CN101857877A - Retrovirus eukaryotic expression vector containing TFPI (Tissue Factor Pathway Inhibiyor) gene and GFP (Green Fluorescence Protein) gene as well as construction method and application thereof - Google Patents
Retrovirus eukaryotic expression vector containing TFPI (Tissue Factor Pathway Inhibiyor) gene and GFP (Green Fluorescence Protein) gene as well as construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a retrovirus eukaryotic expression vector containing a TFPI (Tissue Factor Pathway Inhibiyor) gene and a GFP (Green Fluorescence Protein) gene as well as a construction method and application thereof. The retrovirus eukaryotic expression vector containing the TFPI gene and the GFP gene is prepared by the following steps of: taking a sequence shown by a sequence table SEQ ID NO. 2 and a sequence table SEQ ID NO. 3 as a primer and a pIRES-TEPI as a template, amplifying full length cDNA of a human tissue factor pathway inhibiting factor gene shown by a sequence table SEQ ID NO. 1, inserting the full length cDNA into retrovirus vector pMSCV-IRES-GFP to obtain a pMSCV-TFPI-IRES-GFP recombinant plasmid , thereby realizing non-fused expression of TFPI and the green fluorescence protein in EPCs (Endothelial Progenitor Cells), wherein the functions the two kinds of proteins are not mutually interfered. When the biological functions of the TFPI are normally exerted, GFP expression can be used as an effective tracing method of transgenosis cells and provide great convenience for studying tissue orientation and differentiation condition of the EPCs carrying the TFPI gene in vivo.
Description
Technical field
The invention belongs to gene engineering technology field, specifically, relate to the retroviral eukaryotic expression vector and construction process and the application that contain human tissue factor pathway supressor (TFPI) gene and green fluorescent protein (GFP) Gene Double cistron.
Background technology
Vascular restenosis (restenosis RS) is one of difficult problem of perplexing at present treating cardiovascular disease, the fast development of stem cells technology and gene therapy technology in recent years, and control has brought new hope to vascular restenosis.TFPI is the main anticoagulin of body, is that (tissue factor, TF) unique specific inhibitor belongs to Kunitz type proteinase inhibitor to tissue factor.It participates in body coagulation process and thrombotic adjusting by the function that suppresses FXa, TF/FVIIa mixture.Studies show that in a large number that in recent years TFPI can effectively suppress the propagation and the migration of thrombosis, inflammatory reaction and unstriated muscle behind the blood vessel injury.It causes people's attention day by day in the effect aspect the prevention RS.Preliminary experimentation on animals shows that the intravenous drip TFPI recombinant protein vascular restenosis that damage is brought out to air bag has the obvious suppression effect.Employing is the TFPI gene of carrier through conduit with the liposome to the blood vessel regional perfusion that air bag damages, and the degree of angiostenosis is obviously alleviated.The recombinant adenovirus that will have the TFPI gene is used for the treatment of vascular restenosis animal model, can make the degree of angiostenosis reduce 43%.Directly use the TFPI recombinant protein, because its transformation period is short, using dosage is big, and administration time is long, and deficiency factor such as cost an arm and a leg becomes the bottleneck of clinical application.Therefore gene therapy may be more efficiently method, but general viral genetic vector lacks effective target, is to limit one of its principal element of applying.
Endothelial progenitor cell (endothelial progenitor cells, EPCs) be the precursor cell of vascular endothelial cell, because of it has the potential that height self ability and directional migration are divided into vascular endothelial cell, be the desirable seed cell for the treatment of ischemic angiopathy at present.EPCs can be used as Vectors in Gene Therapy and takes therapeutic gene to target organ at external energy stability and high efficiency ground expression alien gene simultaneously, is the good carrier that carries foreign gene.
The main control strategy of vascular restenosis is: 1, suppress the partial vascular smooth muscle cell proliferation of pathology; 2, suppress partial inflammatory reaction of pathology and thrombosis; 3, promote the partial blood vessel endothelium quickly-healing of pathology.Therefore we imagine, employing has the TFPI gene transformation that suppresses thrombosis, inflammatory reaction and vascular smooth muscle cell curing and has to the directed EPCs that assembles and can be divided into vascular endothelial cell in impaired endothelium position, a kind of collection suppress thrombosis, inflammatory reaction and vascular smooth muscle cell curing and promote the damaged blood vessels endothelium to heal and novel therapeutic technology that can the target vascular therapy damaged tissue thereby form, can obviously improve the prevention effect of vascular restenosis.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, the retroviral eukaryotic expression vector of a kind of TFPI of containing gene and GFP Gene Double cistron is provided.
Second purpose of the present invention provides a kind ofly can express TFPI and two kinds of proteic rat endothelial progenitor cells of GFP simultaneously.
The 3rd purpose of the present invention provides the construction process of the retroviral eukaryotic expression vector of a kind of TFPI of containing gene and GFP Gene Double cistron.
The 4th purpose of the present invention provides the construction process that can express TFPI and two kinds of proteic rat endothelial progenitor cells of GFP simultaneously.
The present invention is applied to the vascular restenosis study on prevention and makes it provide effective tool for being used for the clinical disease control from now on for the endothelial progenitor cells of further carrying out the TFPI genetic modification.
Technical scheme of the present invention is summarized as follows:
The retroviral eukaryotic expression vector that contains human tissue factor pathway supressor gene and green fluorescence protein gene bicistronic mRNA, make with following method: with sequence table SEQ ID NO.2, the described sequence of SEQ ID NO.3 is the PCR primer, pIRES-TFPI is a template, amplify full-length cDNA with the human tissue factor approach inhibition factor gene shown in the sequence table SEQ ID NO.1, be inserted among the retroviral vector pMSCV-IRES-GFP, obtain the pMSCV-TFPI-IRES-GFP recombinant plasmid.Adopt the dna sequencing method to identify.
Can express two kinds of proteic rat endothelial progenitor cells of TFPI and GFP simultaneously, be to make up with following method, with sequence table SEQ ID NO.2, the described sequence of SEQ ID NO.3 is the PCR primer, pIRES-TFPI is a template, amplify full-length cDNA with the human tissue factor approach inhibition factor gene shown in the sequence table SEQ ID NO.1, be inserted among the retroviral vector pMSCV-IRES-GFP, obtain the pMSCV-TFPI-IRES-GFP recombinant plasmid; With described pMSCV-TFPI-IRES-GFP recombinant plasmid calcium phosphate method transfection 293T cell, in the 293T cell, carry out the packing of virus, collection contains the cell conditioned medium liquid of virus, the endothelial progenitor cells of infected rats derived from bone marrow, acquisition can be expressed TFPI and two kinds of proteic rat endothelial progenitor cells of GFP simultaneously.The ELISA method detects the proteic expression of TFPI in the EPCs culture supernatant, and RT-PCR detects the expression of TFPI mRNA in the EPCs.
The construction process that contains the retroviral eukaryotic expression vector of human tissue factor pathway supressor gene and green fluorescence protein gene bicistronic mRNA, comprise the steps: that with sequence table SEQ ID NO.2, the described sequence of SEQ ID NO.3 be the PCR primer, pIRES-TFPI is a template, amplify full-length cDNA with the human tissue factor approach inhibition factor gene shown in the sequence table SEQ ID NO.1, be inserted among the retroviral vector pMSCV-IRES-GFP, obtain the pMSCV-TFPI-IRES-GFP recombinant plasmid.
Can express the construction process of TFPI and two kinds of proteic rat endothelial progenitor cells of 6FP simultaneously, comprise the steps: that with sequence table SEQ ID NO.2, the described sequence of SEQ ID NO.3 be the PCR primer, pIRES-TFPI is a template, amplify full-length cDNA with the human tissue factor approach inhibition factor gene shown in the sequence table SEQ ID NO.1, be inserted among the retroviral vector pMSCV-IRES-GFP, obtain the pMSCV-TFPI-IRES-GFP recombinant plasmid; With described pMSCV-TFPI-IRES-GFP recombinant plasmid calcium phosphate method transfection 293T cell, in the 293T cell, carry out the packing of virus, collection contains the cell conditioned medium liquid of virus, the endothelial progenitor cells of infected rats derived from bone marrow, acquisition can be expressed TFPI and two kinds of proteic rat endothelial progenitor cells of GFP simultaneously.
The present invention has successfully made up the retroviral eukaryotic expression vector of a kind of TFPI of containing gene and GFP Gene Double cistron, has realized TFPI and the green fluorescent protein non-fusion expression in EPCs, and function is not disturbed mutually between two kinds of albumen.In normal performance TFPI biological function, the expression of GFP can be used as effective spike means of transgenic cell, the EPCs tissue positioned and the differentiation situation in vivo of carrying the TFPI gene for research provides very big facility, the research that the EPCs that carries the TFPI gene is used for the vascular restenosis control has important pushing effect, and the EPCs that carries the TFPI gene has great application potential in the control of vascular restenosis.
Description of drawings
Fig. 1 is the TFPI fragment of pcr amplification: clip size is 935bp; (the described nucleotide sequence of sequence table SEQ ID NO.1)
Fig. 2 is the pMSCV-TFPI-IRES-GFP PCR qualification result of the recombinant plasmid of the present invention's structure;
Fig. 3 is the growing state of observation by light microscope rat endothelial progenitor cells;
Fig. 4 observes the expression of green fluorescent protein in each test group EPCs for the fluorescence inverted microscope;
Fig. 5 detects TFPI mRNA expression level in the EPCs cell for RT-PCR;
Fig. 6 detects the protein content (n=5) of TFPI in the EPCs cells and supernatant for the ELISA method.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment.
Contain the preparation of the retroviral eukaryotic expression vector of TFPI gene and GFP Gene Double cistron
1. design TFPI upstream and downstream PCR primer respectively, shown in sequence table SEQ ID NO.2, SEQ ID NO.3, give birth to the worker by Shanghai and be responsible for synthetic and purifying.With recombinant plasmid pIRES-TFPI is that template amplification goes out human tissue factor approach inhibition factor full length cDNA sequence shown in the SEQ ID NO.1.PCR reaction system: cumulative volume 50ul:TaKaRa Ex Taq (0.5U/ μ L) 2.5 μ L; 10 * Ex Taq Buffer, 5 μ L; DNTP Mixture (each 2.5mM) 4 μ L; PIRES-TFPI plasmid 20ng; Upstream primer (20 μ M) 1 μ L; Downstream primer (20 μ M) 1 μ L; Aqua sterilisa polishing to 50 μ L; Amplification condition: 1. 94 ℃, 2min, 2. 94 ℃, 30s; 55 ℃, 30s; 72 ℃, 1min repeats 30 circulations.③72℃，4min。The PCR reaction solution is splined on 1.5% sepharose, and electrophoresis was made a video recording after 30 minutes, and (see Fig. 1, M represents dna molecular amount standard among the figure, 100-6000 Wide Range DNA Marker (TaKaRa): 1 expression pcr amplification product TFPI fragment 935bp).Glue reclaims and obtains the described nucleotide fragments of sequence table SEQ ID NO.1 then.
With restriction enzyme EcoRI and XhoI respectively enzyme cut the TFPI cDNA fragment and the retroviral vector pMSCV-IRES-GFP of above-mentioned pcr amplification, the pcr amplified fragment of reaction system 20ul:TFPI or pMSCV-IRES-GFP 1 μ g; 10 * H Buffer, 2 μ L; EcoRI 1 μ L; XhoI 1 μ L; Aqua sterilisa polishing to 20 μ L; Reaction conditions: 4h is hatched in 37 ℃ of water-baths;
3. above-mentioned TFPIcDNA fragment after enzyme is cut is connected reaction system 25 μ L:10 * T4 DNA Ligase Buffer 2.5 μ L with the T4 ligase enzyme with pMSCV-IRES-GFP; The about 0.3pmol of target gene fragment; The about 0.03pmol of carrier DNA fragment; T4 DNA Ligase (350U/ μ L) 1 μ L; Aseptic deionized water polishing to 25 μ L; Reaction conditions: 16 ℃, reaction overnight 16 hours.With reaction solution transformed competence colibacillus bacillus coli DH 5 alpha, be coated on then on the LB ammonia benzyl agar culture plate, cultivated 12 hours for 37 ℃.
4. the single colony inoculation of picking is to 10mL LB ammonia benzyl substratum (penbritin content is 100 μ g/L), 250rpm, 37 ℃ cultivated 12 hours, use the little extraction reagent kit of plasmid (day root biochemical technology company limited) to extract plasmid DNA in a small amount, increase with PCR method.PCR reaction system: cumulative volume 50 μ L:TaKaRa Ex Taq (0.5U/ μ L) 2.5 μ L; 10 * ExTaq Buffer, 5 μ L; DNTP Mixture (each 2.5mM) 4 μ L; Plasmid 20ng; Upstream primer (20 μ M) 1 μ L; Downstream primer (20 μ M) 1 μ L; (being respectively shown in sequence table SEQ ID NO.2, the SEQ ID NO.3) aqua sterilisa polishing to 50 μ L; Amplification condition: 1. 94 ℃, 2min, 2. 94 ℃, 30s; 55 ℃, 30s; 72 ℃, 1min repeats 30 circulations.③72℃，4min。The PCR reaction solution is splined on 1.5% sepharose, electrophoresis shooting in 30 minutes (is seen Fig. 2, M represents dna molecular amount standard among the figure, 100-6000 Wide Range DNA Marker (TaKaRa): 1-6 represents that the plasmid of positive single bacterium colony extraction carries out the TFPI fragment 935bp of pcr amplification respectively), the amplification of pcr amplification male bacterial strain is preserved, called after DH5 α-pMSCV-TFPI-IRES-GFP, its contained plasmid is called pMSCV-TFPI-IRES-GFP.
5. extract the pMSCV-TFPI-IRES-GFP plasmid, send Beijing Invitrogen company to check order, sequencing result shows that the TFPI sequence among the pMSCV-TFPI-IRES-GFP is in full accord with expection.
In the 22nd the 6th phase of volume of " chitosan carries the research of gene nanoparticle " " biomedical engineering magazine " (2005), the 1171-1176 page or leaf is open for the structure of pIRES-TFPI.
Endothelial progenitor cells (endothelial progenitor cells, separation EPCs) and the cultivation in rat marrow source
Aseptic condition separates the Sprague-Dawley rat femur down, removes soft tissue, and marrow is gone out with the PBS damping fluid in excision femur two ends, and mixing fully scatters it gently.Adopt mononuclearcell parting liquid (Histopaque 1083 Sigma) to carry out density gradient centrifugation, obtain mononuclearcell.Adjusting cell concn is 1 * 10
6Individual/ml, be inoculated in the culture dish that diameter is 10cm, add the EGM2 culture medium culturing, change liquid behind the 72h, changed liquid 1 time, and went down to posterity after 7-9 days in later every 2-3 days.(see Fig. 3, A shows the BMNC that the separation of Histopaque 1083 density gradient centrifugations obtains among the figure; B shows the EPCs clone who forms behind the vitro culture 5d; The C display body is cultured to the 7th day EPCs outward, is single spindle cell sample form.)
Contain the expression of retroviral eukaryotic expression vector in rat EPCs cell of TFPI gene and GFP Gene Double cistron
1. virus is packed
Adopt calcium phosphate method transfection 293T cell: before the transfection with the 293T passage in the 10cm culture dish, when the cell growth converges when reaching 60-80%, with packaging plasmid pMD.MLV 15 μ g, pVSV-G 7.5 μ g, pMSCV-IRES-GFP or
PMSCV-TFPI-IRES-GFP 7.5 μ g, CaCl2 67 μ l, HEPES-buffer salt solution (HBS) 1ml, slowly be added drop-wise in the 293T cell culture fluid behind the mixing, change fresh culture 10ml behind the 8h, behind transfection 48h, collect the 293T cell conditioned medium liquid that contains virus, with 0.45 μ m membrane filtration, that viral supernatant liquor adding polybrene solution (final concentration is 8 μ g/mL) the mixing back that obtains is stand-by.
The EPCs growth converges when reaching 80-90%, adds above-mentioned viral liquid 8ml, EGM2 perfect medium 2ml after suction goes substratum, PBS to wash 2 times, cultivates 24h and changes the EGM2 perfect medium, detects efficiency of infection with fluorescent microscope behind the 48h.(see Fig. 4, A represents that observation under the fluorescent microscope does not add the EPCs of the blank group of plasmid, and B represents to observe under the fluorescent microscope EPCs of transfection pMSCV-IRES-GFP, and C represents the EPCs of observation transfection pMSCV-TFPI-IRES-GFP fluorescent microscope under).
The RT-PCR method detects the expression level of TFPImRNA in the EPCs cell
Collect each experimental group endothelial progenitor cells, experiment is divided into 3 groups: the I group is the experimental group of transfection pMSCV-TFPI-IRES-GFP plasmid; The II group is the control group of transfection pMSCV-IRES-GFP empty carrier; The III group is not for adding the blank group of plasmid.(American I nvitrogen company) extracts total RNA with Trizol reagent, detects the expression of TFPI mRNA with RT-PCR.People TFPI upstream and downstream primer is shown in sequence table SEQ ID NO.4, SEQ ID NO.5 (product 230bp); Rat GAPDH upstream and downstream primer is shown in sequence table SEQ IDNO.6, SEQ ID NO.7 (product 308bp), and the RT reaction conditions is: 1. 50 ℃, and 30min, 2. 99 ℃, 5min, 3. 5 ℃, 5min.The pcr amplification condition is: 1. 94 ℃, and 2min, 2. 94 ℃, 30s; 55 ℃, 30s; 72 ℃, 1min repeats 30 circulations.③72℃，4min。RT-PCR product electrophoresis and taking pictures on 1.5% sepharose with amplification.The result shows that the TFPI amplified fragments of 230bp appears in transfection pMSCV-TFPI-IRES-GFP plasmid group, and normal EPCs group and EPCs transfection pMSCV-IRES-GFP empty plasmid group are not seen people TFPI amplified band; Each group all has the GAPDH mRNA of similar level to express.(the results are shown in Figure 5,1-3 shows the amplified fragments of I-III experimental group TFPI cDNA; M represents dna molecular amount standard, 150bp DNAMarker (TaKaRa); 4-6 shows the amplified fragments of I-III experimental group GAPDH cDNA.)
The ELISA method detects TFPI protein level in the cells and supernatant
Learn from else's experience EPCs culture supernatant behind the retroviral infection 48h that has the pMSCV-TFPI-IRES-GFP gene, the EPCs with EPCs and transfection pMSCV-IRES-GFP is blank and negative control respectively.With TFPI concentration in the ELISA kit detection cell supernatant, every group of sample established five multiple holes.OD with each hole
450Value substitution typical curve, calculate and respectively organize TFPI concentration, ELISA detects demonstration, infect in the pMSCV-TFPI-IRES-GFP plasmid group EPCs cell conditioned medium people TFPI protein expression is arranged, concentration is 26.57 ± 2.72ng/mL, and normal EPCs group and transfection pMSCV-IRES-GFP empty plasmid group all do not detect the proteic expression of people TFPI.(the results are shown in Figure 6,1 normal EPCs groups; 2 EPCs transfection pMSCV-IRES-GFP empty plasmid groups; 3 EPCs transfection pMSCV-TFPI-IRES-GFP plasmid groups.)
Sequence table
＜110〉Inst. of Biomedicine Engineering Chinese Academy of Medicine
＜120〉contain retroviral eukaryotic expression vector and the construction process and the application of TFPI gene and GFP Gene Double cistron
<160>6
<210>1
<211>935
<212>DNA
＜213〉artificial sequence
<220>
<222>(1)..(935)
<400>1
gacggaattc?atgatttaca?caatgaagaa?agtacatgca?ctttgggctt?ctgtatgcct?60
gctgcttaat?cttgcccctg?cccctcttaa?tgctgattct?gaggaagatg?aagaacacac?120
aattatcaca?gatacggagt?tgccaccact?gaaacttatg?cattcatttt?gtgcattcaa?180
ggcggatgat?ggcccatgta?aagcaatcat?gaaaagattt?ttcttcaata?ttttcactcg?240
acagtgcgaa?gaatttatat?atgggggatg?tgaaggaaat?cagaatcgat?ttgaaagtct?300
ggaagagtgc?aaaaaaatgt?gtacaagaga?taatgcaaac?aggattataa?agacaacatt?360
gcaacaagaa?aagccagatt?tctgcttttt?ggaagaagat?cctggaatat?gtcgaggtta?420
tattaccagg?tatttttata?acaatcagac?aaaacagtgt?gaacgtttca?agtatggtgg?480
atgcctgggc?aatatgaaca?attttgagac?actggaagaa?tgcaagaaca?tttgtgaaga?540
tggtccgaat?ggtttccagg?tggataatta?tggaacccag?ctcaatgctg?tgaataactc?600
cctgactccg?caatcaacca?aggttcccag?cctttttgaa?tttcacggtc?cctcatggtg?660
tctcactcca?gcagacagag?gattgtgtcg?tgccaatgag?aacagattct?actacaattc?720
agtcattggg?aaatgccgcc?catttaagta?cagtggatgt?gggggaaatg?aaaacaattt?780
tacttccaaa?caagaatgtc?tgagggcatg?taaaaaaggt?ttcatccaaa?gaatatcaaa?840
aggaggccta?attaaaacca?aaagaaaaag?aaagaagcag?agagtgaaaa?tagcatatga?900
agaaattttt?gttaaaaata?tgtgactcga?ggcgc 935
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<222>(1)..(20)
<400>2
gacggaattc?atgatttaca?20
<210>3
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<222>(1)..(25)
<400>3
gcgcctcgag?tcacatattt?ttaac 25
<210>4
<211>27
<212>DNA
＜213〉artificial sequence
<220>
<222>(1)..(27)
<400>4
ggaagaagat?cctggaatat?gtcgagg 27
<210>5
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<222>(1)..(25)
<400>5
cttggttgat?tgcggagtca?gggag 25
<210>6
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<222>(1)..(20)
<400>6
tccctcaaga?ttgtcagcaa?20
<210>7
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<222>(1)..(20)
<400>7
agatccaca?acggatacatt?20
Claims (4)
1. the retroviral eukaryotic expression vector that contains human tissue factor pathway supressor gene and green fluorescence protein gene bicistronic mRNA, it is characterized in that making with following method: with sequence table SEQ ID NO.2, the described sequence of SEQ ID NO.3 is the PCR primer, pIRES-TFPI is a template, amplify full-length cDNA with the human tissue factor approach inhibition factor gene shown in the sequence table SEQ ID NO.1, be inserted among the retroviral vector pMSCV-IRES-GFP, obtain the pMSCV-TFPI-IRES-GFP recombinant plasmid.
2. can express two kinds of proteic rat endothelial progenitor cells of TFPI and GFP simultaneously, it is characterized in that making up with following method, with sequence table SEQ ID NO.2, the described sequence of SEQ ID NO.3 is the PCR primer, pIRES-TFPI is a template, amplify full-length cDNA with the human tissue factor approach inhibition factor gene shown in the sequence table SEQ ID NO.1, be inserted among the retroviral vector pMSCV-IRES-GFP, obtain the pMSCV-TFPI-IRES-GFP recombinant plasmid; With described pMSCV-TFPI-IRES-GFP recombinant plasmid calcium phosphate method transfection 293T cell, in the 293T cell, carry out the packing of virus, collection contains the cell conditioned medium liquid of virus, the endothelial progenitor cells of infected rats derived from bone marrow, acquisition can be expressed TFPI and two kinds of proteic rat endothelial progenitor cells of GFP simultaneously.
3. the construction process of the retroviral eukaryotic expression vector that contains human tissue factor pathway supressor gene and green fluorescence protein gene bicistronic mRNA of claim 1, it is characterized in that comprising the steps: that with sequence table SEQ ID NO.2, the described sequence of SEQ ID NO.3 be the PCR primer, pIRES-TFPI is a template, amplify full-length cDNA with the human tissue factor approach inhibition factor gene shown in the sequence table SEQ ID NO.1, be inserted among the retroviral vector pMSCV-IRES-GFP, obtain the pMSCV-TFPI-IRES-GFP recombinant plasmid.
4. the construction process that can express TFPI and two kinds of proteic rat endothelial progenitor cells of GFP simultaneously of claim 2, it is characterized in that comprising the steps: that with sequence table SEQ ID NO.2, the described sequence of SEQ ID NO.3 be the PCR primer, pIRES-TFPI is a template, amplify full-length cDNA with the human tissue factor approach inhibition factor gene shown in the sequence table SEQ ID NO.1, be inserted among the retroviral vector pMSCV-IRES-GFP, obtain the pMSCV-TFPI-IRES-GFP recombinant plasmid; With described pMSCV-TFPI-IRES-GFP recombinant plasmid calcium phosphate method transfection 293T cell, in the 293T cell, carry out the packing of virus, collection contains the cell conditioned medium liquid of virus, the endothelial progenitor cells of infected rats derived from bone marrow, acquisition can be expressed TFPI and two kinds of proteic rat endothelial progenitor cells of GFP simultaneously.
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| CN111944849A (en) * | 2020-07-08 | 2020-11-17 | 天津科技大学 | Method for constructing bicistronic expression plasmid by using HPV18 spacer sequence |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1920029A (en) * | 2006-09-14 | 2007-02-28 | 中国医学科学院生物医学工程研究所 | Human tissue factor pathway inhibitory factor mutation gene mTFPI, recombination carrier including the same and host cell thereof |
| CN1924016A (en) * | 2006-09-14 | 2007-03-07 | 中国医学科学院生物医学工程研究所 | Human tissue factor pathway inhibitory factor mutation gene m2TFPI, recombination carrier and recombination microzyme including the same |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1920029A (en) * | 2006-09-14 | 2007-02-28 | 中国医学科学院生物医学工程研究所 | Human tissue factor pathway inhibitory factor mutation gene mTFPI, recombination carrier including the same and host cell thereof |
| CN1924016A (en) * | 2006-09-14 | 2007-03-07 | 中国医学科学院生物医学工程研究所 | Human tissue factor pathway inhibitory factor mutation gene m2TFPI, recombination carrier and recombination microzyme including the same |
Non-Patent Citations (1)
| Title |
|---|
| 《国际生物医学工程杂志》 20100428 王海燕等 双顺反子表达人组织因子途径抑制因子和绿色荧光蛋白重组病毒的构建及其在内皮祖细胞中表达 第65-69页 1-4 第33卷, 第2期 2 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111944849A (en) * | 2020-07-08 | 2020-11-17 | 天津科技大学 | Method for constructing bicistronic expression plasmid by using HPV18 spacer sequence |
| CN111944849B (en) * | 2020-07-08 | 2023-09-15 | 天津科技大学 | Method for constructing bicistronic expression plasmid by using HPV18 spacer sequence |
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