CN103555764B - MUC1 (Mucins) and IL-2 double-gene coexpression recombinant vector as well as preparation method and application thereof - Google Patents
MUC1 (Mucins) and IL-2 double-gene coexpression recombinant vector as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an MUC1 (Mucins) and IL-2 double-gene coexpression recombinant vector which is sequentially connected with an MUC1 gene, an IRES (Internal Ribosome Entry Site) sequence and an IL-2 gene along the transcription direction of the vector, or is sequentially connected with the IL-2 gene, the IRES sequence and the MUC1 gene along the transcription direction of the vector, wherein the nucleotide sequence of the MUC1 gene is as shown in the sequence table SEQ ID NO:1; the nucleotide sequence of the IL-2 gene is shown in the sequence table SEQ ID NO:2; the nucleotide sequence of the IRES sequence is shown in the sequence table SEQ ID NO:3. According to the double-gene coexpression recombinant vector disclosed by the invention, the IRES sequence is adopted to connect the MUC1gene and IL-2 gene, human epithelium mucoprotein and human interleukin-2 can be simultaneously expressed in one same vector, the recombinant vector can be applied to gene immune treatment on tumors, and both the immunomodulatory effects of cell factors can be achieved, and specificity anti-tumor effects can be achieved in a targeted manner.
Description
Technical field
The present invention relates to a kind of recombinant vectors and its preparation method and application, particularly relate to a kind of double gene coexpression recombinant vectors and its preparation method and application.
Background technology
The object of immunotherapy of tumors, not only effectively will activate the immune response eliminating tumour cell, also will set up long-range antitumor memory effect.Immunotherapy of tumors comprises two main strategies: non-specific skeptophylaxis treatment and antigen specific immunotherapy.Non-specific skeptophylaxis treatment carrys out stimulating immune system by the concentration of regulation system or local cytokine or costimulatory molecules.Antigen specific immunotherapy, comprises the transhipment of antigen, expresses and present, the migration of the T cell of antigen activates, and adopts the DC cell therapy etc. of antigen load.
Research shows, by vector expression tumor associated antigen and coexpression cytokine or costimulatory molecules, shows the collaborative effect promoted mutually, contributes to inducing antigen-specific T cell immunne response, thus show significant anti-tumor activity.As can be seen here, the anti-tumor immune response of inducing potent, not only need the expression of tumor associated antigen and present, also need to strengthen costimulatory molecules and cytokine mediated immunization.In majority research, the number of the effect of anti-tumor immune response and the immunological enhancement cytokines of coexpression and costimulatory molecules is proportionate.
Cytokine gene immunity is the focus of current therapy of tumor research, the cytokine gene with antitumor action is imported in host, and make it stable and effectively express, make the endogenous cytokine of sustainable existence certain level in body, to play antineoplastic action.But existing cytokine gene immunotherapy method just improves body immunity, strengthen costimulatory molecules and cytokine mediated immunization, the expression of tumor associated antigen cannot be realized and present.
Human Inter Leukin-2 (Interleukin-2, IL-2), has another name called SCIF (T Cell Growth Factor, TCRF).IL-2 is under mitogenesis element or specific antigens stimulate, a kind of cytokine in organism immune response with important regulative produced by T lymphocyte or T lymphocyte series.Research finds, IL-2 has the effect of anti-malignant tumor.The mechanism of IL-2 antitumor action, carries out lethal effect mainly through the inherent immunity system and acquired immune system stimulating body to tumour cell.IL-2 can promote the Clonal expansion of a certain specific T cells group, it activates cell quantity and the activity of killer cell (LAK) by raising NK cell and lymph, and activate til cell, thus induction body anti tumor immune response, tumor growth stopping or knurl body are disappeared.In addition, IL-2 also plays an important role in regulatory T-cell ripening process, and also can work in coordination with stimulates B cell proliferation and secretion, increases highly active T cell and produce Interferon, rabbit simultaneously.
IL-2 be used alone or with Tumor-infiltrating lymphocytes together with tumor infiltrating lymphocyte, for the treatment of metastasis melanin tumor and kidney, achieve good clinical effectiveness.Meanwhile, also find that it has result for the treatment of for solid malignants such as lymphoma, lung cancer, colorectal carcinoma, ovarian cancers.Conbined usage IL-2 in chemotherapy, is considered to have good result for the treatment of to the gastrointestinal cancer such as late gastric cancer, colorectal carcinoma and nonsmall-cell lung cancer.IL-2 can activate antitumor immune function because of it, is considered to effective selection for the treatment of Digestive tract parenchyma.But current IL-2 Gene immunotherapy method just improves the IL-2 protein expression level of body partly, and its immunoregulation effect, anti-tumor capacity are less, and lack the targeting of immunotherapy.
Saliva Orthana (Mucins, MUC) is mainly present in the multiple epithelium such as gi tract, respiratory tract, urogenital tract and mammary gland in normal human, plays lubrication and provide protection, go back mediated signal transduction and cell adhesion simultaneously to normal epithelium.And in tumor tissues, MUC has more existing unconventionality expression, the change of the amount of showing as and matter, and relevant to the infiltration of tumour, Metastasis and prognosis.
People's epithelium mucoprotein (MUC1) is by a kind of I type transmembrane protein in the Saliva Orthana family of MUC1 genetic expression, its polypeptide backbone forms primarily of extracellular fragment, cross-film section and born of the same parents' inner segment, extracellular region is by variable number (20 ~ 125) tandem repetitive sequence (Variable Number Tandem Repeats, VNTR) form, namely epitope is positioned at VNTR district.This kind of peptide chain epi-position when normal expression, cover by periphery sugar chain, can not be identified; When canceration, cause incomplete glycosylation because glycosyl transferase activity increases, incomplete glycosylation causes again side chain to shorten and core skin exposes, and shows relevant skin epitope, carbohydrate antigen epi-position, thus adaptive immune originality.MUC1 has determined high expression level in kinds of tumor cells, comprises lung cancer, liver cancer, mammary cancer, ovarian cancer, cancer of the stomach, colorectal cancer, carcinoma of the pancreas, bladder cancer, prostate cancer, multiple myeloma etc.
Summary of the invention
Based on this, be necessary the defect existed for prior art, provide a kind of MUC1 and IL-2 double gene coexpression recombinant vectors, this recombinant vectors can be used for the Gene immunotherapy of tumour, can play cytokine immunoregulation effect, specificity antineoplastic effect can be produced in targeting ground again.
Another object of the present invention is, provides the preparation method of described MUC1 and IL-2 double gene coexpression recombinant vectors.
Another object of the present invention is, provides the application of described MUC1 and IL-2 double gene coexpression recombinant vectors in the Gene immunotherapy of tumour.
MUC1 and IL-2 double gene coexpression recombinant vectors, it is connected with MUC1 gene, IRES sequence and IL-2 gene in turn along carrier transcriptional orientation, or is connected with IL-2 gene, IRES sequence and MUC1 gene in turn along carrier transcriptional orientation; The nucleotide sequence of described MUC1 gene is as shown in SEQ ID NO:1 in sequence table, and the nucleotide sequence of described IL-2 gene is as shown in SEQ ID NO:2 in sequence table, and the nucleotide sequence of described IRES sequence is as shown in SEQ ID NO:3 in sequence table.
Wherein in an embodiment, described carrier is pIRES2-EGFP plasmid vector, and described double gene coexpression recombinant vectors is pIRES2-MUC1-IL-2 recombinant vectors; Wherein, MUC1 gene is positioned at the upstream of IRES sequence, and IL-2 gene is positioned at the downstream of IRES sequence.
Wherein in an embodiment, the EGFP sequence in described pIRES2-EGFP plasmid vector is by IL-2 gene substitution.
The preparation method of MUC1 and IL-2 double gene coexpression recombinant vectors of the present invention, comprises the following steps:
1) the MUC1 gene fragment containing specific cleavage site is obtained;
2) MUC1 gene fragment step 1) obtained is connected to carrier, builds the recombinant vectors containing MUC1 gene;
3) the IL-2 gene fragment of cutting sticky end containing enzyme is obtained;
4) IL-2 gene fragment step 3) obtained is connected to step 2) recombinant vectors that obtains, MUC1 and the IL-2 double gene coexpression recombinant vectors described in structure.
Wherein in an embodiment, the preparation method of described pIRES2-MUC1-IL-2 recombinant vectors, comprises the following steps:
A) the MUC1 gene fragment containing specific cleavage site is obtained: obtain cDNA as template from breast cancer cell MCF-7, pcr amplification is carried out with the MUC1 Auele Specific Primer containing Bgl II and EcoR I restriction enzyme site sequence, obtain the MUC1 gene fragment containing Bgl II and EcoR I restriction enzyme site, its nucleotide sequence is as shown in SEQ ID NO:6 in sequence table;
B) build pIRES2-MUC1-EGFP recombinant vectors: with restriction enzyme Bgl II, EcoR I respectively enzyme cut the MUC1 gene fragment that pIRES2-EGFP plasmid and step a) obtain, and adopt T4DNA ligase enzyme to carry out ligation, obtain pIRES2-MUC1-EGFP recombinant vectors;
C) the IL-2 gene fragment of cutting sticky end containing enzyme is obtained: obtain cDNA as template from CIK cell, pcr amplification is carried out with the IL-2 Auele Specific Primer cutting sticky end containing BstX I and Not I enzyme, the PCR reaction product obtained is carried out hybridization PCR again and is reacted, obtain IL-2 gene fragment mixture, a kind of IL-2 gene fragment wherein has BstX I and Not I enzyme cuts sticky end;
D) pIRES2-MUC1-IL-2 recombinant vectors is built: cut with restriction enzyme BstX I, Not I enzyme the pIRES2-MUC1-EGFP recombinant vectors that step b) obtains, the IL-2 gene fragment mixture and the enzyme that step c) are obtained are cut rear linearizing pIRES2-MUC1-EGFP recombinant vectors and are mixed, adopt T4DNA ligase enzyme to carry out ligation, obtain pIRES2-MUC1-IL-2 recombinant vectors.
Wherein in an embodiment, the MUC1 Auele Specific Primer described in step a) is:
MUC1 upstream primer: 5 '-GAAGATCTATGACACCGGGCACCC-3 ',
MUC1 downstream primer: 5 '-TTGAATTCCTACAAGTTGGCAGAAGTGG-3 '.
Wherein in an embodiment, IL-2 Auele Specific Primer described in step c) comprises IL-2 first primer pair and IL-2 second primer pair, described IL-2 first primer pair is made up of IL-2 upstream long primer and IL-2 downstream long primer, and described IL-2 second primer pair is made up of IL-2 upstream short primer and IL-2 downstream short primer:
IL-2 first primer pair is:
IL-2 upstream long primer: 5'-AACCATGTACAGGATGCAACTCCTGTCTT-3',
IL-2 downstream long primer: 5'-GGCCGCTCAAGTCAGTGTTGAGATGAT-3';
IL-2 second primer pair is:
IL-2 upstream short primer: 5'-ATGTACAGGATGCAACTCCTGTCTT-3',
IL-2 downstream short primer: 5'-GCTCAAGTCAGTGTTGAGATGATGC-3'.
The application of MUC1 and IL-2 double gene coexpression recombinant vectors of the present invention in oncogene immunotherapy.
Wherein in an embodiment, after the dendritic cell be separated described MUC1 with IL-2 double gene coexpression recombinant vectors cells transfected patient self or allosome in patients with implantation body, carry out oncogene immunotherapy.
MUC1 and IL-2 double gene coexpression recombinant vectors of the present invention, IRES sequence is adopted to connect MUC1 gene and IL-2 gene, people's mucins (MUC1) and Human Inter Leukin-2 (IL-2) can be expressed in identical carrier simultaneously, the consumption of genophore can be reduced, reduce the introducing of the exogenous gene sequence that non-treatment is correlated with.Wherein, MUC1 is the related antigen of tumor-targeting, IL-2 is the cytokine playing immunoregulation effect, both conbined usage, both can play cytokine immunoregulation effect, specific anti-tumour effect can be produced in targeting ground again, thus better immunotherapy of tumors effect can be obtained.After the dendritic cell that double gene coexpression recombinant vectors cells transfected patient self or allosome are separated in patients with implantation body, the great expression of MUC1, dendritic cell submission MUC1 polypeptide and MHC mixture can be stimulated, and then stimulation body immune system, produce specific killing T cell, targeting kills and wounds the tumour cell of expressing MUC1 polypeptide; And the great expression of IL-2, energy enhancing body immunoregulation capability further, and amplification ability and the cell viability of T lymphocyte specific can be strengthened, increasing highly active T cell produces Interferon, rabbit, collaborative expansion antineoplastic immune effect.
IRES sequence derives from some virus and one section of non-translational region holding of cell mRNA 5 ', the mode that can not rely on cap starts the mRNA translation of far-end, jointly can transcribe with the gene be attached thereto under the control of upstream promoter, same transcript translates different albumen.When IRES connection polygene carries out coexpression, the mRNA of multiple gene is on same transcripton, but the translation process after transcribing is separate, upstream gene is translated in a conventional manner, downstream gene relies on IRES sequence to translate in the mode not relying on cap, ensure that absolute construction and the function of each gene.Utilize IRES to replace internal promoter, polygene co-expression carrier not only can be made greatly to reduce, but also overcome the mutual suppression phenomenon in traditional polygene expression vector between promotor, avoid the generation of fusion rotein.
Accompanying drawing explanation
Fig. 1 is the electrophorogram of the MUC1 gene fragment containing specific cleavage site sequence of embodiment one gained, and wherein, 1 is MUC1 gene, and M is mark;
Fig. 2 is pIRES2-EGFP plasmid map;
Fig. 3 is the pIRES2-MUC1-EGFP recombinant vectors collection of illustrative plates of embodiment two gained;
Fig. 4 is that the PCR of the pIRES2-MUC1-EGFP recombinant vectors of embodiment two gained identifies electrophorogram, and wherein, 1 is PCR reaction product, and M is mark;
Fig. 5 is that the enzyme of the pIRES2-MUC1-EGFP recombinant vectors of embodiment two gained cuts qualification electrophorogram, and wherein, 1 is endonuclease reaction product, and M is mark;
Fig. 6 is the electrophorogram of the IL-2 gene fragment with restriction enzyme sticky end of embodiment three gained, and wherein, 1 is pcr amplification product A, and 2 is that pcr amplification product B, M are for marking;
The schema that Fig. 7 reacts for the hybridization PCR described in embodiment three;
Fig. 8 is the pIRES2-MUC1-IL-2 recombinant vectors collection of illustrative plates of embodiment four gained;
Fig. 9 is that the enzyme of the pIRES2-MUC1-IL-2 recombinant vectors of embodiment four gained cuts qualification electrophorogram, wherein, 1 is BglII/EcoR I double digestion reaction product, and 2 is EcoR I/Not I double digestion reaction product, 3 is Bgl II/Not I double digestion reaction product, and M is mark.
Embodiment
In following embodiment, used experimental technique, such as round pcr, design of primers technology, vector construction technology, detection technique, electrophoretic technique etc. are the routine techniques in genetically engineered, those skilled in the art can according to existing techniques in realizing (such as with reference to works such as J. Pehanorm Brookers, " Molecular Cloning: A Laboratory guide " that Huang Peitang etc. translate, Science Press's third edition; Or carry out according to product description).Used in operation equipment, reagent, carrier, bacterial strain etc., be and purchase available conventional products by market.
Embodiment one: obtain the MUC1 gene fragment containing specific cleavage site
1, design of primers
According to the nucleotide sequence (as shown in SEQ ID NO:1 in sequence table) of MUC1 gene and pIRES2-EGFP plasmid vector being expected the multiple clone site inserted, design Auele Specific Primer is as follows:
MUC1 upstream primer (as shown in SEQ ID NO:4 in sequence table):
5 '-GA
aGATCTaTGACACCGGGCACCC-3 ' (underscore part is Bgl II restriction enzyme site sequence),
MUC1 downstream primer (as shown in SEQ ID NO:5 in sequence table):
5 '-TT
gAATTCcTACAAGTTGGCAGAAGTGG-3 ' (underscore part is EcoR I restriction enzyme site sequence).
2, cDNA template is obtained
TRIzon method extracts RNA(TRIzon total RNA extraction reagent box purchased from Beijing CoWin Bioscience Co., Ltd. from human breast cancer cell line Bcap-37, production code member is CW0580), and reverse transcription becomes cDNA(Reverse Transcription box purchased from precious biotechnology (Dalian) company limited, production code member is RR019A).
3, the MUC1 gene fragment containing specific cleavage site is obtained
With the cDNA of the RNA extracted in human breast cancer cell line Bcap-37 institute reverse transcription for template, under the effect of the above-mentioned upstream and downstream primer containing specific cleavage site, MUC1 gene fragment is obtained by PCR reaction, Bgl II restriction enzyme site sequence is contained in its upstream, EcoR I restriction enzyme site sequence is contained in downstream, and its nucleotide sequence is as shown in SEQ ID NO:6 in sequence table.Identify with 1% agarose gel electrophoresis, electrophoresis result as shown in Figure 1.
PCR reaction system following (50 μ L):
Wherein, 2 × Prime STAR Max DNA Polymerase is archaeal dna polymerase-damping fluid mixture, and purchased from precious biological (Dalian) company limited, production code member is R045A.
PCR reaction conditions is: 95 DEG C of denaturation 5min; 98 DEG C of sex change 10s, 55 DEG C of annealing 5s, 72 DEG C extend 5s, 30 circulations; 72 DEG C finally extend 5min.
The structure of embodiment two: pIRES2-MUC1-EGFP recombinant vectors
Use restriction enzyme Bgl II and EcoR I, enzyme cuts the MUC1 gene fragment of pIRES2-EGFP plasmid (Bgl II, EcoR I restriction enzyme site are contained in the multiple clone site place of this plasmid) and embodiment one gained respectively, obtain enzyme cut rear linearizing pIRES2-EGFP carrier and enzyme cut after MUC1 gene order; Adopt T4DNA ligase enzyme system to carry out ligation, hatch 30 minutes at 22 DEG C, then deactivation 5 minutes at 70 DEG C, builds pIRES2-MUC1-EGFP recombinant vectors (as shown in Figure 3).
From the constructional feature (as shown in Figure 2) of pIRES2-EGFP plasmid, after MUC1 gene inserts the multiple clone site of pIRES2-EGFP plasmid, be positioned at the upstream (as shown in Figure 3) of its own sequence IRES of plasmid vector, MUC1 and the EGFP sequence namely under same promotor starts is expressed respectively.
1, double digestion pIRES2-EGFP plasmid
Endonuclease reaction system following (50 μ L):
Wherein, restriction endonuclease Bgl II and 10 × H Buffer is Bgl II restriction endonuclease buffer solution system, and purchased from precious biotechnology (Dalian) company limited, production code member is 1021A; Restriction endonuclease EcoR I and 10 × H Buffer is EcoR I restriction endonuclease buffer solution system, and purchased from precious biotechnology (Dalian) company limited, production code member is 1040A.
Endonuclease reaction condition: react 6 hours at 37 DEG C.
2, double digestion MUC1 gene fragment
Endonuclease reaction system following (50 μ L):
Endonuclease reaction condition: react 6 hours at 37 DEG C.
3, connect MUC1 gene fragment and pIRES2-EGFP carrier, build pIRES2-MUC1-EGFP recombinant vectors ligation system following (20 μ L):
Wherein, T4DNA ligase enzyme and 10 × Ligation Buffer are DNA ligase buffer solution system, and purchased from Beijing CoWin Bioscience Co., Ltd., production code member is CW0805.
Ligation condition: hatch 30 minutes for 22 DEG C, 70 DEG C of deactivations 5 minutes.
4, the qualification of pIRES2-MUC1-EGFP recombinant vectors
In 200 μ L competent cell JM109 (1 ~ 2 × 10
9bacteria/ml), add the above-mentioned ligation product of 20 μ L, after being placed in cooled on ice 30min, being placed in 42 DEG C of water-bath thermal shock 90s, then being placed in cooled on ice 2min, then add LB liquid nutrient medium 780ul; Under 37 DEG C of conditions, on the shaking table of 150 revs/min, 60min is cultivated in recovery, is coated on the LB flat board containing kalamycin resistance by the bacterium liquid of recovering, is inverted cultivation 16 ~ 18 hours at 37 DEG C; Picking positive bacteria drops down onto in kalamycin resistance LB liquid nutrient medium, 37 DEG C, cultivate 12 ~ 16 hours under the condition of 200rpm.
Get positive bacterium colony bacterium liquid, carry out bacterium liquid PCR preliminary evaluation, PCR identification reaction system following (20 μ L):
Wherein, 2 × Power Taq PCR Master Mix is archaeal dna polymerase-enzyme buffer liquid dNTPs mixture, and purchased from hundred Tykes (Beijing) Bioisystech Co., Ltd, production code member is PR1701.
PCR reaction conditions is: 95 DEG C of denaturation 12min; 95 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, 30 circulations; 72 DEG C of ends extend 5min.
Get bacterium liquid PCR identification reaction product, identify with 1% agarose gel electrophoresis, electrophoresis result as shown in Figure 4.Electrophoresis result shows, at 795bp place appearance one electrophoretic band, consistent with the molecular size range of goal gene MUC1 gene, shows the success of pIRES2-MUC1-EGFP construction of recombinant vector.
Adopt plasmid extraction kit (purchased from Beijing CoWin Bioscience Co., Ltd., production code member is CW0511), according to the operation of test kit specification sheets, extract the plasmid of positive bacterium colony, carry out enzyme and cut qualification.
Enzyme cuts identification reaction system following (10 μ L):
Endonuclease reaction condition: react 1 hour at 37 DEG C.
Get enzyme and cut identification reaction product, identify with 1% agarose gel electrophoresis, electrophoresis result as shown in Figure 5.Electrophoresis result shows, and occur two band after electrophoresis, wherein one appears at 795bp place, consistent with the molecular size range of goal gene MUC1 gene, shows the success of pIRES2-MUC1-EGFP construction of recombinant vector.
Embodiment three: two PCR method obtains the IL-2 gene fragment with restriction enzyme sticky end
According to IL-2 gene order and pIRES2-EGFP plasmid vector being expected the multiple clone site inserted, design two pairs of length differences, primer with restriction enzyme sticky end; With the cDNA of the RNA extracted in CIK cell institute reverse transcription for template, carry out pcr amplification with above-mentioned two pairs of primers respectively, obtain two kinds of pcr amplification products; Sex change and annealing is carried out successively by after two kinds of pcr amplification product mixing, obtain four kinds of IL-2 gene fragments, wherein the two ends of two kinds of IL-2 gene fragments are with restriction enzyme sticky end, cutting without the need to using restriction enzyme to carry out enzyme thus, IL-2 gene fragment orientation can be connected in the expection multiple clone site of plasmid vector.
Compared with traditional PCR primer cloning process, present method (two PCR method) has the advantages such as simple, cost is low, efficiency is high, versatility is good, can be widely used in the quick clone of PCR primer.In the method, do not need to use restriction enzyme ferment treatment PCR primer, the DNA fragmentation containing restriction enzyme sticky end can be obtained, the multiple clone site of genophore can be made full use of, without the need to considering whether contain the restriction enzyme site identical with vector multiple cloning site in goal gene, the directed cloning of goal gene easily can be realized.
1, design of primers
According to the nucleotide sequence (as shown in SEQ ID NO:2 in sequence table) of IL-2 gene and pIRES2-EGFP plasmid vector being expected the multiple clone site inserted, design two pairs of length differences, primer with restriction enzyme sticky end, as follows:
IL-2 first primer pair (FL/RL primer pair) is made up of IL-2 upstream long primer FL and IL-2 downstream long primer RL:
IL-2 upstream long primer FL(is as shown in SEQ ID NO:7 in sequence table):
5'-
AACCATGTACAGGATGCAACTCCTGTCTT-3'
Underscore part is the full sequence in cleavage site downstream in restriction enzyme BstX I recognition sequence, and tilted letter ATG is the initiator codon of IL-2 gene;
IL-2 downstream long primer RL(is as shown in SEQ ID NO:8 in sequence table):
5'-
GGCCGCTCAAGTCAGTGTTGAGATGAT-3'
Underscore part is the full sequence in cleavage site downstream in restriction enzyme Not I recognition sequence.
IL-2 second primer pair (FS/RS primer pair) is made up of IL-2 upstream short primer FS and IL-2 downstream short primer RS:
IL-2 upstream short primer FS(is as shown in SEQ ID NO:9 in sequence table):
5'-
ATGTACAGGATGCAACTCCTGTCTT-3'
Underscore part is the reverse complementary sequence of the full sequence of cleavage site upstream in restriction enzyme BstX I recognition sequence, and AT is a part for the initiator codon of IL-2 gene;
IL-2 downstream short primer RS(is as shown in SEQ ID NO:10 in sequence table):
5'-
GCTCAAGTCAGTGTTGAGATGATGC-3'
Underscore part is the reverse complementary sequence of the full sequence of cleavage site upstream in restriction enzyme Not I recognition sequence.
FL/RL primer pair is identical with the target sequence (namely corresponding with IL-2 gene sequence) of FS/RS primer pair.
The recognition sequence of restriction enzyme BstX I is: 5'-CCANNNNN/NTGG-3'
The recognition sequence of restriction enzyme Not I is: 5'-GC/GGCCGC-3'
2, cDNA template is obtained
TRIzon method is from CIK cell (Cytokine-Induced Killer, cytokine induced kill cell) or human peripheral be separated mononuclearcell in, extract RNA(TRIzon total RNA extraction reagent box purchased from Beijing CoWin Bioscience Co., Ltd., production code member is CW0580), and reverse transcription becomes cDNA(Reverse Transcription box purchased from precious biotechnology (Dalian) company limited, production code member is RR019A).
3, the IL-2 gene fragment with restriction enzyme sticky end is obtained
With obtained cDNA for template, carry out pcr amplification with FL/RL primer pair, obtain pcr amplification product A; Another FS/RS primer pair carries out pcr amplification, obtains pcr amplification product B.Pcr amplification product A and pcr amplification product B is all containing IL-2 gene order.
PCR reaction system following (50 μ L):
PCR reaction conditions is: 95 DEG C of denaturation 5min; 98 DEG C of sex change 10s, 55 DEG C of annealing 5s, 72 DEG C extend 5s, 30 circulations; 72 DEG C finally extend 5min.
Get pcr amplification product A and pcr amplification product B respectively, identify with 1% agarose gel electrophoresis, electrophoresis result as shown in Figure 6.Electrophoresis result shows, and the electrophoretic band of pcr amplification product A appears at 472bp place, and the electrophoretic band of pcr amplification product B appears at 464bp place, all consistent with the molecular size range of goal gene.
Get pcr amplification product A respectively and pcr amplification product B checks order, result shows: the 5' end of pcr amplification product A holds many 4 Nucleotide than the 5' of pcr amplification product B, the 3' end of pcr amplification product A also holds many 4 Nucleotide than the 3' of pcr amplification product B, and all the other sequences are identical.
By pcr amplification product A and pcr amplification product B etc. mole of mixing, carry out hybridization PCR and react, obtain IL-2 gene fragment mixture.
Hybridization PCR reaction conditions is as follows: 95 DEG C of 5min, 80 DEG C of 1min, 70 DEG C of 1min, 65 DEG C of 1min, 60 DEG C of 1min, 55 DEG C of 1min, 40 DEG C of 1min, 4 DEG C of preservations.
After pcr amplification product A and pcr amplification product B sex change, obtain four kinds of DNA single chains, four kinds of DNA single chain random combines, produce the DNA fragmentation of four kinds of equal proportions---as shown in Figure 7, it is the IL-2 gene order shown in sequence table SEQ ID NO:2 that straight line omits region for IL-2 gene fragment I, IL-2 gene fragment II, IL-2 gene fragment III and IL-2 gene fragment IV().Wherein, IL-2 gene fragment III, IL-2 gene fragment IV are hybridizing DNA fragment.The 5' end of IL-2 gene fragment III has BstX I sticky end, and its 3' end has Not I sticky end.
The structure of embodiment four: pIRES2-MUC1-IL-2 recombinant vectors
Use restriction enzyme BstX I and Not I double digestion pIRES2-MUC1-EGFP recombinant vectors.Because restriction endonuclease BstX I is arranged in the terminal of the IRES sequence downstream of pIRES2-MUC1-EGFP recombinant vectors and the section start of EGFP Sequences upstream, and restriction endonuclease Not I is arranged in the downstream termination point place of the EGFP sequence of pIRES2-MUC1-EGFP recombinant vectors, therefore, after inserting IL-2 gene fragment by these two restriction enzyme sites, IL-2 gene is positioned at the downstream of IRES sequence, thus make MUC1 gene and IL-2 gene lay respectively at the both sides (as shown in Figure 8) of IRES sequence, achieve the coordinate expression of two goal gene, and the generation of fusion rotein can be avoided.
1, Not I endonuclease reaction
Not I endonuclease reaction system following (50 μ L):
Endonuclease reaction condition: react 6 hours at 37 DEG C.
Restriction endonuclease Not I, 10 × H Buffer, BSA(bovine serum albumin) and Triton X-100(Triton X-100) be Not I restriction endonuclease reaction kit, purchased from precious biotechnology (Dalian) company limited, production code member is 1166A.
Adopt DNA fast purifying test kit (purchased from Beijing CoWin Bioscience Co., Ltd., production code member is CW2302) to carry out purifying, collect the pIRES2-MUC1-EGFP recombinant vectors after Not I enzyme is cut.
2, BstX I endonuclease reaction
BstX I endonuclease reaction system following (50 μ L):
Wherein, restriction endonuclease BstX I and 10 × H Buffer is BstX I restriction endonuclease buffer solution system, and purchased from precious biotechnology (Dalian) company limited, production code member is 1027A.
Endonuclease reaction condition: react 6 hours at 37 DEG C.
Adopt DNA fast purifying test kit (purchased from Beijing CoWin Bioscience Co., Ltd., production code member is CW2302) to carry out purifying, collect the pIRES2-MUC1-EGFP recombinant vectors after BstX I enzyme is cut.
3, connect IL-2 gene and pIRES2-MUC1-EGFP recombinant vectors, build pIRES2-MUC1-IL-2 recombinant vectors
IL-2 gene fragment mixture obtained for embodiment three is mixed with pIRES2-MUC1-EGFP recombinant vectors linearizing after double digestion (mol ratio of IL-2 gene fragment and pIRES2-MUC1-EGFP recombinant vectors is 4 ︰ 1), add T4DNA ligase enzyme and carry out ligation, 30 minutes are hatched at 22 DEG C, then deactivation 5 minutes at 70 DEG C, constructs pIRES2-MUC1-IL-2 recombinant vectors.
Four kinds of DNA fragmentations in IL-2 gene fragment mixture, IL-2 gene fragment III is only had to have the sticky end with the complementation of pIRES2-MUC1-EGFP recombinant vectors, thus can be connected with pIRES2-MUC1-EGFP recombinant vectors orientation, other three kinds of IL-2 gene fragments all can not be connected with pIRES2-MUC1-EGFP recombinant vectors orientation.
Ligation system following (20 μ L):
Ligation condition: hatch 30 minutes for 22 DEG C, 70 DEG C of deactivations 5 minutes.
4, the qualification of pIRES2-MUC1-IL-2 recombinant vectors
The above-mentioned ligation product of 20 μ L to be joined in 200 μ L competent cell JM109 (1 ~ 2 × 10
9bacteria/ml), after being placed in cooled on ice 30min, being placed in 42 DEG C of water-bath thermal shock 90s, then being placed in cooled on ice 2min; Then add LB liquid nutrient medium 780ul, under 37 DEG C of conditions, on the shaking table of 150 revs/min, 60min is cultivated in recovery, is coated on the LB flat board containing kalamycin resistance by the bacterium liquid of recovering, is inverted cultivation 16 ~ 18 hours at 37 DEG C; Picking positive bacteria drops down onto in kalamycin resistance LB liquid nutrient medium, 37 DEG C, cultivate 12 ~ 16 hours under the condition of 200rpm.
Adopt plasmid extraction kit (purchased from Beijing CoWin Bioscience Co., Ltd., production code member is CW0511), according to the operation of test kit specification sheets, extract the plasmid of positive bacterium colony, carry out enzyme and cut qualification.
Endonuclease reaction system one (10 μ L):
Endonuclease reaction condition: react 1 hour at 37 DEG C.
Get above-mentioned endonuclease reaction product respectively, identify with 1% agarose gel electrophoresis, electrophoresis result as shown in Figure 9.Electrophoresis result shows, and occurs two band after Bgl II/EcoR I double digestion reaction product electrophoresis, and wherein one at 795bp place, consistent with the molecular size range of goal gene MUC1 gene; Occur two band after EcoR I/Not I double digestion reaction product electrophoresis, wherein one at 1047bp place, in the same size with the molecular weight sum of goal gene IL-2 gene and IRES sequence; Occur two band after Bgl II/NotI double digestion reaction product electrophoresis, wherein one at 1842bp place, in the same size with the molecular weight sum of goal gene MUC1 gene, IL-2 gene and IRES sequence.Above-mentioned qualification result shows, the success of pIRES2-MUC1-IL-2 construction of recombinant vector.
PIRES2-MUC1-IL-2 recombinant vectors is delivered to gold only intelligence biotechnology (Beijing) company limited check order, consistent with expected results through order-checking, further proof vector construction is successfully.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (1)
- The preparation method of 1.MUC1 and IL-2 double gene coexpression recombinant vectors pIRES2-MUC1-IL-2, comprises the following steps:A) the MUC1 gene fragment containing specific cleavage site is obtained: obtain cDNA as template from breast cancer cell MCF-7, pcr amplification is carried out with the MUC1 Auele Specific Primer containing Bgl II and EcoR I restriction enzyme site sequence, obtain the MUC1 gene fragment containing Bgl II and EcoR I restriction enzyme site, its nucleotide sequence is as shown in SEQ ID NO:6 in sequence table;B) build pIRES2-MUC1-EGFP recombinant vectors: with restriction enzyme Bgl II, EcoR I respectively enzyme cut the MUC1 gene fragment that pIRES2-EGFP plasmid and step a) obtain, and adopt T4DNA ligase enzyme to carry out ligation, obtain pIRES2-MUC1-EGFP recombinant vectors;C) the IL-2 gene fragment of cutting sticky end containing enzyme is obtained: obtain cDNA as template from CIK cell, pcr amplification is carried out with the IL-2 Auele Specific Primer cutting sticky end containing BstX I and Not I enzyme, the PCR reaction product obtained is carried out hybridization PCR again and is reacted, obtain IL-2 gene fragment mixture, a kind of IL-2 gene fragment wherein has BstX I and Not I enzyme cuts sticky end;D) pIRES2-MUC1-IL-2 recombinant vectors is built: cut step b with restriction enzyme BstX I, Not I enzyme) the pIRES2-MUC1-EGFP recombinant vectors that obtains, by step c) the IL-2 gene fragment mixture and the enzyme that obtain cut rear linearizing pIRES2-MUC1-EGFP recombinant vectors and mix, adopt T4DNA ligase enzyme to carry out ligation, obtain pIRES2-MUC1-IL-2 recombinant vectors;Wherein, step a) described in MUC1 Auele Specific Primer be:MUC1 upstream primer: 5 '-GAAGATCTATGACACCGGGCACCC-3 ',MUC1 downstream primer: 5 '-TTGAATTCCTACAAGTTGGCAGAAGTGG-3 ';The reaction conditions of step a) described pcr amplification is: 95 DEG C of denaturation 5min; 98 DEG C of sex change 10s, 55 DEG C of annealing 5s, 72 DEG C extend 5s, 30 circulations; 72 DEG C finally extend 5min;Step c) described in IL-2 Auele Specific Primer comprise IL-2 first primer pair and IL-2 second primer pair, described IL-2 first primer pair is made up of IL-2 upstream long primer and IL-2 downstream long primer, and described IL-2 second primer pair is made up of IL-2 upstream short primer and IL-2 downstream short primer:IL-2 first primer pair is:IL-2 upstream long primer: 5'-AACCATGTACAGGATGCAACTCCTGTCTT-3',IL-2 downstream long primer: 5'-GGCCGCTCAAGTCAGTGTTGAGATGAT-3';IL-2 second primer pair is:IL-2 upstream short primer: 5'-ATGTACAGGATGCAACTCCTGTCTT-3',IL-2 downstream short primer: 5'-GCTCAAGTCAGTGTTGAGATGATGC-3';Step c) reaction conditions of described pcr amplification is: 95 DEG C of denaturation 5min; 98 DEG C of sex change 10s, 55 DEG C of annealing 5s, 72 DEG C extend 5s, 30 circulations; 72 DEG C finally extend 5min;Step c) reaction conditions that reacts of described hybridization PCR is: 95 DEG C of 5min, 80 DEG C of 1min, 70 DEG C of 1min, 65 DEG C of 1min, 60 DEG C of 1min, 55 DEG C of 1min, 40 DEG C of 1min, 4 DEG C of preservations.
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