CN110066768A - Express the construction method of the RBL-2H3 cell strain of hFc ε RI α - Google Patents
Express the construction method of the RBL-2H3 cell strain of hFc ε RI α Download PDFInfo
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Abstract
The invention discloses a kind of construction methods of RBL-2H3 cell strain for expressing hFc ε RI α, comprising the following steps: recombinant slow virus expression vector of the building containing hFc ε RI α gene；The recombinant slow virus expression vector and slow virus packaging plasmid cotransfection are obtained into packaged slow virus to 293T cell；And with the packaged slow-virus infection RBL-2H3 cell.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to the building side of the RBL-2H3 cell strain of expression hFc ε RI α
Method.
Background technique
In recent years, the disease incidence of allergic disease gets worse the harm of human health in the trend risen, flourishing
The incidence of the national adult's generation food hypersenstivity for having 3%-4% every year, children and infant food allergy is 5%.In addition,
With the fast development of GM food, the safety of GM food especially sensitization becomes hot spot concerned by people.Energy
There are many kinds of the food for enough leading to food hypersenstivity, and Codex Alimentarius food labelling subcommittee (CCFL) lists 8 kinds of main sensitization
Property food: peanut, soybean, milk, egg, fish, shell, wheat and nut.Studies have shown that food hypersenstivity be related to it is various types of
Immune response, especially with type i allergic reaction, i.e. the food hypersenstivity that IgE is mediated is most commonly seen.Mast cell, basophilic granulocyte etc.
Effector cell surface Fc ε RI is the key that triggering allergy in conjunction with IgE.
RBL-2H3 cell is as rat basophilic leukemia granulocyte series (rat basophil leukemia, RBL)
One subbreed, it is similar with the numerous characteristics of mast cell and function, it is usually used in the research of allergic reaction, immune response etc..
RBL-2H3 cell surface expression mouse IgE high-affinity receptor (Fc ε RI) cannot be tied due to species specificity with human serum IgE
It closes, therefore has limitation using RBL-2H3 cell research people IgE and Fc ε RI effect.Therefore building expression people IgE high-affinity by
The humanization RBL-2H3 cell of body (hFc ε RI) is one of the important method for studying food hypersenstivity.HFc ε RI is poly- with α β γ 2 four
The form of body (α, β and two γ subunit compositions) or 2 tripolymer of α γ (α and two γ subunit composition) exists, rodent Fc
ε RI only exists in the form of α β γ 2.People IgE high-affinity receptor α subunit (hFc ε RI α) is the binding site of people IgE, is touching
It sends out key position anaphylactoid, important information is provided the diagnosing and treating of anaphylactia.Therefore it constructs and stablizes expression
The RBL-2H3 cell strain of hFc ε RI α is the hot spot of research.
Summary of the invention
Based on this, it is necessary to which, for due to species specificity, RBL-2H3 cell is only capable of expression mouse IgE high-affinity receptor
The problem of, a kind of construction method of RBL-2H3 cell strain for expressing hFc ε RI α is provided.
A kind of construction method of RBL-2H3 cell strain that expressing hFc ε RI α, comprising the following steps:
Construct the recombinant slow virus expression vector containing hFc ε RI α gene；
The recombinant slow virus expression vector and slow virus packaging plasmid cotransfection are obtained to 293T cell packaged
Slow virus；And
With the packaged slow-virus infection RBL-2H3 cell.
The step of recombinant slow virus expression vector constructed containing hFc ε RI α gene in one of the embodiments,
Include:
The hFc ε RI α gene is provided；
Original Lentiviral is provided；And
The hFc ε RI α gene is connect with the original Lentiviral.
The original Lentiviral includes GV367 carrier in one of the embodiments,.
The slow virus packaging plasmid includes pHelper 1.0 and pHelper 2.0 in one of the embodiments,.
The recombinant slow virus expression vector in one of the embodiments: the matter of pHelper 1.0:pHelper 2.0
Amount is than being (3~5): (2~4): (2~4).
The recombinant slow virus expression vector in one of the embodiments: the matter of pHelper 1.0:pHelper 2.0
Amount is than being 4:3:3.
The recombinant slow virus expression vector contains green fluorescent protein tag in one of the embodiments,.
The step of offer hFc ε RI α gene includes: in one of the embodiments,
People cDNA is provided；
The upstream primer and downstream primer for being used for hFc ε RI α gene magnification, the sequence of the upstream primer such as SEQ are provided
Shown in ID NO.1, the sequence of the downstream primer is as shown in SEQ ID NO.2；And
It is primer by template, the upstream primer and the downstream primer of the people cDNA, obtains institute by PCR amplification
State hFc ε RI α gene.
In one of the embodiments, for infecting the virus of the packaged slow virus of the RBL-2H3 cell
Titre is 1 × 108TU/mL~1 × 109TU/mL。
The cell density of the infected RBL-2H3 cell is 1 × 10 in one of the embodiments,4A/mL~1
×105A/mL.
The embodiment of the present invention is by recombinant slow virus expression vector of the building containing hFc ε RI α gene, in 293T cell,
By the slow virus packaging plasmid, the recombinant slow virus expression vector containing hFc ε RI α gene is packed, is obtained
The packaged slow virus contains hFc ε RI α gene in the packaged slow virus, utilizes the packaged slow virus
Aim cell RBL-2H3 cell is infected, so as to which target gene hFc ε RI α gene integration is thin to host cell RBL-2H3
In born of the same parents, so as to obtain the RBL-2H3 cell strain of expression hFc ε RI α, the RBL-2H3 cell strain of the expression hFc ε RI α can
As the experimental cell of research people's food hypersenstivity, experiment basis is laid for further research.
Detailed description of the invention
It is the electrophoresis result photo for grinding gene PCR amplification that Fig. 1, which is the one of the hFc of one embodiment of the invention,；
Fig. 2 is the electrophoresis result photo of the bacterium colony PCR of one embodiment of the invention；
Fig. 3 is the white light of one embodiment of the invention and the result of the slow-virus infection RBL-2H3 cell under fluorescence microscope
Photo, wherein Fig. 3 A is the blank control group under white light, and Fig. 3 B is the negative control group under white light, and Fig. 3 C is the reality under white light
Group is tested, Fig. 3 D, Fig. 3 E and Fig. 3 F are respectively the photo of Fig. 3 A, Fig. 3 B and Fig. 3 C under fluorescence microscope；
Fig. 4 is the table that the flow cytomery of one embodiment of the invention infects GFP the and hFc ε RI α of RBL-2H3 cell
Up to state photo, wherein Fig. 4 A is blank control group, and Fig. 4 B is negative control group, and Fig. 4 C is experimental group.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, by the following examples, it and combines attached
Figure, is further elaborated the construction method of the RBL-2H3 cell strain of expression hFc ε RI α of the invention.It should be appreciated that
Described herein specific examples are only used to explain the present invention, is not intended to limit the present invention.
The embodiment of the present invention provides a kind of construction method of RBL-2H3 cell strain for expressing hFc ε RI α, including following step
It is rapid:
S10 constructs the recombinant slow virus expression vector containing hFc ε RI α gene；
S20 has been wrapped the recombinant slow virus expression vector and slow virus packaging plasmid cotransfection to 293T cell
The slow virus of dress；And
S30, with the packaged slow-virus infection RBL-2H3 cell.
The embodiment of the present invention is by recombinant slow virus expression vector of the building containing hFc ε RI α gene, in 293T cell,
By the slow virus packaging plasmid, the recombinant slow virus expression vector containing hFc ε RI α gene is packed, is obtained
The packaged slow virus contains hFc ε RI α gene in the packaged slow virus, utilizes the packaged slow virus
Aim cell RBL-2H3 cell is infected, so as to which target gene hFc ε RI α gene integration is thin to host cell RBL-2H3
In born of the same parents, so as to obtain the RBL-2H3 cell strain of expression hFc ε RI α, the RBL-2H3 cell strain of the expression hFc ε RI α can
As the experimental cell of research people's food hypersenstivity, experiment basis is laid for further research.
Lentivirus Retroviridae, slow virus carrier are the important tools of genetic engineering.The benefit of the embodiment of the present invention
With the mode of slow-virus infection by the genome of external source target gene hFc ε RI α gene integration to host cell RBL-2H3 cell
In, it obtains hFc ε RI α gene in RBL-2H3 cell and stablizes lasting expression, relative to traditional plasmid transfection expression side
The ability of formula, the infection host cell of the slow-virus infection mode of the embodiment of the present invention is stronger, and target gene hFc ε RI α gene exists
Expression efficiency in host cell RBL-2H3 cell is higher.Relative to other virus transfection modes, as retroviral infection,
Adenovirus infection, the slow-virus infection of the embodiment of the present invention also have very big advantage.Such as retroviral vector can only be felt
Division cells are contaminated, and capacity is limited, adenovirus cannot be generally integrated on chromosome, can only carry out transient infections.And this
The slow-virus infection mode of inventive embodiments can infect the RBL-2H3 cell in any period, can accommodate allogenic gene piece
Duan great, such as the plasmid of 10kb or even hundred kb can be accommodated, and can be conducive to go deep into pathological phenomenon with long-term expression
Research.
In one embodiment, slow virus packaging system is made of Lentiviral and slow virus packaging plasmid.It is described
Lentiviral includes hereditary information required for packaging, transfection, stable integration.The slow virus packaging plasmid can mention
For all transcriptions and pack required auxilin.For the virion for generating high titre, need to express using slow virus
Carrier and slow virus packaging plasmid while cotransfection cells carry out the packaging of virus, packaged lentiviral particle in cell
It is secreted into extracellular culture medium, after centrifuging and taking obtains supernatant, is used directly for the infection of host cell, target gene
After hFc ε RI α gene enters host cell RBL-2H3 cell strain with packaged slow virus, by reverse transcription, it is integrated into
RBL-2H3 cell strain genome, thus high-caliber expression hFc ε RI α.
In step slo, the step of recombinant slow virus expression vector constructed containing hFc ε RI α gene can wrap
It includes:
S11 provides the hFc ε RI α gene；
S12 provides original Lentiviral；And
The hFc ε RI α gene is connect by S13 with the original Lentiviral.
In one embodiment, the step of providing the hFc ε RI α gene may include:
S111 provides people cDNA；
S112 provides upstream primer and downstream primer for hFc ε RI α gene magnification, and the sequence of the upstream primer is such as
Shown in SEQ ID NO.1, the sequence of the downstream primer is as shown in SEQ ID NO.2；And
S113 is primer by template, the upstream primer and the downstream primer of the people cDNA, by PCR amplification
Obtain the hFc ε RI α gene.
The present embodiment is using people cDNA as template (addgene company), including the overall length sequence of hFc ε RI α gene in people cDNA
Column are obtained by being designed for the upstream primer and downstream primer of specific amplification hFc ε RI α gene by PCR specific amplification
To the amplification duplicate of the hFc ε RI α gene.Obtained hFc ε RI α gene gene as a purpose is expanded, by into one
Step connect the building recombinant slow virus expression vector with the original Lentiviral.
In one embodiment, the product that the PCR amplification obtains with the original Lentiviral for connecting
It before may include S114, verification step.By verification step to examine whether hFc ε RI α gene expands success.The verifying
Step can be verified for electrophoresis, and the overall length of the hFc ε RI α gene is 920bp or so, if electrophoresis result has near 820bp
Band then illustrates that amplification is correct.
The original Lentiviral can be preferably GV367 carrier in one of the embodiments, and GV367 is carried
The recombinant slow virus expression vector that body is connect with target gene hFc ε RI α gene be more advantageous to 293T cell transfection and
Packaging.
In step s 13, can be by the way that the original Lentiviral be carried out digestion, the original slow disease after digestion
Malicious expression vector connect to obtain the recombinant slow virus expression vector by enzyme with target gene hFc ε RI α gene, to make weight
Group Lentiviral carries the hFc ε RI α gene.The enzyme connection product can be made by being transformed into competent cell
Recombinant slow virus expression vector obtains further amplification in vivo.In one embodiment, the original Lentiviral is
GV367 carrier, the restriction enzyme site can be Agel and Nhel double enzyme site.
Further, the enzyme connection product for being transformed into the competent cell may include further verification step.
The verification step to examine hFc ε RI α gene and original Lentiviral whether enzyme successful connection.It is described further
Verification step may include bacterium colony PCR verifying.The primer of the bacterium colony PCR may include as shown in SEQ ID NO.3 and such as
The combination of primer shown in SEQ ID NO.4.By the sequence length for the colony PCR product theory that primer combination amplification obtains
For 1363bp, if the sequence length of colony PCR product near 1363bp, illustrates hFc ε RI α gene and original slow virus
The enzyme successful connection of expression vector.It further, may include gene sequencing step after bacterium colony PCR is proved to be successful, to being proved to be successful
Sample carry out gene sequencing, accurate judgement hFc ε RI α gene and original Lentiviral are capable of according to sequencing result
It does not connect and whether succeeds.
The recombinant slow virus expression vector contains green fluorescent protein (Green in one of the embodiments,
Fluorescence protein, GFP) label, it is convenient for judging the sense of host cell RBL-2H3 cell by measurement GFP label
It contaminates efficiency and calculates the virus titer of packaged slow virus.
In step S20, the slow virus packaging plasmid is transcribed and is packed to the recombinant slow virus for providing
Auxilin.The slow virus packaging plasmid can be a kind of plasmid or two kinds of plasmids.It can select according to actual needs most
Excellent scheme.In one embodiment, the slow virus packaging plasmid may include that pHelper 1.0 and pHelper 2.0 is used
The combination of 2.0 two kinds of slow virus packaging plasmids of pHelper 1.0 and pHelper is so that packaging effect is more preferable, and biological safety is more
It is high.
The recombinant slow virus expression vector for 293T cell described in cotransfection and institute in one of the embodiments,
The mass ratio for stating slow virus packaging plasmid is the key factor of slow virus packaging, influences slow virus quality and further infection place
The efficiency of the RBL-2H3 cell of chief cell.In one embodiment, the slow virus packaging plasmid is 1.0 He of pHelper
The binary slow virus package carrier of pHelper 2.0.The recombinant slow virus expression vector: pHelper 1.0:pHelper
2.0 mass ratio can be (3~5): (2~4): (2~4).In the quality than being expressed for the recombinant slow virus in range
The packaging effect of carrier is more preferable, and the packaged slow virus is higher for the efficiency of infection of RBL-2H3 cell.Preferably, institute
State recombinant slow virus expression vector: the mass ratio of pHelper 1.0:pHelper 2.0 is 4:3:3.
In one embodiment, by culture medium culture 293T cell, by the recombinant lentiviral under conditions of adding transfection liquid
Virus expression carrier and the slow virus packaging plasmid cotransfection carry out slow virus packaging to the 293T cell.It is packaged slow
The recombinant slow virus expression vector containing hFc ε RI α gene that there is virus protein coat and the protein coat to wrap up,
The obtained packaged slow virus has the ability of infection host cell RBL-2H3 cell.Preferably, the 293T cell
It is preferably in logarithmic phase, faster, the state of cell is higher for the growth rate of the cell of logarithmic phase, is conducive to the progress of transfection.
In one embodiment, the transfection liquid may include LipofectamineTM2000, it is described to include
LipofectamineTM2000 transfection liquid is easy to use, can be directly added into the culture medium and be transfected, and the packet
Include LipofectamineTMThe addition of 2000 transfection liquid is smaller for the growth effect of 293T cell itself, is more advantageous to holding
The good growth quality of 293T cell.
In one embodiment, by the recombinant slow virus expression vector and the slow virus packaging plasmid cotransfection described in
When 293T cell, the culture medium of the 293T cell is serum free medium, and the environment of serum free medium is more advantageous to described
Recombinant slow virus expression vector and the slow virus packaging plasmid pass through cell membrane transfection and enter the 293T cell interior.One
In embodiment, the 293T cell after transfection is cultivated using fetal calf serum culture medium, and the nutritional profile of the fetal calf serum is more
The growth of 293T cell after being conducive to transfection.
The cell supernatant of 293T cell after collecting the transfection in one of the embodiments, the cell conditioned medium
Contain the packaged slow virus in liquid, for RBL-2H3 cell described in subsequent infection.
In one embodiment, before infecting the RBL-2H3 cell, may include to the packaged slow virus into
The step of row virus titer measures.The measurement of the virus titer may comprise steps of: to the packaged slow virus
It is diluted；Extract the DNA of the dilution；And carry out real-time fluorescence quantitative PCR.The DNA of the packaged slow virus
Containing green fluorescent protein (GFP) label, the DNA of the packaged slow virus can be detected by real-time fluorescence quantitative PCR
Change in fluorescence obtain the fluorecyte number of the dilution.The calculation formula of the virus titer are as follows: virus titer (TU/mL)
Virus stock solution used amount contained by=fluorecyte number/terminal dilution tube.Preferably, for infecting described in the RBL-2H3 cell
The virus titer of packaged slow virus is 1 × 108TU/mL~1 × 109TU/mL.Within the scope of the virus titer, it is described
The slow virus of packaging is stronger to the infection ability of RBL-2H3 cell.
It in step s 30, will be described infected described before with packaged slow-virus infection RBL-2H3 cell
RBL-2H3 cell culture is to certain cell density.The cell density of the infected RBL-2H3 cell is for slow disease
The efficiency of infection of poison has a significant impact.Preferably, the cell density of the infected RBL-2H3 cell is 1 × 104
A/mL~1 × 105A/mL.Within the scope of the cell density, the growth conditions of the RBL-2H3 cell are preferable.
Further, further include step S40 in step S30, verify infection of the packaged slow virus to RBL-2H3 cell
Situation.
In one embodiment, the packaged slow virus of verifying can pass through reality to the infection conditions of RBL-2H3 cell
When fluorescence quantitative PCR detection hFc ε RI α gene expression, flow cytomery GFP expression rate and flow cytomery hFc
One or more modes among the expression of ε RI α are verified.The table of the real-time fluorescence quantitative PCR detection hFc ε RI α gene
Up to can be by extracting the mRNA of infected RBL-2H3 cell, using the mRNA of the infected RBL-2H3 cell as mould
Plate detects the expression quantity of the hFc ε RI α gene of infected RBL-2H3 cell by real-time fluorescence quantitative PCR.By verifying,
It may determine that whether hFc ε RI α gene can succeed to be expressed in RBL-2H3 cell.
Embodiment
(1) building of recombinant slow virus expression vector
HFc ε RI α gene magnification PCR: reaction system: upstream primer (sequence as shown in SEQ ID NO.1,10 μM) 1 μ L,
Downstream primer (sequence as shown in SEQ ID NO.2,10 μM) 1 μ L, 5 × buffer10 μ L, dNTP mixture (each 2.5mM) 4 μ L,
Template cDNA (10ng/ μ L) 1 μ L, archaeal dna polymerase 0.5 μ L, ddH232.5 μ L of O, totally 50 μ L；Amplification cycles reaction condition: 98 DEG C
10 seconds, 55 DEG C 10 seconds, 72 DEG C 90 seconds, 30 circulation.Agarose gel electrophoresis, agarose gel electrophoresis knot are carried out to PCR product
Fruit illustrates hFc ε RI α gene magnification success referring to Fig. 1, there is the amplified band of 820bp or so.
Carrying out Agel/Nhel double digestion to initial Lentiviral GV367 carrier makes its linearisation, reaction system:
GV367 plasmid (1 μ g/ μ L) 2 μ L, 10 × enzyme cutting buffering liquid 5 μ L, AgeI (10U/ μ L) 1 μ L, NheI (10U/ μ L) 1 μ L, ddH2O
41 μ L, totally 50 μ L；Reaction condition: 37 DEG C of digestion 3h.Agarose gel electrophoresis is carried out to digestion products, recycles digestion purpose item
Band.HFc ε RI α gene magnification PCR product carries out enzyme with the GV367 carrier of AgeI/NheI double digestion and connect to obtain recombinant slow virus
Expression vector.Enzyme coupled reaction system and condition are as follows: 5 × Cell Buffer, 2 μ L, hFc ε RI α gene magnification PCR product (0.1
μ g/ μ L) 1 μ L, the 2.5 μ L of GV367 carrier of double digestion, DNA ligase 1 μ L, ddH2O 3.5μL；37 DEG C of reaction 30min.
Enzyme connection product is then placed in ice-water bath cooling 5min, enzyme connection product and TOP10 competent cell are mixed
It is even, it mixes product and places 30min, 42 DEG C of heat shock 90s on ice, ice-water bath is incubated for 2min.Then the mixing product is added
The LB liquid medium of 500 μ L is placed in 37 DEG C of shaking table shaken cultivation 1h and obtains bacterium solution.The bacterium solution is uniformly coated on containing ammonia
In the screening flat board of parasiticin resistance, culture 12-16h is inverted in 37 DEG C of constant incubators.
Wherein, reagent source: upstream primer and downstream primer are synthesized by JaRa biotech firm；(Shanghai Ji is triumphant for GV367 carrier
Chemical gene Technology Co., Ltd.)；Template cDNA (addgene company).
(2) bacterium colony PCR is verified
In superclean bench, 20 μ L bacterium colony PCR are respectively placed in 8 single colonies in sterile pipette tips picking screening flat board
In verifying system, piping and druming is mixed, and is placed in PCR instrument and is reacted.
Bacterium colony PCR verifies system are as follows: 0.4 μ l of upstream primer (sequence is as shown in SEQ ID NO.3) (10 μM), downstream primer
(sequence is as shown in SEQ ID NO.4) (10 μM) 0.4 μ l, 2 × Taq Plus Master Mix (bacterium colony PCR reaction reagent) 10 μ
L, ddH29.2 μ l of O, totally 20 μ l.Colony PCR amplification circular response condition: 94 DEG C, 30 seconds, 55 DEG C, 30 seconds, 72 DEG C, 30 seconds, 22
A circulation.Colony PCR amplification product identifies positive colony through 1% agarose gel electrophoresis, referring to Fig. 2, swimming lane 1 indicates negative
Compare (ddH2O)；Swimming lane 2 indicates negative control (unloaded to connect GV367 vehicle Control group certainly)；Swimming lane 3 indicates positive control, glycerol
Aldehyde -3- phosphate dehydrogenase (Glyceraldehyde-3-phosphate dehydrogenase, GAPDH)；Swimming lane 4 indicates DNA
Molecular weight marker Marker；Swimming lane 5-12 indicates the colony PCR amplification product of different bacterium colonies.The results show that swimming lane 5-12 exists
1363bp nearby observes clearly amplified band, and connecting GV367 vehicle Control group obtains 661bp's certainly for the zero load of swimming lane 2
Band illustrates the success of recombinant slow virus expression vector establishment.
Wherein, reagent source: upstream primer and downstream primer are synthesized by JaRa biotech firm；Other reagents of bacterium colony PCR
(Takara company).
(3) packaging and purifying of recombinant slow virus expression vector
According to going endotoxin plasmid extraction kit to illustrate, enough recombinant slow virus expression vectors and zero load are extracted respectively
GV367 carrier.
With the 293T cell of trypsin digestion logarithmic growth phase, 293T cell is adjusted with the culture medium containing 10% serum
Cell density about 5 × 106A/15ml, is reinoculated in the Tissue Culture Dish of 10cm, 37 DEG C, 5%CO2Culture in incubator.
It can be used to transfect when cell density is up to 70%~80% for 24 hours.
The culture medium of 293T cell is changed to serum free medium by 2h before transfecting, by recombinant slow virus expression vector or right
According to each 20 μ g of unloaded GV367 carrier respectively with slow virus packaging plasmid (15 μ g pHelper1.0 and 10 μ g pHelper 2.0)
It is added after mixing and contains appropriate LipofectamineTMIn 2000 Opti-MEM transfection liquid, transfection cocktail is obtained, adjustment turns
Dye mixed liquor total volume is 1mL.It is incubated for 15min at room temperature, transfection cocktail is slowly added dropwise to the serum-free training of 293T cell
It supports and is transfected and packed in base culture solution, mix, in 37 DEG C, 5%CO2It is cultivated in cell incubator.
After cultivating 6h, the culture medium containing transfection liquid is discarded, the PBS cleaning that 10mL is added is primary, softly shakes culture dish
To wash abandoning after remaining transfection liquid；It is slowly added to the cell culture medium 20mL containing 10% fetal calf serum, in 37 DEG C, 5%CO2
Incubator continues to cultivate 48h.
The 293T cell supernatant of 48h after transfecting is collected, 4 DEG C, 4000rpm/min is centrifuged 10min, removes cell fragment.
The supernatant of cell fragment is filtered away in 40mL ultracentrifugation pipe with 0.45 μm of filter, in 4 DEG C of supercentrifuges
25000rpm/min is centrifuged 2h.It after centrifugation, discards supernatant, removal as far as possible remains in the liquid on tube wall, PBS is added, gently
Gently piping and druming is resuspended repeatedly, through after completely dissolution, high speed centrifugation 10000rpm/min after being centrifuged 5min, takes after supernatant packing -80 DEG C
It saves backup.Contain packaged slow virus in the supernatant.
Wherein, reagent source: endotoxin plasmid extraction kit (Promega company) is gone to；LipofectamineTM
2000 Opti-MEM transfection liquid (Thermo Fisher Scientific company)；293T cell (the lucky triumphant chemical gene in Shanghai
Technology Co., Ltd.)；PHelper1.0 and pHelper 2.0 (Shanghai JiKai Gene Chemical Technology Co., Ltd).
(4) the virus titer measurement of packaged slow virus
Detection the previous day, pancreatin digest logarithmic growth phase 293T cell, adjustment cell number to 4 × 105A/ml, by 293T
Cell is inoculated into 96 orifice plates by every 100 μ l of hole.10 sterile EP tubes of same day preparation are detected, 90 μ of serum free medium is added in every pipe
The first pipe is added in l, the packaged 10 μ l of slow virus supernatant stoste for taking step to be measured (3) to obtain, and 10 μ l is taken to be added to after mixing
In second pipe, to the last one is managed using same operation, obtain slow virus dilution.The cell hole of the cell containing 293T is chosen,
90 μ l culture mediums are discarded, 90 μ l slow virus dilutions are added, after infection for 24 hours, 100 μ l of complete medium is added in incubator culture,
Careful operation not blow afloat cell.After 4 days, green fluorescent protein (GFP) expression is observed, fluorecyte number is with dilution times
Several increases and reduce.Slow virus titre is calculated, calculation formula is as follows: virus titer (TU/ml)=fluorecyte number/terminal is dilute
Release virus stock solution used amount contained by pipe.The virus titer being calculated is 5 × 108TU/mL。
(5) packaged slow-virus infection RBL-2H3 cell
RBL-2H3 cell (being granted by state food security risk assessment center toxicological experiment room) is incubated at containing 10% tire ox
The RPMI-1640 culture medium of serum.Infection the previous day digests RBL-2H3 cell, adjustment cell number to 4 × 104A/ml, every hole
2ml is seeded to 6 orifice plates.Respectively with the experimental group for having packed slow virus and the unloaded negative control for connecting GV367 carrier slow virus certainly
Group infection RBL-2H3 cell.Conventional medium is replaced after infection 16h, after cultivating 72h, changes purine-containing mycin (2 μ g/ml) into
Culture medium continues to cultivate 48h, screens to slow virus infected cell, infects under final acquisition fluorescence microscope and has packed slow disease
Poison and the aim cell strain for stablizing expression GFP.Referring to Fig. 3, blank control group is uninfected by slow virus, no under fluorescence microscope
Show fluorecyte；Negative control group is unloaded from GV367 carrier slow virus is connected, and the unloaded GV367 carrier that connects certainly contains GFP mark
Label, infection is unloaded to show fluorescence from the RBL-2H3 cell for connecting GV367 carrier；Experimental group is to have packed slow virus, contains GFP
Label, the RBL-2H3 cell that slow virus has been packed in infection show fluorescence.
Referring to Fig. 4, using the expression of flow cytomery GFP expression and hFc ε RI α, the results show that blank control
Expression of the RBL-2H3 cell of group without GFP and hFc ε RI α；Certainly connecting GV367 is not inserted into purpose base for the zero load of negative control group
Because of hFc ε RI α, GFP is only expressed, does not express hFc ε RI α；And the slow virus of packaging of experimental group contains recombinant slow virus expression and carries
Body can express GFP and hFc ε RI α simultaneously.
Table 1 is please referred to, the result statistics of the expression of the hFc ε RI α of flow cytomery transfection RBL-2H3 cell shows
The hFc ε RI alpha expression for having packed slow virus experimental group is significantly raised compared with negative control group and blank control group.Carry out statistics credit
Analysis display, the difference packed in slow virus experimental group is statistically significant (P < 0.05), and in negative control group and blank group
No significant difference.
1 flow cytomery of table stablizes the hFc ε RI alpha expression of infection RBL-2H3 cell
| Grouping | hFcεRIα |
| Blank control group | 1.19±0.17 |
| Negative control group | 0.98±0.14 |
| Experimental group | 8.10±0.96** |
**Indicate P < 0.01
Table 2 is please referred to, the expression of the mRNA of the hFc ε RI α of infection RBL-2H3 cell is detected using Real-time PCR,
The results show that the mRNA expression for having packed the hFc ε RI α of slow virus experimental group obviously rises compared with negative control group and blank control group
It is high.It carries out statistical analysis and shows that the difference packed in slow virus experimental group is statistically significant (P < 0.05), and it is negative right
According to group and the no significant difference in blank group.
The expression of the hFc ε RI α mRNA of table 2Real-time PCR detection infection RBL-2H3 cell
| Grouping | hFcεRIα |
| Blank control group | 0.725±0.003 |
| Negative control group | 1.001±0.049 |
| Experimental group | 10533.522±1165.428** |
**Indicate P < 0.01
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Chinese Center for Disease Control and Prevention nutrition and healthy institute
State food security risk assessment center
<120>construction method of the RBL-2H3 cell strain of hFc ε RI α is expressed
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<213>artificial sequence (Artificial Sequence)
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gaggatcccc gggtaccggt cgccaccatg gctcctgcca tggaatcc 48
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<213>artificial sequence (Artificial Sequence)
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cacacattcc acaggctagt cagttgtttt tggggtttgg c 41
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<213>artificial sequence (Artificial Sequence)
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<213>artificial sequence (Artificial Sequence)
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cgtcgccgtc cagctcgacc ag 22
Claims (10)
1. a kind of construction method for the RBL-2H3 cell strain for expressing hFc ε RI α, comprising the following steps:
Construct the recombinant slow virus expression vector containing hFc ε RI α gene；
The recombinant slow virus expression vector and slow virus packaging plasmid cotransfection are obtained into packaged slow disease to 293T cell
Poison；And
With the packaged slow-virus infection RBL-2H3 cell.
2. the construction method of the RBL-2H3 cell strain of expression hFc ε RI α according to claim 1, which is characterized in that described
The step of constructing recombinant slow virus expression vector containing hFc ε RI α gene include:
The hFc ε RI α gene is provided；
Original Lentiviral is provided；And
The hFc ε RI α gene is connect with the original Lentiviral.
3. the construction method of the RBL-2H3 cell strain of expression hFc ε RI α according to claim 2, which is characterized in that described
Original Lentiviral includes GV367 carrier.
4. the construction method of the RBL-2H3 cell strain of expression hFc ε RI α according to claim 1, which is characterized in that described
Slow virus packaging plasmid includes pHelper 1.0 and pHelper 2.0.
5. the construction method of the RBL-2H3 cell strain of expression hFc ε RI α according to claim 4, which is characterized in that described
Recombinant slow virus expression vector: the mass ratio of pHelper 1.0:pHelper 2.0 is (3~5): (2~4): (2~4).
6. the construction method of the RBL-2H3 cell strain of expression hFc ε RI α according to claim 5, which is characterized in that described
Recombinant slow virus expression vector: the mass ratio of pHelper 1.0:pHelper 2.0 is 4:3:3.
7. the construction method of the RBL-2H3 cell strain of expression hFc ε RI α according to claim 1, which is characterized in that described
Recombinant slow virus expression vector contains green fluorescent protein tag.
8. the construction method of the RBL-2H3 cell strain of expression hFc ε RI α according to claim 2, which is characterized in that described
The step of providing the hFc ε RI α gene include:
People cDNA is provided；
The upstream primer and downstream primer for being used for hFc ε RI α gene magnification, the sequence of the upstream primer such as SEQ ID are provided
Shown in NO.1, the sequence of the downstream primer is as shown in SEQ ID NO.2；And
It is primer by template, the upstream primer and the downstream primer of the people cDNA, is obtained by PCR amplification described
HFc ε RI α gene.
9. the construction method of the RBL-2H3 cell strain of expression hFc ε RI α according to claim 1, which is characterized in that be used for
The virus titer for infecting the packaged slow virus of the RBL-2H3 cell is 1 × 108TU/mL~1 × 109TU/mL。
10. the construction method of the RBL-2H3 cell strain of expression hFc ε RI α according to claim 1, which is characterized in that quilt
The cell density of the RBL-2H3 cell of infection is 1 × 104A/mL~1 × 105A/mL.
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