CN103352052A - Construction and application of multi-cistron double-label expression lentivirus vector - Google Patents
Construction and application of multi-cistron double-label expression lentivirus vector Download PDFInfo
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Abstract
The invention discloses a lentiviral vector, wherein the original hUbc promoter in FUGW is replaced by a U6 promoter or a strong promoter EF1alpha, the original EGFP gene is replaced by a strong fluorescent protein copGFP or a Tomato gene, a eukaryon resistance label gene, multiple clone sites and tag protein Flag are linked to the same cistron of the gene, and splicing protein T2A and P2A are adopted to link a plurality of genes on the same cistron. Compared with the conventional lentivirus vector, the lentiviral vector of the present invention has the following advantages that: a plurality of proteins can be concurrently and efficiently expressed with the same cistron, fluorescence intensity is high, induced function recovery is performed on the interfered gene, the vector is suitable for carrying large fragment genes, and the like. In addition, the lentivirus vector has characteristics of wide application, high efficiency, benefits for applications of the lentiviral vector in clinical and science researches, good practical value and good application prospects.
Description
Technical field
The invention belongs to field of biological pharmacy, particularly the two marks of a kind of polycistron are expressed the construction and application of lentiviral vectors.
Background technology
Lentiviral vectors refers to that lentiviral vectors has comprised packing, transfection, the needed genetic information of stable integration, is the chief component of slow virus carrier system with a kind of virus vector in human immunodeficiency virus-1 (HIV-1) source.The lentiviral vectors that carries foreign gene is lower assisting of slow virus packaging plasmid, clone, becomes infectious virion through the virus packing, and by cells infected or biological tissue, the realization foreign gene is expressed in cell or biological tissue.The infection pedigree of slow virus particle is wide, not only can efficiently infect division cells, and can infect non-division cells, can also efficiently infect primary cell and the embryonic stem cell of the transfection of plasmid difficulty, therefore increasing researchist begins to enter cell or tissue with lentiviral vectors technology transduction purpose fragment.In view of its have foreign gene expression time in the host of carrying long, toxicity is low, portable exogenous genetic fragment large, be difficult for bringing out the advantage such as host immune response, now become desirable gene transfer vector, be widely used in the field of scientific studies such as gene therapy, production of vaccine, transgenic animal, gene knockout, drug research, production target protein clone.
The often result of polygene change occurs in tumour, imports polygene during gene therapy and could realize its value.And when lentiviral vectors is used, in order to realize different scientific research purposes, as positioning simultaneously the different purposes such as tracking and screening, need to be at lentiviral vectors access reporter gene and eucaryon resistance screening gene etc.These all will by making up the polycistron lentiviral vectors, namely be expressed a plurality of genes and realize simultaneously on same carrier.Making up at present slow virus polycistron carrier strategy commonly used has: (1) internal ribosome entry site (internal ribosome entry site, IRES) connect, about 1000bp, self structure is large, upstream and downstream is expressed the defectives such as uneven but this mode exists, and has seriously limited its application; (2) gene fusion, this mode have caused the folding or transportation of abnormal protein and have affected its expression; (3) use a plurality of promotors, each promotor starts independently encoder block.Its shortcoming is the easy phase mutual interference of promotor of series connection.The characteristics of a maximum of lentiviral vectors are the carrier finite capacities, can insert the external source fragment that is no more than 2Kb, and titre increases with Insert Fragment length and reduces.As long as high titre could realize the gene of high effect nontoxic and import, therefore need to express a plurality of albumen on the same cistron by transforming carrier, also be not subjected to simultaneously the restriction of carrier capacity.Have small molecule structure, 18-22 is amino acid whose can to make this purpose become a reality from shear protein 2A polypeptide.
The 2A polypeptide of some positive strand viruses coding can make on the same cistron a plurality of genes express respectively.The higher structure that its " shearing " mechanism is 2A in translation process causes size exclusion to the ribosomal peptidyl transferase center, so that the peptide bond between 2A and the 2B can't form, thereby the release that causes upstream gene starts downstream protein translation (M.J.Osborn, 2005 simultaneously; P.de Felipe, 2006).The 2A small peptide that has now found that has P2A(porcine teschovirus), T2A (Thosea asigna virus).Short and small in view of the 2A structure, shear efficiency is high, the advantage of upstream and downstream genetic expression balance, becomes the desirable instrument of polycistron that makes up.
Summary of the invention
The invention provides a kind of lentiviral vectors, its be with lentiviral vectors FUGW for the carrier that sets out improves, inserted the U6 multiple clone site, label protein Flag, shear protein P2A, label protein HA and shear protein T2A.
Preferably, described lentiviral vectors replaces with U6 promotor or strong promoter EF1 α with original hUbc promotor among the FUGW.
Preferably, described lentiviral vectors is hyperfluorescenceCeng Yongminggaoyingguang albumen copGFP or Tomato gene with original EGFP Gene Replacement among the FUGW.
Preferably, described lentiviral vectors has inserted the eucaryon resistant maker gene.
Preferred, described lentiviral vectors is that original hUbc promotor among the FUGW is replaced with U6 promotor or strong promoter EF1 α, and be hyperfluorescenceCeng Yongminggaoyingguang albumen copGFP or Tomato gene with original EGFP Gene Replacement among the FUGW, and connect eucaryon resistant maker gene and multiple clone site and label protein Flag in the same cistron of this hyperfluorescenceCeng Yongminggaoyingguang albumen copGFP or Tomato gene, and adopt shear protein T2A to be connected a plurality of genes on this same cistron with P2A.
Further preferred, described eucaryon resistant maker gene is Puromycin or Hygromycin resistant gene.
Preferably, described lentiviral vectors is called again pUETP for the fragment with (comprising hUbC promotor and EGFP) between the Pac I of lentiviral vectors FUGW and the EcoR I restriction enzyme site is replaced into the lentiviral vectors that U6-EF1 α-the MCS-FLAG-P2A-Tomato-T2A-Puromycin fragment gets.Described U6-EF1 α-MCS-FLAG-P2A-Tomato-T2A-Puromycin fragment is the U6 promotor, strong promoter EF1 α, multiple clone site MCS, label protein Flag, shear protein P2A, fluorescin Tomato, shear protein T2A, eucaryon resistance marker Puromycin.
Preferably, described lentiviral vectors is called again pUEGH for the fragment with (comprising hUbC Promoter and EGFP) between the Pac I of lentiviral vectors FUGW and the EcoR I restriction enzyme site is replaced into the lentiviral vectors that U6-EF1 α-the MCS-FLAG-P2A-copGFP-T2A-Hygromycin fragment gets.Described U6-EF1 α-MCS-FLAG-P2A-copGFP-T2A-Hygromycin fragment is the U6 promotor, strong promoter EF1 α, multiple clone site MCS, label protein Flag, shear protein P2A, fluorescin copGFP, shear protein T2A, eucaryon resistance marker Hygromycin.
Preferred, the nucleotide sequence of described U6-EF1 α-MCS-FLAG-P2A-Tomato-T2A-Puromycin fragment is shown in SEQ ID NO.2 in the sequence table; The nucleotide sequence of described U6-EF1 α-MCS-FLAG-P2A-copGFP-T2A-Hygromycin fragment is shown in SEQ ID NO.3.
The present invention also provides a kind of recombined lentivirus vector, and it is take lentiviral vectors of the present invention as carrier, has inserted exogenous nucleic acid.
Preferably, described exogenous nucleic acid is the shRNA of goal gene, and described exogenous nucleic acid inserts in the U6 promotor of described lentiviral vectors.
Preferred, described exogenous nucleic acid inserts between the EcoR I and BamH I site of U6 promotor.
Preferably, described exogenous nucleic acid is the cDNA of goal gene, and described exogenous nucleic acid inserts the multiple clone site MCS of described lentiviral vectors.
The present invention also provides a kind of slow virus particle, it is got by the method preparation that may further comprise the steps: with described lentiviral vectors or described recombined lentivirus vector and three kinds of packing helper plasmid Gag pol, VSVG, REV is transfection 293T cell together, by obtaining the slow virus particle in the cell culture fluid.
The present invention also provides described lentiviral vectors in gene clone or the application in expressing.
On the basis that meets this area general knowledge, above-mentioned each optimum condition, but arbitrary combination namely get the preferred embodiments of the invention.
The raw material that the present invention is used or reagent except specifying, equal commercially available getting.
Positive progressive effect of the present invention is:
For the carrier that sets out, the two marks of polycistron that are based on the 2A shear protein after the improvement are expressed lentiviral vectors with known carrier FUGW in the present invention.Utilize original hUbc-promoter and EGFP element among the gene engineering method excision FUGW, and again introduce U6-EF1 α-MCS-T2A-copGFP/Tomato-P2A-hygromycin/puromycin element.This element comprises the U6 promotor, strong promoter EF1 α, multiple clone site MCS, label protein Flag, shear protein P2A, fluorescin copGFP or Tomato, label protein HA, shear protein T2A, eucaryon resistance marker Puromycin or Hygromycin.
U6 promotor of the present invention, it is the promotor that a kind of RNA polymerase III relies on, promotor self element all is positioned at the upstream of transcriptional domain, can make hairpin RNA (small hairpin RNA, shRNA) expression in mammalian cell, point-device from the synthetic RNA of the position transcriptional start of a fixed range of promotor, running into 4-5 continuous U stops, and make transcription product cut next at second uridylic place, but also can in mammalian cell, express more microRNA.After the RNA that transcribes out forms the hair fastener spline structure, can form 2 outstanding uridylics at 3 ' end, this is similar to natural siRNA, thereby extremely is conducive to double-stranded RNA and brings out RNAi.
Promotor EF1 α of the present invention is a kind of promotor of strongly expressed, can make foreign gene wide expression in mammalian cell, even can express in primary cell and stem cell.
Fluorescin Tomato of the present invention, this has the strongest albumen fluorescent brightness.It is a kind of orange derivative of original fruit albumen, comprises two copies of Tomato, by the joint link of 12 amino-acid residues.
Fluorescin copGFP of the present invention is green fluorescent protein, and stability is high, and fluorescent brightness is strong, and its fluorescent brightness can be 3 times of EGFP.
The existing conventional lentiviral vectors of lentiviral vectors of the present invention has more advantage.
1) the shRNA sequence of goal gene is inserted the U6 promotor, can reticent goal gene; Or the cDNA insertion multiple clone site MCS of goal gene is regional, can cross the expression goal gene; Therefore, can carry out to the gene that has disturbed the inducibility function and reply, be used for having more convictive functional verification experiment.
2) fluorescin that uses of the present invention is copGFP and the Tomato of latest generation, and fluorescence intensity is high, and form is more stable, and is monomer.
3) the present invention adopts the short and small 2A polypeptide of structure, so that efficiently expressing simultaneously, same cistron expresses a plurality of albumen, it is uneven to have avoided upstream and downstream to express, P2A has efficiently started the expression of fluorescin (copGFP or Tomato), and T2A has efficiently started resistance protein (puromycin or hygromyin).
4) the present invention adopts the short and small 2A polypeptide of structure, effectively raises virus titer, and the slow virus that is applicable to the large fragment carrier makes up.
5) lentiviral vectors of the present invention is applicable more extensive, and efficient is higher, more is conducive to the application of lentiviral vectors in clinical and scientific research, has good practical value and application prospect.
Description of drawings
Fig. 1 shows the carrier collection of illustrative plates.Wherein, Figure 1A is carrier FUGW, and Figure 1B is carrier pUETP, and Fig. 1 C is carrier pUEGH.
Fig. 2 shows the electrophorogram of the connection product that is used for vector construction.Wherein, Fig. 2 A is U6-EF1 α-MCS-FLAG fragment (1015bp) electrophorogram; Fig. 2 B is amplification P2A-Tomato-T2A-Puromycin fragment (1454bp) electrophorogram; Fig. 2 C is amplification copGFP-Hygromycin fragment (1926bp) electrophorogram.Wherein, the nucleic acid fragment sample in 1,2, the 3 expression revision tests, M represents molecular weight marker.
Fig. 3 shows pUETP-shCDH1 carrier transfection fluorogram.Wherein, A is pUETP light field figure; B is pUETP details in a play not acted out on stage, but told through dialogues figure; C is pUETP-shCDH1 light field figure; D is pUETP-shCDH1 details in a play not acted out on stage, but told through dialogues figure.
Fig. 4 shows the CDH1knockdown checking.Wherein, 1, pUETP; 2, pUETP-shCDH1.
Fig. 5 shows pUEGH-CDH1 carrier transfection fluorogram.Wherein, A is pUEGH light field figure; B is pUEGH details in a play not acted out on stage, but told through dialogues figure; C is pUEGH-CDH1 light field figure; D is pUEGH-CDH1 details in a play not acted out on stage, but told through dialogues figure.
Fig. 6 shows that CDH1 crosses expression checking electrophorogram.1，pUEGH；2，pUEGH-CDH1。a，CDH1(130kD)；b，β-Actin。
Embodiment
Mode below by embodiment further specifies the present invention, but does not therefore limit the present invention among the described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example according to ordinary method and condition, or is selected according to catalogue.
Embodiment 1U6-EF1 α-MCS-FLAG-P2A-Tomato-T2A-Puro(is called for short pUETP) the lentiviral vectors structure
(1) increase respectively U6, EF1 α-MCS-FLAG, Tomato and Puromycin
1. the U6 promotor increases
1) the synthetic U6 sequence of full gene
The synthetic U6 fragment of artificial full gene, sequence is shown in SEQ ID NO.1.
2) take the synthetic U6 fragment of full gene as template, by Asc I and the Nhe I restriction enzyme site in the overlapping extension PCR removal U6 fragment, in sequence, introduce simultaneously EcoR I and BamH I restriction enzyme site.Wherein, in first round PCR, amplify the U6-1 fragment with U6-F1 and U6-R1 primer PCR, go out the U6-2 fragment with U6-F2 and U6-R2 primer amplification.Take turns among the PCR second, take U6-1 and U6-2 fragment as template, take U6-F1 and U6-R2 as primer carries out pcr amplification, obtain the U6 fragment with EcoR I and BamH I restriction enzyme site.Used primer sequence is as follows:
U6-F1：5’-TTAATTAATTTCCCATGATTCC-3’
U6-R1：5’-GAATTCCTCGACGGATCCTCGTCCTTTCCACAAG-3’
U6-F2：5’-GGATCCGTCGAGGAATTCAGTTATTAATAGTAATC-3’
U6-R2：5’-CGCAGATCCTTAATTAAAACGCGGAAC-3’。
Adopt KOD-Plus PCR reagent (available from Toyobo company) to carry out the PCR reaction.Pcr amplification system following (together lower):
PCR program following (together lower):
2. EF1 α-MCS-FLAG increases
Take pMIRNA1 carrier (available from System Biosciences, SBI) as template, take hEF1-F and hEF1-MCS-R as primer, carry out the qPCR amplification, and in the hEF1-MCS-R primer sequence, introduce the MCS fragment, obtain EF1 α-MCS fragment.Simultaneously as pcr template and primer, reaction and the following pcr amplification that carries out of system get the FLAG fragment with Flag – F and Flag – R primer.Take EF1 α-MCS and FLAG as template, carry out pcr amplification with hEF1-F and FLAG-R primer, get EF1 α-MCS-FLAG fragment.
EF1 α-MCS amplimer:
hEF1-F：5’-TTAATTAAGGATCTGCGATCGCTC-3’；
hEF1-MCS-R：
5’-GCTAGCtccTCTAGACGTCGACGGCGCGCCCATGGTGGCgGCGAT?CGCGTAGGCGCCGGTCACAG-3’。
The FLAG amplimer:
FLAG-F：
5’-
CGTCTAGAggaGCTAGCggaTTCGAAGACTACAAAGACCATGACG?GTGATT
ACAAGGATGACGATGAC-3’；
FLAG-R：
5’-GCCTGCTTCAGCAGGCTGAAGTTAGTAGCTCCGCTTCCGTTAAC?CTTGTCATCGTCATCCTTGT-3’。
FLAG fragment PCR reaction system
The PCR program is as follows:
3. P2A-Tomato and T2A-puromycin increase
Take pLVX-IRES-td Tomato carrier (available from Clontech) as template, amplify Tomato with Tomato-F and Tomato-R primer PCR, introduce simultaneously P2A in the Tomato upstream.Take pMIRNA1 carrier (available from System Biosciences, SBI) as template, go out Puromycin with Puro-F and Puro-R primer amplification, introduce simultaneously T2A in the Puromycin upstream.Wherein, primer sequence is as follows:
Tomato-F：
5’-
CAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGG?ACCTATGGTGAGCAAGGGCGAG-3’；
Tomato-R：
5’-CTCTCCGCTTCCATTTAAATaGTTAACGGCGTAGTCAGGCACGTC?GTAAGGATACTTGTACAGCTCGTCC-3’；
Puro-F：
5’-
GAAGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACG?TGGAGGAGAATCCCGGCCCTATGACCGAGTA-3’；
Puro-R：5’-TCAGCTATTTAAATGGCACCGGGCTTGCGGGTC-3’。
(2) overlapping extension PCR amplification U6-EF1 α-MCS-FLAG and P2A-Tomato-T2A-Puromycin
1. U6-EF1 α-MCS-FLAG increases
Take the U6 of above-mentioned preparation and EF1 α-MCS-FLAG as template, go out U6-EF1 α-MCS-FLAG with U6-F1 and P2A-SpeI-R primer amplification, introduce simultaneously the SpeI restriction enzyme site, simultaneously pcr amplification 3 pipes, product is as shown in Figure 2.Wherein, primer sequence is as follows:
U6-F1：5’-TTAATTAATTTCCCATGATTCC-3’；
P2A-SpeI-R：5’-cgat?act?agt?GTT?AAC?CTT?GTC?ATC?GTC?ATC?C-3’。
2. amplification P2A-Tomato-T2A-Puromycin amplification
Take the Tomato of above-mentioned preparation and Puromycin as template, go out Tomato-Puromycin with P2A-SpeI-F and Puro-R primer amplification, and introduce the SpeI restriction enzyme site, simultaneously pcr amplification 3 pipes, product is as shown in Figure 2.Wherein, primer sequence is as follows:
P2A-SpeI-F：
5’-cgat?act?agt?GGA?AGC?GGA?GCT?ACT?AAC?TTC?AGC?CTG?CTG?AAG?C-3’；
Puro-R：5’-TCAGCTATTTAAATGGCACCGGGCTTGCGGGTC-3’。
(3) overlapping extension PCR amplification U6-EF1 α-MCS-FLAG-P2A-Tomato-T2A-Puromycin
1. after respectively U6-EF1 α-MCS-FLAG and P2A-Tomato-T2A-Puromycin enzyme being cut, connect and be cloned in the pJET1.2/blunt carrier (Fermentas).Wherein enzyme is cut system and linked system is as follows:
Enzyme is cut system:
Linked system:
2. U6-EF1 α-MCS-FLAG-P2A-Tomato-T2A-Puromycin increases
The pJET1.2-U6-EF1 α that makes up take previous step-MCS-FLAG carrier and pJET1.2-P2A-Tomato-T2A-Puromycin carrier are together as template, take U6-F1 and Puro-R as primer carries out pcr amplification, get U6-EF1 α-MCS-FLAG-P2A-Tomato-T2A-Puromycin fragment.Wherein, primer is as follows:
U6-F1：5’-TTAATTAATTTCCCATGATTCC-3’；
Puro-R：5’-TCAGCTATTTAAATGGCACCGGGCTTGCGGGTC-3’。
3. U6-EF1 α-MCS-FLAG-P2A-Tomato-T2A-Puromycin is cloned among the lentiviral vectors FUGW, produces pUETP
Enzyme is cut system:
The FUGW enzyme is cut product and is filled:
The FUGW enzyme is cut product and is filled rear dephosphorylation:
U6-EF1 α-MCS-FLAG-P2A-Tomato-T2A-Puromycin product phosphorylation:
Linked system:
Obtain U6-EF1 α-MCS-FLAG-P2A-Tomato-T2A-Puro lentiviral vectors (being called for short pUETP), its collection of illustrative plates is seen Fig. 1.
Embodiment 2U6-EF1 α-MCS-FLAG-P2A-copGFP-T2A-Hygromycin(is called for short pUEGH) the lentiviral vectors structure
(1) increase respectively U6, EF1 α-MCS-FLAG, P2A-copGFP and T2A-Hygromycin
1. U6, EF1 α-MCS-FLAG increase
The same pUETP of method.
2. T2A-Hygromycin increases
Take the linearizing fragment of linear hygro (available from Clontech) as template, by the EcoR I restriction enzyme site among the overlapping extension PCR point mutation removal Hygromycin.Wherein, in first round PCR, take linear hygro as template, be that primer amplification goes out Hygro-1 with Hygro-F and Hygro-R, be that primer amplification goes out Hygro-2 with Hygro-MF and Hygro-MR.Take turns among the PCR second, take Hygro-1 and Hygro-2 as template, take Hygro-F and Hygro-MR as the primer PCR amplification, obtain the Hygromycin without EcoR I restriction enzyme site, and introduce T2A in the Hygromycin upstream.Wherein primer sequence is as follows:
Hygro-F：
5’-GAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAG?AATCCTGGACCTtATTTAAATATGAAAAAGCC-3’；
Hygro-R：
5’-TCAGCTATTTAAATTTCCTTTGCCCTCGGACG-3’；
Hygro-MF：5’-ATTGGGGAGTTCAGCGAG-3’；
Hygro-MR：5’-CTCGCTGAACTCCCCAAT-3’。
3. copGFP increases
Take the pMIRNA1 carrier as template, go out copGFP with copGFP-F and copGFP-R primer amplification, PCR system and program are the same.Wherein, primer sequence is as follows:
copGFP-F：5’-CAGCCTGCTGAAGCAGGCTG-3’；
copGFP-R：
5’-TCCTCTGCCCTCTCCGCTTCCGTTAACGGCGTAGTCAGGCACGT?CGTAAGGATAGCGAGATCCGGTGGAG-3’。
(2) overlapping extension PCR amplification U6-EF1 α-MCS-FLAG and copGFP-T2A-Hygromycin
1. U6-EF1 α-MCS-FLAG increases
The same pUETP of method.
2. copGFP-Hygromycin increases
Take the copGFP of above-mentioned preparation and Hygromycin as template, go out copGFP-Hygromycin with P2A-SpeI-F and Hygro-MR primer amplification, and introduce the SpeI restriction enzyme site, the PCR product is as shown in Figure 2.Wherein, primer sequence is as follows:
P2A-SpeI-F：
5’-cgat?act?agt?GGA?AGC?GGA?GCT?ACT?AAC?TTC?AGC?CTG?CTG?AAG?C-3’；
Hygro-MR：5’-CTCGCTGAACTCCCCAAT-3’。
(3) overlapping extension PCR amplification U6-EF1 α-MCS-FLAG-P2A-copGFP-T2A-Hygromycin
Overlapping extension PCR amplification U6-EF1 α-MCS-FLAG-P2A-copGFP-T2A-Hygromycin, the same pUETP of method is except used primer sequence.Primer sequence is as follows:
U6-F1：5’-TTAATTAATTTCCCATGATTCC-3’；
Hygro-MR：5’-CTCGCTGAACTCCCCAAT-3’。
U6-EF1 α-MCS-FLAG-P2A-copGFP-T2A-Hygromycin is cloned among the lentiviral vectors FUGW that PacI and EcoRI enzyme cut, produces pUEGH, the same pUETP of method.
Obtain U6-EF1 α-MCS-FLAG-P2A-copGFP-T2A-Hygromycin lentiviral vectors (being called for short pUEGH), its collection of illustrative plates is seen Fig. 1.
The application example of embodiment 3pUETP and pUEGH lentiviral vectors
(1) pUETP is applied to the structure that the CDH1 gene RNAi is stablized strain
1.CDH1 gene shRNA design of primers
According to shRNA design of primers principle, for the CDH1 gene (NCBI number: NM_004360.3) design 1 pair of shRNA primer.
shCDH1-F：
5’-GATCCGGCGATTCAAAGTGGGCACAGATGGTACCATCTGTGCC?CACTTTGAATCGTTTTTG-3’；
shCDH1-R：
5’-AATTCAAAAACGATTCAAAGTGGGCACAGATGGTACCATCTGT?GCCCACTTTGAATCGCCG-3’。
2. make up the pUETP-shCDH1 lentiviral vectors
With primers F (10 μ M) 10 μ l, primer R(10 μ M) 10 μ l, 10 * annealing buffer, 5 μ l, ddH
2The reaction system of O25 μ l is positioned over that water-bath naturally cooled to room temperature and anneals after 5 minutes in 100 ℃ of thermostat water baths.After obtaining the CDH1shRNA two strands, be connected on the EcoR I and BamH I site that the present invention transforms lentiviral vectors U6.Reaction system is: shRNA primer 4 μ l, lentiviral vectors 2 μ l, ddH
2O2 μ l, 10 * T4 damping fluid, 1 μ l, Roche T4 ligase enzyme 1 μ l, room temperature was placed 2 hours.Be transformed in the GeneHogs competence Bacillus coli cells, coat on the solid LB culture plate that contains penbritin, cultivate after 14 hours, choose full positive colony and increase for 37 ℃, Promega plasmid purification test kit extracts plasmid, the checking of KpnI single endonuclease digestion.EB gel electrophoresis, visible two bands be the dna fragmentation of 2.8Kb and 7.2Kb clearly, conforms to theory.Obtain the pUETP-shCDH1 lentiviral vectors.
3.pUETP-shCDH1 slow virus packing
With pUETP-shCDH1 lentiviral vectors and three kinds packing helper plasmids (REV is available from Invitrogen for Gag pol, VSVG) together transfection 293T cell, observe the fluorescence situation behind the 24h.As shown in Figure 3, can find out that the red fluorescence expression intensity is high, illustrates that P2A can efficiently start the expression of Tomato.And 48h and 72h collect the thick liquid of viral supernatant after transfection respectively.The virus liquid supernatant of collecting is behind centrifugal removal residual cells and cell debris, and Millex-HV0.45 μ m membrane filtration is with the further cell debris of removing remnants.The titre that fluorometric determination records the thick liquid of virus is 3E7TU/ml.
4.A549 cell pUETP-shCDH1 stablizes the strain screening
When the good purpose cell density of exponential phase growth conditions reaches 70%～80%, the processing of going down to posterity, and be inoculated in 24 orifice plates with suitable number, so that the second day cell density reaches about 30%～40%.Use empty carrier slow virus liquid pUETP and pUETP-shCDH1 slow virus liquid to infect respectively the A549 cell after 96 hours, add 0.4 μ g/ml puromycin and process 1 week of cell, the fluorescence infection proportion reaches 100%.Then puromycin concentration is down to 0.1 μ g/ml and continues enlarged culturing, obtain stable cell line, and collecting cell RNA sample and protein sample.The qPCR result of RNA sample CDH1 as shown in Figure 4, pUETP-shCDH1 slow virus infection group is compared CDH1 inhibition on the mRNA level and is reached more than 97% with empty carrier pUETP.A549 cell CDH1 gene RNAi is stablized the successful screening of strain, and the knockdown that can be effectively applied to goal gene in the pUETP carrier is described, T2A can efficiently start the expression of puromycin simultaneously.
5. sum up
PUETP-shCDH1 stablizes the smooth structure of strain, good authentication and the high-intensity red fluorescence of CDH1 Gene silencing efficacy expressed, element U6 on the pUETP carrier has been described, Tomato, Puromycin all can efficiently express, and it is uneven that the application success of P2A and T2A has been avoided the expression of same cistron upstream and downstream on the carrier.
(2) pUEGH crosses to express for large fragment CDH1 and stablizes the structure of strain
1. make up the pUEGH-CDH1 lentiviral vectors
The cDNA fragment of clone's CDH1 gene (2649bp) is with cDNA fragment 4 μ l, through the pUEGH carrier 2 μ l that Asc I and Nhe I enzyme were cut, Roche T4ligase1 μ l, 10 * T4buffer1 μ l, 2 μ l ddH
2The reaction system of O is positioned over 24 ℃ of water-baths and connected in 2 hours, connects product plasmid extraction after transforming, shaking bacterium and gets the pUEGH-CDH1 lentiviral vectors.
2.pUEGH-CDH1 slow virus packing
With pUEGH-CDH1 lentiviral vectors and three kinds packing helper plasmids (Gag pol, VSVG, REV) together transfection 293T cell, observe the fluorescence situation behind the 24h.As shown in Figure 5, can find out that the green fluorescence expression intensity is high, its fluorescence intensity is 2 times of EGFP on the former FUGW carrier, illustrates that also P2A can efficiently start the expression of copGFP simultaneously.And 48h and 72h collect the thick liquid of viral supernatant after transfection respectively.The virus liquid supernatant of collecting is behind centrifugal removal residual cells and cell debris, and Millex-HV0.45 μ m membrane filtration is with the further cell debris of removing remnants.Utilize GFP fluorescence observation method to measure the thick drop degree of pUEGH-CDH1 virus and be 3X10
6TU/ml, the pFUGW-CDH1 lentiviral vectors is unpackaged to go out the slow virus particle and get take FUGW as carrier.
3.A549 crossing to express, cell CDH1 stablizes the strain screening
When the good purpose cell density of exponential phase growth conditions reaches 70%～80%, the processing of going down to posterity, and be inoculated in 24 orifice plates with suitable number, so that the second day cell density reaches about 30%～40%.Use empty carrier slow virus liquid pUEGH and pUEGH-CDH1 slow virus liquid to infect respectively the A549 cell after 96 hours, add 100 μ g/ml Hygromycin and process 1 week of cell, the fluorescence infection proportion reaches 100%.Then Hygromycin concentration is down to 25 μ g/ml and continues enlarged culturing, obtain stable cell line, and the collecting cell protein sample.The western blot result of albumen sample as shown in Figure 6, the CDH1 of pUEGH-CDH1 group had and significantly crossed expression effect.A549 cell CDH1 gene overexpression is stablized the successful screening of strain, illustrates that can be effectively applied to crossing of large fragment gene in the pUEGH carrier expresses, and has illustrated that also T2A can efficiently start the expression of hygromycin simultaneously.
4. sum up
PUEGH-CDH1 crosses and expresses successful screening and the checking of stablizing strain, has illustrated that EF1 α, coGFP and the Hygromycin on the pUEGH carrier all can efficiently express, and it is uneven that the application success of P2A and T2A has been avoided the expression of same cistron upstream and downstream on the carrier.Simultaneously, the successful packing of CDH1 gene (size is 2649bp) slow virus liquid illustrates that the pUEGH carrier can be used for the slow virus packing of the above large fragment gene of 2Kb.
Should be understood that after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. lentiviral vectors, it is to improve for the carrier that sets out with lentiviral vectors FUGW, it is characterized in that, described lentiviral vectors is that original hUbc promotor among the FUGW is replaced with U6 promotor or strong promoter EF1 α, and be hyperfluorescenceCeng Yongminggaoyingguang albumen copGFP or Tomato gene with original EGFP Gene Replacement among the FUGW, and connect eucaryon resistant maker gene and multiple clone site and label protein Flag in the same cistron of this hyperfluorescenceCeng Yongminggaoyingguang albumen copGFP or Tomato gene, and adopt shear protein T2A to be connected a plurality of genes on this same cistron with P2A.
2. lentiviral vectors as claimed in claim 1 is characterized in that, described eucaryon resistant maker gene is Puromycin or Hygromycin resistant gene.
3. lentiviral vectors as claimed in claim 1 or 2, it is characterized in that described lentiviral vectors is replaced into the lentiviral vectors that U6-EF1 α-the MCS-FLAG-P2A-Tomato-T2A-Puromycin fragment gets for the fragment that will comprise hUbC promotor and EGFP between the Pac I of lentiviral vectors FUGW and the EcoR I restriction enzyme site; Perhaps, described lentiviral vectors is replaced into the lentiviral vectors that U6-EF1 α-the MCS-FLAG-P2A-copGFP-T2A-Hygromycin fragment gets for the fragment that will comprise hUbC Promoter and EGFP between the Pac I of lentiviral vectors FUGW and the EcoR I restriction enzyme site.
4. lentiviral vectors as claimed in claim 3 is characterized in that, the nucleotide sequence of described U6-EF1 α-MCS-FLAG-P2A-Tomato-T2A-Puromycin fragment is shown in SEQ ID NO.2 in the sequence table; The nucleotide sequence of described U6-EF1 α-MCS-FLAG-P2A-copGFP-T2A-Hygromycin fragment is shown in SEQ ID NO.3 in the sequence table.
5. a recombined lentivirus vector is characterized in that, it has inserted exogenous nucleic acid take lentiviral vectors claimed in claim 1 as carrier.
6. recombined lentivirus vector as claimed in claim 5 is characterized in that, described exogenous nucleic acid is the shRNA of goal gene, and described exogenous nucleic acid inserts in the U6 promotor of described lentiviral vectors.
7. recombined lentivirus vector as claimed in claim 6 is characterized in that, described exogenous nucleic acid inserts between the EcoR I and BamH I site of U6 promotor.
8. recombined lentivirus vector as claimed in claim 5 is characterized in that, described exogenous nucleic acid is the cDNA of goal gene, and described exogenous nucleic acid inserts the multiple clone site of described lentiviral vectors.
9. slow virus particle, it is characterized in that, it is got by the method preparation that may further comprise the steps: with lentiviral vectors claimed in claim 1 or recombined lentivirus vector claimed in claim 5 and three kinds of packing helper plasmid Gag pol, VSVG, REV is transfection 293T cell together, by obtaining the slow virus particle in the cell culture fluid.
10. the application of lentiviral vectors as claimed in claim 1 in gene clone or expression.
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| CN116218844B (en) * | 2022-09-07 | 2024-05-14 | 吉满生物科技(上海)有限公司 | Bidirectional promoter, over-expression vector thereof, lentiviral expression plasmid and application |
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