CN103614414A - AFP (Alpha Fetoprotein) and IL-2 (Interleukin-2) double-gene co-expressing recombinant vector as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses an AFP (Alpha Fetoprotein) and IL-2 (Interleukin-2) double-gene co-expressing recombinant vector, wherein an AFP gene, an IRES (Internal Ribosome Entry Site) sequence and an IL-2 gene are sequentially connected along a transcription direction of the vector, or the IL-2 gene, the IRES sequence and the AFP gene are sequentially connected along the transcription direction of the vector; the nucleotide sequence of the AFP gene is shown as SEQ ID NO:1 in a sequence table, the nucleotide sequence of the IL-2 gene is shown as SEQ ID NO:2 in the sequence table, and the nucleotide sequence of the IRES sequence is shown as SEQ ID NO:3 in the sequence table. According to the double-gene co-expressing recombinant vector disclosed by the invention, the AFP gene and the IL-2 gene are connected via the IRES sequence, so that the AFP gene and the IL-2 gene can be simultaneously expressed in the same vector; the recombinant vector can be applied to the genetic immunization treatment of liver cancer, thereby achieving an immunomodulatory effect of cell factors and producing a specific anti-tumor effect to liver cancer in a targeted manner.

Description

AFP and IL-2 double gene coexpression recombinant vectors and its preparation method and application

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

Primary hepatocellular carcinoma (Hepatocellular Carcinoma, HCC) be one of modal malignant tumour, China is the district occurred frequently of liver cancer, etesian liver cancer accounts for global more than 50%, oneself accounts for the second of national tumor mortality rate mortality of liver cancer, is a kind of disease of serious threat people life and health.Excision is the best liver cancer treatment means of current curative effect, Technology of Liver Transplantation in China is also quite ripe at present, but because expense is high, donor source is difficult, postoperative use anti-rejection drugs can make residual cancer growth sooner and occur the problems such as transfer because of immunosuppression, not good enough to the curative effect of mid and late liver cancer.

In recent years, develop rapidly along with subjects such as molecular biology and tumor immunologies, immunotherapy of tumors has become the focus of oncotherapy research, panimmunity methods for the treatment of is for the clinical study of liver cancer, such as cytokine therapy, autologous complete tumour-cell vaccine, effector cell's adoptive therapy, the dendritic cell of take is basic Hepatoma Vaccine etc.At present, multiple different Immunotherapy Strategy has shown biological immune activity and the preliminary clinical efficacy of anti-liver cancer, can affect liver cancer recurrence and treat rear lifetime.Wherein, cytokine gene immunotherapy is the combination of immunotherapy of tumors and gene therapy, it is by importing the cytokine gene with antitumor action in host, and make it stable and effectively express, make the endogenous cell factor of sustainable existence certain level in body, to bring into play antineoplastic action.Yet current cytokine gene immunotherapy method lacks tumor-targeting, its antitumous effect has much room for improvement.

Interleukin II (interleukin-2, IL-2), has another name called SCIF (T cell growth factor, TCRF), is the extensive bioactive cytokine that has mainly being produced by the CD4+T cell activating and CD8+T cell.IL-2 is the somatomedin of all T cell subsets, and can promote activating B cell propagation, therefore for regulating and controlling the important factor of immunne response, it also participates in antibody response, hematopoiesis and oncological surveillance.The mechanism of IL-2 antitumor action is mainly by stimulating inherent immunity system and the acquired immune system of body to carry out lethal effect to tumour cell.IL-2 can promote clone's property amplification of a certain specific T cells group, it can activate by improving NK cell and lymph cell quantity and the activity of killer cell (LAK), and activate til cell, thereby induction body anti tumor immune response stops tumor growth or knurl body disappears.In addition, IL-2 also plays an important role in regulating T cell mature process, also can work in coordination with stimulation B cell proliferation and secretion simultaneously, increase highly active T Cells Interferon Production.

In recent years, IL-2, because it can activate antitumor immune function, is considered to treat effective selection of Digestive tract parenchyma.Combining after using gemcitabine and IL-2 for the treatment of carcinoma of the pancreas hepatic metastases or pancreatic cancer surgery prevents hepatic metastases to have good effect.Research discovery, IL-2 is the important influence prognosis factor of of hepatocarcinoma patient, for some early stage patients, is even also like this; Through portal vein direct injection IL-2, can stimulate better liver natural killer cell (Nature Killer Cell, NK cell), liver neoplasm is played to lethal effect; And combine, use IL-2 and IL-12 to be in the news can to improve Hepatectomy patient body immunity and extend survival rate.Yet current IL-2 Gene immunotherapy method just improves the IL-2 protein expression level of body partly, its immunoregulation effect, anti-tumor capacity are less, and lack the targeting of immunotherapy.

Alpha-fetoprotein (Alpha Fetoprotein, AFP) is a specific alpha-globulin of embryo, is an important component in Mammalian Embryo serum.Under normal circumstances, AFP is produced by the liver of fetus and yolk sac, and after fetal birth, the AFP content in serum declines rapidly, and to become in human body be can't detect or only have an extremely AFP of trace normal.AFP be the mankind find first there is the tumor markers of clinical value, be considered to the important symbol thing of diagnosis of HCC all the time.In adult, approximately have in the serum of 80% hepatocellular carcinoma patients, the expression of AFP can be detected, show that AFP examination is a kind of efficient means of finding early hepatocarcinoma.AFP can be used as tumor markers, target position as immunotherapy of tumors, stimulate body to produce humoral immunization and cellular immunization, tumour cell can be at its surface expression submission AFP polypeptide and MHC mixture, and then stimulation body immune system, produce specific killing T cell, killing tumor cell.Research shows, AFP can with multiple cancer therapy drug combination, and optionally enter tumour cell by receptor-mediated endocytosis, thereby target, kill tumour cell efficiently.

Summary of the invention

Based on this, be necessary the defect existing for prior art, a kind of AFP and IL-2 double gene coexpression recombinant vectors are provided, this recombinant vectors can be used for the Gene immunotherapy of liver cancer, can bring into play cytokine immunoregulation effect, again can targeting ground for liver cancer, produce specificity antineoplastic effect.

Another object of the present invention is that the preparation method of described AFP and IL-2 double gene coexpression recombinant vectors is provided.

A further object of the present invention is that described AFP and the application of IL-2 double gene coexpression recombinant vectors in the Gene immunotherapy of liver cancer are provided.

AFP and IL-2 double gene coexpression recombinant vectors, it is connected with AFP gene, IRES sequence and IL-2 gene in turn along carrier transcriptional orientation, or is connected with IL-2 gene, IRES sequence and AFP gene in turn along carrier transcriptional orientation; The nucleotide sequence of described AFP 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.

In an embodiment, described carrier is pIRES2-EGFP plasmid vector therein, and described double gene coexpression recombinant vectors is pIRES2-AFP-IL-2 recombinant vectors; Wherein, AFP gene is positioned at the upstream of IRES sequence, and IL-2 gene is positioned at the downstream of IRES sequence.

In an embodiment, the EGFP sequence in described pIRES2-EGFP plasmid vector is by IL-2 gene substitution therein.

The preparation method of AFP of the present invention and IL-2 double gene coexpression recombinant vectors, comprises the following steps:

1) obtain the AFP gene fragment that contains specificity restriction enzyme site;

2) AFP gene fragment step 1) being obtained is connected in carrier, builds the recombinant vectors that contains AFP gene;

3) obtain and contain the IL-2 gene fragment that enzyme is cut sticky end;

4) IL-2 gene fragment step 3) being obtained is connected in step 2) recombinant vectors that obtains, build described AFP and IL-2 double gene coexpression recombinant vectors.

In an embodiment, the preparation method of described pIRES2-AFP-IL-2 recombinant vectors, comprises the following steps therein:

A) obtain the AFP gene fragment that contains specificity restriction enzyme site: from liver cancer cell HepG2, obtain cDNA as template, with the AFP Auele Specific Primer that contains Bgl II and EcoR I restriction enzyme site sequence, carry out pcr amplification, obtain the AFP gene fragment that contains 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-AFP-EGFP recombinant vectors: with restriction enzyme Bgl II, EcoR I respectively enzyme cut the AFP gene fragment that pIRES2-EGFP plasmid and step a) obtain, and adopt T4DNA ligase enzyme to carry out ligation, obtain pIRES2-AFP-EGFP recombinant vectors;

C) obtain and contain the IL-2 gene fragment that enzyme is cut sticky end: from CIK cell, obtain cDNA as template, with the IL-2 Auele Specific Primer that contains BstX I and Not I enzyme and cut sticky end, carry out pcr amplification, resulting PCR reaction product is hybridized PCR reaction again, obtain IL-2 gene fragment mixture, a kind of IL-2 gene fragment wherein has BstX I and Not I enzyme is cut sticky end;

D) build pIRES2-AFP-IL-2 recombinant vectors: with restriction enzyme BstX I, Not I enzyme, cut the pIRES2-AFP-EGFP recombinant vectors that step b) obtains, IL-2 gene fragment mixture and the enzyme that step c) is obtained cut rear linearizing pIRES2-AFP-EGFP recombinant vectors and mixed, adopt T4DNA ligase enzyme to carry out ligation, obtain pIRES2-AFP-IL-2 recombinant vectors.

Therein in an embodiment, the AFP Auele Specific Primer described in step a) is:

AFP upstream primer: 5 '-GCAGATCTATGAAGTGGGTGGAA-3 ',

AFP downstream primer: 5 '-TTGAATTCTTAAACTCCCAAAGCAGC-3 '.

Therein in an embodiment, IL-2 Auele Specific Primer described in step c) comprises IL-2 the first primer pair and IL-2 the second primer pair, described IL-2 the first primer pair is comprised of IL-2 upstream long primer and IL-2 downstream long primer, and described IL-2 the second primer pair is comprised of IL-2 upstream short primer and IL-2 downstream short primer:

IL-2 the first primer pair is:

IL-2 upstream long primer: 5'-AACCATGTACAGGATGCAACTCCTGTCTT-3',

IL-2 downstream long primer: 5'-GGCCGCTCAAGTCAGTGTTGAGATGAT-3';

IL-2 the second primer pair is:

IL-2 upstream short primer: 5'-ATGTACAGGATGCAACTCCTGTCTT-3',

IL-2 downstream short primer: 5'-GCTCAAGTCAGTGTTGAGATGATGC-3'.

AFP of the present invention and the application of IL-2 double gene coexpression recombinant vectors in hepatocarcinoma gene immunotherapy.

In an embodiment, in patients with implantation body after the described AFP dendritic cell separated with IL-2 double gene coexpression recombinant vectors transfection liver cancer patient self or allosome, carry out hepatocarcinoma gene immunotherapy therein.

AFP of the present invention and IL-2 double gene coexpression recombinant vectors, adopt IRES sequence to connect AFP gene and IL-2 gene, can in identical carrier, express human a-fetoprotein (AFP) and Human Inter Leukin-2 (IL-2) simultaneously, can reduce the consumption of genophore, reduce the introducing of the exogenous gene sequence that non-treatment is relevant.Wherein, AFP is the related antigen of tumor-targeting, IL-2 is the cytokine of performance immunoregulation effect, both combine use, both can bring into play the immunoregulation effect of cytokine, can for liver cancer, produce specific anti-tumour effect to targeting again, thereby can obtain better liver cancer immunity result for the treatment of.In patients with implantation body after the dendritic cell of double gene coexpression recombinant vectors transfection liver cancer patient self or allosome separation, the great expression of AFP, can stimulate dendritic cell submission AFP polypeptide and MHC mixture, and then stimulation body immune system, produce specific killing T cell, targeting is killed and wounded the tumour cell of expressing AFP polypeptide; And the great expression of IL-2, energy is enhancing body immunoregulation capability further, and can strengthen amplification ability and the cell viability of T lymphocyte specific, increases highly active T Cells Interferon Production, the collaborative antineoplastic immune effect that expands.

IRES sequence is the one section of non-translational region that derives from some virus and cell mRNA 5 ' end, the mode that can not rely on cap starts the mRNA translation of far-end, can under the control of upstream promoter, jointly transcribe with the gene being attached thereto, on same transcript, translate different albumen.When IRES connection polygene carries out coexpression, the mRNA of a plurality of genes 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 not rely on the mode of cap, has guaranteed absolute construction and the function of each gene.Utilize IRES to replace internal promoter, not only can make polygene co-expression carrier greatly dwindle, but also overcome the mutual inhibition phenomenon between promotor in traditional polygene expression vector, avoid the generation of fusion rotein.

Accompanying drawing explanation

Fig. 1 is the electrophorogram that specific enzymes is cut the AFP gene fragment of site sequence that contains of embodiment mono-gained, and wherein, 1 is AFP gene, and M is mark;

Fig. 2 is pIRES2-EGFP plasmid map;

Fig. 3 is the pIRES2-AFP-EGFP recombinant vectors collection of illustrative plates of embodiment bis-gained;

Fig. 4 is that the PCR of the pIRES2-AFP-EGFP recombinant vectors of embodiment bis-gained identifies electrophorogram, and wherein, 1 is PCR reaction product, and M is mark;

Fig. 5 is that the enzyme of the pIRES2-AFP-EGFP recombinant vectors of embodiment bis-gained is cut evaluation 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 tri-gained, and wherein, 1 is pcr amplification product A, and 2 is pcr amplification product B, and M is mark;

Fig. 7 is the schema of the hybridization PCR reaction described in embodiment tri-;

Fig. 8 is the pIRES2-AFP-IL-2 recombinant vectors collection of illustrative plates of embodiment tetra-gained;

Fig. 9 is that the enzyme of the pIRES2-AFP-IL-2 recombinant vectors of embodiment tetra-gained is cut evaluation electrophorogram, wherein, 1 is Bgl II/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, experimental technique used, such as round pcr, design of primers technology, vector construction technology, detection technique, electrophoretic technique etc. is the routine techniques in genetically engineered, those skilled in the art can be according to existing techniques in realizing (such as with reference to the work such as J. Pehanorm Brooker, the < < molecular cloning experiment guide > > that Huang Peitang etc. translate, Science Press's third edition; Or according to product description, carry out).In operating process, equipment used, reagent, carrier, bacterial strain etc., be by market and purchase available conventional products.

Embodiment mono-: obtain the AFP gene fragment that contains specificity restriction enzyme site

1, design of primers

According to the multiple clone site that on the nucleotide sequence of AFP gene (as shown in SEQ ID NO:1 in sequence table) and pIRES2-EGFP plasmid vector, expection is inserted, design Auele Specific Primer is as follows:

AFP upstream primer (as shown in SEQ ID NO:4 in sequence table):

5 '-GC aGATCTaTGAAGTGGGTGGAA-3 ' (underscore is partly Bgl II restriction enzyme site sequence),

AFP downstream primer (as shown in SEQ ID NO:5 in sequence table):

5 '-TT gAATTCtTAAACTCCCAAAGCAGC-3 ' (underscore is partly EcoR I restriction enzyme site sequence).

2, obtain cDNA template

TRIzon method is extracted RNA(TRIzon total RNA extraction reagent box purchased from Kang Wei century bio tech ltd, Beijing from human liver cancer cell HepG2, production code member is CW0580), and reverse transcription becomes cDNA(reverse transcription test kit purchased from precious biotechnology (Dalian) company limited, production code member is RR019A).

3, obtain the AFP gene fragment that contains specificity restriction enzyme site

The cDNA of the RNA institute reverse transcription of extracting in human liver cancer cell HepG2 of take is template, under the effect of the above-mentioned upstream and downstream primer that contains specificity restriction enzyme site, by PCR, react and obtain AFP gene fragment, 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.With 1% agarose gel electrophoresis, identify, 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 ℃ of denaturation 5min; 98 ℃ of sex change 10s, 55 ℃ of annealing 5s, 72 ℃ are extended 5s, 30 circulations; 72 ℃ of final 5min that extend.

Embodiment bis-: the structure of pIRES2-AFP-EGFP recombinant vectors

Use restriction enzyme Bgl II and EcoR I, enzyme is cut the AFP 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 mono-gained respectively, the AFP gene order after obtaining enzyme and cutting rear linearizing pIRES2-EGFP carrier and enzyme and cut; Adopt T4 DNA ligase system to carry out ligation, hatch 30 minutes at 22 ℃, then deactivation 5 minutes at 70 ℃, builds pIRES2-AFP-EGFP recombinant vectors (as shown in Figure 3).

Constructional feature (as shown in Figure 2) from pIRES2-EGFP plasmid, AFP gene inserts after the multiple clone site of pIRES2-EGFP plasmid, be positioned at the upstream (as shown in Figure 3) of self sequence IRES of plasmid vector, AFP and the EGFP sequence under same promotor starts expressed respectively.

1, double digestion pIRES2-EGFP plasmid

Endonuclease reaction system following (50 μ L):

Wherein, restriction endonuclease Bgl II and 10 * H Buffer are 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 are 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 ℃.

2, double digestion AFP gene fragment

Endonuclease reaction system following (50 μ L):

Endonuclease reaction condition: react 6 hours at 37 ℃.

3, connect AFP gene fragment and pIRES2-EGFP carrier, build pIRES2-AFP-EGFP recombinant vectors

Ligation system following (20 μ L):

Wherein, T4 DNA ligase and 10 * Ligation Buffer are DNA ligase buffer solution system, and purchased from Kang Wei century bio tech ltd, Beijing, production code member is CW0805.

Ligation condition: hatch 30 minutes 70 ℃ of deactivations 5 minutes for 22 ℃.

4, the evaluation of pIRES2-AFP-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, be placed in after cooled on ice 30min, be placed in 42 ℃ of water-bath thermal shock 90s, then be placed in cooled on ice 2min, then add LB liquid nutrient medium 780ul; Under 37 ℃ of conditions, on the shaking table of 150 revs/min, 60min is cultivated in recovery, the bacterium liquid of recovering is coated on the LB flat board that contains kalamycin resistance, at 37 ℃, is inverted and cultivates 16～18 hours; Picking positive bacteria drops down onto in kalamycin resistance LB liquid nutrient medium, under 37 ℃, the condition of 200rpm, cultivates 12～16 hours.

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 ℃ of denaturation 12min; 95 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations; 72 ℃ are extended 5min eventually.

Get bacterium liquid PCR identification reaction product, with 1% agarose gel electrophoresis, identify, electrophoresis result as shown in Figure 4.Electrophoresis result shows, occurs an electrophoretic band at 1830bp place, consistent with the molecular size range of goal gene AFP gene, shows the success of pIRES2-AFP-EGFP construction of recombinant vector.

Adopt plasmid extraction kit (purchased from Kang Wei century bio tech ltd, Beijing, 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 evaluation.

Enzyme is cut identification reaction system following (10 μ L):

Endonuclease reaction condition: react 1 hour at 37 ℃.

Get enzyme and cut identification reaction product, with 1% agarose gel electrophoresis, identify, electrophoresis result as shown in Figure 5., there are two bands in electrophoresis result demonstration, wherein one appears at 1830bp place, consistent with the molecular size range of goal gene AFP gene after electrophoresis, show the success of pIRES2-AFP-EGFP construction of recombinant vector.

Embodiment tri-: two PCR methods are obtained the IL-2 gene fragment with restriction enzyme sticky end

The multiple clone site of inserting according to expection on IL-2 gene order and pIRES2-EGFP plasmid vector, designs two pairs of length differences, with the primer of restriction enzyme sticky end; The cDNA of the RNA institute reverse transcription of extracting in CIK cell of take is template, carries out pcr amplification respectively with two pairs of above-mentioned primers, obtains two kinds of pcr amplification products; After being mixed, two kinds of pcr amplification products carry out successively sex change and annealing, 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, without using restriction enzyme to carry out enzyme, cut thus, IL-2 gene fragment orientation can be connected in the expection multiple clone site of plasmid vector.

Compare with traditional PCR product cloning method, 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 product.In the method, do not need to use restriction enzyme to process PCR product, can obtain the DNA fragmentation that contains restriction enzyme sticky end, can make full use of the multiple clone site of genophore, without considering, in goal gene, whether contain the restriction enzyme site identical with carrier multiple clone site, can easily realize the directed cloning of goal gene.

1, design of primers

The multiple clone site of inserting according to expection on the nucleotide sequence of IL-2 gene (as shown in SEQ ID NO:2 in sequence table) and pIRES2-EGFP plasmid vector, designs two pairs of length differences, with the primer of restriction enzyme sticky end, as follows:

IL-2 the first primer pair (FL/RL primer pair) is comprised 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 is partly 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 is partly the full sequence in cleavage site downstream in restriction enzyme Not I recognition sequence.

IL-2 the second primer pair (FS/RS primer pair) is comprised 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 is partly 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 is partly the reverse complementary sequence of the full sequence of cleavage site upstream in restriction enzyme Not I recognition sequence.

The target sequence of FL/RL primer pair and FS/RS primer pair (with the corresponding sequence of IL-2 gene) is identical.

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, obtain cDNA template

TRIzon method is from CIK cell (Cytokine-Induced Killer, cytokine induced kill cell) or in the mononuclearcell of human peripheral separation, extract RNA(TRIzon total RNA extraction reagent box purchased from Kang Wei century bio tech ltd, Beijing, production code member is CW0580), and reverse transcription becomes cDNA(reverse transcription test kit purchased from precious biotechnology (Dalian) company limited, production code member is RR019A).

3, obtain the IL-2 gene fragment with restriction enzyme sticky end

Take resulting cDNA as template, with FL/RL primer pair, carry out pcr amplification, obtain pcr amplification product A; Separately with FS/RS primer pair, carry out pcr amplification, obtain pcr amplification product B.Pcr amplification product A and pcr amplification product B all contain IL-2 gene order.

PCR reaction system following (50 μ L):

PCR reaction conditions is: 95 ℃ of denaturation 5min; 98 ℃ of sex change 10s, 55 ℃ of annealing 5s, 72 ℃ are extended 5s, 30 circulations; 72 ℃ of final 5min that extend.

Get respectively pcr amplification product A and pcr amplification product B, with 1% agarose gel electrophoresis, identify, electrophoresis result as shown in Figure 6.Electrophoresis result demonstration, 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 respectively pcr amplification product A and pcr amplification product B checks order, result shows: the 5' end of pcr amplification product A is held many 4 Nucleotide than the 5' of pcr amplification product B, the 3' end of pcr amplification product A is also held many 4 Nucleotide than the 3' of pcr amplification product B, and all the other sequences are identical.

By mole mixing such as pcr amplification product A and pcr amplification product B, hybridize PCR reaction, obtain IL-2 gene fragment mixture.

Hybridization PCR reaction system following (25 μ L):

Amplified production A(52.5 μ M) 10.8 μ L,

Amplified production B(46.1 μ M) 12.3 μ L,

Sterile purified water 1.9 μ L.

Hybridization PCR reaction conditions is as follows: 95 ℃ of 5min, 80 ℃ of 1min, 70 ℃ of 1min, 65 ℃ of 1min, 60 ℃ of 1min, 55 ℃ of 1min, 40 ℃ of 1min, 4 ℃ 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 hybrid 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.

Embodiment tetra-: the structure of pIRES2-AFP-IL-2 recombinant vectors

Use restriction enzyme BstX I and Not I double digestion pIRES2-AFP-EGFP recombinant vectors.Because restriction endonuclease BstXI is arranged in the terminal in IRES sequence downstream and the section start of EGFP sequence upstream of pIRES2-AFP-EGFP recombinant vectors, and restriction endonuclease Not I is arranged in the downstream termination point place of the EGFP sequence of pIRES2-AFP-EGFP recombinant vectors, therefore, by these two restriction enzyme sites, insert after IL-2 gene fragment, IL-2 gene is positioned at the downstream of IRES sequence, thereby make AFP gene and IL-2 gene lay respectively at the both sides (as shown in Figure 8) of IRES sequence, realize the coordinate expression of two goal gene, and can avoid the generation of fusion rotein.

1, Not I endonuclease reaction

Not I endonuclease reaction system following (50 μ L):

Endonuclease reaction condition: react 6 hours at 37 ℃.

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 Kang Wei century bio tech ltd, Beijing, production code member is CW2302) to carry out purifying, collect the pIRES2-AFP-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 are 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 ℃.

Adopt DNA fast purifying test kit (purchased from Kang Wei century bio tech ltd, Beijing, production code member is CW2302) to carry out purifying, collect the pIRES2-AFP-EGFP recombinant vectors after BstX I enzyme is cut.

3, connect IL-2 gene and pIRES2-AFP-EGFP recombinant vectors, build pIRES2-AFP-IL-2 recombinant vectors

The IL-2 gene fragment mixture that embodiment tri-is made mixes (mol ratio of IL-2 gene fragment and pIRES2-AFP-EGFP recombinant vectors is 4 ︰ 1) with linearizing pIRES2-AFP-EGFP recombinant vectors after double digestion, add T4DNA ligase enzyme to carry out ligation, at 22 ℃, hatch 30 minutes, then deactivation 5 minutes at 70 ℃, constructs pIRES2-AFP-IL-2 recombinant vectors.

Four kinds of DNA fragmentations in IL-2 gene fragment mixture, only have IL-2 gene fragment III to there is the sticky end with the complementation of pIRES2-AFP-EGFP recombinant vectors, thereby can be connected with pIRES2-AFP-EGFP recombinant vectors is directed, other three kinds of IL-2 gene fragments all can not be connected with pIRES2-AFP-EGFP recombinant vectors is directed.

Ligation system following (20 μ L):

Ligation condition: hatch 30 minutes 70 ℃ of deactivations 5 minutes for 22 ℃.

4, the evaluation of pIRES2-AFP-IL-2 recombinant vectors

The above-mentioned ligation product of 20 μ L is joined in 200 μ L competent cell JM109 to (1～2 * 10 ⁹bacteria/ml), be placed in after cooled on ice 30min, be placed in 42 ℃ of water-bath thermal shock 90s, then be placed in cooled on ice 2min; Then add LB liquid nutrient medium 780ul, under 37 ℃ of conditions, on the shaking table of 150 revs/min, 60min is cultivated in recovery, the bacterium liquid of recovering is coated on the LB flat board that contains kalamycin resistance, at 37 ℃, is inverted and cultivates 16～18 hours; Picking positive bacteria drops down onto in kalamycin resistance LB liquid nutrient medium, under 37 ℃, the condition of 200rpm, cultivates 12～16 hours.

Adopt plasmid extraction kit (purchased from Kang Wei century bio tech ltd, Beijing, 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 evaluation.

Endonuclease reaction system one (10 μ L):

Endonuclease reaction system two (10 μ L):

Endonuclease reaction system three (10 μ L):

Endonuclease reaction condition: react 1 hour at 37 ℃.

Get respectively above-mentioned endonuclease reaction product, with 1% agarose gel electrophoresis, identify, electrophoresis result as shown in Figure 9.Electrophoresis result shows, after Bgl II/EcoR I double digestion reaction product electrophoresis, occurs two bands, and wherein one at 1830bp place, consistent with the molecular size range of goal gene AFP gene; After EcoR I/Not I double digestion reaction product electrophoresis, occur two bands, wherein one at 1047bp place, in the same size with the molecular weight sum of goal gene IL-2 gene and IRES sequence; After Bgl II/Not I double digestion reaction product electrophoresis, occur two bands, wherein one at 2877bp place, in the same size with the molecular weight sum of goal gene AFP gene, IL-2 gene and IRES sequence.Above-mentioned qualification result shows, the success of pIRES2-AFP-IL-2 construction of recombinant vector.

By pIRES2-AFP-IL-2 recombinant vectors deliver to gold only intelligence biotechnology (Beijing) company limited check order, consistent with expected results through order-checking, further prove that vector construction successfully.

The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore 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 (9)

1.AFP and IL-2 double gene coexpression recombinant vectors, it is connected with AFP gene, IRES sequence and IL-2 gene in turn along carrier transcriptional orientation, or is connected with IL-2 gene, IRES sequence and AFP gene in turn along carrier transcriptional orientation;

The nucleotide sequence of described AFP 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.

2. AFP according to claim 1 and IL-2 double gene coexpression recombinant vectors, is characterized in that: described carrier is pIRES2-EGFP plasmid vector, and described double gene coexpression recombinant vectors is pIRES2-AFP-IL-2 recombinant vectors; Wherein, AFP gene is positioned at the upstream of IRES sequence, and IL-2 gene is positioned at the downstream of IRES sequence.

3. AFP according to claim 2 and IL-2 double gene coexpression recombinant vectors, is characterized in that: the EGFP sequence in described pIRES2-EGFP plasmid vector is by IL-2 gene substitution.

4. the preparation method of AFP claimed in claim 1 and IL-2 double gene coexpression recombinant vectors, comprises the following steps:

1) obtain the AFP gene fragment that contains specificity restriction enzyme site;

2) AFP gene fragment step 1) being obtained is connected in carrier, builds the recombinant vectors that contains AFP gene;

3) obtain and contain the IL-2 gene fragment that enzyme is cut sticky end;

4) IL-2 gene fragment step 3) being obtained is connected in step 2) recombinant vectors that obtains, build described AFP and IL-2 double gene coexpression recombinant vectors.

5. the preparation method of AFP claimed in claim 2 and IL-2 double gene coexpression recombinant vectors, comprises the following steps:

A) obtain the AFP gene fragment that contains specificity restriction enzyme site: from liver cancer cell HepG2, obtain cDNA as template, with the AFP Auele Specific Primer that contains Bgl II and EcoR I restriction enzyme site sequence, carry out pcr amplification, obtain the AFP gene fragment that contains 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-AFP-EGFP recombinant vectors: with restriction enzyme Bgl II, EcoR I respectively enzyme cut the AFP gene fragment that pIRES2-EGFP plasmid and step a) obtain, and adopt T4DNA ligase enzyme to carry out ligation, obtain pIRES2-AFP-EGFP recombinant vectors;

C) obtain and contain the IL-2 gene fragment that enzyme is cut sticky end: from CIK cell, obtain cDNA as template, with the IL-2 Auele Specific Primer that contains BstX I and Not I enzyme and cut sticky end, carry out pcr amplification, resulting PCR reaction product is hybridized PCR reaction again, obtain IL-2 gene fragment mixture, a kind of IL-2 gene fragment wherein has BstX I and Not I enzyme is cut sticky end;

D) build pIRES2-AFP-IL-2 recombinant vectors: with restriction enzyme BstX I, Not I enzyme, cut the pIRES2-AFP-EGFP recombinant vectors that step b) obtains, IL-2 gene fragment mixture and the enzyme that step c) is obtained cut rear linearizing pIRES2-AFP-EGFP recombinant vectors and mixed, adopt T4DNA ligase enzyme to carry out ligation, obtain pIRES2-AFP-IL-2 recombinant vectors.

6. preparation method according to claim 5, is characterized in that, the AFP Auele Specific Primer described in step a) is:

AFP upstream primer: 5 '-GCAGATCTATGAAGTGGGTGGAA-3 ',

AFP downstream primer: 5 '-TTGAATTCTTAAACTCCCAAAGCAGC-3 '.

7. preparation method according to claim 5, it is characterized in that, IL-2 Auele Specific Primer described in step c) comprises IL-2 the first primer pair and IL-2 the second primer pair, described IL-2 the first primer pair is comprised of IL-2 upstream long primer and IL-2 downstream long primer, and described IL-2 the second primer pair is comprised of IL-2 upstream short primer and IL-2 downstream short primer:

IL-2 the first primer pair is:

IL-2 upstream long primer: 5'-AACCATGTACAGGATGCAACTCCTGTCTT-3',

IL-2 downstream long primer: 5'-GGCCGCTCAAGTCAGTGTTGAGATGAT-3';

IL-2 the second primer pair is:

IL-2 upstream short primer: 5'-ATGTACAGGATGCAACTCCTGTCTT-3',

IL-2 downstream short primer: 5'-GCTCAAGTCAGTGTTGAGATGATGC-3'.

8. the AFP described in claim 1 or 2 and the application of IL-2 double gene coexpression recombinant vectors in hepatocarcinoma gene immunotherapy.

9. application according to claim 8, is characterized in that: in patients with implantation body after the described AFP dendritic cell separated with IL-2 double gene coexpression recombinant vectors transfection liver cancer patient self or allosome, carry out hepatocarcinoma gene immunotherapy.

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