CN105483158A - Lentiviral expression vector, as well as preparation method and application of lentiviral expression vector, and preparation method of recombinant lentivirus - Google Patents
Lentiviral expression vector, as well as preparation method and application of lentiviral expression vector, and preparation method of recombinant lentivirus Download PDFInfo
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Abstract
The invention discloses a lentiviral expression vector, as well as a preparation method and an application of the lentiviral expression vector, and a preparation method of a recombinant lentivirus. The lentiviral expression vector is a pRRLSIN.cPPT.MSCV-Dual.WPRE vector, which is obtained by replacing the GFP sequence of a pRRLSIN.cPPT.MSCV/GFP.WPRE vector with a double gene expression box Dual, wherein the double gene expression box Dual comprises the following structure sequentially arranged from the 5' terminal to the 3' terminal: a first polyclonal site-a furin cleavage site-a V5 label-Spacer-2A peptide-a second polyclonal site, the mark '-' represents the connection relation. According to the lentiviral expression vector, 2A peptide is utilized for realizing the expression of double exogenous genes in cells of mammals, 2A peptide has the 'self-shearing' property, in the translation process of protein, 2A peptide changes the activity of ribosome, promotes the hydrolysis of ester chains between peptide residue Gly and tRNAGly of 2A, releases upstream polypeptides and starts the translation of upstream polypeptides from a transcribed compound, and the realizes double gene expression in the level of translation.
Description
Technical field
The present invention relates to biological technical field, particularly relate to the preparation method of a kind of Lentiviral and its preparation method and application, recombinant slow virus.
Background technology
Genetic expression be cell in vital process, the genetic information that is stored in DNA sequence dna through transcribing and translating, be transformed into the process with bioactive protein molecule.The expression of foreign gene in biomass cells even organism can be realized by genetically engineered, thus reach the object of artificial adjustment cell or real object proterties.
The importing of the foreign gene of eukaryotic cell, especially mammalian cell, completes mainly through physical method, chemical process or biological method.Physical method mainly comprises the methods such as DNA microinjection, electroporation and metallic particles blast technique; Chemical process mainly comprises the methods such as liposome-mediated and receptor-mediated; Biological method is mainly realized, as adenovirus, adeno-associated virus, retrovirus, slow virus etc. by various virus.Physical method and chemical process can a large amount of exogenous genetic fragments of load, and there is Part Methods foreign gene can be realized to be inserted in the genome of cell or organism, but on the whole, the efficiency being realized exogenous gene expression by these two class methods is not high, and foreign gene insertion cell or organism genome time most of, cannot be realized well, and then the stable permanent expression of foreign gene cannot be realized.The methods such as the adeno-associated virus in biological method, slow virus then can be realized this goal well, and they can by exogenous origin gene integrator in the genome of cell or organism, thus realize the stable permanent of foreign gene and express.Wherein, slow virus has with it and can infect non-division cells, holds the favor that the advantages such as allogenic gene fragment is larger are subject to scientific circles.
At present, no matter be in laboratory or in industrial community, the application of slow virus all widely.Along with the continuous expansion of Application Areas, the requirement of slow virus is also being improved constantly.Current, in scientific research and practice, two genes are once imported and to realize the demand of its high expression also increasing by slow virus, such as, first of ten big sciences that " Science " magazine in 2013 is chosen are broken through-immunotherapy for cancer, comprise the technology such as TCR-T.Due to TCR-T technology can the cell of expression specificity receptor target identification specificity as tumour cell, paid close attention to widely and studied, and being changed into present clinical application from the fundamental immunity research at initial stage.Because TCR-T technology needs DNA sequence dna corresponding for φt cell receptor (TCR) two subunits to import in T cell simultaneously, therefore it is very urgent to the demand that can realize dual-gene quick leading-in technique, and existing lentiviral vectors still can not meet this demand well.
Current slow virus technology realizes dual-gene importing mainly through two kinds of modes: build double-promoter or a promotor+one internal ribosome entry site sequence (IRES) in the genetic expression district of lentiviral vectors, realized the expression of two foreign genes by two starting elements at transcriptional level.These two kinds of modes all can realize dual-gene expression, but there are following two main shortcomings: the expression level 1, being positioned at carrier upstream and downstream gene is inconsistent.Due to difference and the interaction that may exist of different starting element efficiency, thus cause the expression level of upstream and downstream gene widely different (differences as 10 times) between the two, even occur the situation that one of them gene is not expressed; 2, slow virus is to the loading capacity of foreign gene generally at about 8000bp ~ 10000bp, and the size of starting element is generally at about 500bp ~ 700bp.Can see, single starting element accounts for the ratio of slow virus loading capacity close to 10%, and therefore many introducings starting element just means the exogenous gene sequence imported by slow virus, just means the minimizing that can be used for importing foreign gene capacity.Therefore the slow virus double gene expression using these two kinds of modes to realize is not all desirable especially.The lentiviral vectors that can overcome the above problems is there is no in prior art.
Summary of the invention
Based on this, be necessary the preparation method that good Lentiviral of a kind of double gene expression effect and its preparation method and application, recombinant slow virus are provided.
A kind of Lentiviral, described Lentiviral is pRRLSIN.cPPT.MSCV-Dual.WPRE carrier, and described pRRLSIN.cPPT.MSCV-Dual.WPRE carrier is that the GFP sequence of pRRLSIN.cPPT.MSCV/GFP.WPRE carrier is replaced by double gene expression box Dual and obtains;
Described double gene expression box Dual comprises from 5 ' to 3 ' and holds the following structure be arranged in order: the first multiple clone site-furin cleavage site-V5 label-Spacer-2A peptide-the second multiple clone site, and wherein, "-" representative connects.
In one embodiment, the sequence of described double gene expression box Dual is as shown in SEQIDNo.1.
In one embodiment, described first multiple clone site comprises from 5 ' to 3 ' and holds the following structure be arranged in order: AscI restriction enzyme site, BstBI restriction enzyme site, BamHI restriction enzyme site and AgeI restriction enzyme site;
Described second multiple clone site comprises from 5 ' to 3 ' and holds the following structure be arranged in order: XhoI restriction enzyme site, SpeI restriction enzyme site, XmaI restriction enzyme site and SalI restriction enzyme site.
In one embodiment, the sequence of described first multiple clone site is as shown in SEQIDNo.2;
The sequence of described second multiple clone site is as shown in SEQIDNo.3;
The sequence of described furin cleavage site is as shown in SEQIDNo.4;
The sequence of described V5 label is as shown in SEQIDNo.5;
The sequence of described Spacer is as shown in SEQIDNo.6;
The sequence of described 2A peptide is as shown in SEQIDNo.7.
In one embodiment, described first multiple clone site is inserted with the first goal gene, and described second multiple clone site is inserted with the second goal gene;
Described first goal gene is mouse TCR α gene, and the NCBI of described mouse TCR α gene is numbered DQ452619, and described second goal gene is mouse TCR β, and the NCBI of described mouse TCR β gene is numbered DQ452620.
A preparation method for Lentiviral described above, comprises the steps:
Design double gene expression box Dual, and entrust the described double gene expression box of synthesis, obtain the Escherichia coli bacteria liquid containing pUC-Dual plasmid, containing described double gene expression box Dual in described pUC-Dual plasmid, described double gene expression box Dual comprises from 5 ' to 3 ' and holds the following structure be arranged in order: the first multiple clone site-furin cleavage site-V5 label-Spacer-2A peptide-the second multiple clone site, and "-" representative connects;
By the described Escherichia coli bacteria liquid containing pUC-Dual plasmid with the mixing of selectivity LB liquid nutrient medium, in 37 DEG C of constant-temperature tables, 300rpm cultivates 12h ~ 16h to OD
600be 0.6 ~ 0.8, centrifugal for the bacterium liquid obtained rear reservation first is precipitated, extract described pUC-Dual plasmid by after described first precipitation cracking;
With AscI restriction endonuclease and SalI restriction endonuclease process pUC-Dual plasmid at 37 DEG C, fully reclaim after reaction, obtain the fragment of described double gene expression box Dual;
With AscI restriction endonuclease and SalI restriction endonuclease process pRRLSIN.cPPT.MSCV/GFP.WPRE carrier at 37 DEG C, the GFP sequence of described pRRLSIN.cPPT.MSCV/GFP.WPRE carrier is excised, reclaim after abundant reaction, obtain linearizing pRRLSIN.cPPT.MSCV/GFP.WPRE carrier;
Be that the fragment of described double gene expression box Dual mixes with described linearizing pRRLSIN.cPPT.MSCV/GFP.WPRE carrier by 3 ~ 10:1 according to mol ratio, then add T4DNA ligase enzyme, connecting at 4 DEG C and spend the night, obtaining the connection product containing connecting plasmid; And
By the described connection product conversion competence Top10 intestinal bacteria containing connecting plasmid, and on even spread to optionally LB flat board, be inverted for 37 DEG C and cultivate 12h ~ 16h, the positive bacterium colony of picking is placed in described selectivity LB liquid nutrient medium, and in 37 DEG C of constant-temperature tables, 300rpm cultivates 12h ~ 16h to OD
600be 0.6 ~ 0.8, centrifugal for the bacterium liquid obtained rear reservation second precipitated, extract described connection plasmid by after described second precipitation cracking, described connection plasmid is described Lentiviral.
In one embodiment, after being also included in the fragment obtaining described double gene expression box Dual, insert the first goal gene in described first multiple clone site of the fragment of described double gene expression box Dual, insert the operation of the second goal gene in described second multiple clone site of the fragment of described double gene expression box Dual;
Described first goal gene is mouse TCR α gene, and the NCBI of described mouse TCR α gene is numbered DQ452619, and described second goal gene is mouse TCR β gene, and the NCBI of described mouse TCR β gene is numbered DQ452620.
A kind of slow virus expresses test kit, comprises Lentiviral described above.
A preparation method for recombinant slow virus, comprises the steps:
Above-mentioned Lentiviral is provided, and insert the first goal gene in described first multiple clone site of the fragment of described double gene expression box Dual, insert the second goal gene in described second multiple clone site of the fragment of described double gene expression box Dual, obtain the Lentiviral carrying goal gene;
Be be transfected in 293FT cell after 2:1:1:1 mixing by described Lentiviral, pMDLg/pRRE carrier, pRSV-Rev carrier and the pMD-G carrier carrying goal gene according to mol ratio, after transfection, 4h ~ 6h is replaced by perfect medium cultivation, nutrient solution is collected after 48h, described supernatant 0.45 μm of filtering head also filters by centrifugal rear reservation supernatant, retain filtrate, described filtrate is the solution of recombinant slow virus.
In one embodiment, after being also included in the solution obtaining described recombinant slow virus, to the step that the titre of the solution of described recombinant slow virus detects, be specially:
First day, is inoculated in porous plate by 293FT cell, each hole inoculation 2 × 10
5individual cell, each hole adds 500 μ L substratum, 37 DEG C, 5%CO
2overnight incubation;
Second day is 1,10 by the Dilution ratio of the solution of described recombinant slow virus
1, 10
2, 10
3, 10
4, 10
5, 10
6, 10
7with 10
8with substratum, the solution gradient of described recombinant slow virus is diluted, then respectively the solution of the described recombinant slow virus of 100 μ L gradient dilutions is mixed transfection from the cell culture fluid in 100 μ L porous plates in the different holes of porous plate, transfection starts rear 24h, suck substratum and change the fresh culture of 500 μ L containing 5UDNaseI into, cultivate 30min at 37 DEG C to remove the remaining plasmid DNA that may be attached to cell surface, then change substratum into 1mL normal incubation medium, continue to cultivate 48h;
4th day, suck the substratum in each hole of described porous plate, add 500 μ L pancreas enzyme-EDTA solution digestion cells, 37 DEG C of reactions 1 minute, then add substratum and stop digestion reaction by under cell purge, the cell in each hole of collected by centrifugation, the total serum IgE of the every porocyte of extracting, then reverse transcription obtains total cDNA of every porocyte; And
Respectively quantitative fluorescent PCR is carried out to total cDNA of the described every porocyte obtained, obtains the Ct value of every porocyte, select the different minimum but experimental group more than 2 with control group Ct value difference, obtain its extension rate, according to following formulae discovery slow virus titre:
T=R × 20 × 10
3, wherein, T is slow virus titre, and the unit of T is TU/mL, R is extension rate.This Lentiviral utilizes 2A peptide to realize the two expression of foreign gene in mammalian cell, and 2A peptide has the characteristic of " self splicing ", and in the translation process of protein, 2A peptide changes ribosomal activity, promotes 2A peptide residue Gly and tRNA
glybetween the hydrolysis of ester chain, from the translation of initial downstream polypeptide again while transcription complex discharges upstream polypeptide, the double gene expression that translation skill realizes.According to the study, 2A Toplink almost realizes shearing completely.Therefore, this Lentiviral can ensure that the equivalent of two foreign gene in mammalian cell is expressed theoretically, and relative to traditional lentiviral vectors for double gene expression, the double gene expression effect of this Lentiviral is better.
Accompanying drawing explanation
Fig. 1 is the structural representation of the Lentiviral of an embodiment;
Fig. 2 is the schema of the preparation method of Lentiviral as shown in Figure 1;
Fig. 3 be in embodiment 5 Flow cytometry TCR in the result of CD4+T lymphocytic cell surface expression;
Fig. 4 be in embodiment 5 Flow cytometry TCR in the result of CD8+T lymphocytic cell surface expression.
Embodiment
Mainly in conjunction with the drawings and the specific embodiments the preparation method of Lentiviral and its preparation method and application, recombinant slow virus is described in further detail below.
Unaccounted medicine in the present invention, reagent is this area commercial product (purchased from Sangon Biotech (Shanghai) Co., Ltd., precious biotechnology (Dalian) company limited, QIAGEN company of Germany, LifeTechnologies company of the U.S., GIBCO company of the U.S. etc.), unaccounted operation all adopts this area ordinary method, concrete steps can be see: " MolecularCloning:ALaboratoryManual " (Sambrook, J., Russell, DavidW., MolecularCloning:ALaboratoryManual, 3rdedition, 2001, NY, ColdSpringHarbor).
The Lentiviral of an embodiment is as shown in Figure 1 pRRLSIN.cPPT.MSCV-Dual.WPRE carrier.PRRLSIN.cPPT.MSCV-Dual.WPRE carrier is that the GFP sequence of pRRLSIN.cPPT.MSCV/GFP.WPRE carrier is replaced by double gene expression box Dual (Dual-geneexpressioncassette) and obtains.
PRRLSIN.cPPT.MSCV/GFP.WPRE carrier is transformed by the business-like plasmid pRRLSIN.cPPT.PGK/GFP.WPRE purchased from addgene, has changed the PGK promotor of plasmid pRRLSIN.cPPT.PGK/GFP.WPRE into MSCV promotor.
GFP sequence is replaced tool and has the following advantages: 1, replace GFP and can hold larger foreign gene (GFP gene itself also has the sequence more than 700bp); 2, after GFP position is just in time in MSCV promotor, its replacement can be improved the expression level of foreign gene.
The GFP sequence of pRRLSIN.cPPT.MSCV/GFP.WPRE carrier can adopt AscI restriction endonuclease and the excision of SalI restriction endonuclease, then be connected with the pRRLSIN.cPPT.MSCV/GFP.WPRE carrier having excised GFP sequence by the fragment of T4DNA ligase enzyme by the double gene expression box Dual through same treatment, build and obtain above-mentioned pRRLSIN.cPPT.MSCV-Dual.WPRE carrier.
Double gene expression box Dual comprises from 5 ' to 3 ' and holds the following structure be arranged in order: the first multiple clone site-furin cleavage site-V5 label-Spacer-2A peptide-the second multiple clone site, and wherein, "-" representative connects.
This Lentiviral utilizes 2A peptide to realize the two expression of foreign gene in mammalian cell, and 2A peptide has the characteristic of " self splicing ", and in the translation process of protein, 2A peptide changes ribosomal activity, promotes 2A peptide residue Gly and tRNA
glybetween the hydrolysis of ester chain, from the translation of initial downstream polypeptide again while transcription complex discharges upstream polypeptide, the double gene expression that translation skill realizes.According to the study, 2A Toplink almost realizes shearing completely.Therefore, this Lentiviral can ensure that the equivalent of two foreign gene in mammalian cell is expressed theoretically, and relative to traditional lentiviral vectors for double gene expression, the double gene expression effect of this Lentiviral is better.
Preferably, the sequence of double gene expression box Dual is as shown in SEQIDNo.1.
First multiple clone site comprises from 5 ' to 3 ' and holds the following structure be arranged in order: AscI restriction enzyme site, BstBI restriction enzyme site, BamHI restriction enzyme site and AgeI restriction enzyme site.
Second multiple clone site comprises from 5 ' to 3 ' and holds the following structure be arranged in order: XhoI restriction enzyme site, SpeI restriction enzyme site, XmaI restriction enzyme site and SalI restriction enzyme site.
Preferably, the sequence of the first multiple clone site is as shown in SEQIDNo.2.
Preferably, the sequence of the second multiple clone site is as shown in SEQIDNo.3.
Preferably, the sequence of furin cleavage site is as shown in SEQIDNo.4.
Preferably, the sequence of V5 label is as shown in SEQIDNo.5.
Preferably, the sequence of Space is as shown in SEQIDNo.6.
Preferably, the sequence of 2A peptide is as shown in SEQIDNo.7.
In actual applications, the first multiple clone site is inserted with the first goal gene, and the second multiple clone site is inserted with the second goal gene.
Preferably, the first goal gene is mouse TCR α gene, and the NCBI of mouse TCR α gene is numbered DQ452619, and the second goal gene is mouse TCR β gene, and the NCBI of mouse TCR β gene is numbered DQ452620.
Present invention also offers a kind of slow virus and express test kit, comprise above-mentioned Lentiviral.
Compared with prior art, this slow virus is expressed test kit and has easy to use, efficient, the high expression of the two foreign gene of mammalian cell can be realized well, and more gene fragment can be held, can be used as strong tool applications in scientific research and industrial community, as the cellular immunotherapy of tumour.Cellular immunotherapy is a kind of emerging, antitumor therapy with significant curative effect, compensate for traditional operation, the drawback of chemicotherapy, being acknowledged as in combined therapy of tumour pattern the most rising, is also the treatment means being uniquely hopeful complete tumors destroyed at present.The immunologic function of cytotoxic T cell (CTL) plays a decisive role in antineoplastic immune, the prerequisite that tumour antigen activates CTL and CTL killing tumor cell is the effective identification of CTL to tumour antigen, in order to enhanced CT L is to the immunne response of tumour cell, multiple therapeutic strategy is in clinical experimental stage, such as T cell is transplanted, the immunity of tumour antigen or DC cell.Tumor-specific CTL conventional at present comprises the CTL of TIL, DC induction and the T cell (TCR-T and CAR-T) of genetic modification, these technology all have specific function of killing and wounding the tumour cell of expressing this antigen, are the important development directions of following cellular immunotherapy.
The preparation method of above-mentioned Lentiviral as shown in Figure 2, comprises the steps:
S10, design double gene expression box Dual, and entrust synthesis double gene expression box Dual, obtain the Escherichia coli bacteria liquid containing pUC-Dual plasmid.
The synthesis of double gene expression box Dual entrusts commercial company to complete, and directly obtains the Escherichia coli bacteria liquid containing pUC-Dual plasmid.
Containing double gene expression box Dual in pUC-Dual plasmid, double gene expression box Dual comprises from 5 ' to 3 ' and holds the following structure be arranged in order: the first multiple clone site-furin cleavage site-V5 label-Spacer-2A peptide-the second multiple clone site, and "-" representative connects.
S20, the Escherichia coli bacteria liquid containing pUC-Dual plasmid obtained by S10 mix with selectivity LB liquid nutrient medium, and in 37 DEG C of constant-temperature tables, 300rpm cultivates 12h ~ 16h to OD
600be 0.6 ~ 0.8, centrifugal for the bacterium liquid obtained rear reservation first is precipitated, extract pUC-Dual plasmid by after the first precipitation cracking.
Selectivity LB liquid nutrient medium is the LB liquid nutrient medium containing 100 μ g/mL penbritins.
S30, at 37 DEG C with the pUC-Dual plasmid that AscI restriction endonuclease and SalI restriction endonuclease treatment S 20 obtain, fully reclaim after reaction, obtain the fragment of double gene expression box Dual.
S40, at 37 DEG C with AscI restriction endonuclease and SalI restriction endonuclease process pRRLSIN.cPPT.MSCV/GFP.WPRE carrier, the GFP sequence of pRRLSIN.cPPT.MSCV/GFP.WPRE carrier is excised, reclaim after abundant reaction, obtain linearizing pRRLSIN.cPPT.MSCV/GFP.WPRE carrier.
PRRLSIN.cPPT.MSCV/GFP.WPRE carrier is transformed by the business-like pRRLSIN.cPPT.PGK/GFP.WPRE purchased from addgene, has changed PGK promotor into MSCV promotor.
S50, be that the fragment of the double gene expression box Dual that S30 obtains by 3 ~ 10:1 mixes with the linearizing pRRLSIN.cPPT.MSCV/GFP.WPRE carrier that S40 obtains according to mol ratio, add T4DNA ligase enzyme again, connecting at 4 DEG C and spend the night, obtaining the connection product containing connecting plasmid.
Preferably, the fragment of double gene expression box Dual and the mol ratio of linearizing pRRLSIN.cPPT.MSCV/GFP.WPRE carrier are 3:1.
S60, the connection product conversion competence Top10 intestinal bacteria containing connection plasmid that S50 is obtained, and on even spread to optionally LB flat board, be inverted for 37 DEG C and cultivate 12h ~ 16h, the positive bacterium colony of picking is placed in selectivity LB liquid nutrient medium, and in 37 DEG C of constant-temperature tables, 300rpm cultivates 12h ~ 16h to OD
600be 0.6 ~ 0.8, centrifugal for the bacterium liquid obtained rear reservation second is precipitated, connecting plasmid by extracting after the second precipitation cracking, connecting plasmid and being Lentiviral.
After the preparation method of this Lentiviral is also included in the fragment obtaining double gene expression box Dual, insert the first goal gene in the first multiple clone site of the fragment of double gene expression box Dual, insert the operation of the second goal gene in the second multiple clone site of the fragment of double gene expression box Dual.
Preferably, the first goal gene is mouse TCR α gene, and the NCBI of mouse TCR α gene is numbered DQ452619, and the second goal gene is mouse TCR β gene, and the NCBI of mouse TCR β gene is numbered DQ452620.
A preparation method for recombinant slow virus, is characterized in that, comprises the steps:
Above-mentioned Lentiviral is provided, and insert the first goal gene in the first multiple clone site of the fragment of double gene expression box Dual, insert the second goal gene in the second multiple clone site of the fragment of double gene expression box Dual, obtain the Lentiviral carrying goal gene; Be be transfected in 293FT cell after 2:1:1:1 mixing by carrying the Lentiviral of goal gene, pMDLg/pRRE carrier, pRSV-Rev carrier and pMD-G carrier according to mol ratio, after transfection, 4h ~ 6h is replaced by perfect medium cultivation, nutrient solution is collected after 48h, supernatant 0.45 μm of filtering head also filters by centrifugal rear reservation supernatant, retains the solution that filtrate is recombinant slow virus.
First goal gene is mouse TCR α gene, and the NCBI of mouse TCR α gene is numbered DQ452619, and the second goal gene is mouse TCR β gene, and the NCBI of mouse TCR β gene is numbered DQ452620.
After the first multiple clone site of the fragment of double gene expression box Dual inserts the first goal gene, need the first goal gene to inserting to check order, sequencing primer is: MCSI upstream primer and MCSI downstream primer, chooses sequencing result and expect that the carrier conformed to completely is for next step after having checked order.
After the second multiple clone site of the fragment of double gene expression box Dual inserts the second goal gene, need the second goal gene to inserting to check order, sequencing primer is: MCSII upstream primer and MCSII downstream primer, chooses sequencing result and expect that the carrier conformed to completely is for next step after having checked order.
The sequence of MCSI upstream primer is as shown in SEQIDNo.8.
The sequence of MCSI downstream primer is as shown in SEQIDNo.9.
The sequence of MCSII upstream primer is as shown in SEQIDNo.10.
The sequence of MCSII downstream primer is as shown in SEQIDNo.11.
PMDLg/pRRE carrier, pRSV-Rev carrier and pMD-G carrier are conventional slow virus assembling carrier.
After the preparation method of this recombinant slow virus is also included in the solution obtaining recombinant slow virus, to the step that the titre of the solution of recombinant slow virus detects, be specially:
First day, is inoculated in porous plate by 293FT cell, each hole inoculation 2 × 10
5individual cell, each hole adds 500 μ L substratum, 37 DEG C, 5%CO
2overnight incubation;
Second day is 1,10 by the Dilution ratio of the solution of recombinant slow virus
1, 10
2, 10
3, 10
4, 10
5, 10
6, 10
7with 10
8with substratum, the solution gradient of recombinant slow virus is diluted, then respectively the solution of the recombinant slow virus of 100 μ L gradient dilutions is mixed transfection from the cell culture fluid in 100 μ L porous plates in the different holes of porous plate, transfection starts rear 24h, suck substratum and change the fresh culture of 500 μ L containing 5UDNaseI into, cultivate 30min at 37 DEG C to remove the remaining plasmid DNA that may be attached to cell surface, then change substratum into 1mL normal incubation medium, continue to cultivate 48h;
4th day, suck the substratum in each hole of porous plate, add 500 μ L pancreas enzyme-EDTA solution digestion cells, 37 DEG C of reactions 1 minute, then add substratum and stop digestion reaction by under cell purge, the cell in each hole of collected by centrifugation, the total serum IgE of the every porocyte of extracting, then reverse transcription obtains total cDNA of every porocyte; And
Be respectively upstream and downstream primer with above-mentioned MCSI upstream primer and MCSII downstream primer, according to following table application of sample, then carry out quantitative fluorescent PCR, obtain the Ct value of each porocyte quantitative fluorescent PCR reaction.
| Reagent | Usage quantity (μ L) | Final concentration |
| Upstream primer (10 μMs) | 0.4μL | 0.2μM |
| Downstream primer (10 μMs) | 0.4μL | 0.2μM |
| ROX dyestuff (50 ×) | 0.4μL | 1× |
| CDNA template | 2.0μL | |
| Pre-mixing Taq enzyme (2 ×) | 10μL | 1× |
| dH 2O | 6.8μL | |
| Total amount | 20.0μL |
According to the Ct value of the every porocyte obtained, select the different minimum but experimental group more than 2 with control group Ct value difference, obtain its extension rate, according to following formulae discovery slow virus titre:
T=R × 20 × 10
3, wherein, T is slow virus titre, and the unit of T is TU/mL, R is extension rate.
It is generally acknowledged, as long as slow virus titre reaches 10
7more than TU/mL, namely thinks and successfully obtains required slow virus liquid.
Specific embodiment.The plasmid used in specific embodiment obtains for pRRLSIN.cPPT.MSCV transforms, and GFP.WPRE, 293FT cell is commercial goods, and the reagent used is commercial goods.
The design of embodiment 1 double gene expression box
The core of double gene expression box expresses 2A peptide (SEQIDNo.7), having the first multiple clone site (SEQIDNo.2), 2A peptide (SEQIDNo.7) and the second multiple clone site (SEQIDNo.3) to deposit namely to can be used in case realizing the equivalent expression of two foreign gene.But the exogenous gene expression amount obtained by which is not high, and can be coupled with at the peptide sequence end of 2A peptide upstream the amino-acid residue that 13 are derived from 2A peptide, this may produce certain impact to the characteristic of this polypeptide.
Further, between first multiple clone site and 2A peptide of the double gene expression box of previous step, the expression level that Spacer (SEQIDNo.6) effectively can improve foreign gene is inserted.
Further, between first multiple clone site and Spacer of double gene expression box obtained in the previous step, insert furin cut site (SEQIDNo.4), can make the cutting of polypeptide again through furin after expression of 2A peptide upstream, its end only can remain 4 amino-acid residues.Unexpected, the expression level of foreign gene has had again further raising.
Optimally, cut between site and Spacer insert the expression level that V5 label (SEQIDNo.5) can promote foreign gene again at the furin of double gene expression box obtained in the previous step, the most efficient equivalent realizing two foreign gene is expressed.
As shown in Figure 1, the sequence of the double gene expression box finally obtained is as shown in SEQIDNo.1.
The structure of embodiment 2pRRLSIN.cPPT.MSCV-Dual.WPRE carrier
Entrust the sequence of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd to double gene expression box to synthesize, the sequence obtained is included in the pUC57-DGEC carrier in intestinal bacteria body.Carry out enlarged culturing to these intestinal bacteria, and extract pUC57-DGEC carrier wherein, then measure its purity and concentration, result is as shown in the table.
The purity of pUC57-DGEC carrier and concentration
| Recombinant vectors | A260/A280 | Concentration (ng/ μ L) |
| pUC57-DGEC | 1.93 | 626.3 |
Respectively double digestion is carried out to restructuring pUC57 carrier and pRRLSIN.cPPT.MSCV/GFP.WPRE carrier with AscI and SalI, after electrophoresis, reclaim the large fragment of small segment in restructuring pUC57 carrier digestion products and pRRLSIN.cPPT.MSCV/GFP.WPRE carrier digestion products respectively.Segment by pUC51 carrier digestion products: the mol ratio of linearizing pRRLSIN.cPPT.MSCV/GFP.WPRE carrier is after 3:1 mixing, and adds T4DNA ligase enzyme, is placed in 4 DEG C of connections and spends the night.
To connect product conversion competence Top10 intestinal bacteria, and even spread is on the LB flat board containing 100 μ g/mL penbritins.Be inverted for 37 DEG C and cultivate 12-16h.The multiple positive bacterium colony of picking, is placed in 5mLLB liquid nutrient medium (containing 100 μ g/mL penbritins) respectively, on Tempeerature-constant air shaking table 37 DEG C, and 300rpm cultivates 12-16h to OD
600=0.6-0.8, is placed in whizzer by the bacterium liquid obtained, and the centrifugal 1min of 10000rpm, abandons supernatant, obtains required thalline, and checks order, extract plasmid wherein after sequence verification.Sequencing result conforms to completely with expection, illustrates and successfully builds pRRLSIN.cPPT.MSCV-Dual.WPRE carrier.
Embodiment 3 expresses the vector construction of mouse targeting melanoma related antigen gp100 (154-162) TCR α and TCR β
According to the gene order provided in NCBI (TCR α and TCR β, numbering is respectively DQ452619 and DQ452620), and add AscI and AgeI restriction enzyme site respectively in the upstream and downstream of TCR α gene, and remove its terminator codon; The upstream and downstream of TCR β gene adds XhoI and SalI restriction enzyme site respectively.Shanghai Sangon Biological Engineering Technology And Service Co., Ltd is entrusted to synthesize.
Synthesis acquisition two strain intestinal bacteria, comprise pUC57-TCR α and pUC57-TCR β carrier respectively.Carry out enlarged culturing to these two strains intestinal bacteria, extract pUC57-TCR α wherein and pUC57-TCR β carrier respectively, then measure its purity and concentration, result is as shown in the table.
The purity of pUC57-TCR α and pUC57-TCR β carrier and concentration
| Recombinant vectors | A260/A280 | Concentration (ng/ μ L) |
| pUC57-TCRα | 1.86 | 813.7 |
| pUC57-TCRβ | 1.97 | 759.4 |
Process pUC57-TCR α carrier with AscI and AgeI enzyme, XhoI and SalI enzyme processes pUC57-TCR β carrier, reclaims the small segment TCR α both obtaining and TCR β respectively after electrophoresis.The pRRLSIN.cPPT.MSCV-Dual.WPRE carrier AscI obtain previous step and AgeI enzyme carry out double digestion process, carrier is reclaimed after electrophoresis, then with T4DNA ligase enzyme, it is connected with TCR α, obtains pRRLSIN.cPPT.MSCV-Dual.WPRE-TCR α carrier.With MCSI upstream primer (SEQIDNo.8) and MCSI downstream primer (SEQIDNo.9), check order, result conforms to completely with expection.
With XhoI and SalI enzyme, double digestion process is carried out to pRRLSIN.cPPT.MSCV-Dual.WPRE-TCR α carrier, carrier is reclaimed after electrophoresis, then with T4DNA ligase enzyme, it is connected with TCR α, obtains pRRLSIN.cPPT.MSCV-Dual.WPRE-TCR α-TCR β carrier.Check order with MCSII upstream primer (SEQIDNo.10) and MCSII downstream primer (SEQIDNo.11), result conforms to completely with expection.So far, the pRRLSIN.cPPT.MSCV-Dual.WPRE-TCR α-TCR β carrier for expressing mouse targeting melanoma related antigen gp100 (154-162) TCR α and TCR β has successfully built.
Embodiment 4 slow virus is packed
Cultivate 293FT cell, get the good cell of growth conditions and be inoculated in 10cm culture dish, each ware inoculation 5 × 10
6individual cell, add after reaching 80-90% to degrees of fusion after DMEM is about 18h without dual anti-culture medium culturing cell, get each 5 μ g of pRRLSIN.cPPT.MSCV-Dual.WPRE-TCR α-TCR β carrier 10 μ g, pMDLg/pRRE, pRSV-Rev and pMD-G carrier, with Lipofectamine2000 transfection in 293FT cell, after transfection, 4-6h is replaced with DMEM perfect medium.Collect the supernatant substratum containing virus after 48h, after the centrifugal 10min of 6000g, get supernatant liquor and filter with 0.45 μm of filtering head again, obtain virus liquid.
Cultivate 293FT cell, get the good 293FT cell of growth conditions and be inoculated in 24 orifice plates, each hole inoculation 2 × 10
5individual cell, adds 500 μ L substratum, 37 DEG C, 5%CO
2overnight incubation.Second day, by virus stock solution used: the Dilution ratio of substratum is 10
0-10
8the each 100 μ L of the sick slow malicious gradient dilution liquid of preparation, then draw each 100 μ L of the former substratum in each hole respectively, then add each 100 μ L of slow virus diluent and start transfection.Transfection starts rear 24h, and sucking-off, containing the substratum of slow virus, changes the fresh culture of 500 μ L containing 5UDNaseI into, cultivates 30min to remove the remaining plasmid DNA that may be attached to cell surface at 37 DEG C.Then change substratum into 1mL normal incubation medium, continue to cultivate 48h.
Carefully siphon away whole substratum in each hole, add 500 μ L pancreas enzyme-EDTA solution digestion cells, 37 DEG C of reactions 1 minute.Then add substratum and stop digestion reaction by under cell purge, the cell in each hole of collected by centrifugation.The total serum IgE of the every porocyte of extracting, then reverse transcription is cDNA.Upstream and downstream primer is respectively, according to following table application of sample with MCSI upstream primer and MCSII downstream primer:
| Reagent | Usage quantity (μ L) | Final concentration |
| Upstream primer (10 μMs) | 0.4μL | 0.2μM |
| Downstream primer (10 μMs) | 0.4μL | 0.2μM |
| ROX dyestuff (50 ×) | 0.4μL | 1× |
| CDNA template | 2.0μL | |
| Pre-mixing Taq enzyme (2 ×) | 10μL | 1× |
| dH 2O | 6.8μL | |
| Total amount | 20.0μL |
Then carry out quantitative fluorescent PCR, obtain the Ct value of each porocyte quantitative fluorescent PCR reaction.Reaction conditions is as follows:
Denaturation: 95 DEG C 30 seconds, circulate 1 time;
PCR react: 95 DEG C 5 seconds, 58 DEG C 30 seconds, circulate 40 times;
Dissociate: 95 DEG C 15 seconds.
It is generally acknowledged, compared with cellular control unit, Ct value difference is different to be reached more than 2 and can think to there is significant difference, therefore finds out the different minimum but experimental group more than 2 with control group Ct value difference, obtains its extension rate, according to following formulae discovery slow virus titre:
T=R × 20 × 10
3, wherein, T is slow virus titre, and the unit of T is TU/mL, R is extension rate.
As calculated, the slow virus titre of this packaging is greater than 10
7tU/mL, shows that the packaging of this slow virus is successful.
Embodiment 5 cell transfecting and expression conditions detect
Utilize the magnetic bead technology of MyltenyiBiotec separation of C D4+ and CD8+T lymphocyte from the peripheral blood lymphocyte (PBL) of tumour patient, then add slow virus according to the ratio of each T cell 3TU and carry out transfection, after 32 DEG C of centrifugal 2h, cell is placed in 37 DEG C of CO
2incubator is cultivated.After 3 days, utilize the tetramer (Tetramer) of the corresponding polypeptide of Flow cytometry TCR to observe the expression of TCR on T cell surface, its result as shown in Figure 3 and Figure 4.
Can be seen by Fig. 3 and Fig. 4, experimental group CD4+ after slow virus process and CD8+T lymphocyte, its TCR is all significantly increased at the expression level of cell surface and undressed control group, illustrates that pRRLSIN.cPPT.MSCV-Dual.WPRE carrier can perform well in the high expression of the two foreign gene of mammalian cell.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. a Lentiviral, it is characterized in that, described Lentiviral is pRRLSIN.cPPT.MSCV-Dual.WPRE carrier, and described pRRLSIN.cPPT.MSCV-Dual.WPRE carrier is that the GFP sequence of pRRLSIN.cPPT.MSCV/GFP.WPRE carrier is replaced by double gene expression box Dual and obtains;
Described double gene expression box Dual comprises from 5 ' to 3 ' and holds the following structure be arranged in order: the first multiple clone site-furin cleavage site-V5 label-Spacer-2A peptide-the second multiple clone site, and wherein, "-" representative connects.
2. Lentiviral according to claim 1, is characterized in that, the sequence of described double gene expression box Dual is as shown in SEQIDNo.1.
3. Lentiviral according to claim 1, is characterized in that, described first multiple clone site comprises from 5 ' to 3 ' and holds the following structure be arranged in order: AscI restriction enzyme site, BstBI restriction enzyme site, BamHI restriction enzyme site and AgeI restriction enzyme site;
Described second multiple clone site comprises from 5 ' to 3 ' and holds the following structure be arranged in order: XhoI restriction enzyme site, SpeI restriction enzyme site, XmaI restriction enzyme site and SalI restriction enzyme site.
4. the Lentiviral according to claim 1 or 3, is characterized in that, the sequence of described first multiple clone site is as shown in SEQIDNo.2;
The sequence of described second multiple clone site is as shown in SEQIDNo.3;
The sequence of described furin cleavage site is as shown in SEQIDNo.4;
The sequence of described V5 label is as shown in SEQIDNo.5;
The sequence of described Spacer is as shown in SEQIDNo.6;
The sequence of described 2A peptide is as shown in SEQIDNo.7.
5. Lentiviral according to claim 1, is characterized in that, described first multiple clone site is inserted with the first goal gene, and described second multiple clone site is inserted with the second goal gene;
Described first goal gene is mouse TCR α gene, and the NCBI of described mouse TCR α gene is numbered DQ452619, and described second goal gene is mouse TCR β, and the NCBI of described mouse TCR β gene is numbered DQ452620.
6. a preparation method for the Lentiviral according to any one of Claims 1 to 5, is characterized in that, comprises the steps:
Design double gene expression box Dual, and entrust the described double gene expression box of synthesis, obtain the Escherichia coli bacteria liquid containing pUC-Dual plasmid, containing described double gene expression box Dual in described pUC-Dual plasmid, described double gene expression box Dual comprises from 5 ' to 3 ' and holds the following structure be arranged in order: the first multiple clone site-furin cleavage site-V5 label-Spacer-2A peptide-the second multiple clone site, and "-" representative connects;
By the described Escherichia coli bacteria liquid containing pUC-Dual plasmid with the mixing of selectivity LB liquid nutrient medium, in 37 DEG C of constant-temperature tables, 300rpm cultivates 12h ~ 16h to OD
600be 0.6 ~ 0.8, centrifugal for the bacterium liquid obtained rear reservation first is precipitated, extract described pUC-Dual plasmid by after described first precipitation cracking;
With AscI restriction endonuclease and SalI restriction endonuclease process pUC-Dual plasmid at 37 DEG C, fully reclaim after reaction, obtain the fragment of described double gene expression box Dual;
With AscI restriction endonuclease and SalI restriction endonuclease process pRRLSIN.cPPT.MSCV/GFP.WPRE carrier at 37 DEG C, the GFP sequence of described pRRLSIN.cPPT.MSCV/GFP.WPRE carrier is excised, reclaim after abundant reaction, obtain linearizing pRRLSIN.cPPT.MSCV/GFP.WPRE carrier;
Be that the fragment of described double gene expression box Dual mixes with described linearizing pRRLSIN.cPPT.MSCV/GFP.WPRE carrier by 3 ~ 10:1 according to mol ratio, then add T4DNA ligase enzyme, connecting at 4 DEG C and spend the night, obtaining the connection product containing connecting plasmid; And
By the described connection product conversion competence Top10 intestinal bacteria containing connecting plasmid, and on even spread to optionally LB flat board, be inverted for 37 DEG C and cultivate 12h ~ 16h, the positive bacterium colony of picking is placed in described selectivity LB liquid nutrient medium, and in 37 DEG C of constant-temperature tables, 300rpm cultivates 12h ~ 16h to OD
600be 0.6 ~ 0.8, centrifugal for the bacterium liquid obtained rear reservation second precipitated, extract described connection plasmid by after described second precipitation cracking, described connection plasmid is described Lentiviral.
7. Lentiviral according to claim 6, it is characterized in that, after being also included in the fragment obtaining described double gene expression box Dual, insert the first goal gene in described first multiple clone site of the fragment of described double gene expression box Dual, insert the operation of the second goal gene in described second multiple clone site of the fragment of described double gene expression box Dual;
Described first goal gene is mouse TCR α gene, and the NCBI of described mouse TCR α gene is numbered DQ452619, and described second goal gene is mouse TCR β gene, and the NCBI of described mouse TCR β gene is numbered DQ452620.
8. slow virus expresses a test kit, it is characterized in that, comprises the Lentiviral according to any one of Claims 1 to 5.
9. a preparation method for recombinant slow virus, is characterized in that, comprises the steps:
Lentiviral according to any one of Claims 1 to 4 is provided, and insert the first goal gene in described first multiple clone site of the fragment of described double gene expression box Dual, insert the second goal gene in described second multiple clone site of the fragment of described double gene expression box Dual, obtain the Lentiviral carrying goal gene;
Be be transfected in 293FT cell after 2:1:1:1 mixing by described Lentiviral, pMDLg/pRRE carrier, pRSV-Rev carrier and the pMD-G carrier carrying goal gene according to mol ratio, after transfection, 4h ~ 6h is replaced by perfect medium cultivation, nutrient solution is collected after 48h, described supernatant 0.45 μm of filtering head also filters by centrifugal rear reservation supernatant, retain filtrate, described filtrate is the solution of recombinant slow virus.
10. the preparation method of recombinant slow virus according to claim 9, is characterized in that, after being also included in the solution obtaining described recombinant slow virus, to the step that the titre of the solution of described recombinant slow virus detects, is specially:
First day, is inoculated in porous plate by 293FT cell, each hole inoculation 2 × 10
5individual cell, each hole adds 500 μ L substratum, 37 DEG C, 5%CO
2overnight incubation;
Second day is 1,10 by the Dilution ratio of the solution of described recombinant slow virus
1, 10
2, 10
3, 10
4, 10
5, 10
6, 10
7with 10
8with substratum, the solution gradient of described recombinant slow virus is diluted, then respectively the solution of the described recombinant slow virus of 100 μ L gradient dilutions is mixed transfection from the cell culture fluid in 100 μ L porous plates in the different holes of porous plate, transfection starts rear 24h, suck substratum and change the fresh culture of 500 μ L containing 5UDNaseI into, cultivate 30min at 37 DEG C to remove the remaining plasmid DNA that may be attached to cell surface, then change substratum into 1mL normal incubation medium, continue to cultivate 48h;
4th day, suck the substratum in each hole of described porous plate, add 500 μ L pancreas enzyme-EDTA solution digestion cells, 37 DEG C of reactions 1 minute, then add substratum and stop digestion reaction by under cell purge, the cell in each hole of collected by centrifugation, the total serum IgE of the every porocyte of extracting, then reverse transcription obtains total cDNA of every porocyte; And
Respectively quantitative fluorescent PCR is carried out to total cDNA of the described every porocyte obtained, obtains the Ct value of every porocyte, select the different minimum but experimental group more than 2 with control group Ct value difference, obtain its extension rate, according to following formulae discovery slow virus titre:
T=R × 20 × 10
3, wherein, T is slow virus titre, and the unit of T is TU/mL, R is extension rate.
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| CN106119287A (en) * | 2016-08-29 | 2016-11-16 | 广东华南联合疫苗开发院有限公司 | A kind of recombinant vector expressing respiratory syncystial virus F protein and method |
| WO2017101244A1 (en) * | 2015-12-17 | 2017-06-22 | 深圳精准医疗科技有限公司 | Method for preparing and using lentivirus expression vector, and method for preparing recombinant lentivirus |
| CN111676243A (en) * | 2020-05-09 | 2020-09-18 | 厦门大学附属中山医院 | Preparation method of mouse model for specific expression of Cre enzyme in gastric mucosal epithelial cells |
| CN114672515A (en) * | 2020-12-24 | 2022-06-28 | 深圳华大临床检验中心 | Transduction plasmid, lentiviral vector system containing same and application thereof |
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| CN109825527A (en) * | 2018-10-23 | 2019-05-31 | 深圳市慧思基因科技有限公司 | A kind of building of MYCT-1 gene Lentiviral and identification method |
| CN113584085B (en) * | 2021-06-30 | 2023-05-12 | 湖南丰晖生物科技有限公司 | Lentiviral vector for suspension cells and application thereof |
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