CN109136270A - A kind of non-viral transfection reagent and its preparation method and application - Google Patents
A kind of non-viral transfection reagent and its preparation method and application Download PDFInfo
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- CN109136270A CN109136270A CN201810887390.7A CN201810887390A CN109136270A CN 109136270 A CN109136270 A CN 109136270A CN 201810887390 A CN201810887390 A CN 201810887390A CN 109136270 A CN109136270 A CN 109136270A
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- transfection reagent
- viral
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- cationic
- transfection
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- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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Abstract
本发明属于基因工程领域,尤其涉及一种转染试剂及其制备方法和应用，所述转染试剂组成为：终浓度为10～50ng/ml的阳离子聚合物、终浓度为0.1～20μg/ml的重组多肽、终浓度为5～80ng/ml的阳离子脂质体。本发明能够显著提高具有单一溶酶体逃逸效应的非病毒载体的转染效率，进而提高核酸的递送效率；此外，本发明采用分子自组装方法，可以方便的大规模制备转染复合物，而且该复合物在生物体内可降解，具有较高的使用安全性。
The invention belongs to the field of genetic engineering, and in particular relates to a transfection reagent, a preparation method and application thereof. cationic liposomes with a final concentration of 5-80 ng/ml. The present invention can significantly improve the transfection efficiency of the non-viral vector with a single lysosome escape effect, thereby improving the delivery efficiency of nucleic acid; in addition, the present invention adopts the molecular self-assembly method, which can conveniently prepare the transfection complex on a large scale, and The complex is degradable in vivo and has high safety in use.
Description
Technical field
The invention belongs to genetic engineering fields, more particularly to a kind of non-viral transfection reagent and its preparation method and application.
Background technique
Cell transfection technique refers to exogenous DNA or RNA incorporation host cell with target gene and obtains cell newly
The method of genetic marker.It is analysis genes within cells, gene product, the important tools of analysis of protein expression function.Cell turns
The main purpose of dye is to study gene, gene product and recombinant protein by enhancing or inhibiting the expression of specific gene in cell
Function.Cell transfection technique has huge application value in genetic engineering field, such as by gene therapy by purpose base
Because transfecting into cell the symptom treated disease or improve patient, pass through people's tissue plasminogen enzyme of Chinese hamster ovary cell acquisition
Former activator etc..
The genophore researched and developed so far mainly has viral vectors and non-virus carrier two major classes, most common non-
Viral transfection medium is liposome and cationic polymer, they the characteristics of it is similar with virus, readily penetrate through cell membrane.Virus
Carrier, such as retrovirus, adenovirus, herpesviral, adeno-associated virus, although transfection efficiency is high, there is in human body
Inside there is the risk of self-replacation；And non-virus carrier due to be easy large scale preparation, it is at low cost, to foreign gene without size limit
System has many advantages, such as low immunogenicity and comparatively safe, due to the limitation of viral agents, cationic polymer transfection reagent day
Benefit is taken seriously.
Cationic polymer can be used as a kind of non-virus carrier, the characteristic with class eucaryotic cell structure and biomembrane, in vivo
Bioactivity that is degradable while can protect its passenger gene segment, and it is easy to operate, reproducible, it is that one kind has clinic
The Gene transfer vector of application potential.It is positively charged polymerization that cationic polymer (cationic polymers), which transfects principle,
Object and the electronegative phosphate group of nucleic acid formed after positively charged compound with the electronegative proteoglycan phase interaction of cell surface
Enter cell with and by endocytosis.Common cationic polymer have polylysine (PLL), polyethyleneimine (PEI),
Tree-shaped viral envelope glycoprotein polymer, chitosan etc..
But because cytotoxicity is very big, the problems such as causing haemocyte to solidify in transfection process in vivo, existing sun from
Sub- polymer cannot be provided simultaneously with two basic demands of safety and low toxicity and high-efficiency transfection and therefore greatly limit it
Using.
Viral envelope glycoprotein B (Envelope glycoprotein B, gB), can be by combining mammalian cell table
On wide general existing receptor, such as pass through heparan mucopolysaccharide (the heparin sulfate on the various cells of mammal
Proglycan, HSPG) mediate retroviral phagocytic process, and also gB glycoprotein can be in the feelings for not depending on other viral envelope proteins
It is functioned alone under condition.The combination of gB and host cell can also not depend on HSPG simultaneously, while gB may also be combined into cell
Integrin (integrin) or EGF-R ELISA (epidermal growth factor receptor, EGFR) to
Promote poisoning intrusion process.Although viral envelope glycoprotein peptide itself quickly and efficiently can enter cell, in conjunction with
Limited with the ability of loading siRNA or Plasmid DNA, transfection polypeptide is used alone can not efficient transportation siRNA or Plasmid DNA.
Summary of the invention
In view of the security risk of virus transfection, the inefficient of cationic polymer and to cellulotoxic side effect, the present invention is public
A kind of non-viral transfection reagent and its preparation method and application has been opened, transfection efficiency can be improved in the non-viral transfection reagent,
The toxic side effect that a kind of cationic polymer is applied alone to cell is reduced simultaneously.
The invention is realized by the following technical scheme:
A kind of non-viral transfection reagent, comprising: the cationic polymer of final concentration of 10~50ng/ml, final concentration of 0.1
The cationic-liposome of the recombinant polypeptide of~20 μ g/ml, final concentration of 5~80ng/ml.
Further, the non-viral transfection reagent, comprising: cationic polymer, the final concentration of final concentration of 20ng/ml
Recombinant polypeptide, final concentration of 40ng/ml cationic-liposome for 8 μ g/ml.
Further, the cationic polymer of the non-viral transfection reagent is one kind based on proton sponge effect or appoints
The a variety of cationic polymers for proportion of anticipating.
Preferably, the cationic polymer includes polyethyleneimine, polylysine, Polyamide amine or chitosan etc..
Further, the recombinant polypeptide is viral envelope glycoprotein；The viral envelope glycoprotein is tree-shaped virus packet
Membrane glycoprotein, preferably recombinant herpesvirus envelope glycoprotein.
Preferably, the amino acid sequence of the recombination gB polypeptide is shown in SEQ ID NO:1 or SEQ ID NO:2.
Further, the cationic-liposome is didodecyldimethylammbromide bromide DDAB, amine cation lipoid
Including the amine salt lipoid plastid such as quaternary ammonium, primary amine, secondary amine, such as the oily alkenyloxy group propyl ammonium DOTMA of chlorination trimethyl -2,3- bis-, bromine
Change the oily alkenyloxy group propyl ammonium DORIE of dimethyl -2- ethoxy -2,3- two, bromination dimethyl -2- ethoxy -2,3- dioleoyl oxygen
The quaternary ammonium salt lipids bodies such as base propyl ammonium DORI.
Further, the transfection reagent further includes solvent；The solvent is HBS (pH7.4).
A kind of preparation method of above-mentioned non-viral transfection, comprising the following steps:
Cationic polymer, recombinant polypeptide, cationic-liposome and solvent mixing, obtain non-viral transfection preparation.
Further, the preparation method of the non-viral transfection reagent, comprising the following steps:
1) cationic polymer solution, is prepared；
2), the preparation of recombinant polypeptide；The preparation of cationic-liposome/recombinant polypeptide compound；
3), the preparation of transfection reagent.
Further, cationic polymer solution formulation operations include: in the step 1)
(1) preparation of HBS (pH7.4): NaCl is dissolved in ultrapure water, HEPES is added, adjusts pH value, constant volume stores up after filtering
Be stored in 4 DEG C it is spare；
(2) cationic polymer powder is dissolved in the HBS (pH7.4) of step (1) preparation, concussion uniformly, is configured to
The PEI solution of required concentration, filtering, be stored in 4 DEG C it is spare.
Further, cationic-liposome/viral envelope glycoprotein compound preparation in the step 2), comprising: take
Equivalent cationic-liposome and viral envelope glycoprotein are dissolved in 1 × HBS (pH7.4), and uniformly, filtering is positive to get arriving for concussion
Cationic liposomal/viral envelope glycoprotein complex solution, be stored in 4 DEG C it is spare.
A method of it is transfected using above-mentioned non-viral transfection reagent, comprising:
(4) pipe is taken, number is No. 1 pipe: non-viral transfection reagent and serum free medium is added in serum free medium,
Room temperature 5min；
(5) pipe is separately taken, number is No. 2 pipes: nucleic acid and Opti-MEM culture medium is added；
(6) No. 1 pipe solution is slowly added in No. 2 pipes by the speed of 1ml per minute, then the mixing that is vortexed；It stands, nothing is added
In blood serum medium；Change complete nutrition culture medium (such as containing 10% serum DMEM culture medium) after 12-16h into；Centrifugation, mistake
Filter.
Further, the serum free medium, including Opti-MEM culture medium, DMEM, α-MEM, EMEM, 1640 equal bases
Basal culture medium,
Further, the nucleic acid be one of Plasmid DNA, genetic fragment, double-strand or Single-stranded DNA fragments, cDNA or
The a variety of combinations arbitrarily matched.
The invention has the advantages that:
Transfection polypeptide of the invention can be used as cell transfecting reinforcing agent, the cell transfecting that auxiliary cationic polymer mediates.
When being used in combination with cationic polymer, cationic polymer and polypeptide are mainly combined together by charge effect, this knot
Conjunction is covalent bond, will not destroy the biological activity of protein.But this combination can by the restriction enzyme site of protein and
Binding site is covered, therefore the enzyme that can not be cultured in base is identified, can so as to effective protection polypeptide without the degradation by enzyme
Improve transfection efficiency.
Envelope glycoprotein B polypeptide provided by the invention keeps DNA poly- when acting on cell membrane by intrusion or integrated structure
It closes object to be adhered on cell, improves efficiency gene transfection, cytotoxicity will not be caused.
Viral envelope glycoprotein B polypeptide provided by the invention does not have the infection potentiality of viral vectors, because multiple without virus
The safety problem of system and infection has the potentiality applied to gene therapy.
The present invention can significantly improve the transfection efficiency of the non-virus carrier with single lysosome escape effect, Jin Erti
The delivery efficiency of high nucleic acid；In addition, the present invention uses molecular self-assembling method, it may be convenient to which large scale preparation transfection is compound
Object, and the compound is degradable in vivo, safety in utilization with higher.
Detailed description of the invention
Fig. 1 is the expression that cation PEI polymer individually transfects GFP gene after Hela cell；Wherein 1 (A) are as follows: bright
Field figure；1 (B) are as follows: fluorogram；1 (C) are as follows: stacking chart.
Fig. 2 is the expression that liposome individually transfects GFP gene after Hela cell；Wherein 2 (A) are as follows: light field figure；2(B)
Are as follows: fluorogram；2 (C) are as follows: stacking chart.
Fig. 3 is the expression that PEI and liposome composition transfect GFP gene after Hela cell；Wherein 3 (A) are as follows: light field
Figure；3 (B) are as follows: fluorogram；3 (C) are as follows: stacking chart.
Fig. 4 is the expression that PEI and peptide composition transfect GFP gene after Hela cell；Wherein 4 (A) are as follows: light field
Figure；4 (A) are as follows: fluorogram；4 (A) are as follows: stacking chart.
Fig. 5 is the expression that liposome and peptide composition transfect GFP gene after Hela cell；Wherein 5 (A) are as follows: bright
Field figure；5 (A) are as follows: fluorogram；5 (A) are as follows: stacking chart.
Fig. 6 is GFP gene after transfection reagent of the invention (cation, polypeptide and liposome composition) transfection Hela cell
Expression；Wherein 6 (A) are as follows: light field figure；6 (A) are as follows: fluorogram；6 (A) are as follows: stacking chart.
Fig. 7 is the expression that commercially available Lipofectamine 2000 transfects GFP gene after Hela cell.
Fig. 8 is the Cell viability and OD value comparison diagram of a reagent transfection after transfecting 48h and 72h；Wherein 8 (A) are living for cell
Rate comparison diagram；8 (B) are OD value comparison diagram.
Fig. 9 bitmap 1 to Fig. 7 transfection experiment transfection efficiency map；Wherein 9-1 is that PEI transfects (Fig. 1), transfection efficiency
It is 2.1%；9-2 is liposome transfection (Fig. 2), transfection efficiency 24.5%；9-3 is 3. cations and liposome complex (group
Close object 4, Fig. 3) transfection efficiency be 38.8%；9-4 is combined (composition 3, Fig. 4) transfection efficiency with polypeptide for 3. cations
30%；9-5 is that liposome and polypeptide are combined (composition 2, Fig. 5), transfection efficiency 66.2%；9-6 is that transfection of the invention tries
Agent (cation, polypeptide and liposome combination (composition 1, Fig. 6)) transfection efficiency is 82.6%；9-7 is Lipofectamine
2000 (Fig. 7), transfection efficiency 60.5%.
Specific embodiment
Technical solution problem to be solved, the technical solution of use and reach beneficial in order to better illustrate the present invention
Effect is further described now in conjunction with specific embodiment.It is worth noting that technical solution of the present invention is including but not limited to following
Embodiment.
Particular technique or condition are not specified in the embodiment of the present invention, according to the literature in the art described technology or
Condition is carried out according to product description.Reagents or instruments used without specified manufacturer, being can be by commercially available etc.
The conventional products that approach obtains.
The preparation of the transfection reagent of the present invention of embodiment 1
It the inducing expression of the 1.1 amphiphilic polypeptides of cation and isolates and purifies
The peptide fragment recombinant polypeptide of host cell membrane is merged and combined in a kind of viral envelope glycoprotein, and it includes following institutes
The amino acid sequence shown: IYNGWYAX and TXQMXTIYQ, and the polypeptide is the recombinant polypeptide of cell secretion.
In above-mentioned amino acid sequence, X is alternative acidic amino acid, and other than Asp, Glu also be can achieve equally
Effect.When X is Asp, the sequence of gB recombinant polypeptide is as shown in SEQ ID NO:1；When X is Glu, polypeptide sequence is SEQ ID
Shown in NO:2.
When X be Asp when, which obtains by the following method: synthesis EBV virus BALF4 gene in adherency
The related one section of encoding gene of host (NCBI Gene ID:3783680), sequence such as SEQ ID NO:3 correctly weigh sequence
Group polypeptide gB gene is connect with eukaryon secreted expression carrier pSecTag2B by T4DNA ligase, and competence large intestine is transformed into
Bacillus, screening positive clone filter out the recombinant plasmid pSecTag2B-gB correctly connected, the recombinant plasmid through digestion
PSecTag2B-gB is transformed into HEK293T cell and expresses, expression product is in culture supernatant, through examining after linearisation
It surveys, the expression quantity of recombinant polypeptide gB of the invention is 5mg/mL~12mg/mL.
When X be Glu when, sequence such as SEQ ID NO:4, other methods with X be Asp when method.
The preparation of 1.2 cationic polymers
The preparation of 1 × HBS (pH7.4): 8.76g NaCl is dissolved in 900ml ultrapure water, is added 20ml 1M (4.766g)
HEPES, adjust pH value to 7.4, be settled to 1L, be stored in after filtering (0.2 μm of filter membrane) 4 DEG C it is spare.
It weighs 50mg PEI powder to be dissolved in 1 × HBS of 50ml (pH7.4), concussion uniformly, is configured to 1.0mg/ml's
PEI solution, 0.2 μm of membrane filtration, be stored in 4 DEG C it is spare.
The preparation of 1.3 cationic-liposomes/recombination gB polypeptide complex
4mg commercialization cationic-liposome didodecyldimethylammbromide bromide (DDAB) is taken, 1 × HBS of 10mL is dissolved in
(pH7.4), concussion uniformly, 0.2 μm of membrane filtration obtains the liposome solutions of 400 μ g/ml, 1.1 described in 8mg/ml in it is made
Liposome solutions are diluted 10 times by the recombination gB polypeptide solution obtained, and configuration contains 40 μ g/ml cationic-liposomes and 8mg/ml gB
The cationic-liposome of polypeptide/recombination gB polypeptide complex solution, be stored in 4 DEG C it is spare.
The preparation of 1.4 transfection reagents of the present invention
1:50 is mixed the compound that cationic polymer solution made from 1.2 and 1.3 are prepared in proportion.
Embodiment 2 is transfected with transfection reagent of the present invention
2 1.5ml EP pipes are taken, wherein No. 1 pipe: transfection reagent+0.25mL Opti-MEM culture medium 2 μ L of the invention,
Room temperature 5min；No. 2 pipes: (plasmid is convenient for containing the carrier pcDNA3.1 (+) of green fluorescent protein (GFP) to 2ul 1ug plasmid
The transfection efficiency of cell is observed by luciferase expression situation, pcDNA3.1 (+) is bought from addgene company)+0.25ml Opti-
MEM culture medium；It manages for No. 1 and slowly (1ml per minute) is added in No. 2 pipes, then be mixed by inversion 5 seconds.20min is stood, is added long extremely
In the cell of 80% convergence degree.12-16h is changed containing 10% serum DMEM culture medium.1300rpm is centrifuged 5min, 0.45 μm of filter membrane
Filtering.
The detection of 3 transfection of embodiment
Cationic polymer transfection efficiency and toxicity detection；With different cell detection PEI transfection efficiencies and toxicity.
Positively charged amino group can be with phosphorus negatively charged on plasmid in amino acids cationic-liposome structure
Acid groups are compressed completely by electrostatic adsorption, stable cationic liposome complex are formed, by cationic-liposome pressure
Shorten the particle of dispersion into.Primary structure at this time does not change, can also avoid being degraded by internal enzyme and salt etc..By certain
After amino acids cationic-liposome and PEI or peplos recombinant polypeptide are prepared into compound suspension by ratio, observation discovery
No matter liposome combines in any proportion can all occur phenomenon of flocculating, polypeptide not only has protection to Plasmid DNA and promotees infiltration effect, together
When, it can also reduce the toxicity of cationic-liposome.Polypeptide head rich in hydrophilic amino acid
(MTRRRVLSVVVLLAALACRLGAQT, wherein it is hydrophilic amino acid at underscore), it, can in the aqueous solution of low concentration
To form hydrophilic barrier in surface of liposome, to increase stability of the liposome under transfection reagent system, flexible liposomes
Character.
From Fig. 1 (Fig. 9-1) and Fig. 2 (Fig. 9-2) as can be seen that the intracellular GFP gene expression of PEI, liposome transfection
Situation is seldom.
By the cell that DNA plasmid transfects, for the transfection method of Plasmid DNA with embodiment 2, transfection reagent is to combine in table 1
Object 1-4, and each composition transfection results are observed, see Fig. 3-Fig. 6 (Fig. 9-3 to 9-6).
Each transfection reagent of table 1 composition and final concentration
To the expression of GFP gene after HeLa cytosis, the realization result of 3d such as Fig. 3-Fig. 6 after transfection (Fig. 9-3 to
Shown in 9-6).From Fig. 3-Fig. 6 (Fig. 9-3 to 9-6) as can be seen that compared with the cell that PEI, liposome complex transfect respectively,
Cation and liposome complex (composition 4, Fig. 3), cation and polypeptide combination (composition 3, Fig. 4), liposome and polypeptide
It is combined (composition 2, Fig. 5), cation of the invention, polypeptide and liposome and is combined (composition 1, Fig. 6), Lipofectamine
2000 (Fig. 7, Fig. 9-7) turn reagent compound wink and can enter aim cell with high-efficiency delivery Plasmid DNA and inhibit associated with these types
The expression of target gene, intracellular GFP expression conditions are remarkably reinforced, wherein cation of the invention, polypeptide and lipid
It is most strong that body is combined (Fig. 6, Fig. 9-6) effect, and is better than commercially available Lipofectamine 2000 (Fig. 7, Fig. 9-7), that is, passes through
The synergy of PEI, gB polypeptide and liposome, transfection efficiency of the GFP gene in HeLa cell significantly improve, and show that PEI/ is more
The Plasmid DNA system of peptidoliposome compound delivering can be with stable transfection HeLa cell.
4 cytotoxicity experiment of embodiment
It is inoculated with 4000/100 μ L cell suspensions respectively in 2 96 orifice plates.Culture plate is put into preculture in the incubator
(37 DEG C, 5%CO2), are transfected respectively with the composition of table 1, and after transfecting 48h and 72h again, measure cell survival with cck8 method
Rate is added 20 μ L CCK solution to every hole, culture plate is incubated for 4 hours in incubator, with microplate reader measurement at 450nm
Absorbance.As a result as shown in Fig. 8 (8 (A), 8 (B)).
Cck8 decoration method surveys cell quantity and motility rate
It is demonstrated experimentally that this basic no cytotoxicity of carrier, biodegradable, there is higher efficiency gene transfection: with
Cervical cancer cell Hela is that target cell is better than commercially available cationic-liposome when GFP is that target gene carries out transfection experiment
The transfection efficiency of Lipofectamine 2000, under the highest concentration of transfection efficiency, the transfection effect of Lipofectamine 2000
Rate is 60.5%, and transfection efficiency of the present invention is 82.6%；Under the highest concentration of transfection efficiency, PEI, liposome transfection
Cell survival rate is respectively 71.59%, 78.47% after 48h, and cytotoxicity is stronger, and commercially available Lipofectamine 3000 is deposited
Motility rate is higher, is 90.13%, and the survival rate of the cell of cationic polymer gene transfection agent of the present invention transfection is
93.66%, toxicity is minimum.
Non-virus carrier lysosome escape mechanism is mainly proton sponge effect, film fusion effect, film perforation or film at present
Disturbing effect.There are two types of lysosome escape effects for transfection composite tool in the present invention: the proton sponge effect of cationic polymer
Film perforation or film disturbing effect with viral envelope glycoprotein.Both effects, which are used in combination, can significantly improve with single molten
The transfection efficiency of the non-virus carrier of enzyme body escape effect, and then improve the delivery efficiency of nucleic acid.The present invention uses molecule from group
Dress method, it may be convenient to large scale preparation transfection composite, and also the compound is degradable in vivo, is not easy in vivo
Accumulation, safety in utilization with higher.
The above experiment and the experimental results showed that, the novel transgenosis that gB polypeptide, cationic polymer and nucleic acid are formed is multiple
Closing object not only can be with high-efficiency delivery DNA, but also cytotoxicity is small.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Guangzhou Hui Yuanyuan Pharmaceutical Technology Co., Ltd
<120>a kind of non-viral transfection reagent and its preparation method and application
<130> HPCNP201810060
<141> 2018-08-06
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Arg Val Cys Glu Leu Ser Ser His Gly Asp Leu Phe Arg Phe Ser Ser
50 55 60
Asp Ile Gln Cys Pro Ser Phe Gly Thr Arg Glu Asn His Thr Glu Gly
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Leu Leu Met Val Phe Lys Asp Asn Ile Ile Pro Tyr Ser Phe Lys Val
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Arg Ser Tyr Thr Lys Ile Val Thr Asn Ile Leu Ile Tyr Asn Gly Trp
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Tyr Ala Asp Ser Val Thr Asn Arg His Glu Glu Lys Phe Ser Val Asp
115 120 125
Ser Tyr Glu Thr Asp Gln Met Asp Thr Ile Tyr Gln Cys Tyr Asn Ala
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Val Lys Met Thr Lys Asp Gly Leu Thr Arg Val Tyr Val Asp Arg Asp
145 150 155 160
Gly Val Asn Ile Thr Val Asn Leu Lys Pro Thr Gly Gly Leu Ala Asn
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Gly Val Arg Arg Tyr Ala Ser Gln Thr Glu Leu Tyr Asp Ala Pro Gly
180 185 190
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195 200 205
Thr Asp Met Met Ala Lys Ser Asn Ser Pro Phe Asp Phe Phe Val Thr
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Thr Thr Gly Gln Thr Val Glu Met Ser Pro Phe Tyr Asp Gly Lys Asn
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cgtcagcaaa ccagctttcc tttccgagtc tgcgagctct ccagccacgg cgacctgttc 180
cgcttctcct cggacatcca gtgtccctcg tttggcacgc gggagaatca cacggagggc 240
ctgttgatgg tgtttaaaga caacattatt ccctactcgt ttaaggtccg ctcctacacc 300
aagatagtga ccaacattct catctacaat ggctggtacg cggactccgt gaccaaccgg 360
cacgaggaga agttctccgt tgacagctac gaaactgacc agatggatac catctaccag 420
tgctacaacg cggtcaagat gacaaaagat gggctgacgc gcgtgtatgt agaccgcgac 480
ggagttaaca tcaccgtcaa cctaaagccc accgggggcc tggccaacgg ggtgcgccgc 540
tacgccagcc agacggagct ctatgacgcc cccgggtggt tgatatggac ttacagaaca 600
agaactaccg tcaactgcct gataactgac atgatggcca agtccaacag ccccttcgac 660
ttctttgtga ccaccaccgg gcagactgtg gaaatgtccc ctttctatga cgggaaaaat 720
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cgagagcaaa ccagctttcc tttccgagtc tgcgagctct ccagccacgg cgacctgttc 180
cgcttctcct cggagatcca gtgtccctcg tttggcacgc gggagaatca cacggagggc 240
ctgttgatgg tgtttaaaga gaacattatt ccctactcgt ttaaggtccg ctcctacacc 300
aagatagtga ccaacattct catctacaat ggctggtacg cgggacccgt gaccaaccgg 360
cacgaggaga agttctccgt tgagagctac gaaactgagc agatggagac catctaccag 420
tgctacaacg cggtcaagat gacaaaagag gggctgacgc gcgtgtatgt agagcgcgag 480
ggagttaaca tcaccgtcaa cctaaagccc accgggggcc tggccaacgg ggtgcgccgc 540
tacgccagcc agacggagct ctatgaggcc cccgggtggt tgatatggac ttacagaaca 600
agaactaccg tcaactgcct gataactgag atgatggcca agtccaacag ccccttcgac 660
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129367A (en) * | 2019-05-28 | 2019-08-16 | 苏州博特龙免疫技术有限公司 | A kind of cationic transfection reagent and its preparation method and application |
| CN111154805A (en) * | 2020-02-11 | 2020-05-15 | 苏州吉恒基因科技有限公司 | Cationic polymer DNA complex and method for promoting target plasmid to transfect cells and express |
| CN116083491A (en) * | 2023-01-05 | 2023-05-09 | 上海利康瑞生物工程有限公司 | Cationic transfection reagent composition and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101199858A (en) * | 2007-10-18 | 2008-06-18 | 广州拓谱基因技术有限公司 | Multiple target point small interference RNA cocktail agent for treating ophthalmic disease and preparing method thereof |
| CN103865942A (en) * | 2012-12-18 | 2014-06-18 | 常州碳宇纳米科技有限公司 | Nano-particles capable of improving gene transfection efficiency and preparation method of gene transfection reagent based on particles |
| CN107875400A (en) * | 2017-11-20 | 2018-04-06 | 南通大学 | A kind of siRNA echoviruses delivery system of core-shell structure and application |
-
2018
- 2018-08-06 CN CN201810887390.7A patent/CN109136270B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101199858A (en) * | 2007-10-18 | 2008-06-18 | 广州拓谱基因技术有限公司 | Multiple target point small interference RNA cocktail agent for treating ophthalmic disease and preparing method thereof |
| CN103865942A (en) * | 2012-12-18 | 2014-06-18 | 常州碳宇纳米科技有限公司 | Nano-particles capable of improving gene transfection efficiency and preparation method of gene transfection reagent based on particles |
| CN107875400A (en) * | 2017-11-20 | 2018-04-06 | 南通大学 | A kind of siRNA echoviruses delivery system of core-shell structure and application |
Non-Patent Citations (2)
| Title |
|---|
| GABRIELE D. SCHMIDT-WOLF等: "Non-viral and hybrid vectors in human gene therapy: an update", 《TRENDS IN MOLECULAR MEDICINE》 * |
| M MORILLE等: "Progress in developing cationic vector for non-viral systematic gene therapy against cancer", 《BIOMATERIALS》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129367A (en) * | 2019-05-28 | 2019-08-16 | 苏州博特龙免疫技术有限公司 | A kind of cationic transfection reagent and its preparation method and application |
| CN110129367B (en) * | 2019-05-28 | 2020-04-07 | 苏州博特龙免疫技术有限公司 | Cationic transfection reagent and preparation method and application thereof |
| CN111154805A (en) * | 2020-02-11 | 2020-05-15 | 苏州吉恒基因科技有限公司 | Cationic polymer DNA complex and method for promoting target plasmid to transfect cells and express |
| CN111154805B (en) * | 2020-02-11 | 2024-03-12 | 苏州吉恒基因科技有限公司 | Cationic multimeric DNA complexes and methods for promoting transfected cells and expression of plasmids of interest |
| CN116083491A (en) * | 2023-01-05 | 2023-05-09 | 上海利康瑞生物工程有限公司 | Cationic transfection reagent composition and application thereof |
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