CN101787373A

CN101787373A - Foreign gene-carrying recombinant virus vector efficiently produced in packaging cell and construction method and application thereof

Info

Publication number: CN101787373A
Application number: CN200910045994A
Authority: CN
Inventors: 钱其军; 金华君; 李琳芳; 吕赛群; 吴孟超
Original assignee: Oriental Hepatobiliary Surgery Hospital Second Military Medical University Of Chinese Pla
Current assignee: Oriental Hepatobiliary Surgery Hospital Second Military Medical University Of Chinese Pla
Priority date: 2009-01-23
Filing date: 2009-01-23
Publication date: 2010-07-28
Anticipated expiration: 2029-01-23
Also published as: CN101787373B

Abstract

The invention provides a foreign gene-carrying recombinant virus vector efficiently produced in a packaging cell and a construction method and application thereof. The foreign gene-carrying recombinant virus vector is characterized in that at least one target point nucleotide sequence of the following micro RNA is inserted into the 3' non-translational zone of a foreign gene connected with a virus in an operable way; and the micro RNA expresses at a high level in a virus packaging cell, and does not express or expresses at a low level in a target cell. Therefore, the invention realizes the selective inhibition of the expression of the foreign gene in the virus packaging cell, avoids the negative effect of the foreign gene on the virus packaging and production, and improves the production efficiency and titer of the virus. Simultaneously, the invention can not lower the expression level of the foreign gene in the target cell, and can not affect the therapeutic effect or the research on the function of the foreign gene.

Description

A kind of recombinant viral vector that carries foreign gene High-efficient Production in packing cell, its construction process and uses thereof

Invention field

Relate generally to recombinant viral vector of the present invention.More particularly, the present invention relates to a class carry foreign gene in packing cell High-efficient Production recombinant viral vector, its construction process and uses thereof.

Background technology

Virus is to specific cells infection rate height, can efficiently foreign gene be transduceed in the cell, and long-time the expression, the overexpression of instrument mediate foreign gene in target cell of Chang Zuowei transgenosis, the mode that obtains (gain-of-function) by function is studied the function of gene.On the other hand, virus also is the most frequently used carrier of gene therapy, by in September, 2008, existing in the world 1472 gene therapy schemes are used for clinical trial, wherein totally 1006 clinical trials adopt different types of virus as carrier, account for 68.3% (referring to database http://www.wiley.co.uk/genmed/clinical/) of sum.

The packing of recombinant virus needs suitable package cell line with amplification, as 293 cells, promptly contains the HEKC in 5 type adenovirus E 1 districts, is usually used in the packing and the production of recombinant adenovirus, adeno-associated virus; 293T and 293FT cell (the quick growth mutation of 293T) are derived from by 293 cells, express the SV40 large T antigen simultaneously, are usually used in the packing and the production of recombinant slow virus; The GP293 cell is derived from by 293 cells, expresses Gag-pol albumen simultaneously, is usually used in retroviral packing and production.Carry the recombinant virus of foreign gene and can in packing cell, assemble or duplicate propagation, impel a large amount of amplifications of the foreign gene that comprises in the recombinant virus genomes and express.Yet, the foreign gene pair cell of overexpression has certain toxicity on the one hand, especially suicide gene, apoptosis gene, cell cycle genes involved etc., cause viral packing cell dead ahead of time, shortened the opportunity that each functional gene of recombinant virus is transcribed and translated in packing cell, and cause the part virion to have little time assembling; On the other hand, transcribing in a large number and translate also of foreign gene can take hypercellular rna transcription, protein translation involved enzyme class and resource, thereby suppressed transcribing and translating of each functional gene of recombinant virus indirectly.The influence of two aspects has reduced the production efficiency of recombinant virus in packing cell, can't obtain enough virus titers.

Therefore, obtain higher virus titer, be starved of the method that in packing cell, to carry out the High-efficient Production recombinant virus for making the recombinant virus that carries foreign gene.

Microrna (microRNA) is the endogenous small fragment RNA of organism, by combining with target site nucleotide sequence in the 3 ' non-translational region of the ripe mRNA of target gene, suppresses the translation of target gene protein.Microrna has the tissue specificity and the sequential of height, expression level difference is big in dissimilar cells, thereby can be by the regulatory mechanism and the expression tissue specificity of Microrna, utilize viral packing cell high level expression and do not express in the target cell or the low micro RNA regulation and control expression of exogenous gene of expressing, foreign gene is not expressed in packing cell, improve virus production efficient and titre, and in target cell, do not reduce the expression of exogenous gene level, keep original result of treatment or study the foreign gene function by the mode that function obtains.

Summary of the invention

The invention provides a kind of recombinant viral vector, it is characterized in that inserting in the 3 ' non-translational region of the foreign gene that this virus is operably connected the target site nucleotide sequence of at least one following small RNAs (microRNA), described Microrna high level expression and not expressing in the target cell or low expression the in viral packing cell.

In one embodiment, described virus is selected from adenovirus (Adenovirus), adeno-associated virus (Adeno-associated virus), retrovirus (Retrovirus), slow virus (Lentivirus), vaccinia virus (Vaccinia virus), vaccinia virus (Poxvirus), hsv (Herpes simplex virus), Chinese mugwort Pasteur's virus (Epstein-Barr virus), people's papillary tumor virus (Human Papillomavirus), baculovirus (Baculoviruses), flavivirus (Flavivirus), Measles virus (Measlesvirus), Avian pneumo-encephalitis virus (Newcastle disease virus), Semliki forest virus (Semliki forest virus), Sendai virus (Sendai virus), at least a in simian virus 40 (Simian virus 40) and the alphavirus (Alphaviruses).Described virus is preferably selected from adenovirus, adeno-associated virus, retrovirus, slow virus, vaccinia virus, and more preferably described virus is selected from adenovirus.

In one embodiment, the foreign gene that is operably connected of described virus is selected from least a in apoptosis gene, prodrug conversion enzyme gene, cell cycle genes involved, cytokine gene, signaling genes, transcription factor gene, oncogene, cancer suppressor gene, blood vessel suppressor gene, antibody gene (comprising full length antibody, chimeric antibody, Fab fragment, Fv fragment), the vaccine gene.Preferred described gene is selected from apoptosis gene, prodrug conversion enzyme gene, cancer suppressor gene, blood vessel suppressor gene, antibody gene, vaccine gene, and more preferably described gene is selected from apoptosis gene and antibody gene.

In one embodiment, described viral target cell can be selected from least a in myocardial cell, Skeletal Muscle Cell, inoblast, vascular endothelial cell, liver cell, lymphocyte, neurocyte, adipocyte, embryonic stem cell, adult stem cell, the tumour cell.Described target cell is preferably selected from liver cell, tumour cell.

In one embodiment of the invention, described Microrna target site sequence is made of in series connection multiple mode Microrna target site sequence identical more than one or more.Wherein, the unit number of Microrna target site sequence is 100,90,80,70,60,50,40,30,20,10,9,8,7,6,5,4,3,2 or 1.Preferred 1-3,1-5,1-7,1-9,1-11,1-15,1-20.

In one embodiment, described Microrna target site sequence is made of in the placed in-line mode of continuous series connection or interval Microrna target site sequences different more than one or more.Wherein, the unit number of Microrna target site sequence is respectively 100,90,80,70,60,50,40,30,20,10,9,8,7,6,5,4,3,2 or 1.Preferred 1-3,1-5,1-7,1-9,1-11,1-15,1-20.

In one embodiment, described viral packing cell is selected from 293,293T, 293FT, 293E, 293ET, 293KB, 293Eco, at least a among GP293 and the FIP293.Described packing cell is preferably selected from 293,293FT.

In one embodiment, described Microrna target site sequence is selected from miR-15a, miR-16, miR-17, miR-18a, at least a among miR-19b and the miR-196a.Described Microrna target site sequence preference is selected from miR-15a, miR-18a, miR-16.

In one embodiment of the invention, selected virus is human adenovirus carrier, and it comprises A, B, C, D, an E and F6 subgenus.

In one embodiment of the invention, selected adenovirus carrier derives from the adenovirus of human C subgenus 5 types.

In one embodiment, the invention still further relates to the construction process of recombinant viral vector, it is characterized in that: in 3 ' non-translational region of the foreign gene that described virus vector is operably connected, inserted the target site nucleotide sequence of at least one following small RNAs, described Microrna high level expression and not expressing in the target cell or low expression the in viral packing cell.

In one embodiment, the foreign gene that described recombinant virus is used for recombinant virus is operably connected is not expressed at viral packing cell, and in target cell normal expression.In one embodiment, described recombinant virus is used to suppress growth of tumour cell.

The accompanying drawing summary

The carrier collection of illustrative plates of Fig. 1: pPE3-F11.Mainly by pBHGlox (delta) E1,3Cre transforms and forms, E1 district disappearance, can with homologous recombination in the plasmid born of the same parents that comprise the adenovirus left arm, acquisition has the adenovirus of infection ability; Comprise the E3 district that derives from pBHGE3, its 5 type cilia protein gene is replaced by 5/11 fiber.

Fig. 2: expression virus vector Ad11-RTX and the replication comparative result of Ad11-RTX-15T in the HEK293 cell.

Fig. 3: the expression amount analytical results of expression Rituxan in HEK293 and L02 cell.

Fig. 4: expression control group and treatment group mouse different time sections transplanted tumor volume statistic analysis result.

Detailed Description Of The Invention

As indicated above, the present invention relates to a kind of recombinant viral vector, it is characterized in that comprising at least high level expression in the Viral packaging cell in the 3 ' non-translational region of the foreign gene that this virus is operably connected and do not express in the target cell or the target site nucleotide sequence of low Microrna (microRNA) of expressing.

Microrna (microRNA, miRNA) be that organism is endogenous, length is about the little RNA of non-coding of 19-25 nucleotides, it by with target gene mRNA on target complement sequence pairing combination, negative regulation is carried out in expression to gene on post-transcriptional level, cause degraded or the translation of mRNA to suppress (Lai, 2002). According to estimates, have about 1000 miRNAs in human genome, most of Micrornas are positioned at the subarea that includes of intergenic region or protein coding gene, and minority is positioned at exon 1 or mRNA non-translational region. Most of Microrna individualism, the part Microrna flocks together, and becomes a Microrna gene cluster. Bioinformatic data shows, human about 1/3 protein coding gene is subjected to the regulation and control of Microrna, and each Microrna can be regulated 100-200 target gene, and a plurality of Microrna can be regulated same gene, the regulated and control network (Berezikov et al., 2006) that has formed a complexity.

Microrna is quite conservative in spore, only expresses at specific tissue and stage of development, has tissue specificity and the timing of height. The specifically expressing in heart and skeletal muscle tissue such as miR-1 and miR-133, miR-1 can be regulated and control the balance of cardiac muscle cell between differentiation and propagation, and miR-133 can regulate and control the differentiation of muscle cell, and it is expressed to descend and can promote the regeneration of zebra fish fin; MiR-122 is a large amount specifically expressing in the liver of people and mouse, and its content can account for 70% of all miRNA of liver; MiR-126 is specifically expressing in the heart, lung endothelial cell, can suppress the interaction of lymphocyte and endothelial cell, prevents the generation of vascular inflammation; MiR-142, miR-181, miR-223 specifically expressing in hematopoietic tissue, wherein miR-181 is at the CD4 of mouse chest cell growth⁺CD8 ⁺Two positive phases are specific expressed, the maturation of regulation and control B cell, and miR-223 expresses high in marrow, can regulate and control granulocytic maturation; MiR-143 is specifically expressing in adipose tissue, can regulate and control the differentiation of adipocyte, knocks out the accumulation that miR-143 will cause triglycerides; MiR-371-373 bunch (comprises miR-271, miR-272, miR-273) specifically expressing in people's embryo, and mir-290-295 bunch (comprise miR-290, miR-291, miR-292, miR-293, miR-294, miR-295) specifically expressing in the embryo of mouse then, they play an important role in the atomization of embryonic stem cell; MiR-375 is specifically expressing in the mouse islet cells, the regulation and control islet cells is grown and insulin secretion, and in addition, miR-375 also efficiently expresses at the zebra fish pituitary gland, regulate the secretion (referring to summary Landgraf et al., 2007) of other hormones and neuroendocrine product.

Have at the higher Microrna of 293 serial cells levels: miR-15a, miR-16, miR-17, miR-18a, miR-19b, is miR-196a (referring to Microrna database http://www.microrna.org/microrna/get ExprForTissues.do? tissue=hsa_Kidney-embryo-HEK293+). Wherein miR-15a and miR-16 are closely adjacent, are positioned at human genome 13q14 district, belong to same Microrna bunch; MiR-17, miR-18a and miR-19b are closely adjacent, are positioned at human genome 13q31 district, belong to miR-17-92 bunch; MiR-196a has two copies in human genome, wherein miR-196a-1 is positioned at the 17th chromosome, and miR-196a-2 is positioned at the 12nd chromosome. Except higher level in 293 serial cells is expressed, miR-15a and miR-16 also have higher expression in multiple T cell, B cell, and 65% B cell chronic lymphatic type leukaemia (CLL) patient, 50% lymphoma mantle cell patient, the myeloma patient of 16-40%, the disappearance that the 13q14 district is all arranged among 60% the prostate cancer patient causes the forfeiture (Calin et al., 2005) of miR-15a and miR-16 gene; The genome 13q31 district at miR-17, miR-18a and miR-19b place is the normal amplification that the site takes place in the tumours such as the B cell lymphoma that scatters, follicular lymphoma, lymphoma mantle cell, causes its abnormal expression to raise (He et al., 2005); MiR-196a expression in breast cancer cell MCF7 is higher. Above Microrna expression level in the organ-tissues such as the heart, lung, liver, spleen, ovary, uterus, prostate, thyroid gland is lower or do not express.

High level expression in application packages cell of the present invention and do not express in the target cell or the target site nucleotide sequence of low Microrna of expressing inserts 3 ' non-translational region of the foreign gene that comprises in the recombinant virus genomes, thereby make foreign gene can not be in incasing cells synthetic proteins, do not affect normal packing and the propagation of recombinant virus in incasing cells, thereby improve the production efficiency of recombinant virus, improve virus titer. And in target cell, do not reduce the expression of foreign gene, keep original result for the treatment of or by gain-of-function Study of Exogenous gene function.

" recombinant virus " comprises adenovirus (Adenovirus), adeno-associated virus (Adeno-associated virus), retroviruse (Retrovirus), slow virus (Lentivirus), vaccinia virus (Vacciniavirus), vaccinia virus (Poxvirus), herpes simplex virus (Herpes simplexvirus), Chinese mugwort Pasteur's virus (Epstein-Barrvirus), people's papillary tumor virus (Human Papillomavirus), baculoviral (Baculoviruses), flavivirus (Flavivirus), measles virus (Measles virus), NDV (Neweastle disease virus), Semliki forest virus (Semlikiforest virus), sendai virus (Sendai virus), simian virus 40 (Simianvirus 40) and alphavirus (Alphaviruses). " incasing cells " refers to that it can be the clone that goes down to posterity of stablizing of cultivating for the clone of virus restructuring, amplification from n cell, also can be the clone that comprises virus packing indispensable gene in its genome through transforming. Can be used for restructuring and the amplification of adenovirus such as 293 cells.

" target cell " refers to the target infection cell of recombinant virus, both can be functional impairment or disorder, the cell that needs the recombinant virus foreign gene-carrying to proofread and correct also can be that function is normal, be suitable for expression alien gene behind the recombinant virus infection, the cell of Study of Exogenous gene function.

" in the incasing cells high level expression and do not express in the target cell or low Microrna of expressing " refers to the big Microrna of expression difference between incasing cells and target cell, namely at incasing cells expression height, and low or do not have at the target cell expression. Such as miR-15a, it is at 293 cells level height, and is that the L02 expression is extremely low the normal liver cell. Usually, described " low express " refers to compare with incasing cells, in the target cell 1/3 of microrna expression quantity not sufficient incasing cells.

" the target site nucleotide sequence of Microrna " thus refer to Microrna can be with it in conjunction with the dna sequence dna of performance regulating and controlling effect. It both can be the dna sequence dna with the pairing of the complete reverse complemental of ripe Microrna, also can be to be combined with Microrna in the known Microrna target gene, with the dna sequence dna of the incomplete reverse complemental pairing of Microrna.

" foreign gene " refer to comprise in the recombinant virus genomes but do not belong to the genomic protein coding gene of wild-type virus, it can be the native protein encoding gene of human or other species, also can be artificial synthetic protein coding gene.

" 3 ' non-translational region of foreign gene " refers to the zone that does not translate in the coded sequence downstream among the ripe mRNA of foreign gene, and it is between foreign gene terminator codon and polyA tail.

The series connection of the identical Microrna target site sequence " repeat " refers to more than 2 not joining end to end of interval of identical Microrna target site sequence, perhaps identical Microrna target site sequence several nucleotide sequences but the centre is separated by that join end to end more than 2.

" different Microrna target site sequences are connected continuously " is meant that a kind of Microrna target site sequence series connection repeats the series connection repetition that the back connects another kind of Microrna target site sequence.Different Microrna target site sequences connect another kind of Microrna target site sequence after connecting at interval and being meant a kind of Microrna target site sequence, and repeat more than 2 times as the unit.

In one embodiment of the invention, packing cell is selected from people's kidney embryo HEK293 clone, and other clone of deriving from HEK293 and cultivating, as 293T, and 293FT, 293E, 293ET, 293KB, 293Eco, GP293, FIP293 etc.

In one embodiment of the invention, described Microrna target site nucleotide sequence can be selected from the miR-15a of high level expression in the 293 serial cells, miR-16, miR-17, miR-18a, miR-19b, at least a among the miR-196a.

The practical use of the recombinant adenoviral vector, its construction process and the described recombinant adenovirus that adopt layout strategy of the present invention is also disclosed in the present invention illustratively.For example by carry total length Mabthera Rituxan full length antibody gene 3 ' non-translational region insert the dna fragmentations of 5 copy miR-15a target site sequences, make this recombinant adenovirus that contains the foreign gene Mabthera in packing cell 293, obtain high titre, and behind the virus infection target cell-liver cell by this method amplification, can in liver cell, express activated Rituxan antibody effectively, and make it to be secreted into the extracellular, finally act on CD20 male tumour cell, cause death of neoplastic cells.

The practical use of the recombined lentivirus vector, its construction process and the described recombinant slow virus that adopt layout strategy of the present invention is also disclosed in the present invention illustratively.For example insert the dna fragmentations of 4 copy miR-15a target site sequences, make it in packing cell 293FT cell, to have obtained the recombinant slow virus of high titre by 3 ' non-translational region of the people P53 gene that is operably connected at recombinant slow virus.

Compare with existing recombinant viral vector, the present invention has following beneficial effect:

The present invention inserts high level expression at least one packing cell and does not express in the target cell or the target site nucleotide sequence of low Microrna of expressing in 3 ' non-translational region of the foreign gene that recombinant viral vector is operably connected, make foreign gene can not be in the packaging series cell along with duplicating of virus great expression, the competition that relevant enzymes and resource were transcribed, translated to cytotoxicity and the pair cell of having avoided the foreign gene overexpression to cause has improved the production efficiency and the virus titer of recombinant virus.And in target cell, do not reduce the expression of exogenous gene level, keep original result of treatment or study the foreign gene function by gain-of-function.

Embodiment among the present invention only is used for illustrative specific embodiments of the present invention, and it does not limit the application's protection domain, and the improvement of the equivalence that any those skilled in the art can carry out according to prior art is included within the application's the protection domain.

Embodiment

One. embodiment 1: carry Mabthera (Rituxan) gene and can be in 293 cells the structure of the recombinant adenoviral vector of High-efficient Production

1. carry the structure of the replication-defective adenoviral vector of Mabthera (Rituxan) gene

The pDC315 carrier is purchased in Canadian Microbix Biosystem Inc. (Toronto), contain 5 type adenovirus left arm E1 districts, 1417 to 2344bp sequence fragments, and reverse terminal repeat (ITR), can with the skeleton plasmid generation homologous recombination that comprises the adenovirus right arm, obtain replication-defective adenoviral.The pDC315 carrier is cut transformation through enzyme, replace the mCMV promotor, connect EF1 α (eukaryotic translation elongation factor 1 alpha) promotor and intron, new multiple clone site, keep original SV40 PolyA signal sequence, naming this carrier is pDC338.Use polymerase chain reaction,PCR (PCR) technology increase respectively Mabthera (Rituxan) gene light chain, heavy chain gene sequence, and with ribosome entry site(RES) sequence (IRES) connection weight chain gene, called after pDC338-RTX.

Primer 1:CCG GAATTCACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGG (SEQ ID NO:1)

Primer 2: TGC GTCGACTTATCAACACTCTCCCCTGTTGAA (SEQ ID NO:2)

Primer 3:CGC GGATCCACCATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTA (SEQ ID NO:3)

Primer 4:CCC GCTAGCTTACTATTTACCCGGAGACAGGGA (SEQ ID NO:4)

Use primer 3 and primer 4 amplifications, obtain the light chain gene of 720bp, head and the tail add EcoRI and SalI restriction enzyme site respectively, use primer 5 and primer 6 amplifications, obtain the heavy chain gene of 1431bp, and head and the tail add BamHI and NheI restriction enzyme site respectively.EF1 α promotor is as follows to the sequencing result (5 '-3 ') of sequence between the SV40 PolyA signal:

TCTAGAGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAG

TTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTG

GGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATA

TAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG

GTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGT

GCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTG

GAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGA

GTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGC

CTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGA

CGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATT

TCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGC

GAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCC

GGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGG

CTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGC

AGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACAC

AAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGG

GCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTG

GGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTA

GGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCT

TGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTC

GTGAGGAATTAGCTTGGTACTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGGT

AAGCTC GAATTCACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCT

CCCAGATACCACCGGACAAATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTC

CAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTACATCCACTGG

TTCCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGC

TTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACTTCTTACTCTCTCACCA

TCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGACTAGTAAC

CCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATCAAACGTACTGTGGCTGCACCATC

TGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGT

GCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCC

CTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTA

CAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACG

CCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA

GAGTGTTGATAA GTCGACCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGC

GGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTCGATCGTTCTGACATCCGG

CGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGTTTCTGACATCCGGCGGGT

TTCTGACATCCGGCGGGT AAGCTTGGATCCACCATGGAGTTTTGGCTGAGCTGGGTTTT

CCTTGTTGCTATTTTAAAAGGTGTCCAGTGTCAGGTACAACTGCAGCAGCCTGGGGCTG

AGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTT

ACCAGTTACAATATGCACTGGGTAAAACAGACACCTGGTCGGGGCCTGGAATGGATTGG

AGCTATTTATCCCGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCA

CATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCT

GAGGACTCTGCGGTCTATTACTGTGCAAGATCGACTTACTACGGCGGTGACTGGTACTT

CAATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCAGCCTCCACCAAGGGCCCAT

CGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGC

TGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCT

GACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCA

GCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTG

AATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAA

AACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCC

TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC

GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGG

CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACC

GTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAG

TGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAA

AGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCA

AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTG

GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA

CTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC

AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAG

AAGAGCCTCTCCCTGTCTCCGGGTAAATAGTAA GCTAGCACTAGTCTCGACTTCGAGCA

ACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACA

AATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATC

TTATCATGTCTGGATCGTCTAGCATCGAAGATCC(SEQ?ID?NO：5)

2. carry Mabthera (Rituxan) gene and be subjected to the structure of the replication-defective adenoviral vector of miR-15a regulation and control

The synthetic dna fragmentation (5 * miR-15T) that comprises 5 copy miR-15a target site sequences, and at 5 '-terminal and the 3 '-terminal restriction enzyme site that adds NheI and SpeI respectively, this dna fragmentation carries out full-length gene by the living worker's biotechnology in Shanghai company limited and synthesizes.Correct through checking order, called after pUC57-miR-15T, its nucleotide sequence following (5 '-3 '):

GCTAGCCACAAACCATTATGTGCTGCTAGATCCACAAACCATTATGTGCTGCTAACGAT

CACAAACCATTATGTGCTGCTACTCCACAAACCATTATGTGCTGCTACGATCGCACAAA

CCATTATGTGCTGCTA ACTAGT(SEQ?ID?NO：6)

Utilize NheI and SpeI double digestion pUC57-miR-15T plasmid, reclaim 5 * miR-15T fragment of 146bp, be inserted into the 3 ' non-translational region (behind the heavy chain gene, before the SV40 Poly A tailing signal) of the Mabthera gene of pDC338-RTX carrier, called after pDC338-RTX-15T.

3. carry Mabthera (Rituxan) gene and be subjected to the reorganization of the replication-defective adenoviral of miR-15a regulation and control

The HEK293 cell strain is purchased in Canadian Microbix Biosystem Inc. (Toronto), is to transform the human embryonic kidney cell by the 5 type adenovirus DNAs of shearing to form, and it contains and expresses 5 type adenovirus E 1 districts, and adenovirus DNA has high transfection efficiency to it.The plasmid that will contain 5 type adenovirus left arms is united plasmid co-transfection 293 cells that contain 5 type adenovirus right arms, can produce the adenovirus with infectivity by homologous recombination.We pass through the thin strain of Lipofectamine cotransfection to 293 with the plasmid pPE3-F11 that contains 5 type adenovirus right arms respectively with pDC338-RTX and pDC338-RTX-15T, and its concrete grammar is referring to the operation instructions of GIBCO BRL company.PPE3-F11 is by pBHGlox (delta) E1,3Cre transforms and forms, E1 district disappearance, can with homologous recombination in the shuttle plasmid born of the same parents that comprise the adenovirus left arm, acquisition has the adenovirus of infection ability, contain the E3 district that derives from pBHGE3, its 5 type cilia protein gene is replaced by 5/11fiber, and concrete carrier collection of illustrative plates is seen accompanying drawing 1.PBHGlox (delta) E1,3Cre and pBHGE3 purchase in Canadian Microbix Biosystems Inc. (Ontario).The adenovirus plaque appearred behind the cotransfection in 9-14 days, through three adenovirus plaque purifying, the recombinant adenovirus that promptly gets the recombinant adenovirus that carries Rituxan full length antibody gene and carry Rituxan full length antibody gene and regulated and control by miR-15a, difference called after Ad11-RTX and Ad11-RTX-15T (CCTCC-V200813, on January 9th, 2009, Chinese typical culture collection center).

Adenovirus is breeding in a large number in the HEK293 cell, uses the method large-scale purification adenovirus of caesium chloride density gradient centrifugation.

4. the replication of recombinant adenovirus in the HEK293 cell of carrying the recombinant adenovirus of Rituxan gene and carrying the Rituxan gene and regulated and control by miR-15a compares

The HEK293 cell strain is pressed 5 * 10 ⁵Cells/well is layered in the 6-orifice plate, at 37 ℃ of incubator 5%CO ₂Cultivate, changed serum free medium 1ml in second day, then, add recombinant adenovirus Ad11-RTX respectively, Ad11-RTX-15T, its adenovirus amount is MOI=5, cultivate after 90 minutes, (PBS) washes twice with phosphate buffered saline buffer, with the adenovirus flush away, and the nutrient solution that adds 5% foetal calf serum cultivates, and collects freeze thawing three times at 0 hour and 48 hours respectively, detect adenovirus titre (method is referring to the AdEasyTM operational manual of U.S. Qbiogene company) with the TCID50 method, found that the adenoviral replication ability Ad11-RTX-15T in HEK293 is 100 times of (see figure 2)s of Ad11-RTX.Show that the insertion of miR-15aT target site nucleotide sequence can significantly improve the production efficiency of recombinant adenovirus.

5. the recombinant adenovirus that carries the recombinant adenovirus of Rituxan gene and carry the Rituxan gene and regulated and control by miR-15a is expressed Rituxan in the HEK293 cell ability compares

The HEK293 cell is pressed 1 * 10 ⁵Cells/well is layered in the 24-orifice plate, at 37 ℃ of incubator 5%CO ₂Cultivate.Added recombinant adenovirus Ad11-RTX, Ad11-RTX-15T in second day respectively, its adenovirus amount is MOI=5, flush away virus after two hours.Respectively 3 days with 7 days after collecting cell, use enzyme-linked immunosorbent assay (enzyme linked immunosorbentassay, ELISA) expression level of Rituxan antibody in the detection supernatant, found that at the 3rd day, the concentration of antibody is respectively 2.19 ± 0.2 μ g/ml, 0.31 ± 0.1 μ g/ml behind Ad11-RTX and the Ad11-RTX-15T infection HEK293 cell.At the 7th day, the concentration that Ad11-RTX and Ad11-RTX-15T infect back antibody was respectively 3.43 ± 0.3 μ g/ml, 0.39 ± 0.2 μ g/ml.As seen in each time period, the proteic expression level Ad11-RTX-15T of Rituxan had reduced by 7.0 times and 8.8 times of (see figure 3)s respectively all far below Ad11-RTX after Ad11-RTX and Ad11-RTX-15T infected the HEK293 cell.The insertion that shows miR-15a target site nucleotide sequence can make the expression of foreign gene in viral packing cell 293 decline that is changed significantly.

6. the expressing antibodies efficiency ratio is in normal liver cell for the recombinant adenovirus that carries the recombinant adenovirus of Rituxan gene and carry the Rituxan gene and be subjected to miR-15a regulation and control

People's normal liver cell strain L02 miR-15a expresses negative, and it is pressed 1 * 10 ⁵Cells/well is layered in the 24-orifice plate, at 37 ℃ of incubator 5%CO ₂Cultivate.Added recombinant adenovirus Ad11-RTX, Ad11-RTX-15T in second day respectively, its adenovirus amount is MOI=5, flush away virus after two hours.Respectively at the 3rd day and the 7th day collecting cell, use enzyme-linked immunosorbent assay (enzymelinked immunosorbent assay, ELISA) expression level of detection antibody, found that at the 3rd day, the concentration of antibody is respectively 1.55 ± 0.1 μ g/ml, 1.67 ± 0.2 μ g/ml behind Ad11-RTX and the Ad11-RTX-15T infection L02 cell.At the 7th day, the concentration that Ad11-RTX and Ad11-RTX-15T infect back antibody was respectively 3.21 ± 0.4 μ g/ml, 3.35 ± 0.2 μ g/ml.As seen in each time period, the similar (see figure 3) of the proteic expression level of Rituxan behind Ad11-RTX and the Ad11-RTX-15T infected liver cell L02.The insertion that shows miR-15a target site nucleotide sequence does not cause that the expression of foreign gene in target cell changes.

7. the recombinant adenovirus that carries the Rituxan gene and be subjected to miR-15a regulation and control is in nude mouse internal therapy transplantation tumor

With the 4-5 SCID mouse hypodermic inoculation CD20 male Raji cell strain 1 * 10 in age in week ⁷, after two weeks, once give the recombinant adenovirus Ad11-RTX that the tumor-bearing mice tail vein injection carries the Rituxan gene, and the recombinant adenovirus Ad11-RTX-15T that carries the Rituxan gene and regulated and control by miR-15a, dosage respectively is 5 * 10 ⁸Pfu, the situation over time of statistics gross tumor volume.Found that the back gross tumor volume increase of 3 week of its not treatment group is more than 3 times, and the treatment group can significantly be dwindled the size of tumour, and the effect of recombinant adenovirus Ad11-RTX and Ad11-RTX-15T inhibition tumor growth there is not the significant difference (see figure 4).Show that the insertion of miR-15a target site nucleotide sequence does not reduce result of treatment in animal body.

Two. embodiment 2: carrier P53 gene and can be in the 293FT cell structure of the recombinant slow virus of High-efficient Production

1. the structure that comprises the slow virus packaging expression plasmid of people P53 gene

PKCDNA-CMV--EF1 α-GFP plasmid is purchased in health and is become biotech firm, and it contains the GFP expression of gene box under the control of EF1 α promotor, and foreign gene can be expressed under the control of CMV promotor.Extract human total rna, use the synthesizing single-stranded cDNA of reversed transcriptive enzyme, and use polymerase chain reaction,PCR (PCR) technology amplifies people P53 gene from strand cDNA library open reading frame.Called after pKCDNA-P53.

Primer 5:CCG TCTAGAACCATGGAGGAGCCGCAGTCAGA (SEQ ID NO:7)

Primer 6:TGC GAATTCTCAGTCTGAGTCAGGCCC (SEQ ID NO:8)

Primer 5 and primer 6 amplifications obtain the coding region sequence of the P53 gene of 1197bp, and head and the tail add XbaI and NheI restriction enzyme site respectively.Its sequencing result (5 '-3 ') is as follows:

TCTAGAACCATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTGAGTCAGGA

AACATTTTCAGACCTATGGAAACTACTTCCTGAAAACAACGTTCTGTCCCCCTTGCCGT

CCCAAGCAATGGATGATTTGATGCTGTCCCCGGACGATATTGAACAATGGTTCACTGAA

GACCCAGGTCCAGATGAAGCTCCCAGAATGCCAGAGGCTGCTCCCCCCGTGGCCCCTGC

ACCAGCAGCTCCTACACCGGCGGCCCCTGCACCAGCCCCCTCCTGGCCCCTGTCATCTT

CTGTCCCTTCCCAGAAAACCTACCAGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCAT

TCTGGGACAGCCAAGTCTGTGACTTGCACGTACTCCCCTGCCCTCAACAAGATGTTTTG

CCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGATTCCACACCCCCGCCCGGCA

CCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGG

CGCTGCCCCCACCATGAGCGCTGCTCAGATAGCGATGGTCTGGCCCCTCCTCAGCATCT

TATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAACACTTTTCGAC

ATAGTGTGGTGGTGCCCTATGAGCCGCCTGAGGTTGGCTCTGACTGTACCACCATCCAC

TACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGGAGGCCCATCCTCAC

CATCATCACACTGGAAGACTCCAGTGGTAATCTACTGGGACGGAACAGCTTTGAGGTGC

GTGTTTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGAAGAGAATCTCCGCAAGAAA

GGGGAGCCTCACCACGAGCTGCCCCCAGGGAGCACTAAGCGAGCACTGCCCAACAACAC

CAGCTCCTCTCCCCAGCCAAAGAAGAAACCACTGGATGGAGAATATTTCACCCTTCAGA

TCCGTGGGCGTGAGCGCTTCGAGATGTTCCGAGAGCTGAATGAGGCCTTGGAACTCAAG

GATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGGCTCACTCCAGCCACCTGAAGTC

CAAAAAGGGTCAGTCTACCTCCCGCCATAAAAAACTCATGTTCAAGACAGAAGGGCCTG

ACTCAGACTGAGCTAGC(SEQ?ID?NO：9)

2. comprise people P53 gene and be subjected to the structure of the slow virus packaging expression plasmid of miR-15a regulation and control

The synthetic dna fragmentation (4 * miR-15T) that comprises 4 copy miR-15a target site sequences, and at 5 '-terminal and the 3 '-terminal restriction enzyme site that adds NheI and EcoRI respectively, this dna fragmentation carries out full-length gene by the living worker's biotechnology in Shanghai company limited and synthesizes, and correct through sequence verification.4 * miR-15T fragment is inserted the 3 ' non-translational region (after the terminator codon, before the SV40 Poly A tailing signal) of the people P53 gene of pKCDNA-P53 carrier, called after pKCDNA-P53-15T.The segmental nucleotide sequence of 4 * miR-15T following (5 '-3 '): GCTAGCCACAAACCATTATGTGCTGCTAGATCCACAAACCATTATGTGCTGCTAACGATCAC AAACCATTATGTGCTGCTACTCCACAAACCATTATGTGCTGCTA GAATTC(SEQID NO:10)

3. comprise people P53 gene and recombinated by the packing of the slow virus of miR-15a regulation and control

The 293FT cell is pressed 7.5 * 10 ⁵Be layered on the 10cm dish 5%CO2 incubator incubated overnight.The 10%DMEM substratum changed in second day into 2% DMEM substratum, after 4 hours with 20 μ g expression plasmids (pKCDNA-P53 and pKCDNA-P53-15T), 10 μ g packaging plasmid pCMVdelta8.91 mix with 5 μ g packaging plasmid pMD.G, add sterilized water to 250 μ l, the concussion mixing.The 0.5MCaCl that in above-mentioned mixed solution, adds 250 μ l successively ₂, then in above-mentioned mixed solution, dropwise add 2 * HBS of 500 μ l, the concussion of limit edged.Left standstill 30 minutes, above-mentioned mixed solution is evenly added in the cell culture medium.After the transfection 12 hours, outwell the 2%DMEM substratum, PBS cleans twice, gains the 10%DMEM substratum, respectively at 36 hours, collects cell culture medium after 60 hours.The cell culture medium that collection is contained virion is collected supernatant liquor with the centrifugal 10min of 4000g, and filters with 0.45 μ m filter, filterable liquid places 40mL ultracentrifugation pipe, 4 ℃, centrifugal 20 minutes of 25000r/min uses the resuspended virus precipitation of 500ul ice PBS liquid.The slow virus of the carrier P53 gene that packing obtains, and carrier P53 gene and the slow virus that is subjected to the miR-15a regulation and control called after Le-P53 and Le-P53-15T respectively.

4. comprise people P53 gene and be subjected to the titer determination of the slow virus of miR-15a regulation and control

In six orifice plates, press 2x10 ⁵Shop ,/hole Hela cell uses the 10%DMEM culture medium culturing, places the 5%CO2 incubator to spend the night.With virus to be measured (Le-P53 and Le-P53-15T) gradient dilution is 10 ^-2-10 ^-6Doubly, the viral liquid of each concentration is diluted in the 2%DMEM substratum, final volume is 1ml.After dilution is finished, substratum original in six orifice plates is outwelled, changed into the substratum that adds viral dilution liquid, place 5%CO ₂The incubator incubated overnight.The 2%DMEM substratum that will contain virus in second day changes the 10%DMEM substratum into, infected the back the 4th to five day, clean cell with PBS, the digestion back is resuspended, detect the luciferase expression situation through flow cytometer, calculation formula: the per-cent/viral liquid of virus titer (TU/mL)=cell quantity x fluorescencepositive cell amasss (ml).Cells were tested by flow cytometry shows that the virus titer of Le-P53-15T is 8.75x10 ⁹TU/mL, and the virus titer of Le-P53 is 1.37x10 ⁸TU/mL, the virus titer of Le-P53-15T are 64 times of Le-P53.The insertion that shows miR-15a target site nucleotide sequence can significantly provide the virus titer of recombinant slow virus at packing cell 293FT.

5. comprise people P53 gene and be subjected to the slow virus of miR-15a regulation and control in tumour cell, to express the level determination of P53

Hepatoma cell strain Hep3B, lung cancer cell line A549, breast carcinoma cell strain MCF7 purchases in ATCC, and miR-15a expresses all negative.Above-mentioned tumour cell is pressed 5 * 10 ⁴Cells/well is layered in the 24-orifice plate, at 37 ℃ of incubator 5%CO ₂Overnight incubation.Added recombinant slow virus Le-P53, Le-P53-15T in second day respectively, the MOI value is 10.At the 1st, 2,3 day, extract total RNA respectively, use primer 7 and primer 8, detect the expression level of P53 by the method for real-time quantitative RT-PCR.Found that in each time period Le-P53 and Le-P53-15T express the level of P53 does not have significant difference.The insertion that shows miR-15a target site nucleotide sequence does not cause that the expression amount of external source P53 in the target cell changes.

Primer 7:GCGCACAGAGGAAGAGAATC (SEQ ID NO:11)

Primer 8:CAAGGCCTCATTCAGCTCTC (SEQ ID NO:12)

By above-mentioned evidence, recombinant virus of the present invention has higher proliferate efficiency or production efficiency in packing cell, and does not influence the expression of exogenous gene level in target cell.

Reference:

1.Lai?EC.MicroRNAs?are?complementary?to?3’UTR?sequencemotifs?that?mediate?negative?post-transcriptional?regulation.Nat?Genet.2002；30(4)：363-4

Berezikov?E，Cuppen?E，Plasterk?R.Approaches?tomicroRNAdiscovery.Nat?Genet.2006，38(6)：2-6

2.Landgraf?P，Rusu?M，Sheridan?R，Sewer?A，Iovino?N，AravinA，Pfeffer?S，Rice?A，Kamphorst?AO，Landthaler?M，Lin?C，SocciND，Hermida?L，Fulci?V，Chiaretti?S，FoàR，Schliwka?J，FuchsU，Novosel?A，M?üller?RU，Schermer?B，Bissels?U，Inman?J，PhanQ，Chien?M，Weir?DB，Choksi?R，De?Vita?G，Frezzetti?D，TrompeterHI，Horhung?V，Teng?G，Hartmann?G，Palkovits?M，Di?Lauro?R，Wernet?P，Macino?G，Rogler?CE，Nagle?JW，Ju?J，Papavasiliou?FN，Benzing?T，Lichter?P，Tam?W，Brownstein?MJ，Bosio?A，BorkhardtA，Russo?JJ，Sander?C，Zavolan?M，Tuschl?T.A?mammalian?microRNAexpression?atlas?based?on?small?RNA?library?sequencing.Cell.2007；129(7)：1401-14

3.Calin?GA，Ferracin?M，Cimmino?A，Di?Leva?G，Shimizu?M，Wojcik?SE，Iorio?MV，Visone?R，Sever?NI，Fabbri?M，Iuliano?R，Palumbo?T，Pichiorri?F，Roldo?C，Garzon?R，Sevignani?C，RassentiL，Alder?H，Volinia?S，Liu?CG，Kipps?TJ，Negrini?M，Croce?CM.A?MicroRNA?signature?associated?with?prognosis?and?progressionin?chronic?lymphocytic?leukemia.N?Engl?J?Med.2005；353(17)：1793-801

4.He?L，Thomson?JM，Hemann?MT，Hernando-Monge?E，Mu?D，Goodson?S，Powers?S，Cordon-Cardo?C，Lowe?SW，Hannon?GJ，HammondSM.A?microRNA?polycistron?as?a?potential?human?oncogene.Nature.2005；435(7043)：828-33

Sequence table

＜110〉The 2nd Army Medical College east hospital of liver and gall surgical department

＜120〉a kind of recombinant viral vector that carries foreign gene High-efficient Production in packing cell, its construction process and

Purposes

<130>IDC080120

<160>12

<170>PatentIn?version?3.2

<210>1

<211>54

<212>DNA

＜213〉primer 1

<400>1

ccggaattca?ccatggaagc?cccagctcag?cttctcttcc?tcctgctact?ctgg 54

<210>2

<211>33

<212>DNA

＜213〉primer 2

<400>2

tgcgtcgact?tatcaacact?ctcccctgtt?gaa 33

<210>3

<211>54

<212>DNA

＜213〉primer 3

<400>3

cgcggatcca?ccatggagtt?ttggctgagc?tgggttttcc?ttgttgctat?ttta 54

<210>4

<211>33

<212>DNA

＜213〉primer 4

<400>4

cccgctagct?tactat?ttac?ccggagacag?gga 33

<210>5

<211>3691

<212>DNA

＜213〉promotor+light chain+IRES+ heavy chain+SV40 polyA signal sequence

<400>5

tctagagctc?cggtgcccgt?cagtgggcag?agcgcacatc?gcccacagtc?cccgagaagt 1

tggggggagg?ggtcggcaat?tgaaccggtg?cctagagaag?gtggcgcggg?gtaaactggg 61

aaagtgatgt?cgtgtactgg?ctccgccttt?ttcccgaggg?tgggggagaa?ccgtatataa 121

gtgcagtagt?cgccgtgaac?gttctttttc?gcaacgggtt?tgccgccaga?acacaggtaa 181

gtgccgtgtg?tggttcccgc?gggcctggcc?tctttacggg?ttatggccct?tgcgtgcctt 241

gaattacttc?cacctggctg?cagtacgtga?ttcttgatcc?cgagcttcgg?gttggaagtg 301

ggtgggagag?ttcgaggcct?tgcgcttaag?gagccccttc?gcctcgtgct?tgagttgagg 361

cctggcctgg?gcgctggggc?cgccgcgtgc?gaatctggtg?gcaccttcgc?gcctgtctcg 421

ctgctttcga?taagtctcta?gccatttaaa?atttttgatg?acctgctgcg?acgctttttt 481

tctggcaaga?tagtcttgta?aatgcgggcc?aagatctgca?cactggtatt?tcggtttttg 541

gggccgcggg?cggcgacggg?gcccgtgcgt?cccagcgcac?atgttcggcg?aggcggggcc 601

tgcgagcgcg?gccaccgaga?atcggacggg?ggtagtctca?agctggccgg?cctgctctgg 661

tgcctggcct?cgcgccgccg?tgtatcgccc?cgccctgggc?ggcaaggctg?gcccggtcgg 721

caccagttgc?gtgagcggaa?agatggccgc?ttcccggccc?tgctgcaggg?agctcaaaat 781

ggaggacgcg?gcgctcggga?gagcgggcgg?gtgagtcacc?cacacaaagg?aaaagggcct 841

ttccgtcctc?agccgtcgct?tcatgtgact?ccacggagta?ccgggcgccg?tccaggcacc 901

tcgattagtt?ctcgagcttt?tggagtacgt?cgtctttagg?ttggggggag?gggttttatg 961

cgatggagtt?tccccacact?gagtgggtgg?agactgaagt?taggccagct?tggcacttga 1021

tgtaattctc?cttggaattt?gccctttttg?agtttggatc?ttggttcatt?ctcaagcctc 1081

agacagtggt?tcaaagtttt?tttcttccat?ttcaggtgtc?gtgaggaatt?agcttggtac 1141

taatacgact?cactataggg?agacccaagc?tggctaggta?agctcgaatt?caccatggaa 1201

gccccagctc?agcttctctt?cctcctgcta?ctctggctcc?cagataccac?cggacaaatt 1261

gttctctccc?agtctccagc?aatcctgtct?gcatctccag?gggagaaggt?cacaatgact 1321

tgcagggcca?gctcaagtgt?aagttacatc?cactggttcc?agcagaagcc?aggatcctcc 1381

cccaaaccct?ggatttatgc?cacatccaac?ctggcttctg?gagtccctgt?tcgcttcagt 1441

ggcagtgggt?ctgggacttc?ttactctctc?accatcagca?gagtggaggc?tgaagatgct 1501

gccacttatt?actgccagca?gtggactagt?aacccaccca?cgttcggagg?ggggaccaag 1561

ctggaaatca?aacgtactgt?ggctgcacca?tctgtcttca?tcttcccgcc?atctgatgag 1621

cagttgaaat?ctggaactgc?ctctgttgtg?tgcctgctga?ataacttcta?tcccagagag 1681

gccaaagtac?agtggaaggt?ggataacgcc?ctccaatcgg?gtaactccca?ggagagtgtc 1741

acagagcagg?acagcaagga?cagcacctac?agcctcagca?gcaccctgac?gctgagcaaa 1801

gcagactacg?agaaacacaa?agtctacgcc?tgcgaagtca?cccatcaggg?cctgagctcg 1861

cccgtcacaa?agagcttcaa?caggggagag?tgttgataag?tcgacccggc?gggtttctga 1921

catccggcgg?gtttctgaca?tccggcgggt?ttctgacatc?cggcgggttt?ctgacatccg 1981

gcgggtcgat?cgttctgaca?tccggcgggt?ttctgacatc?cggcgggttt?ctgacatccg 2041

gcgggtttct?gacatccggc?gggtttctga?catccggcgg?gtaagcttgg?atccaccatg 2101

gagttttggc?tgagctgggt?tttccttgtt?gctattttaa?aaggtgtcca?gtgtcaggta 2161

caactgcagc?agcctggggc?tgagctggtg?aagcctgggg?cctcagtgaa?gatgtcctgc 2221

aaggcttctg?gctacacatt?taccagttac?aatatgcact?gggtaaaaca?gacacctggt 2281

cggggcctgg?aatggattgg?agctatttat?cccggaaatg?gtgatacttc?ctacaatcag 2341

aagttcaaag?gcaaggccac?attgactgca?gacaaatcct?ccagcacagc?ctacatgcag 2401

ctcagcagcc?tgacatctga?ggactctgcg?gtctattact?gtgcaagatc?gacttactac 2461

ggcggtgact?ggtacttcaa?tgtctggggc?gcagggacca?cggtcaccgt?ctctgcagcc 2521

tccaccaagg?gcccatcggt?cttccccctg?gcaccctcct?ccaagagcac?ctctgggggc 2581

acagcggccc?tgggctgcct?ggtcaaggac?tacttccccg?aaccggtgac?ggtgtcgtgg 2641

aactcaggcg?ccctgaccag?cggcgtgcac?accttcccgg?ctgtcctaca?gtcctcagga 2701

ctctactccc?tcagcagcgt?ggtgaccgtg?ccctccagca?gcttgggcac?ccagacctac 2761

atctgcaacg?tgaatcacaa?gcccagcaac?accaaggtgg?acaagaaagt?tgagcccaaa 2821

tcttgtgaca?aaactcacac?atgcccaccg?tgcccagcac?ctgaactcct?ggggggaccg 2881

tcagtcttcc?tcttcccccc?aaaacccaag?gacaccctca?tgatctcccg?gacccctgag 2941

gtcacatgcg?tggtggtgga?cgtgagccac?gaagaccctg?aggtcaagtt?caactggtac 3001

gtggacggcg?tggaggtgca?taatgccaag?acaaagccgc?gggaggagca?gtacaacagc 3061

acgtaccgtg?tggtcagcgt?cctcaccgtc?ctgcaccagg?actggctgaa?tggcaaggag 3121

tacaagtgca?aggtctccaa?caaagccctc?ccagccccca?tcgagaaaac?catctccaaa 3181

gccaaagggc?agccccgaga?accacaggtg?tacaccctgc?ccccatcccg?ggatgagctg 3241

accaagaacc?aggtcagcct?gacctgcctg?gtcaaaggct?tctatcccag?cgacatcgcc 3301

gtggagtggg?agagcaatgg?gcagccggag?aacaactaca?agaccacgcc?tcccgtgctg 3361

gactccgacg?gctccttctt?cctctacagc?aagctcaccg?tggacaagag?caggtggcag 3421

caggggaacg?tcttctcatg?ctccgtgatg?catgaggctc?tgcacaacca?ctacacgcag 3481

aagagcctct?ccctgtctcc?gggtaaatag?taagctagca?ctagtctcga?cttcgagcaa 3541

cttgtttatt?gcagcttata?atggttacaa?ataaagcaat?agcatcacaa?atttcacaaa 3601

taaagcattt?ttttcactgc?attctagttg?tggtttgtcc?aaactcatca?atgtatctta 3661

tcatgtctgg?atcgtctagc?atcgaagatc?c 3691

<210>6

<211>140

<212>DNA

<213>pUC57-miR-15T

<400>6

gctagccaca?aaccattatg?tgctgctaga?tccacaaacc?attatgtgct?gctaacgatc 60

acaaaccatt?atgtgctgct?actccacaaa?ccattatgtg?ctgctacgat?cgcacaaacc 120

attatgtgct?gctaactagt 140

<210>7

<211>32

<212>DNA

＜213〉primer 5

<400>7

ccgtctagaa?ccatggagga?gccgcagtca?ga 32

<210>8

<211>27

<212>DNA

＜213〉primer 6

<400>8

tgcgaattct?cagtctgagt?caggccc 27

<210>9

<211>1197

<212>DNA

<213>P53

<400>9

tctagaacca?tggaggagcc?gcagtcagat?cctagcgtcg?agccccctct?gagtcaggaa 60

acattttcag?acctatggaa?actacttcct?gaaaacaacg?ttctgtcccc?cttgccgtcc 120

caagcaatgg?atgatttgat?gctgtccccg?gacgatattg?aacaatggtt?cactgaagac 180

ccaggtccag?atgaagctcc?cagaatgcca?gaggctgctc?cccccgtggc?ccctgcacca 240

gcagctccta?caccggcggc?ccctgcacca?gccccctcct?ggcccctgtc?atcttctgtc 300

ccttcccaga?aaacctacca?gggcagctac?ggtttccgtc?tgggcttctt?gcattctggg 360

acagccaagt?ctgtgacttg?cacgtactcc?cctgccctca?acaagatgtt?ttgccaactg 420

gccaagacct?gccctgtgca?gctgtgggtt?gattccacac?ccccgcccgg?cacccgcgtc 480

cgcgccatgg?ccatctacaa?gcagtcacag?cacatgacgg?aggttgtgag?gcgctgcccc 540

caccatgagc?gctgctcaga?tagcgatggt?ctggcccctc?ctcagcatct?tatccgagtg 600

gaaggaaatt?tgcgtgtgga?gtatttggat?gacagaaaca?cttttcgaca?tagtgtggtg 660

gtgccctatg?agccgcctga?ggttggctct?gactgtacca?ccatccacta?caactacatg 720

tgtaacagtt?cctgcatggg?cggcatgaac?cggaggccca?tcctcaccat?catcacactg 780

gaagactcca?gtggtaatct?actgggacgg?aacagctttg?aggtgcgtgt?ttgtgcctgt 840

cctgggagag?accggcgcac?agaggaagag?aatctccgca?agaaagggga?gcctcaccac 900

gagctgcccc?cagggagcac?taagcgagca?ctgcccaaca?acaccagctc?ctctccccag 960

ccaaagaaga?aaccactgga?tggagaatat?ttcacccttc?agatccgtgg?gcgtgagcgc 1020

ttcgagatgt?tccgagagct?gaatgaggcc?ttggaactca?aggatgccca?ggctgggaag 1080

gagccagggg?ggagcagggc?tcactccagc?cacctgaagt?ccaaaaaggg?tcagtctacc 1140

tcccgccata?aaaaactcat?gttcaagaca?gaagggcctg?actcagactg?agctagc 1197

<210>10

<211>112

<212>DNA

<213>4×miR-15T

<400>10

gctagccaca?aaccattatg?tgctgctaga?tccacaaacc?attatgtgct?gctaacgatc 60

acaaaccatt?atgtgctgct?actccacaaa?ccattatgtg?ctgctagaat?tc 112

<210>11

<211>20

<212>DNA

＜213〉primer 7

<400>11

gcgcacagag?gaagagaatc 20

<210>12

<211>20

<212>DNA

＜213〉primer 8

<400>12

caaggcctca?ttcagc?tctc 20

Claims

1. recombinant viral vector, the be operably connected target site nucleotide sequence of foreign gene and Microrna of this carrier, it is characterized in that: in 3 ' non-translational region of the foreign gene that this virus vector is operably connected, be inserted with the target site nucleotide sequence of at least one following small RNAs, described Microrna high level expression and not expressing in the target cell or low expression the in viral packing cell.

2. according to the described recombinant viral vector of claim 1, it is characterized in that: described virus is selected from adenovirus (Adenovirus), adeno-associated virus (Adeno-associated virus), retrovirus (Retrovirus), slow virus (Lentivirus), vaccinia virus (Vacciniavirus), vaccinia virus (Poxvirus), hsv (Herpes simplexvirus), Chinese mugwort Pasteur's virus (Epstein-Barr virus), people's papillary tumor virus (HumanPapillomavirus), baculovirus (Baculoviruses), flavivirus (Flavivirus), Measles virus (Measles virus), Avian pneumo-encephalitis virus (Newcastle disease virus), Semliki forest virus (Semliki forest virus), Sendai virus (Sendai virus), at least a in simian virus 40 (Simian virus 40) and the alphavirus (Alphaviruses).

3. according to the described recombinant viral vector of claim 1, it is characterized in that foreign gene that described virus is operably connected is that coding is selected from least a in apoptosis gene, prodrug conversion enzyme gene, cell cycle genes involved, cytokine gene, signaling genes, transcription factor gene, oncogene, cancer suppressor gene, blood vessel suppressor gene, antibody gene, the vaccine gene.

4. according to the described recombinant viral vector of claim 1, it is characterized in that described virus vector packing cell is selected from 293,293T, 293FT, 293E, 293ET, 293KB, 293Eco, at least a among GP293 and the FIP293.

5. recombinant viral vector according to claim 4 is characterized in that described Microrna target site sequence is selected from miR-15a, miR-16, miR-17, miR-18a, at least a among miR-19b and the miR-196a.

6. recombinant viral vector according to claim 1 is characterized in that described Microrna target site sequence is made of in series connection multiple mode one or more identical Microrna target site sequences.

7. recombinant viral vector according to claim 1 is characterized in that described Microrna target site sequence is made of in the placed in-line mode of continuous series connection or interval one or more different Microrna target site sequences.

8. the purposes of the described recombinant viral vector of claim 1, its foreign gene that is used for recombinant virus is operably connected is not expressed at viral packing cell, and in target cell normal expression.

9. the recombinant viral vector purposes of claim 8, the target cell that it is characterized in that described virus vector are selected from least a in myocardial cell, Skeletal Muscle Cell, inoblast, vascular endothelial cell, liver cell, lymphocyte, neurocyte, adipocyte, embryonic stem cell, adult stem cell and the tumour cell.