CN106434753A - TAT-RGD-KDR siRNA fusion gene vector, and construction method and application thereof - Google Patents

TAT-RGD-KDR siRNA fusion gene vector, and construction method and application thereof Download PDF

Info

Publication number
CN106434753A
CN106434753A CN201610590302.8A CN201610590302A CN106434753A CN 106434753 A CN106434753 A CN 106434753A CN 201610590302 A CN201610590302 A CN 201610590302A CN 106434753 A CN106434753 A CN 106434753A
Authority
CN
China
Prior art keywords
rgd
tat
kdr
kdr sirna
sirna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201610590302.8A
Other languages
Chinese (zh)
Inventor
王杰
郑旭
吴涛
张俭丽
王晓艳
孙莹莹
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenyang Medical College
Original Assignee
Shenyang Medical College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenyang Medical College filed Critical Shenyang Medical College
Priority to CN201610590302.8A priority Critical patent/CN106434753A/en
Publication of CN106434753A publication Critical patent/CN106434753A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
    • C12N2810/405—Vectors comprising RGD peptide

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention discloses a TAT-RGD-KDR siRNA fusion gene vector. The TAT-RGD-KDR siRNA fusion gene vector is obtained through inserting two oligonucleotide chains for encoding three serially connected RGD-4C to a PGCL-TAT-KDR siRNA GFP vector, and the nucleotide sequences of the two oligonucleotide chains for encoding three serially connected RGD-4C are represented by SEQ ID NO.2 and SEQ ID NO3. Experiments prove that the TAT-RGD-KDR siRNA fusion gene vector has substantial tumor targeting and antitumor activity (can effectively inhibit nude mouse transplantation tumor model tumor tissue growth and has no toxic or side effects), and can effectively solve the problems of in-vivo non-specific distribution and poor tumor targeting of siRNA as an antitumor drug targeting vector.

Description

A kind of TAT-RGD-KDR siRNA fusion gene carrier and its construction method and application
Technical field
The present invention relates to a kind of TAT-RGD-KDR siRNA fusion gene carrier, and its construction method, and it is in conduct Application in the targeting vector of antineoplastic, belongs to technical field of bioengineering.
Background technology
The current treatment to late tumor patient is based on chemotherapy, but the toxic and side effect of medicine limits the further of curative effect Improve.Molecular targeted therapy under new techniques support is the development trend of future tumors drug therapy.
Content of the invention
For above-mentioned prior art, the invention provides a kind of TAT-RGD-KDR siRNA fusion gene carrier, and its structure Construction method, and its application in the targeting vector as antineoplastic.The present invention is on the basis of early-stage Study, will There is the tat peptide sequence promoting nucleic acid transduction function and RGD sequence and the early-stage Study confirmation with cancer target evident characteristics The siRNA sequence of energy effective reticence KDR gene expression（This siRNA sequence is as shown in SEQ ID NO.1；This previous research Paper publishing in JOURNAL OF MICROBIOLOGY, 2013,33（6）：37-42；2014 International Workshop on Biology, Chemistry and Medical Engineering, WuHan, China（EI, ISTP include）；Clinical doctor Engineering, 2012,19（10）：1664-1666；Chinese factories and miness medical science, 2009,6：644-646；Chinese organizational project and clinical health Complicated will, 2007,11（27）：5303-5306）In conjunction with structure is with more the siRNA of vivo biodistribution availability and tumor-targeting Transduction platform, can efficiently solve the problem that siRNA non-specificity in vivo is distributed, tumor-targeting is poor, have more For significant tumor-targeting and antitumor activity, it is that the biotherapeutics with KDR siRNA as core further applies The exploration of oncotherapy provides solid experiment basis.
The present invention is achieved by the following technical solutions：
A kind of TAT-RGD-KDR siRNA fusion gene carrier, is in PGCL-TAT-KDR siRNA GFP carrier（PGCL- The structure of TAT-KDR siRNA GFP carrier and construction method, be disclosed in document " JOURNAL OF MICROBIOLOGY, 2013,33（6）：37- 42；2014 International Workshop on Biology, Chemistry and Medical Engineering, WuHan, China（EI, ISTP include " in）2 oligonucleotide chains of upper 3 series connection RGD-4C of insertion coding, institute The nucleotides sequence stating 2 oligonucleotide chains of 3 series connection RGD-4C of coding is classified as：
Sense strand sequence（5'-3'）：
CATGGTACCTGCGATTGTCGCGGAGATTGCTTCTGCGGTGGAGGCGGGTCTTGCGATTGTCGCGGAGATTGCT TCTGCGGTGGAGGCGGGTCTTGCGATTGTCGCGGAGATTGCTTCTGCGGTGGAGGCGGGTCTAAGCTTCG；As SEQ Shown in ID NO.2；
Wherein, "GGTACC" for Kpn I restriction enzyme site, "AAGCTT" for Hind III restriction enzyme site；
Antisense strand sequence（5'-3'）：
GTAAAGCTTACGCTAACAGCGCCTCTAACGAAGACGCCACCTCCGCCCGAACGCTAACAGCGCCTCTAACGAA GACGCCACCTCCGCCCGAACGCTAACAGCGCCTCTAACGAAGACGCCACCTCCGCCCGAGGTACCGC；As SEQ ID Shown in NO.3；
Wherein, "AAGCTT" for Hind III restriction enzyme site, " "GGTACC" for Kpn I restriction enzyme site.
The construction method of described TAT-RGD-KDR siRNA fusion gene carrier, as follows：
（1）RGD-4C sequent synthesis：By equimolar two oligonucleotide chains（Introduce restriction enzyme site Kpn I and Hind thereon Ⅲ）Mixing, the rearmounted 72 DEG C of annealing of 95 DEG C of denaturation 10min overnight, obtain the double-stranded DNA of 3 series connection RGD-4C of coding；
（2）PGC/TAT-RGD-KDR siRNA carrier builds：With Kpn I and Hind III respectively to pGC/TAT KDR siRNA matter The double-stranded DNA of grain DNA and 3 series connection RGD-4C of coding carries out double digestion reaction, purifies digestion products；Connect enzyme effect in T4 Lower 16 DEG C connect overnight（10～14 hours）；Connection product proceeds to DH5 α competent cell, and routine carries out antibiotic-screening；Screening Positive bacterium colony, clone PCR, obtain final product.
The TAT-RGD-KDR siRNA fusion gene carrier of the present invention, the experiment proved that, it has significantly more tumour Targeting and antitumor activity, as the targeting vector of antineoplastic, can efficiently solve siRNA in vivo non- The poor problem of distribution of specific, tumor-targeting.The present invention has advantages below：
（1）Take the lead at home the RNA interference sequence of KDR gene is set up the skill of RNA interference with the complex form of TAT and RGD Art platform, fills the domestic gaps；TAT-RGD-KDR siRNA slow virus carrier can more effectively suppress Nude Mouse Model to swell Tumor tissue grows, and has no toxic side effect.
（2）This technology platform is used for the target Therapy study of tumour cell and its nude mice model, can effectively suppress nude mice shifting Plant knurl Model Tumor tissue growth, there is siRNA distribution of specific in vivo, tumor-targeting, and nontoxic secondary make With.
Brief description
Fig. 1：TAT-RGD-KDR siRNA fusion gene carrier builds schematic diagram.
Fig. 2：A549 cell KDR mRNA expression.
Fig. 3：A549 cell KDR protein expression level.
Fig. 4：TAT-RGD-KDR siRNA slow virus carrier suppresses A549 cell proliferation.
Fig. 5：TAT-RGD- KDR siRNA slow virus carrier induces A549 Apoptosis, wherein, Fig. 5 A：Duochrome method streaming Cell instrument testing result；Fig. 5 B：The statistics of duochrome method flow cytomery.
Specific embodiment
With reference to embodiment, the present invention is further illustrated.
Involved instrument, reagent, material etc. in following embodiments, unless otherwise noted, are in prior art existing Conventional instrument, reagent, material etc., can be either commercially available by regular.Involved experimental technique in following embodiments, inspection Survey method etc., unless otherwise noted, is existing normal experiment method, detection method etc. in prior art.
The structure of embodiment 1 TAT-RGD-KDR siRNA fusion gene carrier
（1）RGD-4C sequences Design and synthesis：
Design and 3 series connection RGD-4C of composite coding（CDCRGDCFCGGGGS）Two oligonucleotide chains.In positive-sense strand with instead The two ends of adopted chain introduce restriction enzyme site Kpn I and Hind III respectively, as follows：
Sense strand sequence（5'-3'）：
CATGGTACCTGCGATTGTCGCGGAGATTGCTTCTGCGGTGGAGGCGGGTCTTGCGATTGTCGCGGAGATTGCT TCTGCGGTGGAGGCGGGTCTTGCGATTGTCGCGGAGATTGCTTCTGCGGTGGAGGCGGGTCTAAGCTTCG；
Antisense strand sequence（5'-3'）：
GTAAAGCTTACGCTAACAGCGCCTCTAACGAAGACGCCACCTCCGCCCGAACGCTAACAGCGCCTCTAACGAA GACGCCACCTCCGCCCGAACGCTAACAGCGCCTCTAACGAAGACGCCACCTCCGCCCGAGGTACCGC.
By the mixing of two oligonucleotide chains of equimolar, the rearmounted 72 DEG C of annealing of 95 DEG C of denaturation 10min overnight, obtain coding 3 The double-stranded DNA of series connection RGD-4C.
（2）PGC/TAT-RGD-KDR siRNA carrier builds
With Kpn I and Hind III double-stranded DNA to pGC/TAT KDR siRNA DNA and 3 series connection RGD-4C of coding respectively Carry out double digestion reaction, purify digestion products.Under T4 connection enzyme effect, 16 DEG C connect overnight（12 hours）.Connection product proceeds to DH5 α competent cell, routine carries out antibiotic-screening.The censorship sequencing after clone PCR identification of positive bacterium colony.Sequencing result warp Blast compares, the RGD sequence in recombinant vector consistent with design it was demonstrated that pGC/TAT-RGD-KDR siRNA construction of recombinant vector Success, its structure is as shown in Figure 1.
The impact to A549 cell KDR gene expression for the embodiment 2 TAT-RGD- KDR siRNA slow virus carrier
By pGC/TAT-KDR siRNA recombinant virus, pGC/TAT-RGD-KDR siRNA recombinant virus, and the infection of equivalent PBS After three groups of A549 cell culture afterwards reach 80% to adherent rate, extract cell total rna and total protein respectively, apply Real-time PCR and Western blot detects the expression of KDR in A549 cell.Real-time PCR result shows, pGC/TAT- KDR siRNA infected group is equal compared with control group with the KDR mRNA expression of pGC/TAT-RGD-KDR siRNA infected group It is remarkably decreased, statistically significant（p<0.01）.And pGC/TAT-RGD-KDR siRNA infected group and pGC/TAT-KDR SiRNA infected group is compared, and also has significant difference（p<0.01）, see Fig. 2.Western blot result shows, and is uninfected by group Compare, pGC/TAT-KDR siRNA group and pGC/TAT-RGD-KDR siRNA group KDR protein expression level are also remarkably decreased.And The latter's decline is more notable, also has significant difference compared with the former（p<0.01）, see Fig. 3.Prompting pGC/TAT-RGD-KDR SiRNA group plays the role of higher suppression A549 cell KDR gene expression compared with pGC/TAT-KDR siRNA group.
The impact to A549 cell proliferation for the embodiment 3 TAT-RGD-KDR siRNA slow virus carrier
Effect in suppression A549 cell growth for the MTT colorimetric determination pGC/TAT-RGD KDR siRNA slow virus carrier.Figure 4 is visible, and pGC/TAT-KDR siRNA group and pGC/TAT-RGD KDR siRNA group are inhibited to A549 cell proliferation, Longer with incubation time, inhibitory action is more obvious, has the dependence to the time.Additionally, pGC/TAT-RGD KDR siRNA Inhibitory action after cultivating 3 days for the group is more notable compared with pGC/TAT-KDR siRNA group, has statistical significance（p<0.01）. Prompting TAT-RGD- KDR siRNA slow virus carrier has the inhibitory action of stronger suppression A549 cell proliferation.
Embodiment 4 TAT-RGD-KDR siRNA slow virus carrier apoptotic impact on A549
It is apoptotic whether duochrome method flow cytomery TAT-RGD-KDR siRNA slow virus carrier has induction A549 Effect.Fig. 5 is visible, and pGC/TAT-RGD KDR siRNA group apoptosis rate is（52.79±4.39）%, and pGC/TAT-KDR The apoptosis rate of siRNA group is only（24.36±1.37）%, both have significant difference（p<0.01）.Prompting TAT-RGD- KDR siRNA slow virus carrier has the stronger apoptotic effect of induction A549.
The impact to A549 cell invasion power for the embodiment 5 TAT-RGD- KDR siRNA slow virus carrier
The 1 of 50 μ L is added in room on Transwel:30 Matrigel, then every hole addition sterilized water 200 μ L, aseptic 2d is placed, the water in Transwell is killed inside workbench.Preheat Transwell to 37 DEG C in 24 orifice plates, Each Transwell adds 200 μ L invasion buffer, 37 DEG C of culture 1h.Cultured cells 80% merges, free serum culture Base washed cell, trypsin-EDTA vitellophag, collected after centrifugation cell.Move the invasion abandoning Transwell epicoele Buffer, adds 800 μ L fibronectin solution to cavity of resorption by gap.The cell adding 200 μ L in epicoele hangs Liquid, it is to avoid produce bubble, make cell equably be covered with filter, places 36 ~ 48 h in 37 DEG C of incubators.The fixing Soviet Union of formaldehyde Lignin, Yihong counterstain.Counted under microscope cell, counts 8 high power fields（×200）Under through filter membrane cell number. Experiment repeats 3 times.
Result shows（Table 1）, compared with control group, pGC/TAT-KDR siRNA group and pGC/TAT-RGD KDR siRNA Group is significantly reduced by the A549 cell number that matrigel reaches filter membrane bottom, and the invasion and attack rate of cell is remarkably decreased, and is respectively 42.23% and 17.76%.The invasion and attack rate of particularly pGC/TAT-RGD KDR siRNA group declines more notable, with pGC/TAT-KDR There were significant differences for siRNA group（p<0.01）.Prompting pGC/TAT-RGD KDR siRNA group has higher suppression A549 cell body The ability of outer invasion and attack.
The invasion and attack rate of table 1 each group cell
Control group PGC/TAT-KDR siRNA group PGC/TAT-RGD- KDR siRNA group
Cell number （202.67±8.50） （85.59±6.03） （36.0±5.29）
The invasion and attack rate of cell（%） 100% 42.23%* 17.76%*/**
Note：*PGC/TAT-KDR siRNA group, pGC/TAT-RGD KDR siRNA group compared with control group,p<0.01；**pGC/ TAT-RGD KDR siRNA group compared with pGC/TAT-KDR siRNA group,p<0.01.
The experiment of embodiment 6 TAT-RGD-KDR siRNA slow virus carrier suppression Nude Mouse Model tumor tissue growth With toxic and side effect research
1. Nude Mouse Model preparation
TypeⅡ pneumocyte is in 37 DEG C, 5% CO2Under the conditions of cultivate, with the high sugar RPMI1640 culture containing 10% hyclone Base Secondary Culture, rise period cell of taking the logarithm, make single cell suspension.Adjustment oncocyte concentration is 2 × 106/ ml, 80 mouse Every dorsal part waist flank hypodermic injection oncocyte 0.1ml.The maximum major diameter of vernier caliper measurement tumour and vertical minor axis（mm）, Represented with a and b respectively, by formula V（mm3）=（π/6）× a× b2Calculate gross tumor volume, reaching 50～70mm with knurl volume is Become knurl standard.
2. experiment packet, treatment and blood, urine biochemical analysis
Randomly draw 30 mice with tumor for this experiment.30 mice with tumor are randomly divided into 3 groups, every group 10：①TAT-RGD- KDR siRNA experimental group；2. RGD-KDR siRNA group；3. control group.Each group mouse per injection 50 g, the next day 1 time, totally 7 Secondary.
3. the research of pair transplanted tumor inhibitory action
Weigh after 14 days and put to death animal, weigh.Dissect and peel off tumor mass, claim knurl weight, calculate tumour inhibiting rate by average knurl weight.Tumor suppression Rate（%）=[（The average knurl weight of the average knurl weight-administration group of model group）The average knurl weight of/model group] × 100%.
Result：
The impact to Nude Mouse Model body weight for the 1.TAT-RGD-KDR siRNA slow virus carrier
Control group mice with tumor growth conditions are good, and body weight increase is normal, and TAT-RGD-KDR siRNA group and RGD-KDR SiRNA group body weight increases not as control group substantially, and TAT-RGD-KDR siRNA group is compared with RGD-KDR siRNA group, body weight Increase less obvious, point out the TAT-RGD-KDR siRNA carrier of this research and establishment can more effectively suppress the life of mice with tumor Long（It is shown in Table 2）.
The impact to Nude Mouse Model body weight for the table 2 TAT-RGD-KDR siRNA slow virus carrier
Group Sample number Mouse Weight before administration（g） Mouse Weight after administration（g）
TAT-RGD-KDR siRNA group 10/10 18.7±0.17 23.1±5.4*.**
RGD-KDR siRNA group 10/9 18.7±0.30 25.4±2.1*
Normal group 10/10 18.9±0.20 29.3±2.7
Note:*Compared with Normal group,P<0.01；**Compared with RGD-KDR siRNA group,P<0.05
The impact that 2.TAT-RGD-KDR siRNA slow virus carrier grows to subcutaneous transplantation knurl
TAT-RGD-KDR siRNA group and RGD-KDR siRNA group knurl body increases slowly, and tumor weight is significantly less than control group, And the knurl body growth rate of TAT-RGD-KDR siRNA group is significantly lower than RGD-KDR siRNA group slowly（It is shown in Table 3）, point out TAT-RGD-KDR siRNA group has the antitumor action becoming apparent from.
The impact that table 3 TAT-RGD-KDR siRNA slow virus carrier grows to subcutaneous transplantation knurl
Group Sample number Knurl weight（g） Inhibiting rate（%）
TAT-RGD-KDR siRNA group 10/10 0.56±0.31 58.5%*.**
RGD-KDR siRNA group 10/10 0.93±0.29 33.1 %*
Normal group 10/10 1.35±0.27
Note:*Compared with Normal group,P<0.01；**Compared with RGD-KDR siRNA group,P<0.01
Embodiment 7 TAT-RGD-KDR siRNA slow virus carrier is tested to the toxic and side effect of Nude Mouse Model
Randomly draw 30 mice with tumor for this experiment.30 mice with tumor are randomly divided into 3 groups, every group 10：①TAT-RGD- KDR siRNA experimental group；2. RGD-KDR siRNA group；3. control group.Each group mouse per injection 50 g, the next day 1 time, totally 7 Secondary.Periodically leave and take urine and blood sample, carry out biochemical analysis, observe the toxic and side effect to internal organs.
The Nude Mouse Model that TAT-RGD-KDR siRNA group is processed UCr, urea, sky compared with Normal group AST, ALT no significant difference；Hepatic and renal function no significant difference； Erythrocyte, leucocyte, hemoglobin, blood platelet no significant difference, point out TAT-RGD-KDR siRNA that nude mice is moved Plant knurl model important organ to have no significant effect.
SEQUENCE LISTING
<110>Shenyang Medical College
<120>A kind of TAT-RGD-KDR siRNA fusion gene carrier and its construction method and application
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 55
<212> DNA
<213>Artificial sequence
<400> 1
tcctggagaa tcagacgaca tcaagagtgt cgtctgattc tccaggtttt ttttc 55
<210> 2
<211> 143
<212> DNA
<213>Artificial sequence
<400> 2
catggtacct gcgattgtcg cggagattgc ttctgcggtg gaggcgggtc ttgcgattgt 60
cgcggagatt gcttctgcgg tggaggcggg tcttgcgatt gtcgcggaga ttgcttctgc 120
ggtggaggcg ggtctaagct tcg 143
<210> 3
<211> 140
<212> DNA
<213>Artificial sequence
<400> 3
gtaaagctta cgctaacagc gcctctaacg aagacgccac ctccgcccga acgctaacag 60
cgcctctaac gaagacgcca cctccgcccg aacgctaaca gcgcctctaa cgaagacgcc 120
acctccgccc gaggtaccgc 140

Claims (3)

1. a kind of TAT-RGD-KDR siRNA fusion gene carrier it is characterised in that：It is in PGCL-TAT-KDR siRNA GFP 2 oligonucleotide chains of 3 series connection RGD-4C of insertion coding on carrier, 2 of described 3 series connection RGD-4C of coding The nucleotides sequence of oligonucleotide chain is classified as：
Sense strand sequence（5'-3'）：
CATGGTACCTGCGATTGTCGCGGAGATTGCTTCTGCGGTGGAGGCGGGTCTTGCGATTGTCGCGGAGATTGCT TCTGCGGTGGAGGCGGGTCTTGCGATTGTCGCGGAGATTGCTTCTGCGGTGGAGGCGGGTCTAAGCTTCG；As SEQ Shown in ID NO.2；
Antisense strand sequence（5'-3'）：
GTAAAGCTTACGCTAACAGCGCCTCTAACGAAGACGCCACCTCCGCCCGAACGCTAACAGCGCCTCTAACGAA GACGCCACCTCCGCCCGAACGCTAACAGCGCCTCTAACGAAGACGCCACCTCCGCCCGAGGTACCGC；As SEQ ID Shown in NO.3.
2. the TAT-RGD-KDR siRNA fusion gene carrier described in claim construction method it is characterised in that：Including with Lower step：
（1）RGD-4C sequent synthesis：By equimolar two oligonucleotide chains mixing, the rearmounted 72 DEG C of annealing of 95 DEG C of denaturation 10min Overnight, obtain the double-stranded DNA of 3 series connection RGD-4C of coding；
（2）PGC/TAT-RGD-KDR siRNA carrier builds：With Kpn I and Hind III respectively to pGC/TAT KDR siRNA matter The double-stranded DNA of grain DNA and 3 series connection RGD-4C of coding carries out double digestion reaction, purifies digestion products；Connect enzyme effect in T4 Lower 16 DEG C connect overnight；Connection product proceeds to DH5 α competent cell, and routine carries out antibiotic-screening；The positive bacterium colony of screening, gram Grand PCR, obtains final product.
3. the answering as the targeting vector of antineoplastic of the TAT-RGD-KDR siRNA fusion gene carrier described in claim With.
CN201610590302.8A 2016-07-26 2016-07-26 TAT-RGD-KDR siRNA fusion gene vector, and construction method and application thereof Pending CN106434753A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610590302.8A CN106434753A (en) 2016-07-26 2016-07-26 TAT-RGD-KDR siRNA fusion gene vector, and construction method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610590302.8A CN106434753A (en) 2016-07-26 2016-07-26 TAT-RGD-KDR siRNA fusion gene vector, and construction method and application thereof

Publications (1)

Publication Number Publication Date
CN106434753A true CN106434753A (en) 2017-02-22

Family

ID=58185161

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610590302.8A Pending CN106434753A (en) 2016-07-26 2016-07-26 TAT-RGD-KDR siRNA fusion gene vector, and construction method and application thereof

Country Status (1)

Country Link
CN (1) CN106434753A (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101684478A (en) * 2009-06-26 2010-03-31 武汉大学 Method for constructing tandem expression small interfering RNA recombinant lentiviral vector

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101684478A (en) * 2009-06-26 2010-03-31 武汉大学 Method for constructing tandem expression small interfering RNA recombinant lentiviral vector

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
RYSZARD SMOLARCZYK ET AL.: "Antitumor effect of RGD-4C-GG-D(KLAKLAK)2 peptide in mouse B16(F10) melanoma model", 《ACA BIOCHIMICA POLONICA》 *
张俭丽 等: "RGD-TAT-KDRsiRNA慢病毒载体构建及抗肿瘤活性", 《中国公共卫生》 *

Similar Documents

Publication Publication Date Title
CN107893076A (en) CRISPR Cas9 targeting knock outs human breast cancer cell RASSF2 genes and its specific sgRNA
KR101724218B1 (en) Extraction preparation and application of plant microribonucleic acid
CN108148835A (en) The sgRNA of CRISPR-Cas9 targeting knock out SLC30A1 genes and its specificity
CN108064153A (en) For adjusting the RNA agent of GST-PI genes
CN103789309B (en) A kind of long-chain non-coding RNA lncRNA-BcrAR and the application in anti-cell canceration thereof
US20220073921A1 (en) Aptamer and use of the aptamer in the diagnosis and treatment of cancer
CN108220293A (en) A kind of small RNA and medical composition and its use
CN104946646A (en) Small nucleic acid molecules, DNA molecules and proteins for preventing and/or treating Ebola viral hemorrhagic fever and applications
CN104225617B (en) The purposes and its related drugs of people&#39;s AURKA gene therapy tumours
CN105132424A (en) MicroRNA inhibitor, microRNA inhibitor expression vector, building method of microRNA inhibitor expression vector and application of microRNA inhibitor expression vector
CN105734075A (en) Carrier for interfering with expression of gene ABCB5 and application of carrier in tumor stem cell treatment
CN102125698A (en) Method for establishing mouse transplanting tumor model with normal immunologic function
CN106434753A (en) TAT-RGD-KDR siRNA fusion gene vector, and construction method and application thereof
CN103215267B (en) Suppress siRNA and its application of influenza virus related gene
CN103305596B (en) The purposes and its related drugs of people&#39;s RNF138 genes
CN104630221B (en) Suppress shRNA and its recombinant vector and the application of growth of tumour cell
CN104225619B (en) The purposes and its related drugs of people&#39;s ILK gene therapy tumours
CN104922695B (en) A kind of preparation and its application of long-chain non-coding RNA nanoparticle
CN106191118A (en) A kind of slow virus interference carrier and construction method thereof and application
CN102533982B (en) The novelty teabag of people KLF8 gene in oncotherapy
CN103215270B (en) DNA (deoxyribonucleic acid) segment with function of inhibiting cell migration and invasion of glioma, and application of DNA segment
CN107217054A (en) The application of G6PD genes and its expression product in treatment colorectal cancer
CN104745635B (en) A kind of method of OASL genes in silence DF-1 cell lines
CN101787368B (en) siRNA for restraining Z38 gene expression of human being and application thereof in preparing breast-tumor resisting medicine
CN105177000A (en) SiRNA inhibiting Survivin gene expression and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination