CN105018500A - Application of codon-optimized trichosanthin gene in gene therapy - Google Patents
Application of codon-optimized trichosanthin gene in gene therapy Download PDFInfo
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Abstract
The invention relates to a codon-optimized trichosanthin gene and application thereof in gene therapy. According to the invention, after codon optimization, the trichosanthin gene is subjected to drug delivery through a viral or non-viral vector, can express protein at a higher level in mammalian cells so as to play a greater biological role. In the invention, the drug delivery carrier is optimized to improve its transgenic expression level in mammalian cells and play a maximum antitumor role, thus having important significance to research and promotion of tumor gene therapy.
Description
Technical field
The present invention relates to field of gene, specifically, is codon optimized Gene Encoding Trichosanthin and the application in gene therapy thereof.
Background technology
Trichosanthin (Trichosanthin, TCS) derives from traditional Chinese medicine Snakegourd Root (Radicestrichosanthis).Snakegourd Root is the perennial root climbing up by holding on to herbaceous plant snakegourd (Trichosantheskirilowii Maxim) or trichosanthes rosthornii Harms (Trichosanthes rosthornii Herms) of Curcurbitaceae snake gourd.Effect of Snakegourd Root is: heat-clearing and fire-purging, promote the production of body fluid to quench thirst, detumescence and apocenosis, cure mainly: quench one's thirst, jaundice, pyreticosis are thirsty, dryness of the lung hemoptysis etc.TCS is made up of 247 amino-acid residues, belong to I type ribosome inactivating protein (Ribosome-inactivating proteins, RIP), molecular weight is about 27kDa, has multiple pharmacologically actives such as suppressing kinds of tumor cells growth, antiviral, immunomodulatory, AIDS virus resisting.Clinically TCS is used for induced labor and ectopic pregnancy has many years of experience.Some nearest pharmacological researches find that it also has multiple other biological active, mainly comprise: have RNA N-glycosidase activity, DNA enzymatic activity, cell death inducing, the effect of enhancing chemokine etc.The pharmacologically active of its Tumor suppression is more taken seriously.Wherein also have the lethal effect of hepatocellular carcinoma and repeatedly report.TCS has entered II clinical trial phase in the U.S., only observed slight side effect when intravenous injection maximum dose level (1.2mg/ time/week).But research anticancer to TCS mostly at present is directly its protein of application, also TCS gene transfer is not entered tumour cell and carries out anticancer report in vivo.
Between different organisms, significantly, codon preference is the important factor affecting protokaryon and genetic expression between eukaryote and different eukaryote to the difference of genetic codon frequency of utilization.Therefore, gene order codon optimized is that the gene of heterologous protein expresses conventional optimization method, to be increased in the transgene expression in mammalian host cell in mammalian cell.There is multiple research codon optimized about foreign gene, all obtained significantly improving of related protein expression amount.But yet there are no report about a kind of codon optimized Gene Encoding Trichosanthin (codon-optimized Trichosanthin, the coTCS) application in gene therapy.
Adeno-associated virus (Adeno-Associated Virus, AAV) is a kind of parvovirus of diameter 23 nanometer, and protein enclosure is icosahedron, and packaging has length to be about the single stranded DNA genome of 4.7kb.Due to advantages such as its non-virulents, present one of the important carrier becoming gene therapy of recombinant adeno-associated virus (Recombinant Adeno-AssociatedVirus, rAAV).Next our problems faced is exactly: the Gene Encoding Trichosanthin that the son that accesses to your password is optimized, and enters tumour cell by rAAV carrier transduction, the object of therapy of tumor can be reached?
Summary of the invention
The object of the invention is for deficiency of the prior art, provide codon optimized Gene Encoding Trichosanthin preparing the application in gene therapy medicament.
Second object of the present invention provides a kind of codon optimized Gene Encoding Trichosanthin, and carry adeno-associated virus or the plasmid of described codon optimized Gene Encoding Trichosanthin.
For achieving the above object, the technical scheme that the present invention takes is: codon optimized Gene Encoding Trichosanthin is preparing the application in gene therapy medicament.
Preferably, described gene therapy is the gene therapy to tumour.
Preferably, described tumour is liver cancer or cervical cancer.
Preferably, described codon optimized Gene Encoding Trichosanthin is the Gene Encoding Trichosanthin comprising the mammalian sources such as codon humanization, dog source, cat source.
Preferably, described gene therapy be carry codon optimized Gene Encoding Trichosanthin adeno-associated virus for carrier is to the gene therapy of tumour.
Preferably, described gene therapy be carry codon optimized Gene Encoding Trichosanthin plasmid for carrier is to the gene therapy of tumour.
Preferably, described codon optimized Gene Encoding Trichosanthin has the arbitrary described nucleotide sequence of SEQ ID NO.1-SEQ IDNO.21.
For realizing above-mentioned second object, the technical scheme that the present invention takes is: a kind of codon optimized Gene Encoding Trichosanthin, and described codon optimized Gene Encoding Trichosanthin has the arbitrary described nucleotide sequence of SEQ ID NO.1-SEQID NO.21.
Carry virus or the non-virus carrier of described codon optimized Gene Encoding Trichosanthin.
Preferably, described virus is adeno-associated virus, and described non-virus carrier is plasmid.
The invention has the advantages that:
1, after Gene Encoding Trichosanthin codon of the present invention is optimized, then by virus or non-virus carrier administration, can in mammalian cell higher level marking protein, to play larger biological action;
2, the present invention is by drug administration carrier optimization, improves its transgene expression level in mammalian cell, significant to the research and extension of therapy of tumor.
Accompanying drawing explanation
Fig. 1 contains the dose-dependently of Flag-Tag protein expression after the plasmid transfection Huh7-FLuc cell of TCS.
Fig. 2 contains the plasmid of TCS albumen difference amount to the inhibited proliferation of Huh7-FLuc cell.
Fig. 3 contains the plasmid of TCS albumen to the inhibited proliferation of Huh7 cell.
Fig. 4 contains the plasmid of TCS albumen to the inhibited proliferation of Huh7-FLuc cell.
Fig. 5 contains the plasmid of TCS albumen to the inhibited proliferation of LH-86 cell.
The different plasmids of Fig. 6 containing TCS albumen under different promoters are to the inhibited proliferation of Huh7-FLuc cell.
Fig. 7,8,9 expression of EGFP after plasmid transfection Huh7-FLuc cell under different promoters.
The different plasmids of Figure 10 containing TCS albumen under different promoters are to the inhibited proliferation of HEK293 cell.
Figure 11,12,13 expression of EGFP after plasmid transfection HEK293 cell under different promoters.
Figure 14 contains the plasmid of TCS albumen in vivo to the restraining effect of people's liver cancer.
Figure 15 contains the virus of TCS albumen in vivo to the restraining effect of people's liver cancer.
Figure 16 contains PARP protein expression situation after the plasmid transfection human liver cancer cell of TCS albumen.
Interpretation of result after the optimization of Figure 17 Life Technologies company's T CS gene codon.
The time-dependent manner of Flag-Tag protein expression after Figure 18 plasmid coTCS transfection.
The expression of Gaussia Luciferase in cell after Figure 19 plasmid coTCS transfection 48h.
Figure 20 contains the inhibited proliferation of plasmid to tumour cell of coTCS albumen: A. contains the plasmid of coTCS albumen to the inhibited proliferation of HeLa cell; B. the plasmid containing coTCS albumen is to the inhibited proliferation of HepG2 cell; C. the plasmid containing coTCS albumen is to the inhibited proliferation of Huh7 cell.
Embodiment
Below in conjunction with accompanying drawing, embodiment provided by the invention is elaborated.
Embodiment 1
One, experiment material and method
1. plasmid, primer
PdsAAV-AFPp-EGFP plasmid builds (Mingozzi F by Glushakova etc., High KA:Therapeutic in vivo gene transfer for genetic disease using AAV:progress andchallenges.Nature reviews Genetics 2011,12 (5): 341-355.).Wherein AFPp is afp promoter, and EGFP is enhanced green fluorescence protein.TCS gene is synthesized by Life Technologies company, based on sequence (the http://www.ncbi.nlm.nih.gov/nuccore/M34858.1 announced; T.kirilowiitrichosanthin (TCS) mRNA, complete cds; GenBank:M34858.1), and carry out subclone with restriction enzyme A ge I and Hind III, put in pdsAAV-AFPp-EGFP, pdsAAV-CBAp-EGFP plasmid.Wherein CBAp is avian beta-actin promotor.All plasmids check order all before the use.CoTCS is the kinds of schemes that the software provided through Integrated DNA Technologies and LifeTechnologies company respectively obtains TCS gene optimization, in scheme, coTCS has the arbitrary described nucleotide sequence of SEQ ID NO.1-SEQ ID NO.21, wherein ATG is initiator codon, TGA or TAA or TAG is terminator codon.Wherein SEQ ID NO.1-SEQ ID NO.20 is the TCS gene optimization scheme that the software provided by Integrated DNA Technologies company obtains, and SEQ ID NO.21 is the TCS gene optimization scheme that the software provided by Life Technologies company obtains.Build and multiplely comprise the plasmid that different schemes optimizes TCS gene, then after transfection HeLa and HEK293 cell, Western Blot detects Flag-Tag protein expression content.
2. clone is cultivated
Human cervical cancer cell lines HeLa, embryonic kidney cell system HEK293, hepatoma cell line Huh7, HepG2, be all purchased from American Type Culture Collection (Manassas, VA, USA).Photinus pyralis LUC (Firefly Luciferase, FLuc) marker gene mark Bel7402 Huh7-FLuc provides (Ling C by University of Florida's cell and molecular therapy center, Wang Y, Zhang Y, Ejjigani A, YinZ, Lu Y, Wang L, Wang M, Li J, Hu Z, Aslanidi GV, Zhong L, Gao G, Srivastava A, Ling C.Selective in vivo targeting of human liver tumors by optimized AAV3vectors in a murine xenograft model.Hum Gene Ther.2014Dec, 25 (12): 1023-34.).Novel liver cancer clone LH86 provides (Zhu H by Pathology Deparment of medical college of University of Florida, Dong H, Eksioglu E, Hemming A, Cao M, Crawford JM, Nelson DR, Liu C:Hepatitis Cvirus triggers apoptosis of a newly developed hepatoma cell line through antiviraldefense system.Gastroenterology 2007,133 (5): 1649-1659.).Above cell is by complete Dulbecco ' s modified Eagle medium nutrient solution (C-DMEM, Mediatech Inc.Herndon, USA) 10% heat-inactivated foetal calf serum (FBS is added, Sigma-Aldrich Co.LLC, St.Louis, USA), 1% penicillin and Streptomycin sulphate (Lonza, Walkersville, MD).Cell attachment is cultivated at humidification, 37 DEG C, 5%CO
2under environment, the resuspended rear Secondary Culture of pancreatin-ethylenediamine tetraacetic acid (EDTA) (EDTA) mixed solution (Lonza, Walkersville, MD, USA) room temperature digestion 2-5 minute, C-DMEM.
The preparation of 3.rAAV
Packaging: highly purified rAAV storage liquid adopts three plasmid transfection method (Ling C, Lu Y, Cheng B, McGoogan KE, Gee SW, Ma W, Li B, Aslanidi GV, Srivastava A:High-efficiencytransduction of liver cancer cells by recombinant adeno-associated virus serotype 3vectors.Journal of visualized experiments:JoVE2011 (49) .): use polymine (polyethelenimine, PEI, linear, MW 25000, Polysciences, Inc.Warrington, PA, USA) HEK293 cell is entered in three kinds of common transfections of plasmid, cell culture fluid is changed after 6 hours.Transfection is harvested cell after 72 hours, after 3 multigelations, cut destruction cell genomic dna with restriction enzyme Benzonase (Life Technologies, Grand Island, USA) enzyme.
Purifying: Visipaque 320 (iodixanol, Sigma-Aldrich Co.LLC, St.Louis, USA) layering ultracentrifugation method is centrifugal, then uses HiTrap SP/Q HP Filter column (GE Healthcare, Piscataway, USA) ion exchange methods virion, then uses phosphate buffered saline buffer (PBS, Mediatech Inc.Herndon, USA) rinse, and concentrate with 150K molecular weight rotary-classify technology device.
Qualification: the genome of virus is measured by Southern Blot.Method is as follows: 10 μ l viral stock are diluted to 100 μ l and add Benzonase restriction enzyme (Novagen, Darmstadt, Germany) 1 hour is cut at 37 DEG C of enzymes, destroy DNA beyond all virus capsid proteins, add the 100mM NaOH of same volume 65 DEG C of effects 30 minutes, make Benzonase enzyme deactivation and destroy coat protein releasing virus genomic dna, and unwinding, putting in mixture of ice and water and cool fast.A kind of concentration known and with the sheet segment DNA of viral genome identical sequence simultaneously same procedure unwind.By the solution containing DNA and carry out gel electrophoresis, ssAAV genome selects alkaline 1.2% sepharose, and scAAV genome selects neutral 1.2% sepharose.DNA is transferred on nylon membrane, detect by the method (1975) that Southern announces.First glue is balanced in No. 1 solution (0.25M HCl), room temperature, 20 minutes; No. 2 solution (1M NaCl, 0.5M NaOH), room temperature, 40 minutes; No. 3 solution (1M NaCl, 0.5M Tris-HCl), room temperature, 40 minutes.In 20 × SSC solution, the DNA on film is transferred on Immobilon-NY+TM film (Millipore, Billerica, MA).Through crosslinked, film is placed in 25ml hybridization solution (the protamine DNA that 6 × SSC, 100ug/ml boiled, 0.5% sodium lauryl sulphate (SDS), and 5 × Denhardt ' s solution), 68 DEG C, 6 hours prehybridizations.Film is placed in the hybridization solution of the DNA probe containing the 32P-mark boiled, 68 DEG C of hybridization 18-20 hour.With No. 1 washing fluid of 50ml (2 × SSC, 0.1%SDS) flushing membrane 2 times, room temperature, each 15 minutes; Use 50ml No. 2 washing fluids (0.1 × SSC, 0.1%SDS) to rinse 2 times, 68 DEG C, 30 minutes again, at-70 DEG C, use BIOMAX MRTM X-ray sensitive film (Kodak, Rochester, NY) exposure.
Titer determination: titre is recorded by quantitative DNA Slot Blot method, specific as follows: the physical particles titre of highly purified rAAV is detected by quantitative DNA Slot Blot method.10 μ l viral stock are diluted to 100 μ l and add Benzonase restriction enzyme and cut 1 hour at 37 DEG C of enzymes, destroy the DNA beyond all virus capsid proteins, add the 100mM NaOH of same volume 65 DEG C of effects 30 minutes, make Benzonase enzyme deactivation and destroy coat protein releasing virus genomic dna, and unwinding.Then put in mixture of ice and water and cool fast.A kind of concentration known and containing with the plasmid DNA of viral genome identical sequence simultaneously same procedure unwind.All DNA double dilution method point samples unwind are to (Millipore, Bedford, MA) on Immobilon-NY+TM film.Through crosslinked, film is placed in 25ml hybridization solution (the protamine DNA that 6 × SSC, 100ug/ml boiled, 0.5% sodium lauryl sulphate (SDS), and 5 × Denhardt ' s solution), 68 DEG C, 6 hours prehybridizations.Film is placed in the hybridization solution of the DNA probe containing the 32P-mark boiled, 68 DEG C of hybridization 18-20 hour.No. 1 washing fluid of 50ml (2 × SSC, 0.1%SDS) flushing membrane 2 times, room temperature, each 15 minutes; Use 50ml No. 2 washing fluids (0.1 × SSC, 0.1%SDS) to rinse 2 times, 68 DEG C, 30 minutes again, at-70 DEG C, use BIOMAX MRTM X-ray sensitive film (Kodak, Rochester, NY) exposure.
4. plasmid transfection Bel7402, HEK293, HeLa cell
Use Life Technologies company Lipofectamine
tMlTX Reagent test kit does transfection, specific experiment step reference reagent box description operation.
(1) before transfection 1d, cell is laid on 96 orifice plates 1 × 10
4cell/well.
(2) being diluted with 20 μ l plasma-free DMEM medium by plasmid after 24h is 100ng/well.
(3) at use PLUS
tMbefore Reagent, mixing, adds 0.1 μ l PLUS
tMreagent/well, PLUS
tMreagent and plasmid are that 1:1 mixes by ratio, left at room temperature 15min.
(4) add 0.4 μ l Lipofectamine
tMlTX to PLUS
tMin the mixture of Reagent and plasmid, mixing, left at room temperature 25min.
(5) discard DMEM perfect medium, add new perfect medium DMEM 50 μ l/well.
(6) add 20 μ l/well Lipofectamine
tMthe mixture of LTX and plasmid, in 96 orifice plates, jiggles mixing.
(7) change perfect medium DMEM after 4h, 100 μ l/well.
4. cell survival component analysis
At 37 DEG C, 5%CO
2cultivate 24 in incubator, 48, use Dojindo MolecularTechnologies company Cell Counting Kit-8 kit detection cell to breed after 72h, specific experiment step reference reagent box description operation:
(1) after plasmid transfection 24h, 48h, 72h, change perfect medium DMEM, 90 μ l/well, add the CCK-8 solution of 10 μ l to every hole, culture plate is hatched 4h in incubator.
(2) in incubator, hatch 96 orifice plate 4h.
(3) the absorbancy at 450nm place after 4h, is measured with VERSAmax tunable microplate reader.
5. in cell, the detection of Gaussia Luciferase uses New England Biolabs company
gaussia Luciferase Assay Kit test kit, specific experiment step reference reagent box description operation is after plasmid transfection after 48h, different sample cultivation liquid supernatant is added in 96 orifice plates of White-opalescent, 20 μ l nutrient solution supernatant/well, add 50 μ l/well Gluc assay solution in sample cultivation liquid supernatant, each detection hole, uses FLUOstar optima to detect absorbancy fast.
6. laboratory animal
All experimentation on animalies are protected by U.S. Animal and are ratified with the council of use, and operate according to University of Florida (Gainesville, FL, USA) or Shanghai The 2nd Army Medical College protection of animal specification.6-10 mouse age in week, nonobese diabetic/severe combined immunodeficientgamma (NSG) of body weight 15-20g, male or female mice is purchased from Jackson laboratory (Bar Harbor, ME, USA), feed at medical college of University of Florida (Gainesville, FL, USA), room temperature, mouse is freely fetched water and takes food.
7. people's liver cancer xenogenesis Mice Inoculated model
Detect containing the luciferase of planting subcutaneous tumors after TCS plasmid transfection human liver cancer cell.Cell Huh7-FLuc is laid on 6 15cm
2in culture dish.By plasmid pAAV-CMVp-IRES-hrGFP, pAAV-CMVp-TCS-Flag-IRES-hrGFP transfection Huh7-FLuc cell after 24h.Wherein CMVp is people's cytomegalovirus promoter, and IRES is Internal ribosome entry site, and hrGFP is humanization green fluorescent protein.Use Lipofectamine
tMlTX Reagent does transfection, and experimental procedure is according to test kit description operation.Add the digestion of trysinization liquid after 48h and collecting cell, the centrifugal 5min of 1000rpm, abandons supernatant, adds PBS re-suspended cell, adjustment cell concn to 1.0 × 10
8/ ml, is only inoculated in NSG mouse armpit by 50 μ l/ subcutaneous, often organizes each 6.The fluorescent value of tumour cell is detected weekly after inoculation.Working method is with reference to the operation of Caliper Life Sciences company Preparation of Luciferin for In Vivo BioluminescentAssays test kit.Concrete operation method is every mouse 10 μ l/g.Take pictures with small animal living body imager IVIS Lumina II after abdominal injection luciferin10 ~ 15min.
After intra-tumoral injection contains the virus of TCS, luciferase detects.Cell Huh7-FLuc is laid on 6 15cm
2in culture dish.Add the digestion of trysinization liquid after 24h and collecting cell, the centrifugal 5min of 1000rpm, abandons supernatant, adds PBS re-suspended cell, adjustment cell concn to 1.0 × 10
8/ ml, is only inoculated in NSG mouse armpit by 50 μ l/ subcutaneous, often organizes each 6.The fluorescent value of tumour cell is detected weekly after inoculation.Working method is with reference to the operation of Caliper Life Sciences company Preparation of Luciferin for In VivoBioluminescent Assays test kit.Concrete operation method is every mouse 10 μ l/g.Take pictures with small animal living body imager IVIS Lumina II after abdominal injection luciferin 10 ~ 15min.Observed tumor growth situation every 3 days, observe to tumour and can touch, intra-tumoral injection adeno-associated virus rAAV3-2M-AFPp-TCS, rAAV3-2M-AFPp-EGFP.Wherein 2M is the AAV3 of two sudden change.Detect fluorescent value before intra-tumoral injection virus, after injecting virus, detect weekly the fluorescent value of secondary tumour cell, measure secondary tumor size weekly.
8. containing PARP Protein Detection after the plasmid transfection human liver cancer cell of TCS
Plasmid pAAV-CMVp-IRES-hrGFP, pAAV-CMVp-TCS-Flag-IRES-hrGFP transfection Huh7, HepG2 cell (6 orifice plate).
(1) sample preparation uses Lipofectamine
tMlTX Reagent does transfection, and experimental procedure is according to test kit description operation.Before transfection 1d, Huh7, HepG2 cell being in logarithmic phase is made single cell suspension, being diluted to concentration after cell counting is 5 × 10
5the cell suspension of/well, is inoculated in 6 well culture plates by 3ml/well inoculation respectively, by 6 plasmids, 500 μ l plasma-free DMEM medium dilution 2500ng/well after 24h.Concrete transfection method is the same.At 37 DEG C, 5%CO
2extract proteins after cultivating 48 in incubator.Protein Extraction process is as follows: suck nutrient solution, and cold PBS washes twice, blots liquid in 6 orifice plates, adds the precooling RIPA cell pyrolysis liquid of 150 μ l containing 1 × protease inbibitor, is scraped off by cell rapidly.4 DEG C, after placing 10min, the centrifugal 15min of 12000rpm, collect supernatant liquor, by Bio-RadProtein Assay kit Quantitative Western concentration, adjustment protein concentration is 5mg/ml, add 2 × Laemmlisample buffer albumen sample-loading buffer, in boiling water, boil 10min, make protein denaturation ,-20 DEG C save backup.
(2) SDS-PAGE polyacrylate hydrogel electrophoresis
According in the separation gel implantation glass interlayer of formulated 10%, use isopropylcarbinol front cover, after polymerization, remove isopropylcarbinol, with deionized water rinsing several, pour into spacer gel, insert loading comb, take out loading comb, rinse, sample to be analyzed is drawn respectively with micro sample adding appliance, injection volume is 15 μ l, switches on power, voltage 70V, until sample after spacer gel partial concentration is into a line, 100V constant voltage electrophoresis 60min.
(3) western blot analysis
Take out gel, excision spacer gel, is soaked in glue, Nitrocellulose film and filter paper in the transfering buffering liquid of precooling simultaneously.Put well fixing according to anode, filter paper, Nitrocellulose film, gel, filter paper, negative electrode order, insert electrotransfer groove, put into mixture of ice and water box, 100V, 2h.Take off Nitrocellulose film, dyeing 2min, protein band washes dye liquor with water after occurring.TBST solution room temperature containing 5% skim-milk is closed, shaking table vibration 60rpm, 1h.TBST washs 3 times, and vibrate 60rpm, 10min at every turn.Just PARP primary antibodie press 1:1000 dilution proportion in containing 5ml 5% skim-milk TBST solution in, evenly add fill Nitrocellulose film canoe in, 4 DEG C, vibration, 50rpm, spends the night.TBST washs 3 times.Add horseradish peroxidase-labeled against murine two anti-are diluted in the TBST solution containing 5% skim-milk by 1:5000, shaken at room temperature, 60rpm, 1h.TBST washs 3 times.Get the ratio mixing of ChemiluminescentSubstrate colouring reagents two kinds of substrate 1:1, drop on Nitrocellulose film, room temperature lucifuge hatches 5min.Draw unnecessary reaction solution, preservative film is wrapped, and puts into X-ray folder.In darkroom, X-ray sensitive film is put on film, shut magazine, expose about 1min, development.
9. add up
Result represents with median ± standard deviation, adopts the analysis of SPSS 17.0 statistical software, and many group means compare with one-way analysis of variance, and two groups of means compare with t inspection, and P<0.05 has been considered as statistical significance.
Two, result
1. the result of the plasmid Function detection containing TCS
According to known TCS gene order SEQ ID NO.22, its sequence is synthesized.Utilize the technology of molecular cloning that TCS gene fragment is put into pAAV-CMV-IRES-hrGFP carrier.Adopt Western Blot method, the expression of Flag-Tag albumen whether is had after observing plasmid pAAV-CMVp-TCS-Flag-IRES-hrGFP transfecting hepatoma cells Huh7-FLuc 72h, the plasmid amount that wherein during plasmid pAAV-CMVp-TCS-Flag-IRES-hrGFP transfection, transfection is different respectively, is respectively 1/2,1/10 of standard plasmid transfection amount.Result display is compared with control plasmid pAAV-CMVp-IRES-hrGFP, after TCS plasmid transfection Huh7-FLuc containing Flag, Flag-Tag albumen has expression, and Flag-Tag expressing quantity raises along with the rising of transfection amount, points out and can use plasmid pAAV-CMVp-TCS-Flag-IRES-hrGFP in follow-up experiment.See Fig. 1.
The external impact of 2.TCS Hepatocarcinoma Cells
After plasmid pAAV-CMVp-TCS-Flag-IRES-hrGFP transfection Huh7-FLuc cell 24h, 48h, 72h, detect cell proliferative conditions.Huh7-FLuc cell is after giving plasmid pAAV-CMVp-TCS-Flag-IRES-hrGFP transfection 24h, 48h, 72h, number of viable cells has remarkable minimizing compared with control plasmid pAAV-CMVp-IRES-hrGFP, and cell number slippage raises along with the rising of plasmid transfection amount, prompting TCS albumen has restraining effect to Huh7-FLuc cell.After control plasmid pAAV-CMVp-IRES-hrGFP transfection Huh7-FLuc cell 72h, the expression of hrGFP in cell.See Fig. 2.
Plasmid pAAV-CMVp-TCS-Flag-IRES-hrGFP, pAAV-CMVp-TCS-IRES-hrGFP be transfecting hepatoma cells Huh7, Huh7-FLuc, LH-86 respectively.After 2 plasmid transfection Huh7 cells 24h, 48h, 72h, detect cell proliferative conditions.Huh7, Huh7-FLuc, LH-86 cell is after giving 2 plasmid transfections 24h, 48h, 72h, number of viable cells has remarkable minimizing compared with control plasmid pAAV-CMVp-IRES-hrGFP, and the TCS plasmid lethal effect that the TCS plasmid ratio not containing Flag-Tag contains Flag-Tag is stronger.Prompting TCS albumen all has restraining effect to Huh7, Huh7-FLuc, LH-86 cell.The expression of GFP in cell is detected after control plasmid pAAV-CMVp-IRES-hrGFP transfection Huh7 cell 72h.See Fig. 3,4,5.
TCS plasmid transfection is contained to the inhibited proliferation of human liver cancer cell under 2 kinds of different promoters.After 4 plasmid transfection Huh7-FLuc cells 48h, 72h, detect number of viable cells in cell.Huh7-FLuc cell is after giving plasmid transfection 48h, 72h containing TCS, and number of viable cells has remarkable minimizing compared with control plasmid pdsAAV-CB-EGFP, pdsAAV-AFPp-EGFP, and prompting TCS albumen has restraining effect to Huh7-FLuc cell.After control plasmid pdsAAV-CB-EGFP transfection Huh7-FLuc cell 72h, the expression of EGFP in cell.Due to the liver cancer cell that Huh7-FLuc is the AFP positive, when giving plasmid pdsAAV-AFPp-EGFP transfection, the expression amount of EGFP is more than HEK293.See Fig. 6,7,8,9.
The present invention in order to clear and definite 4 plasmids to normal cell with or without lethal effect, carry out transfected HEK 293 with 4 plasmids.After 4 plasmid transfection HEK293 cells 48h, 72h, detect cell proliferative conditions.HEK293 cell is after giving plasmid pdsAAV-CBAp-TCS-Flag, pdsAAV-CBAp-TCS, pdsAAV-AFPp-TCS-Flag, pdsAAV-AFPp-TCS transfection 48h, 72h respectively, number of viable cells is without remarkable minimizing compared with control plasmid pdsAAV-CBAp-EGFP, pdsAAV-AFPp-EGFP, points out TCS albumen to HEK293 cell without lethal effect.After control plasmid pdsAAV-CBAp-EGFP transfected HEK 293 72h, the expression of EGFP in cell.See Figure 10,11,12,13.
Comprehensive above plasmid transfection result, we find that TCS has lethal effect to liver cancer cell Huh7-FLuc, and to HEK293 cell, then lethal effect is very little.
Impact in the body of 3.TCS Hepatocarcinoma Cells
Containing the restraining effect of TCS plasmid transfection to human liver cancer cell plantation subcutaneous tumors.After plasmid transfection liver cancer cell Huh7-FLuc containing TCS, subcutaneous implantation enters mouse oxter, and along with the prolongation of detection time, the expression amount giving the mouse FLuc of subcutaneous vaccination plasmid pAAV-CMVp-TCS-Flag-IRES-hrGFP declines.TCS albumen is in vivo pointed out to have lethal effect to liver cancer cell Huh7-FLuc.See Figure 14.
Virus intra-tumoral injection contains the virus of TCS to the restraining effect of mouse subcutaneous tumors.Along with the prolongation of detection time after intra-tumoral injection adeno-associated virus rAAV3-2M-AFPp-TCS, compared with the viral rAAV3-2M-AFPp-EGFP of contrast, the expression amount giving the mouse FLuc of injecting virus rAAV3-2M-AFPp-TCS declines.TCS albumen is in vivo pointed out to have lethal effect to liver cancer cell Huh7-FLuc.See Figure 15.
Plasmid transfection containing TCS is on the impact of human liver cancer cell PARP protein expression.Adopt WesternBlot method, the expression of PARP albumen after observation plasmid pAAV-CMVp-TCS-Flag-IRES-hrGFP transfecting hepatoma cells Huh7, HepG248h.Result display has expression containing cleaved PARP after TCS plasmid transfection Huh7, HepG2 cell of Flag-Tag, and prompting Trichosanthin can induce liver cancer cell Huh7 apoptosis.See Figure 16.
4. the result of the plasmid Function detection containing coTCS
According to the scheme of the TCS gene codon optimization that Life Technologies company provides, the present invention successfully constructs the expression plasmid containing coTCS.CoTCS in plasmid has the arbitrary described nucleotide sequence of SEQ ID NO.1-SEQ IDNO.21.
See Figure 17, the coTCS described in Figure 17 has the nucleotide sequence described in SEQ ID NO.21, and wherein ATG is initiator codon, and TGA is terminator codon.
Adopt Western Blot method, after observing plasmid pAAV-CMVp-coTCS-Flag-IRES-hrGFP transfection HeLa and HEK293 cell 16h, 24h, whether have the expression of Flag-Tag albumen.Result display is compared with control plasmid pAAV-CMVp-TCS-IRES-hrGFP, and after plasmid contains the coTCS plasmid transfection of Flag-Tag, Flag-Tag albumen has expression, and Flag-Tag expressing quantity raises along with the prolongation of time.The coTCS plasmid coTCS containing Flag-Tag pointing out us to build can normal expression.See Figure 18.
TCS belongs to I type Single Chain Ribosome-Inactivating Proteins, and in order to verify that coTCS has the function of ribosome inactivating protein, the present invention is by plasmid pAAV-CMVp-IRES-hrGFP, pAAV-CMVp-coTCS-IRES-hrGFP, pdsAAV-CB-Gluc transfection HeLa cell.After plasmid transfection HeLa cell 48h, detect the expression of cell Gluc.HeLa cell is giving the plasmid containing coTCS together with the plasmid containing Gluc after transfection 48h, and gives pAAV-CMVp-IRES-hrGFP and to compare Gluc with the plasmid containing Gluc together transfection and express and significantly reduce.Prompting coTCS has ribosome inactivating protein function, can the synthesis of T suppression cell Gluc.See Figure 19.
The external impact of 5.coTCS Gene on Tumor Cells
After plasmid pAAV-CMVp-coTCS-Flag-IRES-hrGFP transfecting hepatoma cells Huh7, HepG2 cell and cervical cancer cell HeLa cell 48h, detect each cell proliferative conditions.Huh7, HepG2 cell and HeLa cell are after giving the plasmid transfection 48h containing coTCS, number of viable cells has remarkable minimizing compared with control plasmid pAAV-CMVp-TCS-IRES-hrGFP, prompting coTCS albumen all has restraining effect to Huh7, HepG2, HeLa cell, and coTCS is stronger than general T CS albumen restraining effect to the restraining effect of tumour cell.See Figure 20.
TCS as a kind of heterologous protein of plant origin, its gene codon through optimization after, then by virus or non-virus carrier administration, can in mammalian cell higher level marking protein, to play larger biological action.The present invention, by drug administration carrier optimization, improves it in the intracellular transgene expression level of mammalian cell, plays maximum biological function, such as antitumor action.This is by significant to the research and extension of therapy of tumor.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.
Claims (10)
1. codon optimized Gene Encoding Trichosanthin is preparing the application in gene therapy medicament.
2. application according to claim 1, is characterized in that, described gene therapy is the gene therapy to tumour.
3. application according to claim 2, is characterized in that, described tumour is liver cancer or cervical cancer.
4. application according to claim 1, is characterized in that, described codon optimized Gene Encoding Trichosanthin is the Gene Encoding Trichosanthin comprising the mammalian sources such as codon humanization, dog source, cat source.
5. application according to claim 1, is characterized in that, described gene therapy be carry codon optimized Gene Encoding Trichosanthin adeno-associated virus for carrier is to the gene therapy of tumour.
6. application according to claim 1, is characterized in that, described gene therapy be carry codon optimized Gene Encoding Trichosanthin plasmid for carrier is to the gene therapy of tumour.
7. application according to claim 1, is characterized in that, described codon optimized Gene Encoding Trichosanthin has the arbitrary described nucleotide sequence of SEQ ID NO.1-SEQ ID NO.21.
8. a codon optimized Gene Encoding Trichosanthin, is characterized in that, described codon optimized Gene Encoding Trichosanthin has the arbitrary described nucleotide sequence of SEQ ID NO.1-SEQ ID NO.21.
9. carry virus or the non-virus carrier of codon optimized Gene Encoding Trichosanthin according to claim 8.
10. carrier according to claim 9, is characterized in that, described virus is adeno-associated virus, and described non-virus carrier is plasmid.
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| CN112824528A (en) * | 2019-11-20 | 2021-05-21 | 成都医学院 | Alpha-charantin gene and overexpression method thereof in mammalian cells |
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