clone), substantially increase cloning efficiency, integrate large-scale DNA fragmentation
In expression vector pLenti4/V5-DESTTM Vector is packaged into not no replication capacity by incasing cells
Pseudovirus；Then pseudovirus infects target cell BHK, obtains the expression functional areas Slam/V by 2-3 resistance pressure screening
Positive cell；Monoclonal cell is obtained by limiting dilution again, expanding is the permanent expression constructed after cultivating and passing on verifying
The positive cell of the functional areas Slam/V.The success of the cell is configured to PPR virus separation, identification provides reliable tools,
Also it lays the foundation for researchs such as PPRV course of infection, pathogenic mechanisms, all has to the further investigation for carrying out PPRV and vaccine virus production
There is important application value.
It embodies as follows:
The first, RNA is extracted using innovation centrifugal column method, feature are as follows: unique genomic DNA is removed column 1 minute and removed
DNA is not necessarily to DNase enzymatic treatment, effectively removes genomic DNA；Phenol, the chloroform used without TRIzol class reagent etc. is toxic to be had
Evil reagent；The advantages that guanidinium isothiocyanate/phenol one-step method reagent stability is good, with high purity, centrifugal column is convenient and efficient is combined, is not necessarily to
Isopropanol precipitating process, RNA can be eluted directly from centrifugal column to be avoided over-drying not readily dissolving problem；It can obtain within 15 minutes
To instant high-quality total serum IgE.
The second, using high-throughput gene clone technology, slow virus expression system and packaging and stable integration system, fastly
Speed, efficient, accurate, low cost construction of expression vector, are packaged into pseudovirus, finally realize stable integration, establish complete thin
Born of the same parents system, this good tool can be completely used for small lot or use of large-scale production.Completely instead of transient transfection before,
It solves and is not easy the problems such as passing on.
Third, building BHK/Slam/V sensitive cells compared with normal bhk cell, slight change in form, through cell
The verifying of a series of experiments such as growth curve, cytotoxicity experiment, biological characteristics are without abnormal.But shorten cytopathogenic effect
The malicious time (shortening to 3-4d from 4-7d) of receipts afterwards, improving PPR virus poison valence, (ct value is increased to from 20.19
17.01), same volume viral copy number is also increased considerably (copy number is increased to 4.9X108 from 5.6X106), substantially
Degree has saved cost.
In conclusion the present invention selects goat slam/V structural domain as target spot, system is expressed and packed by slow virus
System is integrated in target cell, establishes the sensitive cells for being easy to PPRV culture proliferation, for carry out PPRV further investigation and
Vaccine virus production all has important application value.
Detailed description of the invention
Fig. 1 is goat peripheral blood lymphocytes TotalRNA integrality electrophoretogram；
Genetic stability nucleic acid electrophoresis reflects after target gene and genome conformity cell that Fig. 2 is expanded when being carrier construction
Fixed figure；
Fig. 3 is replication defect type slow virus like-particles (packaging slam/v receptor) infection bhk cell after blasticidin-S screens
Monoclonal cell figure；
Fig. 4 is normal BHK and BHK/slam/v cell growth curve figure；
Fig. 5 is cell survival figure when normal BHK and BHK/slam/v cell make growth curve；
Fig. 6 is that BHK/slam/v cell expresses slam/v receptor and anti-goat slam secondary antibody marking figure；
Fig. 7 is that PPRV low virulent strain is inoculated with cytopathogenic effect proof diagram after BHK and BHK/slam/v cell；
Fig. 8 is different time points after inoculation PPRV low virulent strain, and the slam/V of BHK/slam/V cell expression is in transcriptional level
To the proof diagram of PPRV cultivation effect.
M is 250bpDNA molecular weight Marker in Fig. 1；1 is RNA sample.
M is DL2000 molecular mass Marker in Fig. 2；1 arrives slam/v receptor for amplification in goat peripheral blood lymphocytes
Gene；2 and 3 be respectively the F10 generation of BHK/slam/v and the slam/v receptor base that F50 is expanded for cell extraction postgenome
Cause；4 extract postgenome amplification for normal bhk cell.
Cell survival figure when bhk cell counts when A is 254h in Fig. 5；When B is 254h when BHK/slam/v cell count
Cell survival figure.
A is normal bhk cell in Fig. 7；B is normal BHK/slam/v cell；C is bhk cell after inoculation PPRV Attenuate vaccine；
D is BHK/slam/v cell after inoculation PPRV Attenuate vaccine
Specific embodiment
A kind of preparation and verification method based on PPR virus recipient cell BHK/slam/v of the present invention it is specific
Steps are as follows:
I, was designed and synthesized and is cloned according to the characteristics of PPR virus receptor itself and its encoding function area V gene
Carrier pDONRTM221 P5-P2 and expression vector pLenti4/V5-DESTTM What Vector was mutually matched draws
Object；
II, acquires healthy goat anticoagulation, utilizes goat peripheral blood lymphocytes separating liquid (ocean Tianjin Hao) separation lymph
Cell, Moibio Plus RNA Kit cell RNA rapidly extracting kit extract Total RNA, and measurement concentration, RNA are complete
Property and DNA residual (Fig. 1)；
III, one step amplification PPR virus receptor V function area gene, positive findings carry out benefit after T clone purification
Use BPII Enzyme Mix enzyme and donor vehicle pDONRTM221P5-P2 carries out the site B, P homologous recombination, obtains
To Entry Clone；
IV, extracts above-mentioned Entry Clone plasmid and pLenti4/V5-DESTTM Vector expression carries
Body passes through LRII plus Enzyme Mix enzyme carries out the site L, R homologous recombination, obtains expression skeleton；
V, largely extracts expression skeleton positive plasmid and packaging plasmid pLP1, pLP2 and VSV-G cotransfection 293-FT is thin
Born of the same parents obtain the functional areas PPR virus receptor V replication defect type slow virus like-particles, infect bhk cell with it；
VI, carries out the screening of resistance pressure to above-mentioned bhk cell using blasticidin S blasticidin S-HCI, limited
Dilution method carries out monoclonal and selects, and obtains the BHK positive cell subclone of the expression functional areas PPR virus receptor V；
VII. target gene amplification method, Western-Blot immune blotting detection method, cytopathogenic effect is respectively adopted
Effect and real time fluorescent quantitative calculate the methods of relative expression quantity, and it is whole to separately verify PPR virus acceptor gene group
The proliferation that conjunction, transcription, protein expression, reactivity and PPRV are fastened in the positive cell of expression PPR virus receptor
Effect.
This research is quasi- using PPRV susceptible goat as animal model, and selecting the functional areas V of its immunoglobulin SLAM is target area,
Establish the BHK sensitive cells that PPR virus separation rate can be improved.
The present invention is described in further detail with reference to the accompanying drawings and embodiments.
Embodiment 1
1, according to (SEQ ID NO.1) after PPR virus receptor itself and its encoding function area V gene translation
Feature designs and synthesizes and cloning vector pDONRTM221P5-P2 and expression vector pLenti4/V5-DESTTM
The primer SEQ ID NO.2 and SEQ ID NO.3 that Vector is mutually matched, three sequences are as follows:
SEQ ID NO.1:LDLRKGDSPRLEDGYEFHLENLSLRILKSRKED EGWYFISLEENVSVQHFSLQLK
LYEQVSTPQIKVLNSTQEDGNCSLMLACVVEKGDHVTYNWSEEAGAPLLSPT NSSHLLYLTLGPQHA；
SEQ ID NO.2:GGGGACAACTTTGTATACAAAAGTTGTAATG
CTAGATCTGCGGAAAGGTGACT；
SEQ ID NO.3:GGGGACCACTTTGTACAAGAAAGCTGGGTT
GGCATGCTGAGGGCCAAGAGTGAG。
2, prepared by PPR virus receptor correlation total serum IgE
Healthy goat is chosen, jugular vein acquires the glass container before 50ml whole blood is added added with anti-coagulants, shakes up, equivalent
Dilution is added, shakes up, goat lymphocyte separation medium, centrifugation is added in 1:3 ratio, and precipitating is suspended with cell washing solution, it is centrifuged,
It is repeated twice, precipitating is suspended with a small amount of RNAPlus, the goat peripheral blood lymphocytes as obtained.
Moibio Plus RNA Kit tissue/cell RNA rapidly extracting kit extracts Total RNA: lysate first
Rapid lytic cell and killed cells RNA enzyme, cleavage mixture remove column by genomic DNA, remove genomic DNA, RNA
Then penetrate filtration.Then the RNA filtered with 70% ethyl alcohol adjust combine, RNA in the case where height is from sequence salt state selective absorption in from
Silicon substrate plasma membrane in stem, then a series of the step of by quick cleaning-centrifugations, protein liquid removal and cleaning solution are by cell metabolism
Object, the impurity such as albumen removal, is finally eluted Total RNA without DNA/RNA enzyme water with less salt from silicon substrate plasma membrane.
3, PPR virus acceptor gene is expanded
Establish 50ul reaction system amplification acceptor gene: One Step RT-PCR bufferIII 25ul, Ex Taq HS
1ul, PrimerScript RT Enzyme Mix II 1ul, without RNA enzyme water 11ul, Total RNA 10ul；Amplification condition:
50℃30min；94℃2min；94℃1min；58.5℃1min；72℃1min；30cycles； 72℃8min；4℃5min；
Sepharose Purification of the PCR product with 1%, recycling, product carry out T clone, and the suitable plasmid of sequence verification carries out downstream work.
4, Entry clone carrier is constructed
Above-mentioned plasmid and pDONRTM221P5-P2 carrier establish recombining reaction, 10ul system are as follows: positive plasmid 6.5ul, carry
The enzyme 2ul of body pDONRTM221P5-P2 1.5ul, B, P recombining reaction.It clicks and mixes, after 25 DEG C of water-baths recombinate 4h, add
Enter proteinase k, 37 DEG C of reaction 10min, 10ul recombinant products full doses convert One Mach1TMT1R
Chemically Competent Cells competent cell, bacterium solution are applied to card that resistance LB plate, next day, the random picking of rifle point
Single colonie shakes training and upgrading grain, and amplification, sequence verification are that positive plasmid is that the entry vector-Entry clone obtained is carried
Body.
5, building expression skeleton
By above-mentioned Entry clone carrier and pLenti4/V5-DESTTM Vector expression vector is established
10ul L, R recombining reaction system are as follows: Entry clone carrier 6.5ul, pLenti4/V5-DESTTM
Vector expression vector 1.5ul, LRII plus Enzyme Mix enzyme 2ul.Centrifugation is clicked, is mixed, 25 DEG C of weights
Proteinase k, 37 DEG C of reaction 10min is added after group reaction 16h, product full dose converts One Stab13TM
Chemically Competent Cells competent cell, bacterium solution are coated on the LB plate of ammonia benzyl resistance, next day, random picking
Single colonie, small upgrading grain, amplification and the suitable positive plasmid of sequence verification are the expression skeleton obtained, a large amount of with endotoxin-free
Plasmid extraction kit large quantity extracting plasmid.
6, expression skeleton and helper plasmid cotransfection 293-FT cell acquisition replication defect type slow virus like-particles are with above-mentioned,
Tri- helper plasmids of pLP1, pLP2 and VSV-G, 750ul opti- are largely extracted with a large amount of plasmid extraction kits of endotoxin-free
It is separately added into expression skeleton, each 15ug of pLP1, pLP2 and VSV-G in MEM, is added in another 750ul opti-MEM
P3000 30ul successively carries out transfection 293-FT cell step according to 3000 specification of liposome；8h has been replaced after transfection
Full culture medium, 48h sterile collection 293-FT cell supernatant.4 DEG C of 5000rpm are centrifuged 5min, abandon pellet cell debris, collect
Supernatant be to have the replication defect type slow virus like-particles of PPR virus receptor, -70 DEG C of refrigerators are stored in, wait feel
Contaminate target cell.
7, PPR virus receptor integrates target cell BHK-21 and screening
Prepare health BHK-21 cell, after 48h, abandon complete culture solution, maintaining liquid is washed cell face, is repeated once, addition 3ml
The replication defect type slow virus like-particles with PPR virus receptor of above-mentioned collection add 7ml after 1h is made in 37 DEG C of senses
Maintaining liquid, the fresh MEM culture medium replaced after 2d include the blasticidin-S (blasticidin) that concentration is 3.0ug/ml, continuous to train
It supports.Normal BHK-21 cell is under blasticidin resistance pressure, and the cell of not infected PPR virus receptor is gradually
Death, the cell for having infected receptor is survived due to tolerance blasticidin resistance, and forms cell island, cell island pancreas
Neoblast culture bottle is gone to after enzymic digestion, and continues the screening of resistance pressure.Under pressure no longer dead 24 orifice plate of cell into
Row limiting dilution, observes subcloned cells island, and pancreatin digestion, continuous passage simultaneously carry out stable integration, genetic stability, reaction
Property, the verifying such as function, by the 10th generation cyropreservation of stable integration in Wuhan University's collection, be named as BHK/slam/v.
8, BHK/slam/v cellular integration and its genetic stability identification
After the passage of BHK/slam/v cell is stablized, takes BHK/slam/v/F10 and BHK/slam/v/F50 for cell, abandon
Cell culture fluid after pancreatin digests several minutes, abandons pancreatin, claps bottle, is detached from cell and bottle, then with 500ul PBS weight
Outstanding cell, extracts cell genomic dna, with SEQ IDNO.2, SEQ IDNO.3 primer amplification PPR virus receptor.
9, result
As shown in Figure 1, the RNA for extracting healthy goat peripheral blood lymphocytes is completely, to provide for amplification target gene
It ensures.In Fig. 2, target fragment, about 400bp or so, normal bhk cell are arrived in the F10 generation of BHK/slam/v and the amplification of F50 generation
Middle target gene band is exceptionally weak.Illustrate that PPR virus Slam/V receptor has been stably integrated in bhk cell genome
On chromosome, in 50 generations of continuous biography, do not lose, and are not difficult to infer, BHK/slam/v genetic stability is good.Fig. 3 shows that bhk cell passes through
Occur expected monoclonal cell island after blasticidin S resistance screening, provides further guarantor to establish stable cell lines
Card.
Embodiment 2
1-5 step is the same as embodiment 1.
6, expression skeleton and helper plasmid cotransfection 293-FT cell acquisition replication defect type slow virus like-particles are with above-mentioned,
Tri- helper plasmids of pLP1, pLP2 and VSV-G, 750ul opti- are largely extracted with a large amount of plasmid extraction kits of endotoxin-free
It is separately added into expression skeleton, each 12ug of pLP1, pLP2 and VSV-G in MEM, is added in another 750ul opti-MEM
P3000 30ul successively carries out transfection 293-FT cell step according to 3000 specification of liposome；8h has been replaced after transfection
Full culture medium, 48h sterile collection 293-FT cell supernatant.4 DEG C of 5000rpm are centrifuged 5min, abandon pellet cell debris, collect
Supernatant be to have the replication defect type slow virus like-particles of PPR virus receptor, -70 DEG C of refrigerators are stored in, wait feel
Contaminate target cell.
7, PPR virus receptor, which is integrated target cell BHK-21 and screened, prepares health BHK-21 cell, after 48h,
Complete culture solution is abandoned, maintaining liquid is washed cell face, is repeated once, and the above-mentioned collection of 3ml is added has PPR virus receptor
Replication defect type slow virus like-particles, 37 DEG C sense make 3h after, add 7ml maintaining liquid, the fresh MEM culture medium replaced after 2d is interior
The blasticidin-S (blasticidin) for being 3.0ug/ml containing concentration, it is continuous to cultivate.Normal BHK-21 cell is anti-in blasticidin
Property pressure under, the cell of not infected PPR virus receptor is gradually dead, has infected the cell of receptor due to tolerance
Blasticidin resistance and survive, and form cell island, go to neoblast culture bottle after the digestion of cell island pancreatin, and after
It is continuous to carry out the screening of resistance pressure.24 orifice plate of cell no longer dead carries out limiting dilution under pressure, observes subcloned cells island, pancreas
Enzymic digestion, continuous passage and the verifying such as carry out stable integration, genetic stability, reactivity, function, by the 10th generation of stable integration
Cyropreservation is named as BHK/slam/v in Wuhan University's collection.
8, by the good normal BHK and BHK/slam/v cell of growth conditions point kind in 35m2 plate, respectively at 0d, 1d,
2d, 3d, 4d, 5d, 6d, 7d are digested using common propagating method, are collected cell, are carried out cytometer after trypan blue two-fold dilution
Number (counstar company full-automatic cell calculating instrument), draws cell growth curve, and purpose is thin after observing normal cell and integrating
The biological characteristics of born of the same parents.
9, result
It is not poor both it can be seen from cell survival figure (Fig. 5) when by cell growth curve (Fig. 4) and 254h cell count
Not, when 254h, BHK total cell concentration are as follows: 8.88x105/ml；Viable cell concentrations are 8.83x105/ml；Dead cell concentration is
5.26x103/ml；Cell viability 99.41%；Average diameter 15.96um；Average roundness 0.85；Conglomeration rate 4.73%.BHK/
Slam/v total cell concentration are as follows: 6.2x105/ml；Viable cell concentrations are 6.12x105/ml；Dead cell concentration is 7.88x103/
ml；Cell viability 98.73%；Average diameter 16.16um；Average roundness 0.83；Conglomeration rate 1.69%.Bhk cell integration front and back
The two growth curve is almost the same, and living cells, dead cell number, Cell viability do not reduce, further illustrates and surely turns cell
BHK/slam/v has good biological characteristics.
Embodiment 3
1-7 step is the same as embodiment 1.
8, BHK/slam/V cell expression slam/V and its activity analysis
The good BHK and BHK/slam/V cell of growth conditions is chosen respectively to inhale after normal point of kind of a cell is digested with pancreatin
Pancreatin is abandoned, maintaining liquid collects cell, and refrigerator multigelation is added 500ul RIPA cell pyrolysis liquid (Suo Laibao), is blown and beaten with rifle
For several times, come into full contact with lysate with cell, 12000rpm after cracking is centrifuged 5min, above resets and add 4x sample-loading buffer, boil
10min, loading carry out sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%SDS-PAGE), and protein adhesive utilizes BIO-
RAD company electroporation (Turbo it) is transferred to PVDF (polyvinylene dual oxide film) transfer film, is transferred
7min, PBS are washed film 5 times, and each shaking table acts on 5min, and the TBST closing containing 5% skimmed milk power is added dropwise overnight.TBST is washed film 3 times,
Each shaking table acts on 5min, and the anti-slam primary antibody of the diluted goat of 1:200 is added, and 37 DEG C combine 1.5h.Film 5 times are washed with TBST, often
1: the 500 diluted anti-goat ELIAS secondary antibody of donkey is added in secondary 10min, and jog is incubated for 1h at room temperature, and TBST washes film 5 times, every time
5min, through four chloro- 1-, how phenol substrate develops the color, amino black staining analysis BHK/slam/V whether expresses slam/V albumen and it is exempted from
Epidemic disease activity.
9, result
After Western-Blotting, the slam/V foreign protein of BHK/slam/V expression occurs one in 14kDa or so
Band (such as Fig. 6), normal bhk cell do not see band substantially, and the functional areas illustration purpose albumen slam/V can be by slam specificity
Primary antibody and secondary antibody are identified.This result confirms that stable cell strain BHK/slam/V can not only in the application from protein level
Specific expressed slam/V albumen, and can identify have immunologic competence with specific antibody.
Embodiment 4
1-7 step is the same as embodiment 1.
8, BHK/slam/v cellular sensitivity
It chooses the good normal BHK of growth conditions and BHK/slam/v cell is each one bottle (25m2), abandon culture solution, maintaining liquid
It washes cell face twice, is inoculated with the diluted PPRV Attenuate vaccine 2ml (Xinjiang Tian Kang) of serum free medium respectively, 1.5h is made in 37 DEG C of senses
After add maintaining liquid 8ml；One bottle of blank control is respectively set simultaneously, is abandoned and is directly added serum free medium 10ml after nutrient solution.37 DEG C are set,
It observes cytopathogenic effect situation and receives malicious restrovirus copy number.
9, result
A is normal bhk cell in Fig. 7；B is normal BHK/slam/v cell；C is bhk cell after inoculation PPRV Attenuate vaccine；
D is BHK/slam/v cell after inoculation PPRV Attenuate vaccine.As can be seen that after inoculation PPRV Attenuate vaccine, BHK/slam/v cytopathy
It cashes as obvious, 3d can receive poison；Bhk cell 4-7d, cytopathy are not particularly evident.When real time fluorescent quantitative detects, BHK
Cell toxicant ct value is 20.19, copy number 5.6x106/3ul；BHK/slam/v cell toxicant ct value is 17.01, and copy number is
4.9x108/3ul.Illustrate BHK/slam/v cell due to expressing slam/v cell receptor, so that the sensibility of PPRV on it
It improves, and significant effect.
Embodiment 5
1-7 step is the same as embodiment 1.
8, by inoculation PPRV Attenuate vaccine, real-time RT-PCR method verifies BHK/slam/
Influence of the slam/V of V cell expression in transcriptional level to PPRV cultivation effect
The good BHK and BHK/slam/V cell of growth conditions is chosen respectively, abandons nutrient solution, is inoculated with PPRV Attenuate vaccine, point
Not in 12h, for 24 hours, 36h, 48h, 60h (lesion occurs in cell later), collect cell, extract its geneome RNA, real-time quantitative
When, the control of RNA concentration is in 100-200ng/ul, and in 10uM, fluorescent quantitation reagent makes for PPRV and the control of house-keeping gene GAPDH primer
With precious biotech firm One Step Primer ScriptTM RT-PCR Kit (Perfect Real time), according to using
Bright book proportion, 96 orifice plate synchronous amplification PPRV and GAPDH genes, each sample repeat 3 times.Then, with compare threshold method analysis
The Relative copy number of PPRV in different samples.
9, result
When relative expression analysis, 12h, for 24 hours, 36h, 48h, 60h different time points collect cell, therefrom expand PPRV and
Found after GAPDH, no matter overall time or some time point, PPRV Relative copy number is obviously high in BHK/slam/V cell
In bhk cell, and significant effect.Illustrate that BHK/slam/V surely turns the property that there is the slam/V of cell expression enhancing PPRV to breed
Energy.This result further confirms the slam of stable cell strain BHK/slam/V expression in the application from subgenomic transcription level
The functional areas V have effects that specificity enhancing PPRV duplication.Effect is shown in Fig. 8.
Organization Applicant
----------------------
Street: the Chengguan District, Lanzhou City, Gansu Province saltern level ground Bao Xujia 1
City: Lanzhou
State: Gansu
Country: China
PostalCode : 73046
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 cell BHK/slam/v based on PPR virus receptor
<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 :
LDLRKGDSPR LEDGYEFHLE NLSLRILKSR KEDEGWYFIS LEENVSVQHF SLQLKLYEQV 60
STPQIKVLNS TQEDGNCSLM LACVVEKGDH VTYNWSEEAG APLLSPTNSS HLLYLTLGPQ 120
HA 122
<212> Type : PRT
<211> Length : 122
SequenceName : SEQ ID NO.1
SequenceDescription :
Sequence
--------
<213> OrganismName : Ovis aries
<400> PreSequenceString :
ggggacaact ttgtatacaa aagttgtaat gctagatctg cggaaaggtg act 53
<212> Type : DNA
<211> Length : 53
SequenceName : SEQ ID NO.2
SequenceDescription :
Sequence
--------
<213> OrganismName : Ovis aries
<400> PreSequenceString :
ggggaccact ttgtacaaga aagctgggtt ggcatgctga gggccaagag tgag 54
<212> Type : DNA
<211> Length : 54
SequenceName : SEQ ID NO.3
SequenceDescription :
Claims (6)
1. a kind of cell BHK/slam/v based on PPR virus receptor, deposit number are as follows: CCTCC NO:C201739.
2. a kind of cell BHK/slam/v based on PPR virus receptor according to claim 1, feature exist
Sequence after the translation of the functional areas PPR virus recipient cell V are as follows:
SEQ ID NO.1:LDLRKGDSPRLEDGYEFHLENLSLRILKSRKED
EGWYFISLEENVSVQHFSLQLKLYEQVSTPQIKVLNSTQEDGNCSLMLACVVEKGDHVT
YNWSEEAGAPLLSPTNSSHLLYLTLGPQHA。
3. a kind of cell BHK/slam/v based on PPR virus receptor according to claim 1, feature exist
In using SEQ ID NO.1, by constructing Lentiviral, replication defect type slow virus is obtained, with the slow virus of acquisition
The bhk cell of Supernatant infection bhk cell, the screening expression functional areas slam/v clones and verifies it and replicates to PPR virus
Reinforcing effect.
4. a kind of cell BHK/slam/v based on PPR virus receptor according to claim 1 or 2 or 3,
It is characterized in that constructing the cloning vector of the expression skeleton of the PPR virus receptor is pDONRTM221 P5-P2(2-
Fragment), expression vector pLenti4/V5-DESTTM Vector；It is carried with above-mentioned cloning vector and expression
The matched primer sequence of body such as SEQ ID NO.2 and SEQ ID NO.3:
SEQ ID NO.2:
GGGGACAACTTTGTATACAAAAGTTGTAATG
CTAGATCTGCGGAAAGGTGACT,
SEQ ID NO.3:
GGGGACCACTTTGTACAAGAAAGCTGGGTT
GGCATGCTGAGGGCCAAGAGTGAG。
5. a kind of cell BHK/slam/v based on PPR virus receptor according to claim 4, feature exist
It is packed in the expression skeleton cotransfection 293-FT cell, selects 100 square meter cell bottles, divided in 750ul opti-MEM
Skeleton Jia Ru not expressed, the amount of pLP1, pLP2 and VSV-G plasmid is 12-15ug；Target cell BHK is infected, sense is as the time
1-3h；Screening surely turns cell, makes the minimum concentration 3.0ug/ml of the blasticidin blasticidin S of bhk cell death.
6. a kind of cell BHK/slam/v based on PPR virus receptor according to claim 2 or 4, feature
It is that the cell BHK/slam/v is verified using following methods: target gene amplification；Western-Blot immunoblotting；
Induced cytopathic effect and real time fluorescent quantitative calculate Relative copy number.
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Cited By (1)
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| CN110724690A (en) * | 2019-09-03 | 2020-01-24 | 中国农业科学院兰州兽医研究所 | Cloning and prokaryotic expression method of goat SLAM gene |
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