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CN107794265A - The purposes and its related drugs of CPA4 genes - Google Patents

The purposes and its related drugs of CPA4 genes Download PDF

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Publication number
CN107794265A
CN107794265A CN201610784576.0A CN201610784576A CN107794265A CN 107794265 A CN107794265 A CN 107794265A CN 201610784576 A CN201610784576 A CN 201610784576A CN 107794265 A CN107794265 A CN 107794265A
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cpa4
genes
plko
slow virus
sequence
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谭畅
陈琳
王庆亮
沈浩
瞿红花
曹跃琼
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SHANGHAI GENECHEM CO Ltd
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SHANGHAI GENECHEM CO Ltd
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Abstract

The invention discloses the purposes and its related drugs of people's CPA4 genes.The invention discloses purposes of the people CPA4 genes in anti-tumor medicine preparation.The present invention also further constructs people's CPA4 genes small molecules interference RNA, people's CPA4 gene RNAs construct, people CPA4 genes interference slow virus and discloses their purposes.SiRNA provided by the invention or nucleic acid construct comprising the siRNA sequence, slow virus are capable of the expression that specificity suppresses people's CPA4 genes, especially slow virus, target cell can efficiently be infected, expeditiously suppress the expression of CPA4 genes in target cell, and then suppress the growth of tumour cell, promote apoptosis of tumor cells, it is significant in oncotherapy.

Description

The purposes and its related drugs of CPA4 genes
Technical field
The present invention relates to biological technical field, relate more specifically to the purposes and its related drugs of CPA4 genes.
Background technology
The short double-stranded RNA (dsRNA) that RNA interference (RNA interference, RNAi) is formed with nucleotides is carried out PTGS.It can efficiently, specifically block the expression of internal specific gene, cause its degraded, so as to cause biology The silence of internal specific gene, makes cells show go out the missing of certain gene phenotype, is emerging in recent years a kind of conventional grind Study carefully gene function, find the laboratory technique of methods for the treatment of diseases.Research shows, the double-stranded RNA that length is 21-23nt can be Transcription and post-transcriptional level is specific causes RNAi (Tuschl T, Zamore PD, Sharp PA, Bartel DP.RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21to 23nucleotide intervals.Cell 2000；101:25-33.).Though tumor patient through chemotherapy, radiotherapy and complex treatment, To be able to be that the treatment of tumour is opened if the gene relevant with progress to tumor invasion is intervened but five year survival rate is still very low Ward off new way.In recent years, RNAi has turned into the available strategy of the gene therapy of tumour.It can suppress former cancer base using RNAi technology Suppress tumor progression because of, the expression of the tumor suppressor gene of mutation, Cell cycle-related genes, anti-apoptotic related gene etc. (Uprichard,Susan L.The therapeutic potential of RNA interference.FEBS Letters 2005；579:5996-6007.).
Carboxypeptidase A 4 (CPA4, Carboxypeptidase A4), it is one of Carboxypeptidase A/BYA subfamily members.
But effects of the CPA4 in the cancer of the esophagus has no report.
The content of the invention
It is an object of the invention to open treatment method and medicine with people's CPA4 gene-correlations, disturbed (RNAi) with RNA For effect of the means research CPA4 genes in the survival and apoptotic process of tumour cell.
First aspect present invention, using RNA interference as means, it have studied work of the CPA4 genes in tumour occurrence and development With disclosing a kind of suppression or reduction growth of tumour cell, propagation, differentiation and/or the method for survival, this method includes：To swollen Oncocyte applies a kind of table be capable of the transcription or translation of specificity suppression CPA4 genes, or be capable of specificity suppression CPA4 albumen Reach or the molecule of activity, the growth of tumour cell, propagation, differentiation and/or survival are suppressed with this.
The tumour cell is selected from the expression of its growth and CPA4 albumen or the tumour cell that activity is related.Preferably, institute State tumour cell and be selected from the cancer of the esophagus.
In suppression or the reduction growth of tumour cell, propagation, differentiation and/or the method for survival, the administration of the molecule Measure to reduce the transcription or translation of CPA4 genes enough, or reduce the expression of CPA4 albumen or the dosage of activity enough.Enter one Step, the expression of the CPA4 genes is at least lowered 50%, 80%, 90%, 95% or 99%.
The molecule may be selected from but be not limited to：Nucleic acid molecules, carbohydrate, lipid, small-molecule chemical medicine, antibody medicine, Polypeptide, albumen or interference slow virus.
The nucleic acid includes but is not limited to：ASON, double-stranded RNA (dsRNA), ribozyme, endoribonuclease SiRNA (esiRNA) or short hairpin RNA (shRNA) prepared by III.
The double-stranded RNA, ribozyme, esiRNA or shRNA contain the promoter sequence or CPA4 genes of CPA4 genes Information sequence.
Further, the double-stranded RNA is siRNA (siRNA).The siRNA includes the first chain and second Chain, first chain and the second chain complementation are collectively forming RNA dimers, and the sequence of first chain and CPA4 genes Middle 15-27 continuous nucleotide sequences are essentially identical.The small molecules interference RNA can be specifically bound coded by target sequence MRNA fragments, and the expression of specific silence people CPA4 genes.
Further, the first chain-ordering of the siRNA and the target sequence in CPA4 genes are essentially identical.It is more excellent , the target sequence in the CPA4 genes contains SEQ ID NO:Sequence shown in 1.
When target sequence in the CPA4 genes is the small molecules interference RNA specificity silence CPA4 gene expressions, With described small molecules interference RNA it is complementary with reference to mRNA fragments corresponding to CPA4 genes in fragment.
Preferably, the CPA4 gene sources are in people.
First aspect present invention also discloses a kind of people CPA4 genes of separation in preparing or screening anti-tumor medicine Purposes.
Further, the tumour is selected from the cancer of the esophagus.
The CPA4 genes by separation include both sides content for preparing or screening anti-tumor medicine：First, It is applied to prepare anti-tumor medicine or preparation for the action target of tumour cell using CPA4 genes as medicine or preparation；Its Two, it is applied to screening anti-tumor medicine or system for the action target of tumour cell using CPA4 genes as medicine or preparation Agent.
It is described to be applied to prepare oncotherapy for the action target of tumour cell using CPA4 genes as medicine or preparation Medicine or preparation specifically refer to：Target using CPA4 genes as RNA interference effects, come develop for tumour cell medicine or Preparation, so as to reduce the expression of CPA4 genes in tumour cell.
It is described to be applied to screening oncotherapy for the action target of tumour cell using CPA4 genes as medicine or preparation Medicine or preparation specifically refer to：Using CPA4 genes as effective object, medicine or preparation are screened, can be suppressed with finding Or promote the medicine of people's CPA4 gene expressions as oncotherapy drug candidate.CPA4 gene small molecules as described in the present invention are done It is to be obtained by effective object screening of people CPA4 genes to disturb RNA (siRNA), can be used as that there is suppression tumor cell proliferation to make Medicine.In addition, such as antibody drug, small-molecule drug etc. also can be using CPA4 genes and its albumen as effect pair As.
The anti-tumor medicine is to be capable of transcription or translation that specificity suppresses CPA4 genes, or being capable of specificity suppression The expression of CPA4 albumen or the molecule of activity, so as to reduce the expression of CPA4 genes in tumour cell, reach suppression tumour Propagation, growth, differentiation and/or the purpose of survival of cell.
The anti-tumor medicine or diagnosing tumor medicine bag that prepared by the CPA4 genes by separation or screening obtains Include but be not limited to：Nucleic acid molecules, carbohydrate, lipid, small-molecule chemical medicine, antibody medicine, polypeptide, albumen or the slow disease of interference Poison.
The nucleic acid includes but is not limited to：ASON, double-stranded RNA (dsRNA), ribozyme, endoribonuclease SiRNA (esiRNA) or short hairpin RNA (shRNA) prepared by III.
The amount of application of the anti-tumor medicine is enough transcriptions or translation for reducing people's CPA4 genes, or is reduced enough The expression of people's CPA4 albumen or the dosage of activity.At least be lowered 50% to make one the expression of CPA4 genes, 80%, 90%, 95% or 99%.
Using the method for forgoing neoplasms medicine treatment tumour, the mainly expression by reducing people's CPA4 genes Suppress breeding to reach the purpose for the treatment of for tumour cell.Specifically, during treatment, people's CPA4 gene expression water can be effectively reduced Flat administering substances are in patient.
Second aspect of the present invention discloses a kind of nucleic acid molecules for reducing the separation of CPA4 gene expressions in tumour cell, institute Nucleic acid molecules are stated to include：
A) double-stranded RNA, in the double-stranded RNA containing can be under stringent condition with CPA4 gene recombinations nucleotides sequence Row；Or
B) nucleotide sequence that can be under stringent condition with CPA4 gene recombinations is contained in shRNA, the shRNA.
Further, the double-stranded RNA includes the first chain and the second chain, and first chain and second chain are complementary common Form RNA dimers, and the sequence of first chain and 15-27 in the CPA4 genes basic phases of continuous nucleotide sequence Together.Preferably, the sequence of first chain and 19-23 in CPA4 genes continuous nucleotide sequences are essentially identical；More preferably, The sequence of first chain and 19,20 or 21 in CPA4 genes continuous nucleotide sequences are essentially identical.
Further, the double-stranded RNA includes the first chain and the second chain, and first chain and second chain are complementary altogether With formation RNA dimers, and the sequence of first chain and the target sequence in CPA4 genes are essentially identical.
The length of the chain of double-stranded RNA first and the second chain is 15-27 nucleotides；Preferably, length is 19-23 Individual nucleotides；Optimal, length is 19,20 or 21 nucleotides.
Further, the double-stranded RNA is siRNA (siRNA).Further, the chain of siRNA first Sequence such as SEQ ID NO：Shown in 13, specially 5 '-gcuuagaguacgcagugacaa-3 '.
SEQ ID NO：SiRNA shown in 13 is with SEQ ID NO:Sequence shown in 1 disturbs target sequence design for RNA , a chain of siRNA for people's CPA4 genes, another chain be the second chain sequence and the first chain-ordering it is complementary, The siRNA can play a part of endogenous CPA4 gene expressions in specific silence tumour cell.
Further, the shRNA includes positive-sense strand fragment and antisense strand fragment, and the connection positive-sense strand fragment and The sequence of the loop-stem structure of antisense strand fragment, the positive-sense strand fragment and the antisense strand fragment is complementary, and the positive-sense strand The sequence of fragment and 15-27 in CPA4 genes continuous nucleotide sequences are essentially identical.It can turn into after the shRNA is processed SiRNA (siRNA) and then play a part of endogenous CPA4 gene expressions in specific silence tumour cell.
Further, the shRNA includes positive-sense strand fragment and antisense strand fragment, and the connection positive-sense strand fragment and The sequence of the loop-stem structure of antisense strand fragment, the positive-sense strand fragment and the antisense strand fragment is complementary, and the positive-sense strand The sequence of fragment and the target sequence in CPA4 genes are essentially identical.
Preferably, the positive-sense strand fragment and 19-23 in CPA4 genes continuous nucleotide sequences are essentially identical；More preferably , the positive-sense strand fragment and 19,20 or 21 in CPA4 genes continuous nucleotide sequences are essentially identical.
Further, the sequence of the loop-stem structure of the shRNA may be selected from following any：UUCAAGAGA、AUG、CCC、 UUCG, CCACC, CUCGAG, AAGCUU and CCACACC.
Further, the sequence of the shRNA such as SEQ ID NO:Shown in 14, it is specially：5’- gcuuagaguacgcagugacaauucaagagauugucacugcguacucuaagc-3’。
ShRNA can turn into siRNA after digestion is processed, and then play endogenous people CPA4 in specific silence tumour cell The effect of gene expression.
The interference slow virus carrier for encoding shRNA of the present invention genetic fragment contains SEQ ID NO:Sequence shown in 1 And its complementary series.
First chain of the double-stranded RNA or the positive-sense strand fragment of the shRNA and the basic phase of target sequence in CPA4 genes Together, when the target sequence of the CPA4 genes is that siRNA is used for specific silence CPA4 gene expressions, identified by the siRNA And the fragment in the CPA4 genes corresponding to the mRNA fragments of silence.
Preferably, the target sequence in the CPA4 genes contains SEQ ID NO:Sequence shown in 1.
Further, the CPA4 gene sources are in people.
Third aspect present invention, disclose a kind of CPA4 genes RNA construct, containing coding of the present invention point From nucleic acid molecules in shRNA genetic fragment, the shRNA can be expressed.
Described people's CPA4 gene RNA constructs can will encode foregoing people CPA4 genes shRNA gene piece Section is cloned into known carrier acquisition.Further, the CPA4 genes RNA construct is that CPA4 genes disturb slow virus Carrier.
The CPA4 genes interference slow virus carrier of the present invention is to clone the DNA fragmentation for encoding foregoing CPA4 genes shRNA Enter known carrier acquisition, the known carrier is mostly slow virus carrier, and the CPA4 genes interference slow virus carrier is by virus After packaging turns into infectious virion, infected tumor's cell, and then shRNA of the present invention is transcribed out, pass through digestion The steps such as processing, finally obtain the siRNA, the expression for specific silence CPA4 genes.
Further, the CPA4 genes interference slow virus carrier also contains promoter sequence and/or codes for tumor cell In the nucleotide sequence of label that can be detected；Preferably, the label the being detected such as green fluorescent protein (GFP)。
Further, the slow virus carrier can be selected from：pLKO.1-puro、pLKO.1-CMV-tGFP、pLKO.1- puro-CMV-tGFP、pLKO.1-CMV-Neo、pLKO.1-Neo、pLKO.1-Neo-CMV-tGFP、pLKO.1-puro-CMV- TagCFP、pLKO.1-puro-CMV-TagYFP、pLKO.1-puro-CMV-TagRFP、pLKO.1-puro-CMV- TagFP635、pLKO.1-puro-UbC-TurboGFP、pLKO.1-puro-UbC-TagFP635、pLKO-puro-IPTG- 1xLacO、pLKO-puro-IPTG-3xLacO、pLP1、pLP2、pLP/VSV-G、pENTR/U6、pLenti6/BLOCK-iT- DEST、pLenti6-GW/U6-laminshrna、pcDNA1.2/V5-GW/lacZ、pLenti6.2/N-Lumio/V5-DEST、 Any in pGCSIL-GFP or pLenti6.2/N-Lumio/V5-GW/lacZ.
The people CPA4 genes that the embodiment of the present invention specifically lists using pGCSIL-GFP as vector construction disturb slow virus to carry Body, it is named as pGCSIL-GFP-CPA4-siRNA.
The nucleic acid molecules that the present invention separates can be used for the medicine for preparing prevention or treatment tumour, and the tumour is the cancer of the esophagus.
The CPA4 gene siRNAs of the present invention can be used for the propagation for suppressing tumour cell, and it is swollen further to may be used as treatment The medicine or preparation of knurl.CPA4 genes interference slow virus carrier then can be used for preparing the CPA4 gene siRNAs.When as treatment It is that the nucleic acid molecules of safe and effective amount are applied to mammal when the medicine or preparation of tumour.Specific dosage is also taken an examination Method of administration, the factor such as patient health situation are considered, within the scope of these are all skilled practitioners technical ability.
Fourth aspect present invention, a kind of CPA4 genes interference slow virus is disclosed, slow virus is disturbed by foregoing CPA4 genes Carrier slow virus packaging plasmid, cell line auxiliary under, by virus packaging form.The slow virus can infected tumor's cell simultaneously The small molecules interference RNA for CPA4 genes is produced, so as to suppress the propagation of cancer of the esophagus tumour cell.CPA4 genes interference is slow Virus can be used for the medicine for preparing prevention or treatment tumour.
Fifth aspect present invention, discloses a kind of pharmaceutical composition for being used to preventing or treating tumour, and its active principle contains There are the nucleic acid molecules of foregoing separation, CPA4 gene RNA constructs or CPA4 genes disturb one kind or more in slow virus The combination of kind.
Further, described pharmaceutical composition contains double-stranded RNA described in 1~99wt%, shRNA, CPA4 gene interference core Acid con-struct or CPA4 genes interference slow virus, and pharmaceutically acceptable carrier, diluent or excipient.
When preparing these compositions, generally active component is mixed with excipient, or with figuration dilution agent, or Bao Ke With in carrier existing for capsule or sachet.When excipient plays diluent, it can be solid, semisolid or liquid Medium of the material as excipient, carrier or active component.Therefore, composition can be tablet, pill, pulvis, solution, sugar Starch agent, sterilizing injecting solution etc..The example of suitable excipient includes:Lactose, glucose, sucrose, sorbierite, mannitol, shallow lake Powder, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, etc..Preparation may also include:Wetting agent, emulsifying agent, preservative (such as methyl hydroxybenzoate and propyl ester), sweetener.
The invention also discloses application of the described pharmaceutical composition in the anti-tumor medicine for preparing the treatment cancer of the esophagus.
The application of the pharmaceutical composition provides a method that for the treatment of tumour, is specially a kind of prevention or treatment target The method of in-vivo tumour, including the described pharmaceutical composition of effective dose is applied in object.Further, the tumour Selected from the cancer of the esophagus.
Described pharmaceutical composition is used to prevent or during treatment target in-vivo tumour, it is necessary to described medicine by effective dose Composition is applied in object.Using this method, growth, propagation, recurrence and/or the transfer of the tumour are suppressed.Further , the growth of the tumour, propagation, at least the 10% of recurrence and/or transfer, 20%, 30%, 40%, 50%, 60%, 70%, 80%th, 90%, 95% or 99% part is suppressed.
The object of methods described can be people.
Sixth aspect present invention, disclose a kind of kit for being used to reduce the CPA4 gene expressions in tumour cell, institute Stating kit includes：The nucleic acid molecules for the separation being present in container, CPA4 gene RNA constructs, and/or institute The CPA4 genes interference slow virus stated.
In summary, the present invention devises the RNAi target sequences for people's CPA4 genes, builds corresponding CPA4RNAi Carrier, wherein coded sequence SEQ ID NO：1 RNAi carrier pGCSIL-GFP-CPA4-siRNA can significantly lower CPA4 bases Because of the expression in mRNA level in-site and protein level.Genetic manipulation instrument is used as using slow virus (lentivirus, being abbreviated as Lv) Carry RNAi carrier pGCSIL-GFP-CPA4-siRNA and the RNAi sequences for CPA4 genes are efficiently imported into people with can targetting Esophageal cancer cell, the expression of CPA4 genes is reduced, significantly inhibit the multiplication capacity of above-mentioned tumour cell.Therefore slow virus is situated between The CPA4 gene silencings led are malignant tumour potentially clinical non-operative treatment modes.
SiRNA provided by the invention or nucleic acid construct comprising the siRNA sequence, slow virus can specificity suppress The expression of people's CPA4 genes, especially slow virus, can efficiently infect target cell, expeditiously suppress CPA4 genes in target cell Expression, promote Apoptosis, the invasion and attack for reducing tumour cell and transfer ability etc., and then suppress the growth of tumour cell, promote Enter apoptosis of tumor cells, it is significant in oncotherapy.
Brief description of the drawings
Fig. 1：PGCSIL-GFP Plasmid diagrams.
Fig. 2：CPA4-RNAi slow virus infected human esophagus cancer TE-1 cells after 5 days, and CPA4mRNA expression significantly drops It is low.
Fig. 3：CPA4-RNAi slow virus infected human esophagus cancer TE-1 cells after 5 days, caused cell inhibitory effect.
Embodiment
The present inventor is by in-depth study discovery extensively, in human esophagus cancer tumor tissues, CPA4 genes Significantly high expression.Inventor has found, can effectively suppress tumour cell after the expression using CPA4 genes of being transferred person under RNAi methods Propagation, the growth course of tumour can be efficiently controlled, this achievement in research shows that CPA4 genes are proto-oncogenes, can conduct The target spot of oncotherapy.Inventor further synthesizes and tested a variety of small molecules interference RNAs (siRNA) for CPA4 genes Sequence, rna interference vector and RNA interference slow virus.Choose target site of the people CPA4mRNA coding region sequences as siRNA, root According to the individual base sequence design siRNA target sequences of continuous 10-30 (preferably 15-27, more preferably 19-23) in target site.Pass through Gene cloning, the above-mentioned siRNA of construction expression nucleic acid construct, the above-mentioned siRNA of packaging expression slow virus.Cell experiment is demonstrate,proved It is bright, above-mentioned siRNA sequence can in specific silence human tumor cells endogenous CPA4 genes expression.So as to which having filtered out can Effectively suppress CPA4 expression and then suppress the siRNA of human esophagus cancer TE-1 cells propagation and growth, complete on this basis The present invention.
The present invention is expanded on further with reference to embodiment.It should be understood that embodiment is merely to illustrate the present invention, and it is unrestricted The scope of the present invention.The reagent of the experimental method of unreceipted actual conditions and undeclared formula is according to conventional strip in embodiment Part, such as [U.S.] Sambrook.J write；Huang Peitang etc. is translated.Molecular cloning texts guide, the third edition.Beijing：Science Press The condition that condition or manufacturer described in 2002 are suggested carries out or configuration.
Embodiment 1 is directed to the preparation of people CPA4 gene RNAi slow virus
1. screening is for the effective siRNA target spots of people's CPA4 genes
CPA4 (NM_016352) gene information is transferred from Genbank；Effective siRNA target of the design for CPA4 genes Point.Table 1 lists the effective siRNA target sequences for being wherein directed to CPA4 genes.
Table 1 targets the siRNA target sequences of people's CPA4 genes
SEQ ID NO Target Seq
1 gcttagagtacgcagtgacaa
2. the preparation of slow virus carrier
For siRNA target spots (with SEQ ID NO：Exemplified by 1) synthesis both ends I containing Age and EcoR I restriction enzyme site cohesive ends Double-stranded DNA Oligo sequences (table 2)；PGCSIL-GFP carriers (Shang Haiji is acted on Age I and EcoR I restriction enzymes Triumphant chemical gene Technology Co., Ltd. provides, Fig. 1), make its linearisation, agarose gel electrophoresis identification endonuclease bamhi.
The double-stranded DNA Oligo of both ends I containing Age and EcoR the I restriction enzyme site cohesive ends of table 2
Double digestion is linearized to the carrier DNA of (digestion system is as shown in table 4,37 DEG C, reacts 1h) by T4DNA ligases Connect with purified double-stranded DNA Oligo, connected in appropriate buffer system (linked system is as shown in table 5) in 16 DEG C At night, reclaim connection product.Fresh competent escherichia coli cell prepared by connection product conversion calcium chloride (is joined by conversion operation Examine：55-56 pages of the Molecular Cloning:A Laboratory guide second edition).Bacterium clone surface is grown in connection converted product to be stained with, is dissolved in 10 μ l LB culture mediums, mixing take 1 μ l as template；The upstream and downstream of RNAi sequences in slow virus carrier, designs general PCR primer, Upstream primer sequence：5’-CCTATTTCCCATGATTCCTTCATA-3’(SEQ ID NO：6)；Downstream primer sequence：5’- GTAATACGGTTATCCACGCG-3’(SEQ ID NO：7) performing PCR identification experiment (PCR reaction systems such as table 6-1, reaction, are entered Condition such as table 6-2).The clone positive to PCR identifications is sequenced and compared analysis, and it is to successfully construct to compare correctly clone Be directed to SEQ ID NO：1 expression RNAi carrier, is named as pGCSIL-GFP-CPA4-siRNA.
Build pGCSIL-GFP-Scr-siRNA negative control plasmids, negative control siRNA target sequences be 5 '- TTCTCCGAACGTGTCACGT-3’(SEQ ID NO：8).When building pGCSIL-GFP-Scr-siRNA negative control plasmids, pin The double-stranded DNA Oligo sequences (table 3) of both ends I containing Age and EcoR I restriction enzyme site cohesive ends are synthesized to Scr siRNA target spots, its The same pGCSIL-GFP-CPA4-siRNA of remaining construction method, authentication method and condition.
The double-stranded DNA Oligo of both ends I containing Age and EcoR the I restriction enzyme site cohesive ends of table 3
Double digestion is linearized to the carrier of (digestion system is as shown in table 4,37 DEG C, reacts 1h) by T4DNA ligases
Table 4pGCSIL-GFP plasmid enzyme restriction reaction systems
Reagent Volume (μ l)
PGCSIL-GFP plasmids (1 μ g/ μ l) 2.0
10×buffer 5.0
100×BSA 0.5
Age I(10U/μl) 1.0
EcoR I(10U/μl) 1.0
dd H2O 40.5
Total 50.0
The carrier DNA of table 5 and double-stranded DNA Oligo coupled reaction systems
Reagent Positive control (μ l) From even control (μ l) Connection group (μ l)
The carrier DNA (100ng/ μ l) of linearisation 1.0 1.0 1.0
The double-stranded DNA Oligo (100ng/ μ l) of annealing 1.0 - 1.0
10 × T4 phage DNA ligase buffer solutions 1.0 1.0 1.0
T4 phage DNA ligases 1.0 1.0 1.0
dd H2O 16.0 17.0 16.0
Total 20.0 20.0 20.0
Table 6-1PCR reaction systems
Table 6-2PCR reaction system program settings
3. pack CPA4-siRNA slow virus
With the plasmid extraction kit extraction RNAi plasmids pGCSIL-GFP-CPA4-siRNA of Qiagen companies DNA, match somebody with somebody 100ng/ μ l storing liquids are made.
24h before transfection, with the human embryonic kidney cells 293T cells of Trypsin Induced exponential phase, with containing 10% tire ox blood Clear DMEM complete mediums adjustment cell density is 1.5 × 105Cell/ml, is inoculated in 6 orifice plates, 37 DEG C, 5%CO2Incubator Interior culture.It can be used to transfect when cell density reaches 70%-80%.2h before transfection, original culture medium is suctioned out, add 1.5ml Fresh complete medium.According to the MISSION Lentiviral Packaging Mix reagents of Sigma-aldrich companies The explanation of box, the μ l of Packing Mix (PVM) 20 μ l, PEI 12, plasma-free DMEM medium are added into a sterile centrifugation tube 400 μ l, the DNA of the 20 above-mentioned extractings of μ l is taken, adds to above-mentioned PVM/PEI/DMEM mixed liquors.
Above-mentioned transfection mixture is incubated 15min at room temperature, is transferred in the culture medium of human embryonic kidney cells 293T cells, 37 DEG C, 5%CO2Culture 16h in incubator.The culture medium containing transfection mixture is discarded, PBS solution washing, is added complete Culture medium 2ml, continue to cultivate 48h.Collect cell supernatant, Centricon Plus-20 centrifugal ultrafiltration units (Millipore) Purifying and concentration slow virus, step are as follows：(1) 4 DEG C, 4000g centrifugation 10min, remove cell fragment；(2) 0.45 μm of filter mistakes Supernatant is filtered in 40ml ultracentrifugation pipes；(3) 4000g is centrifuged, 10-15min, to the viral concentration volume needed；(4) centrifuge After end, filter cup and following filtered solution collection cups are separated, filter cup is tipped upside down on sample collection cup, centrifugation 2min from Mental and physical efforts are no more than 1000g；(5) Centrifuge Cup is removed from sample collection cup, in sample collection cup is viral concentration liquid.Will The packing of viral concentration liquid is after -80 degrees Celsius of preservations.The sequence of the siRNA contained in viral concentration liquid the first chain such as SEQ ID NO:Shown in 13.The packaging process of slow virus is compareed with CPA4-siRNA slow virus, only with pGCSIL-GFP-Scr-siRNA carriers Instead of pGCSIL-GFP-CPA4-siRNA carriers.
The real-time fluorescence quantitative RT-PCR method of embodiment 2 detects the silence efficiency of CPA4 genes
Human esophagus cancer TE-1 cells in exponential phase carry out pancreatin digestion, and cell suspension is made, and (cell number is about 5 ×104/ ml) it is inoculated in 6 orifice plates, cultivate to cell fusion degree and reach about 30%.According to infecting plural number (MOI, TE-1：20) Value, virus prepared by the embodiment 1 of Sq is added, culture medium is changed after cultivating 24h, after time of infection reaches 5 days, is collected Cell.According to the Trizol operational manuals of Invitrogen companies, extracted total RNA.Grasped according to the M-MLV of Promega companies Book is explained, RNA reverse transcriptions are obtained into cDNA, and (reverse transcription reaction system is shown in Table 7,42 DEG C of reaction 1h, then in 70 DEG C of water-baths Middle water-bath 10min inactivates reverse transcriptase).
Real_time quantitative detection is carried out using TP800 type Real time PCR instruments (TAKARA).The primer of CPA4 genes is such as Under：- the AGGTGGATACTGTTCATTGGGG-3 ' of sense primer 5 ' (SEQ ID NO：And anti-sense primer 5 ' 9)- TTGCTGATCTCGTCTCCATTTC-3’(SEQ ID NO：10).Using house-keeping gene GAPDH as internal reference, primer sequence is as follows：On Swim-the TGACTTCAACAGCGACACCCA-3 ' of primer 5 ' (SEQ ID NO：And anti-sense primer 5 ' 11)- CACCCTGTTGCTGTAGCCAAA-3’(SEQ ID NO：12).By the proportional arrangement reaction system in table 8.
The reverse transcription reaction system of table 7
Reagent Volume (μ l)
5×RT buffer 4.0
10mM dNTPs 2.0
RNasin 0.5
M-MLV-RTase 1.0
DEPC H2O 3.5
Total 11.0
Table 8Real-time PCR reaction systems
Reagent Volume (μ l)
SYBR premix ex taq: 10.0
Sense primer (2.5 μM)： 0.5
Anti-sense primer (2.5 μM)： 0.5
cDNA 1.0
ddH2O 8.0
Total 20.0
Setting program is two-step method Real-time PCR：95 DEG C of pre-degeneration, 15s；Each step is denatured 95 DEG C afterwards, 5s；Move back 60 DEG C of fire extension, 30s；45 circulations are carried out altogether.Every time light absorption value is read in the extension stage.After PCR terminates, 95 DEG C of denaturation 1min, 55 DEG C are subsequently cooled to, DNA double chain is fully combined.To 95 DEG C since 55 DEG C, each step increases by 0.5 DEG C, keeps 4s, while light absorption value is read, make melting curve.Using 2-ΔΔCtAnalytic approach calculates the gene expression abundance for having infected CPA4mRNA.Invade The cell of comparison virus (Lv-Scr-siRNA) is contaminated as control.Experimental result (Fig. 2) shows, human esophagus cancer TE-1 cell mRNAs Expression lowered 87.3%.
Embodiment 3 detects the multiplication capacity for the tumour cell for having infected CPA4-siRNA slow virus
Human esophagus cancer TE-1 cells in exponential phase carry out pancreatin digestion, and cell suspension is made, and (cell number is about 5 ×104/ ml) it is inoculated in 6 orifice plates, cultivate to cell fusion degree and reach about 30%.According to infecting plural number (MOI, TE-1:20), The virus of Sq is added, culture medium is changed after cultivating 24h, after time of infection reaches 5 days, is collected in exponential phase Each experimental group cell.Complete medium is resuspended into cell suspension (2 × 104/ ml), it is about 2000/hole with cell density, inoculation 96 orifice plates.Every group of 5 multiple holes, per the μ l of hole 100.After completing plate, 37 DEG C, 5%CO are put2Incubator culture.Second day after bed board Start, it is daily with Cellomics instruments (Thermo Fisher) detection read plate once, continuous to detect read plate 5 days.Pass through adjustment Cellomics arrayscan input parameter, the cell with green fluorescence in scanning orifice plate every time is calculated exactly Quantity, statistics drawing is carried out to data, draw cell growth curve (result such as Fig. 3).As a result show, it is each swollen that slow virus infects group After cell injuring model 5 days, growth rate is significantly slowed knurl, and the multiplication rate of experimental group TE-1 cells is significantly inhibited, Far below the growth rate of control group tumour cell, vigor cell number have dropped 59.5% respectively, show CPA4 gene silencings Cause tumor cell proliferation ability to be suppressed, prompt the multiplication capacity of target gene and TE-1 cells significantly correlated.
It is described above, only presently preferred embodiments of the present invention, it is not any to the present invention in form and substantial limitation, It should be pointed out that for those skilled in the art, on the premise of the inventive method is not departed from, can also make Some improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art, Without departing from the spirit and scope of the present invention, when made using disclosed above technology contents it is a little more Dynamic, modification and the equivalent variations developed, it is the equivalent embodiment of the present invention；Meanwhile all substantial technologicals pair according to the present invention The variation, modification and evolution for any equivalent variations that above-described embodiment is made, still fall within the scope of technical scheme It is interior.

Claims (14)

  1. A kind of 1. purposes of the people CPA4 genes of separation in preparing or screening anti-tumor medicine.
  2. 2. purposes as claimed in claim 1, it is characterised in that the tumour is selected from the cancer of the esophagus.
  3. 3. a kind of nucleic acid molecules for reducing the separation of CPA4 gene expressions in tumour cell, the nucleic acid molecules include：
    A) double-stranded RNA, in the double-stranded RNA containing can be under stringent condition with CPA4 gene recombinations nucleotide sequence；Or Person
    B) nucleotide sequence that can be under stringent condition with CPA4 gene recombinations is contained in shRNA, the shRNA.
  4. 4. the nucleic acid molecules separated as claimed in claim 3, it is characterised in that the double-stranded RNA includes the first chain and second Chain, first chain and the second chain complementation are collectively forming RNA dimers, and the sequence of first chain and CPA4 genes In target sequence it is essentially identical；The shRNA includes positive-sense strand fragment and antisense strand fragment, and the connection positive-sense strand fragment With the loop-stem structure of antisense strand fragment, the sequence of the positive-sense strand fragment and the antisense strand fragment is complementary, and the justice The sequence of chain fragment and the target sequence in CPA4 genes are essentially identical.
  5. 5. the nucleic acid molecules separated as described in claim 3 or 4, it is characterised in that the target sequence of the CPA4 genes contains SEQ ID NO:Sequence shown in 1.
  6. 6. the nucleic acid molecules separated as described in claim 3 or 4, it is characterised in that the double-stranded RNA is siRNA, and this is small The sequence of the chain of RNA interfering first such as SEQ ID NO：Shown in 13.
  7. 7. the nucleic acid molecules separated as described in claim 3 or 4, it is characterised in that the sequence of the shRNA such as SEQ ID NO： Shown in 14.
  8. 8. a kind of CPA4 genes RNA construct, contain the core separated described in coding claim 3-7 any claims The genetic fragment of shRNA in acid molecule, the shRNA can be expressed.
  9. 9. CPA4 genes RNA construct as claimed in claim 8, it is characterised in that the CPA4 genes RNA structure Body is built as interference slow virus carrier.
  10. 10. CPA4 genes RNA construct as claimed in claim 9, it is characterised in that it is described interference slow virus carrier by Obtained after the gene fragment clone for encoding the shRNA is entered into slow virus carrier, the slow virus carrier is selected from：pLKO.1- puro、pLKO.1-CMV-tGFP、pLKO.1-puro-CMV-tGFP、pLKO.1-CMV-Neo、pLKO.1-Neo、pLKO.1- Neo-CMV-tGFP、pLKO.1-puro-CMV-TagCFP、pLKO.1-puro-CMV-TagYFP、pLKO.1-puro-CMV- TagRFP、pLKO.1-puro-CMV-TagFP635、pLKO.1-puro-UbC-TurboGFP、pLKO.1-puro-UbC- TagFP635、pLKO-puro-IPTG-1xLacO、pLKO-puro-IPTG-3xLacO、pLP1、pLP2、pLP/VSV-G、 pENTR/U6、pLenti6/BLOCK-iT-DEST、pLenti6-GW/U6-laminshrna、pcDNA1.2/V5-GW/lacZ、 Any in pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti6.2/N-Lumio/V5-GW/lacZ.
  11. 11. a kind of CPA4 genes disturb slow virus, slow virus carrier is disturbed to exist as described in claim 9-10 any claims Slow virus packaging plasmid, cell line auxiliary under, by virus packaging form.
  12. 12. a kind of pharmaceutical composition for being used to preventing or treating tumour, its active principle contains any rights of claim 3-7 will The nucleic acid molecules of described separation are sought, CPA4 genes RNA construct described in claim 8-10 any claims, and/ Or the CPA4 genes interference slow virus described in claim 11, and pharmaceutically acceptable carrier, diluent or excipient.
  13. 13. application of the pharmaceutical composition as claimed in claim 12 in the anti-tumor medicine for preparing the treatment cancer of the esophagus.
  14. 14. a kind of kit for being used to reduce CPA4 gene expressions in tumour cell, it is characterised in that the kit includes： It is present in container, the nucleic acid molecules of the separation described in claim 3-7 any claims, any power of claim 8-10 Profit requires the CPA4 genes RNA construct, and/or the CPA4 genes interference slow virus described in claim 11.
CN201610784576.0A 2016-08-30 2016-08-30 The purposes and its related drugs of CPA4 genes Pending CN107794265A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040077001A1 (en) * 2002-08-02 2004-04-22 Millennium Pharmaceuticals Inc. Use for carboxypeptidase-A4 in the diagnosis and treatment of metabolic disorders
FR2904223A1 (en) * 2006-07-26 2008-02-01 Oreal Composition, useful to treat and/or prevent scalp/skin disorder e.g. keratinocytes, skin aging, rehydration disorder of skin and abnormal scaling, comprises polypeptide sequence of amino acids encoded by nucleic acid sequence, in medium

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040077001A1 (en) * 2002-08-02 2004-04-22 Millennium Pharmaceuticals Inc. Use for carboxypeptidase-A4 in the diagnosis and treatment of metabolic disorders
FR2904223A1 (en) * 2006-07-26 2008-02-01 Oreal Composition, useful to treat and/or prevent scalp/skin disorder e.g. keratinocytes, skin aging, rehydration disorder of skin and abnormal scaling, comprises polypeptide sequence of amino acids encoded by nucleic acid sequence, in medium

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