CN100503833C - Expression plasmid for rep gene, RBE cis element fixed point integration system and its preparation method and uses - Google Patents

Abstract

The invention discloses a rep gene expression plasmid, RBE cis-form element fixed-point integrated system and producing method and application for gene treatment in the biological and gene treatment domain, which is characterized by the following: the fixed-point integrated system is composed of expression plasmid with RBE cis-form element and EGFP report gene and rep gene expression plasmid to act in common; or the fixed-point integrated system consists of expression plasmid with RBE cis-form element and human blood clotting factor IX to act in common. The fixed-point integrated system can be used in leading exogenesis gene in human cell strain, which operates together with other therapeutic equipment genes.

Description

The rep gene expression plasmid, RBE cis element site-directed integration system and its production and application

Technical field

The invention belongs to biotechnology and gene therapy technology field, be specifically related to a kind of rep gene expression plasmid, RBE cis element site-directed integration system and preparation method thereof and the application in gene therapy.

Background technology

Gene therapy has the irreplaceable advantage of additive method for the treatment of heredopathia, but gene therapy need modify patient's genome, this makes people show worry to its security.

The route of metastasis that several genetic manipulations are arranged at present, virus vector gene transfering efficiency height, but aspect security, there are many deficiencies:, can in host genome, cause inserting sudden change by random integration as adenovirus, adeno-associated virus, retroviral vector and lentiviral vectors.The gene transfering efficiency of non-virus carrier is low, and exogenous gene expression is of short duration, and the safety issue of random integration is also arranged.

Insert genomic danger at random about foreign gene and once caused great disturbance.2000, ALAIN FISHER etc. successfully cures three patients that suffer from fatal severe complex immunity defective disease SCID-XI, become the most successful example on the gene therapy history, yet, 2002, wherein two tested finding of SCID-XI patient of accepting gene therapy have been suffered from leukemia, and canceration be since when treatment the retroviral vector that uses be inserted near in the position or this proto-oncogene of LMO2 proto-oncogene, activated LMO2 and expressed and cause.This incident has caused more disputes on to the gene therapy feasibility, also to the security of gene therapy proposed more strict requirement (

CE,

A,

MA,

.

.2003; 4:346-358).

Be 2002 equally, according to MILLER etc., removed the recombined glandulae correlation viral vectors of rep gene owing to lost the ability of site-directed integration, made its random integration have polytropy, non-precision, and cause consequences such as chromosome deletion, rearrangement, functional gene are interrupted (

DG, EA, DW..

2002; 30:147-148), the security as gene therapy vector also throws doubt upon to adeno-associated virus to make people.

Because adeno-associated virus is unique human provirus that can site-directed integration of finding up to now, and the integrated mechanism of adeno-associated virus can be used to instruct fixed point integration of foreign gene to human genome, the Nonstructural Protein Nonstructural Protein REP of adeno-associated virus genome encoding plays a part crucial in this process, has caused the research interest that people are suitable in recent years.

The research in past thinks all that except that Rep albumen AAV ITR also is that foreign DNA is integrated necessary composition.Yet, act synergistically as reorganization AAV carrier and the Rep albumen that cis element makes up with ITR, even can site-directed integration to host, the virus of carrying foreign gene that after the host is infected once more by auxilliary poison, in the body generation is had replication, constitute potentially dangerous (Linden, R.M., E.Winocour, and K.I.Berns.1996.The recombination signals foradeno-associated virus site-specific integration.Proc.Natl.Acad.Sci.USA 93:7966-7972).Philpott in 2002 etc. discover if exist the P5 promoter sequence of 138bp (to be called P5 integration efficiency element: P5IEE) as cis element, then only Rep78/68 albumen need be arranged, not need ITR that site-directed integration at AAVS1 also can take place.P5IEE is the proteic regulation and control promotor of Rep78/68 as a multifunctional element, is again the main component of mediated targeted integration

Summary of the invention

The objective of the invention is to propose a kind of safe, fixed point integration of foreign gene system and preparation method thereof and application in gene therapy easily.

Site-directed integration system provided by the invention, comprise by the expression plasmid that carries RBE cis element and EGFP reporter gene (for the commercialization plasmid), the site-directed integration system of the foreign gene that constitutes with the acting in conjunction of rep gene expression plasmid, and by the expression plasmid that carries RBE cis element and human blood coagulation IX, the site-directed integration system of the foreign gene that constitutes with the acting in conjunction of rep gene expression plasmid; Wherein, the dna sequence dna of coding RBE is SEQ.NO1.

Site-directed integration system provided by the invention also can be by carrying RBE cis element (comprising other sequence that contains the RBE cis element), constituting jointly with the expression plasmid for the treatment of integrator gene and REP gene expression plasmid.

The present invention uses RBE and rep integration system can avoid virus packets dressing quantitative limitation, and the loaded down with trivial details and virus infection potential hidden danger of virus preparation imports by the gymnoplasm grain, and is succinct convenient; Can overcome potentially dangerouss such as functional gene fracture that insertion at random caused, proto-oncogene activation, chromosome deletion, rearrangement, reach and make external source therapeutic gene site-directed integration, the purpose of long-term expression, improving in the past, the gymnoplasm grain imports therapeutic gene expression time weak point, the unabiding defective of result of treatment.

The above-mentioned rep gene expression plasmid that the present invention proposes and the preparation method of RBE cis element site-directed integration system, concrete steps are as follows:

1, the design of RBE cis element

5 ' end in the normal chain of RBE sequence increases AseI viscosity restriction enzyme site, increases AseI viscosity restriction enzyme site at 3 ' end of minus strand.The dna sequence dna of designed coding RBE is seen SEQ.NO1.

2, the clone of RBE cis element and order-checking and pRBE-CMV-EGFP construction of recombinant plasmid

Synthetic carries the positive minus strand homologous segment annealing of the RBE renaturation of AseI sticky end, become double chain DNA fragment, insert the pEGFPN2 carrier of cutting through the AseI enzyme by the DNA recombinant technology, use enzyme ligation liquid transformed into escherichia coli DH5a host bacterium again, being coated on card at last receives on the LB culture medium flat plate of mycin resistance, screening may be the bacterium colony of recombinant clone, obtains the pRBE-CMV-EGFP recombinant plasmid.Recombinant plasmid has determined to insert the positive colony of RBE cis element by the handsome Bioisystech Co., Ltd in Shanghai sequencing analysis.

3, pRBE-CMV-FIX construction of recombinant plasmid

Cut out the FIX gene fragment with BamHI restriction enzyme enzyme from the pBS/FIX plasmid of identifying through dna sequencing, with the T4DNA ligase enzyme pRBE-CMV-EGFP (removing the EGFP gene) carrier is inserted in its reorganization then.Use enzyme ligation liquid transformed into escherichia coli DH5a host bacterium again, some extractive recombinant plasmids are cut evaluation through enzyme, determine that it is the recombinant vectors of desired insertion human blood coagulation IX gene.The dna sequence dna of coding FIX is SEQ.NO2.

The result shows, the present invention has successfully obtained pRBE-CMV-EGFP and pRBE-CMV-FIX recombinant expression plasmid, with pRBE-CMV-EGFP recombinant expression plasmid and pRep gene expression plasmid cotransformation embryo gastric cells 293 cells, can long-term expression EGFP and Neo gene, can produce Neo resistant cell clone expeditiously through the G418 screening; The genome of these cell clones of extracting is identified by southern blot, determines that the site-directed integration of foreign gene has taken place for 37% cell clone, and integration site is No. 19 karyomit(e) AAVS1 of people site (Fig. 1,2).Inject the transgenic mice that carries the AAVS1 site with pRBE-CMV-FIX recombinant expression plasmid and pRep plasmid altogether by tail vein hydraulic method, from mice plasma, detect human blood coagulation IX gene product long-term existence, inject and still maintained more than the 100ng/ml concentration in back 100 days.

Experiment shows that PRBE-CMV-EGFP provided by the invention and PRBE-CMV-FIX recombinant expression plasmid constitute the site-directed integration system with the PREP plasmid respectively.This site-directed integration system is used in human cell's strain and carries that fixed point imports foreign gene in the transgenic mice in AAVS1 site, and preparation procedure is simple.RBE cis element and other reporter genes are united utilization and can be reached and make fixed point integration of foreign gene, and the purpose of long-term expression is convenient to obtain the genetically modified cell strain of carrying of stable integration.Unite utilization with the other treatment gene and go for the treatment of multiple disease gene, improving in the past the gymnoplasm grain, to import therapeutic gene expression time short, and the unabiding defective of result of treatment is significant for the development gene therapy vector.

Description of drawings

Fig. 1: the synoptic diagram of pRBE-CMV-EGFP recombinant plasmid and pRep integration system.Wherein, A is the pRep plasmid, and B is the pRBE-CMV-EGFP recombinant plasmid.

Fig. 2: pRBE-CMV-EGFP recombinant plasmid and pRep integration system rotaring redyeing 293 cell, clone through the stabilized cell that the G418 screening obtained after 14 days; Wherein, the clone that sees under fluorescent microscope after 14 days for the EGFP control plasmid rotaring redyeing 293 cell screening that does not contain any element of A forms situation; B is that the clone behind pRBE-CMV-EGFP recombinant plasmid and the pRep integration system rotaring redyeing 293 cell forms situation; C is the EGFP control plasmid rotaring redyeing 293 cell screening that the do not contain any element methylene blue observed cell clone that dyes after 14 days; D be the screening of pRBE-CMV-EGFP recombinant plasmid and pRep integration system rotaring redyeing 293 cell after 14 days the methylene blue observed clone that dyes form situation.

Fig. 3: the stabilized cell clone extracting genome southern blot that pRBE-CMV-EGFP recombinant plasmid and pRep integration system rotaring redyeing 293 cell obtain detects integration site.Wherein, A is the AAVS1 probe hybridization, and B is the Neo probe hybridization.

Embodiment

Material and method

1. restriction enzyme A seI, BamHI, T4DNA ligase enzyme, the klenow archaeal dna polymerase is a Britain Biolabs product.

2. clone and sequencing vector pEGFPN2 are Clontech company products.

3.Promega the plasmid extraction test kit is a promega company product.It is vast Imtech product that vast Tyke glue reclaims test kit.

4. the positive minus strand nucleotide base fragment of designed RBE cis element is synthetic by Shanghai Ying Jun Bioisystech Co., Ltd.

Above material all has commercially available

1.RBE the design clone of cis element and order-checking and pRBE-CMV-EGFP construction of recombinant plasmid

5 ' end in the normal chain of RBE sequence increases AseI viscosity restriction enzyme site, increases AseI viscosity restriction enzyme site at 3 ' end of minus strand.The positive minus strand nucleotide base of RBE cis element fragment is synthetic by Shanghai Ying Jun Bioisystech Co., Ltd, synthetic carries the positive minus strand homologous segment annealing of the RBE renaturation of AseI sticky end, become double chain DNA fragment, by the T4DNA ligase enzyme the double-stranded annealing of RBE product is inserted the pEGFPN2 carrier of cutting through the AseI enzyme, use enzyme ligation liquid transformed into escherichia coli DH5 α host bacterium again, be coated on card at last and receive that screening may be the bacterium colony of recombinant clone on the LB culture medium flat plate of mycin resistance.Recombinant plasmid has determined to insert the positive colony of RBE cis element by the handsome Bioisystech Co., Ltd in Shanghai sequencing analysis.

2.pRBE-CMV-FIX construction of recombinant plasmid

Cut out the FIX gene fragment with BamHI restriction enzyme enzyme from the pBS/FIX plasmid of identifying through dna sequencing, mend flat terminal with the klenow archaeal dna polymerase, pRBE-CMV-EGFP is with BamHI/Not excision EGFP gene and mend flat terminal, reclaim carrier, with the T4DNA ligase enzyme FIX fragment is linked to each other with carrier segments then, use enzyme ligation liquid transformed into escherichia coli DH5 α host bacterium again, some extractive recombinant plasmids are cut evaluation through enzyme, determine the recombinant vectors of desired insertion human blood coagulation IX gene.

3.pRBE-CMV-EGFP with pRep plasmid co-transfection 293 cells

Inoculation 293 cells in six orifice plates, second day cell density reaches at 70%～80% o'clock and carries out transfection.PRBE-CMV-EGFP2 μ g+pRep0.4 μ g/ hole, the Lipofecamine of use invitrogen company ^TMReagent carries out transfection.Transfection was cleaned cell after 5 hours, renewed bright nutrient solution.Cell suspension was got up in 24 hours after the transfection, suitably dilute, be transferred in the 5cm culture dish, the nutrient solution that adds the G418 that contains 500 μ g/ml screens, and screens the cell clone that can obtain EGFP and the integration of Neo stable gene after 14 days.

4. detect the integration situation of foreign gene in cell clone

With the above-mentioned cell clone enlarged culturing that obtains, the genome of these cell clones of extracting after about two weeks, identify by southern blot, hybridize with fluorescently-labeled Neo gene specific probe earlier, can learn the distribution of Neo gene each cell clone from obtaining the x-ray slice, thin piece, then film is cleaned 3 times in the 0.1%SDS that boils, remove the Neo probe, hybridize with fluorescently-labeled Neo gene specific probe again, can learn the distribution in AAVS1 site in each cell clone, the x-ray slice, thin piece contrast of twice hybridization is if the special identical just explanation with AAVS1 specific band position of the band Neo gene in this cell clone of Neo has inserted No. 19 chromosomal AAVS1 specific sites of people.In the cell clone that pRBE-CMV-EGFP and pRep cotransfection obtain, there is 37% cell clone that the site-directed integration (Fig. 1,2) of foreign gene has taken place.

5. tail vein hydraulic method imports foreign gene in transgenic mice

Use the transgenic mice that carries the AAVS1 site in 6～8 ages in week, pRBE-CMV-FIX 25 μ g+pRep 25 μ g are dissolved in 2.5ml Ringer ' s solution, and (composition is 0.9%NaCl, 0.03%KCl, and 0.016%CaCl2) in, 2.5ml solution is injected mouse tail vein within 7 seconds with No. 27 syringe needles.But eye socket is gathered mouse blood sample (adding 3.8% sodium citrate anticoagulant) after 1 day, detects human blood coagulation IX concentration in the mice plasma with the ELISA method.After testing, inject that human blood coagulation IX concentration still maintains more than the 100ng/ml concentration in back 100 days transgenic mice blood plasma.

The sequence that the present invention relates to:

The SEQ.NO1:RBE sequence

5’-ATCAGCGAGCGAGCGAGCTAAT-3’

5’-ATGCTCGCTCGCTCGCTGTAAT-3’

SEQ.NO2: the dna sequence dna of coding FIX

5’-ATGCAGCGCGTGAACATGATCATGGCAGAATCACCAGGCCTCATCACCATCTGCCTTTTAGGATATCTACTCA

GTGCTGAATGTACAGGTTTGTTTCCTTTTTTAAAATACATTGAGTATGCTTGCCTTTTAGATATAGAAATATCTGAT

GCTGTCTTCTTCGCTAAATTTTGATTACATGATTTGACAGCAATATTGAAGAGTCTAACAGCCAGCACGCAGGTTGG

TAAGTACTGGTTCTTTGTTAGCTAGGTTTTCTTCTTCTTCATTTTTAAAACTAAATAGATCGACAATGCTTATGATG

CATTTATGTTTAATAAACACTGTTCAGTTCATGATTTGGTCATGTAATTCCTGTTAGAAAACATTCATCTCCTTGGT

TTAAAAAAATTAAAAGTGGGAAAACAAAGAAATAGCAGAATATAGTGAAAAAAAATAACCACATTATTTTTGTTTGG

ACTTACCACTTTGAAATCAAAATGGGAAACAAAAGCACAAACAATGGCCTTATTTACACAAAAAGTCTGATTTTAAG

ATATATGACATTTCAAGGTTTCAGAAGTATGTAATGAGGTGTGTCTCTAATTTTTTAAATTATATATCTTCAATTTA

AAGTTTTAGTTAAAACATAAAGATTAACCTTTCATTAGCAAGCTGTTAGTTATCACCAAAGCTTTTCATGGATTAGG

AAAAAATCATTTTGTCTCTATGTCAAACATCTTGGAGTTGATATTTGGGGAAACACAATACTCAGTTGAGTTCCCTA

GGGGAGAAAAGCAAGCTTAAGAATTGACATAAAGAGTAGGAAGTTAGCTAATGCAACATATATCACTTTGTTTTTTC

ACAACTACAGTGACTTTATGTATTTCCCAGAGGAAGGCATACAGGGAAGAAATTATCCCATTTGGACAAACAGCATG

TTCTCACAGGAAGCATTTATCACACTTACTTGTCAACTTTCTAGAATCAAATCTAGTAGCTGACAGTACCAGGATCA

GGGGTGCCAACCCTAAGCACCCCCAGAAAGCTGACTGGCCCTGTGGTTCCCACTCCAGACATGATGTCAGCTGGACC

ATAATTAGGCTTCTGTTCTTCAGGAGACATTTGTTCAAAGTCATTTGGGCAACCATATTCTGAAAACAGCCCAGCCA

GGGTGATGGATCACTTTGCAAAGATCCTCAATGAGCTATTTTCAAGTGATGACAAAGTGTGAAGTTAACCGCTCATT

TGAGAACTTTCTTTTTCATCCAAAGTAAATTCAAATATGATTAGAAATCTGACCTTTTATTACTGGAATTCTCTTGA

CTAAAAGTAAAATTGAATTTTAATTCCTAAATCTCCATGTGTATACAGTACTGTGGGAACATCACAGATTTTGGCTC

CATGCCCTAAAGAGAAATTGGCTTTCAGATTATTTGGATTAAAAACAAAGACTTTCTTAAGAGATGTAAAATTTTCA

TGATGTTTTCTTTTTTGCTAAAACTAAAGAATTATTCTTTTACATTTCAGTTTTTCTTGATCATGAAAACGCCAACA

AAATTCTGAATCGGCCAAAGAGGTATAATTCAGGTAAATTGGAAGAGTTTGTTCAAGGGAACCTTGAGAGAGAATGT

ATGGAAGAAAAGTGTAGTTTTGAAGAAGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACTGAATTTTGGAAGCA

GTATGTTGATGGAGATCAGTGTGAGTCCAATCCATGTTTAAATGGCGGCAGTTGCAAGGATGACATTAATTCCTATG

AATGTTGGTGTCCCTTTGGATTTGAAGGAAAGAACTGTGAATTAGATGTAACATGTAACATTAAGAATGGCAGATGC

GAGCAGTTTTGTAAAAATAGTGCTGATAACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGCAGAAAACCA

GAAGTCCTGTGAACCAGCAGTGCCATTTCCATGTGGAAGAGTTTCTGTTTCACAAACTTCTAAGCTCACCCGTGCTG

AGGCTGTTTTTCCTGATGTGGACTATGTAAATTCTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAAGCACC

CAATCATTTAATGACTTCACTCGGGTTGTTGGTGGAGAAGATGCCAAACCAGGTCAATTCCCTTGGCAGGTTGTTTT

GAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTATCGTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTG

AAACTGGTGTTAAAATTACAGTTGTCGCAGGTGAACATAATATTGAGGAGACAGAACATACAGAGCAAAAGCGAAAT

GTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTATTAATAAGTACAACCATGACATTGCCCTTCTGGAACT

GGACGAACCCTTAGTGCTAAACAGCTACGTTACACCTATTTGCATTGCTGACAAGGAATACACGAACATCTTCCTCA

AATTTGGATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACAAAGGGAGATCAGCTTTAGTTCTTCAGTACCTT

AGAGTTCCACTTGTTGACCGAGCCACATGTCTTCGATCTACAAAGTTCACCATCTATAACAACATGTTCTGTGCTGG

CTTCCATGAAGGAGGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCCATGTTACTGAAGTGGAAGGGACCAGTT

TCTTAACTGGAATTATTAGCTGGGGTGAAGAGTGTGCAATGAAAGGCAAATATGGAATATATACCAAGGTATCCCGG

TATGTCAACTGGATTAAGGAAAAAACAAAGCTCACTTAATGAAAGATGGATTTCCAAGGTTAATTCATTGGAATTGA

AAATTAACAGTGCAGGTCGACTCTAGA-3’

Claims (2)

1, a kind of plasmid of fixed point integration of foreign gene combination, expression plasmid and the acting in conjunction of rep gene expression plasmid that carries RBE cis element and EGFP reporter gene that it is characterized in that serving as reasons constitutes, and perhaps constitutes by carrying RBE cis element and human blood coagulation IX expression of gene plasmid and the acting in conjunction of rep gene expression plasmid; The dna sequence dna of RBE of wherein encoding is SEQ.NO:1.

2, a kind of plasmid of fixed point integration of foreign gene as claimed in claim 1 is combined in the application in cell strain reconstruction and the preparation gene therapy medicament.

Images