CN101787373B - Foreign gene-carrying recombinant virus vector efficiently produced in packaging cell and construction method and application thereof - Google Patents
Foreign gene-carrying recombinant virus vector efficiently produced in packaging cell and construction method and application thereof Download PDFInfo
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Abstract
本发明提供一种携带外源基因在包装细胞中高效生产的重组病毒载体、其构建方法及其用途。其特征在于：在该病毒可操作地连接的外源基因的3’非翻译区中插入有至少一个下述微小RNA的靶位点核苷酸序列：所述微小RNA在病毒包装细胞中高水平表达而靶细胞中不表达或低表达。从而实现选择性地抑制外源基因在病毒包装细胞中的表达，避免外源基因对病毒包装与生产的负面影响，提高病毒的生产效率和滴度，同时在靶细胞中不降低外源基因的表达水平，不影响治疗效果或研究外源基因的功能。
The invention provides a recombinant viral vector carrying foreign genes and efficiently produced in packaging cells, its construction method and application. It is characterized in that: at least one target site nucleotide sequence of the following microRNA is inserted into the 3' untranslated region of the foreign gene operably linked to the virus: said microRNA is expressed at a high level in virus packaging cells No expression or low expression in target cells. In this way, the expression of foreign genes in virus packaging cells can be selectively inhibited, the negative impact of foreign genes on virus packaging and production can be avoided, and the production efficiency and titer of viruses can be improved without reducing the expression of foreign genes in target cells. Expression level, does not affect the therapeutic effect or study the function of exogenous gene.
Description
Invention field
Relate generally to recombinant viral vector of the present invention.More particularly, the present invention relates to a class foreign gene-carrying in packing cell High-efficient Production recombinant viral vector, its construction process and uses thereof.
Background technology
Virus is high to the specific cells infection rate, can efficiently foreign gene be transduceed in cell, and long-time the expression, the overexpression of instrument mediate foreign gene in target cell of Chang Zuowei transgenosis, study the function of gene by the mode of gain-of-function (gain-of-function).On the other hand, virus is also the most frequently used carrier of gene therapy, end in September, 2008, existing 1472 gene therapy schemes are for clinical trial in the world, 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, contains the HEKC in 5 type adenovirus E 1 districts, is usually used in packing and the production of recombinant adenovirus, adeno-associated virus; 293T and 293FT cell (the Fast Growth mutation of 293T), derived from by 293 cells, expresses the SV40 large T antigen simultaneously, is usually used in packing and the production of recombinant slow virus; The GP293 cell, derived from by 293 cells, expresses Gag-pol albumen simultaneously, is usually used in retroviral packing and production.The recombinant virus of foreign gene-carrying can assemble or copy propagation in packing cell, impels a large amount of amplifications of the foreign gene comprised in recombinant virus genomes and expresses.Yet, the foreign gene of overexpression has certain toxicity to cell on the one hand, especially suicide gene, apoptosis gene, Cell cycle-related genes etc., cause Viral packaging cell dead ahead of time, shortened each functional gene of recombinant virus on the opportunity of packing cell transcription and translation, and cause the part virion to have little time assembling; On the other hand, transcribing in a large number of foreign gene can take hypercellular rna transcription, protein translation involved enzyme class and resource with translating also, thereby indirectly suppressed transcribing and translating of each functional gene of recombinant virus.The impact of two aspects has reduced the production efficiency of recombinant virus in packing cell, can't obtain enough virus titers.
Therefore, for the recombinant virus that makes foreign gene-carrying obtains higher virus titer, be starved of and can in packing cell, carry out the method for High-efficient Production recombinant virus.
Microrna (microRNA) is the endogenous small fragment RNA of organism, by the target site nucleotide sequence in the 3 ' non-translational region with the ripe mRNA of target gene, is combined, and suppresses the translation of target gene protein.Microrna has tissue specificity and the timing of height, large at dissimilar cells level difference, thereby can be by regulatory mechanism and the expression tissue specificity of Microrna, the expression that utilizes the Viral packaging cell high level expression and do not express or hang down the micro RNA regulation and control foreign gene of expressing in target cell, foreign gene is not expressed in packing cell, improve virus production efficiency and titre, and do not reduce the expression level of foreign gene in target cell, keep original result for the treatment of or by the mode Study of Exogenous gene function of gain-of-function.
Summary of the invention
The invention provides a kind of recombinant viral vector, it is characterized in that inserting in 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 target cell or low expression the in Viral packaging 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 one in simian virus 40 (Simian virus 40) and 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 described virus is operably connected is selected from least one in apoptosis gene, prodrug conversion enzyme gene, Cell cycle-related genes, 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), 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 one in myocardial cell, Skeletal Muscle Cell, inoblast, vascular endothelial cell, liver cell, lymphocyte, neurocyte, adipocyte, embryonic stem cell, adult stem cell, tumour cell.Described target cell is preferably selected from liver cell, tumour cell.
In one embodiment of the invention, the mode that described Microrna target site sequence is repeated with series connection by Microrna target site sequence identical more than one or more forms.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.Preferably 1-3,1-5,1-7,1-9,1-11,1-15,1-20.
In one embodiment, described Microrna target site sequence consists of in the mode of connecting continuously or connect in 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.Preferably 1-3,1-5,1-7,1-9,1-11,1-15,1-20.
In one embodiment, described Viral packaging cell is selected from 293,293T, 293FT, 293E, 293ET, 293KB, 293Eco, at least one in GP293 and 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 one in miR-19b and 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 mankind's adenovirus carrier, and it comprises A, B, C, D, E and 6 subgenus of F.
In one embodiment of the invention, selected adenovirus carrier derives from the adenovirus of mankind 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: inserted the target site nucleotide sequence of at least one following small RNAs in 3 ' non-translational region of the foreign gene be operably connected at described virus vector, described Microrna high level expression and not expressing in target cell or low expression the in Viral packaging cell.
In one embodiment, described recombinant virus is for expressing the foreign gene that recombinant virus is operably connected at Viral packaging cell, and in target cell normal expression.In one embodiment, described recombinant virus is for the inhibition tumor cell growth.
The accompanying drawing summary
The carrier collection of illustrative plates of Fig. 1: pPE3-F11.Mainly by pBHGlox (delta) E1, the 3Cre transformation forms, E1 district disappearance, can with homologous recombination in the plasmid born of the same parents that comprise the adenovirus left arm, obtain the adenovirus that infection ability is arranged; Comprise the E3 district that derives from pBHGE3, its 5 type cilia protein gene is replaced by 5/11 fiber.
Fig. 2: mean virus vector Ad11-RTX and the replication comparative result of Ad11-RTX-15T in the HEK293 cell.
Fig. 3: mean the expression analysis result of Rituxan in HEK293 and L02 cell.
Fig. 4: mean control group and treatment group mouse different time sections transplanted tumor volume statistic analysis result.
Detailed Description Of The Invention
As described above, the present invention relates to a kind of recombinant viral vector, it is characterized in that in 3 ' non-translational region of the foreign gene that this virus is operably connected at least comprising high level expression in a Viral packaging cell and do not express in 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 Nucleotide, 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 approximately 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 Individual existence, the part Microrna flocks together, and becomes a Microrna gene cluster.Bioinformatic data shows, the mankind approximately 1/3 protein coding gene are subject 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 at specific tissue and etap, expresses, and has tissue specificity and the timing of height.The specifically expressing in heart and skeletal muscle tissue as miR-1 and miR-133, miR-1 can regulate and control the balance of myocardial cell between differentiation and propagation, and miR-133 can regulate and control the differentiation of muscle cell, 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 endotheliocyte, can suppress the interaction of lymphocyte and endotheliocyte, prevents the generation of vascular inflammation; MiR-142, miR-181, miR-223 specifically expressing in hemopoietic tissue, the CD4 that wherein miR-181 grows at mouse chest cell
+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 fatty tissue, can regulate and control the differentiation of adipocyte, knocks out the accumulation that miR-143 will cause triglyceride level; 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, 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 is also at the high efficient expression of zebra fish pituitary body, regulate the secretion (referring to summary Landgraf et al., 2007) of other hormones and neuroendocrine product.
At the higher Microrna of 293 serial cells level, have: 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, and wherein miR-196a-1 is positioned at the 17th karyomit(e), and miR-196a-2 is positioned at the 12nd karyomit(e).Except higher level in 293 serial cells is expressed, miR-15a and miR-16 also have higher expression amount in multiple T cell, B cell, and 65% B cell chronic lymphatic type leukemia (CLL) patient, 50% lymphoma mantle cell patient, the myelomatosis patient of 16-40%, the disappearance in 13q14 district is all arranged in 60% prostate cancer patient, cause 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 site occurs in the tumours such as the B cell lymphoma scattered, follicular lymphoma, lymphoma mantle cell, causes its abnormal expression to raise (He et al., 2005); MiR-196a expression level in breast cancer cell MCF7 is higher.Above Microrna expression level level in the organ-tissues such as the heart, lung, liver, spleen, ovary, uterus, prostate gland, Tiroidina is lower or do not express.
High level expression in application packages cell of the present invention and do not express in target cell or the target site nucleotide sequence of low Microrna of expressing inserts 3 ' non-translational region of the foreign gene comprised in recombinant virus genomes, thereby make foreign gene can not be in packing cell synthetic proteins, do not affect normal packing and the propagation of recombinant virus in packing cell, thereby improve the production efficiency of recombinant virus, improve virus titer.And do not reduce the expression level of foreign gene in target cell, 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), retrovirus (Retrovirus), slow virus (Lentivirus), vaccinia virus (Vaccinia virus), vaccinia virus (Poxvirus), hsv (Herpes simplex virus), Chinese mugwort Pasteur's virus (Epstein-Barrvirus), people's papillary tumor virus (Human Papillomavirus), baculovirus (Baculoviruses), flavivirus (Flavivirus), Measles virus (Measles virus), Avian pneumo-encephalitis virus (Newcastle disease virus), Semliki forest virus (Semlikiforest virus), Sendai virus (Sendai virus), simian virus 40 (Simianvirus 40) and alphavirus (Alphaviruses)." packing cell " refers to that it can be the clone gone down to posterity of stablizing of cultivating from n cell for the clone of virus restructuring, amplification, can be also the clone that comprises virus packing indispensable gene in its genome of transformation.As 293 cells can be used for restructuring and the amplification of adenovirus.
" target cell " refers to the target cells infected of recombinant virus, can be both functional impairment or disorder, the cell that needs the recombinant virus foreign gene-carrying to be proofreaied and correct, can be also that function is normal, be suitable for expression alien gene after recombinant virus infection, the cell of Study of Exogenous gene function.
" in packing cell high level expression and in target cell, do not express or low Microrna of expressing " refers to the large Microrna of expression level difference between packing cell and target cell, high at the packing cell expression level, and at the target cell expression level low or nothing.As miR-15a, it is high in 293 cells levels, and is that the L02 expression level is extremely low the normal liver cell.Usually, described " low express " refer to packing cell and compare, in target cell 1/3 of microrna expression quantity not sufficient packing cell.
" 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 can be both the DNA sequence dna with the pairing of the complete reverse complemental of ripe Microrna, can be also with Microrna, to be combined in known Microrna target gene, with the DNA sequence dna of the incomplete reverse complemental pairing of Microrna.
" foreign gene " refer in recombinant virus genomes, comprise but do not belong to the genomic protein coding gene of wild-type virus, it can be the native protein encoding gene of the mankind or other species, can be also the protein coding gene of synthetic.
" 3 ' non-translational region of foreign gene " refers to the zone that in the ripe mRNA of foreign gene, does not translate in the encoding sequence downstream, 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, or identical Microrna target site sequence several nucleotide sequences but centre is separated by that join end to end more than 2.
" different Microrna target site sequences are connected continuously " refers to after a kind of Microrna target site sequence series connection repeats that the series connection that connects another kind of Microrna target site sequence repeats.The series connection of different Microrna target site sequences interval connects another kind of Microrna target site sequence after referring to a kind of Microrna target site sequence, and repeats more than 2 times as unit.
In one embodiment of the invention, packing cell is selected from people's kidney embryo HEK293 clone, and derives other clone of cultivating from HEK293, 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 293 serial cells, miR-16, miR-17, miR-18a, miR-19b, at least one in 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 obtain high titre in packing cell 293, and after the virus infection target cell-liver cell of increasing by the method, can effectively in liver cell, express activated Rituxan antibody, and make it to be secreted into extracellular, finally act on the tumour cell of the CD20 positive, 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 by 3 ' non-translational region of the people P53 gene that is operably connected at recombinant slow virus, insert the DNA fragmentations of 4 copy miR-15a target site sequences, make it to have obtained the recombinant slow virus of high titre in packing cell 293FT cell.
With existing recombinant viral vector, compare, the present invention has following beneficial effect:
Insert high level expression at least one packing cell in 3 ' non-translational region of the foreign gene that the present invention is operably connected at recombinant viral vector and do not express in target cell or the target site nucleotide sequence of low Microrna of expressing, make foreign gene can not be in the packaging series cell along with copying of virus great expression, the cytotoxicity of having avoided the foreign gene overexpression to cause reaches the competition to cell transcription, translation relevant enzymes and resource, has improved production efficiency and the virus titer of recombinant virus.And do not reduce the expression level of foreign gene in target cell, keep original result for the treatment of or by gain-of-function Study of Exogenous gene function.
Embodiment in the present invention is only for illustrative specific embodiment of the invention scheme, and it does not limit the application's protection domain, within the improvement of the equivalence that any those skilled in the art can carry out according to prior art is included in the application's 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 from Canadian Microbix Biosystem Inc. (Toronto), contain 5 type adenovirus left arm E1 district 1417 to 2344bp sequence fragments, and inverted 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, retain original SV40 PolyA signal sequence, naming this carrier is pDC338.Application 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
gAATTCaCCATGGAAGCCCCAGCTCAGCTTCTC
TTCCTCCTGCTACTCTGG(SEQ?ID?NO：1)
Primer 2: TGC
gTCGACtTATCAACACTCTCCCCTGTTGAA (SEQ ID NO:2)
Primer 3:CGC
gGATCCaCCATGGAGTTTTGGCTGAGCTGGGTT
TTCCTTGTTGCTATTTTA(SEQ?ID?NO：3)
Primer 4:CCC
gCTAGCtTACTATTTACCCGGAGACAGGGA (SEQ ID NO:4)
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. the structure of the replication-defective adenoviral vector that carries Mabthera (Rituxan) gene and regulated and controled by miR-15a
The synthetic DNA fragmentation (5 * miR-15T) that comprises 5 copy miR-15a target site sequences, and adding respectively the restriction enzyme site of NheI and SpeI at 5 '-end and 3 '-end, this DNA fragmentation is given birth to work biotechnology company limited by Shanghai, and to carry out full-length gene synthetic.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 (after heavy chain gene, before SV40Poly A tailing signal) of the Mabthera gene of pDC338-RTX carrier, called after pDC338-RTX-15T.3. the restructuring of the replication-defective adenoviral that carries Mabthera (Rituxan) gene and regulated and controled by miR-15a
The HEK293 cell strain is purchased from Canadian Microbix Biosystem Inc. (Toronto), is to be formed by the 5 type adenovirus DNA transformation of human embryonic kidney cells of shearing, 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 combined plasmid co-transfection 293 cells that contain 5 type adenovirus right arms, by homologous recombination, can produce the adenovirus with infectivity.We pass through the thin strain of Lipofectamine cotransfection to 293 with the plasmid pPE3-F11 that contains 5 type adenovirus right arms respectively by 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, the 3Cre transformation 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 accompanying drawing 1 is shown in by concrete carrier collection of illustrative plates.PBHGlox (delta) E1,3Cre and pBHGE3 are purchased from Canadian Microbix Biosystems Inc. (Ontario).The adenovirus plaque appears in after cotransfection 9-14 days, through three adenovirus plaque purifying, the recombinant adenovirus that must carry the recombinant adenovirus of Rituxan full length antibody gene and carry Rituxan full length antibody gene and regulated and controled by miR-15a, difference called after Ad11-RTX and Ad11-RTX-15T (CCTCC-V200813, on January 9th, 2009, Chinese Typical Representative culture collection center).
Adenovirus is amount reproduction in the HEK293 cell, the method large-scale purification adenovirus of application caesium chloride density gradient centrifugation.
4. carry the recombinant adenovirus of Rituxan gene and carry the Rituxan gene and compared by the replication of recombinant adenovirus in the HEK293 cell of miR-15a regulation and control
The HEK293 cell strain is pressed to 5 * 10
5cells/well is layered in the 6-orifice plate, at 37 ℃ of incubator 5%CO
2cultivate, second day changes serum free medium 1ml, then, add respectively recombinant adenovirus Ad11-RTX, Ad11-RTX-15T, its adenovirus amount is MOI=5, cultivate after 90 minutes, with phosphate buffered saline buffer (PBS), wash twice, adenovirus is washed away, and add the nutrient solution of 5% foetal calf serum to cultivate, at 0 hour and 48 hours, collect respectively, freeze thawing three times, detect adenovirus titre (method is referring to the AdEasyTM operational manual of U.S. Qbiogene company) by the TCID50 method, found that the adenoviral replication ability Ad11-RTX-15T in HEK293 is Ad11-RTX 100 times (seeing Fig. 2).Show that the insertion of miR-15aT target site nucleotide sequence can significantly improve the production efficiency of recombinant adenovirus.
5. carry the recombinant adenovirus of Rituxan gene and carry the Rituxan gene and compared in the ability of HEK293 cells Rituxan by the recombinant adenovirus of miR-15a regulation and control
The HEK293 cell is pressed to 1 * 10
5cells/well is layered in the 24-orifice plate, at 37 ℃ of incubator 5%CO
2cultivate.Second day adds respectively recombinant adenovirus Ad11-RTX, Ad11-RTX-15T, and its adenovirus amount is MOI=5, after two hours, washes away virus.Respectively 3 days with 7 days after collecting cell, application enzyme-linked immunosorbent assay (enzyme linked immunosorbentassay, ELISA) detect the expression level of Rituxan antibody in supernatant, found that at the 3rd day, after Ad11-RTX and Ad11-RTX-15T infection HEK293 cell, the concentration of antibody is respectively 2.19 ± 0.2 μ g/ml, 0.31 ± 0.1 μ g/ml.At the 7th day, after Ad11-RTX and Ad11-RTX-15T infect, the concentration of antibody was respectively 3.43 ± 0.3 μ g/ml, 0.39 ± 0.2 μ g/ml.Visible in each time period, Ad11-RTX and Ad11-RTX-15T infect the expression level Ad11-RTX-15T of Rituxan albumen after the HEK293 cell all far below Ad11-RTX, have reduced respectively 7.0 times and 8.8 times (seeing Fig. 3).The insertion that shows miR-15a target site nucleotide sequence can make the expression of foreign gene in Viral packaging cell 293 decline that is changed significantly.
6. carry the recombinant adenovirus of Rituxan gene and carry the Rituxan gene and be subject to the recombinant adenovirus of miR-15a regulation and control to express the antibody efficiency ratio in normal liver cell
Human normal hepatocyte strain L02 miR-15a expresses negative, and it is pressed to 1 * 10
5cells/well is layered in the 24-orifice plate, at 37 ℃ of incubator 5%CO
2cultivate.Second day adds respectively recombinant adenovirus Ad11-RTX, Ad11-RTX-15T, and its adenovirus amount is MOI=5, after two hours, washes away virus.Respectively at the 3rd day and the 7th day collecting cell, application enzyme-linked immunosorbent assay (enzymelinked immunosorbent assay, ELISA) detect the expression level of antibody, found that at the 3rd day, after Ad11-RTX and Ad11-RTX-15T infection L02 cell, the concentration of antibody is respectively 1.55 ± 0.1 μ g/ml, 1.67 ± 0.2 μ g/ml.At the 7th day, after Ad11-RTX and Ad11-RTX-15T infect, the concentration of antibody was respectively 3.21 ± 0.4 μ g/ml, 3.35 ± 0.2 μ g/ml.Visible in each time period, the expression level similar (seeing Fig. 3) of Ad11-RTX and Rituxan albumen after 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 regulated and controled by miR-15a is in nude mouse internal therapy transplantation tumor
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 from health and becomes biotech firm, the expression cassette that it contains the GFP gene under the control of EF1 α promotor, and foreign gene can be expressed under the control of CMV promotor.Extract human total rna, the synthesizing single-stranded cDNA of application reversed transcriptive enzyme, and application polymerase chain reaction,PCR (PCR) technology amplifies the open reading frame of people P53 gene from the strand cDNA library.Called after pKCDNA-P53.
Primer 5:CCG
tCTAGAaCCATGGAGGAGCCGCAGTCAGA (SEQ ID NO:7)
Primer 6:TGC
gAATTCtCAGTCTGAGTCAGGCCC (SEQ ID NO:8)
TCTAGAACCATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTGAGTCAGGA
AACATTTTCAGACCTATGGAAACTACTTCCTGAAAACAACGTTCTGTCCCCCTTGCCGT
CCCAAGCAATGGATGATTTGATGCTGTCCCCGGACGATATTGAACAATGGTTCACTGAA
GACCCAGGTCCAGATGAAGCTCCCAGAATGCCAGAGGCTGCTCCCCCCGTGGCCCCTGC
ACCAGCAGCTCCTACACCGGCGGCCCCTGCACCAGCCCCCTCCTGGCCCCTGTCATCTT
CTGTCCCTTCCCAGAAAACCTACCAGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCAT
TCTGGGACAGCCAAGTCTGTGACTTGCACGTACTCCCCTGCCCTCAACAAGATGTTTTG
CCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGATTCCACACCCCCGCCCGGCA
CCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGG
CGCTGCCCCCACCATGAGCGCTGCTCAGATAGCGATGGTCTGGCCCCTCCTCAGCATCT
TATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAACACTTTTCGAC
ATAGTGTGGTGGTGCCCTATGAGCCGCCTGAGGTTGGCTCTGACTGTACCACCATCCAC
TACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGGAGGCCCATCCTCAC
CATCATCACACTGGAAGACTCCAGTGGTAATCTACTGGGACGGAACAGCTTTGAGGTGC
GTGTTTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGAAGAGAATCTCCGCAAGAAA
GGGGAGCCTCACCACGAGCTGCCCCCAGGGAGCACTAAGCGAGCACTGCCCAACAACAC
CAGCTCCTCTCCCCAGCCAAAGAAGAAACCACTGGATGGAGAATATTTCACCCTTCAGA
TCCGTGGGCGTGAGCGCTTCGAGATGTTCCGAGAGCTGAATGAGGCCTTGGAACTCAAG
GATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGGCTCACTCCAGCCACCTGAAGTC
CAAAAAGGGTCAGTCTACCTCCCGCCATAAAAAACTCATGTTCAAGACAGAAGGGCCTG
ACTCAGACTGA
GCTAGC(SEQ?ID?NO：9)
2. the structure of the slow virus packaging expression plasmid that comprises people P53 gene and regulated and controled by miR-15a
The synthetic DNA fragmentation (4 * miR-15T) that comprises 4 copy miR-15a target site sequences, and add respectively the restriction enzyme site of NheI and EcoRI at 5 '-end and 3 '-end, this DNA fragmentation carries out full-length gene by Shanghai living work biotechnology company limited and synthesizes, and correct through sequence verification.4 * miR-15T fragment is inserted to the 3 ' non-translational region (after terminator codon, before SV40Poly A tailing signal) of the people P53 gene of pKCDNA-P53 carrier, called after pKCDNA-P53-15T.The nucleotide sequence of 4 * miR-15T fragment following (5 '-3 '):
GCTAGCCACAAACCATTATGTGCTGCTAGATCCACAAACCATTATGTGCTGCTAACGAT
CACAAACCATTATGTGCTGCTACTCCACAAACCATTATGTGCTGCTA
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 to 7.5 * 10
5be layered on 10cm dish 5%CO2 incubator incubated overnight.Second day changes the 10%DMEM substratum into 2% DMEM substratum, after 4 hours by 20 μ g expression plasmids (pKCDNA-P53 and pKCDNA-P53-15T), 10 μ g packaging plasmid pCMVdelta 8.91 mix with 5 μ g packaging plasmid pMD.G, add sterilized water to 250 μ l, and concussion mixes.To the 0.5MCaCl that adds successively 250 μ l in above-mentioned mixed solution
2, then to the 2 * HBS that dropwise adds 500 μ l in above-mentioned mixed solution, the concussion of limit edged.Standing 30 minutes, above-mentioned mixed solution is evenly added in cell culture medium.After transfection 12 hours, outwell the 2%DMEM substratum, PBS cleans twice, gains the 10%DMEM substratum, respectively at 36 hours, after 60 hours, collects cell culture medium.Collection is closed to the cell culture medium of virion with the centrifugal 10min of 4000g, collect supernatant liquor, and filter with 0.45 μ m filter, the liquid filtered is placed in 40mL ultracentrifugation pipe, 4 ℃, centrifugal 20 minutes of 25000r/min, used the resuspended virus precipitation of 500ul ice PBS liquid.The slow virus of the carrier P53 gene that packing obtains, and carrier P53 gene the slow virus that is subject to the miR-15a regulation and control called after Le-P53 and Le-P53-15T respectively.
4. comprise people P53 gene and be subject to the titer determination of the slow virus of miR-15a regulation and control
Press 2 * 10 in six orifice plates
5/ hole paving Hela cell, used the 10%DMEM culture medium culturing, is placed in the 5%CO2 incubator and spends the night.By virus to be measured (Le-P53 and Le-P53-15T) gradient dilution, be 10
-2-10
-6doubly, the virus liquid of each concentration is diluted in to the 2%DMEM substratum, final volume is 1ml.After having diluted, substratum original in six orifice plates is outwelled, changed into the substratum that adds viral dilution liquid, be placed in 5%CO
2the incubator incubated overnight.Second day will change the 10%DMEM substratum into containing virulent 2%DMEM substratum, infect latter the 4th to five days, clean cell with PBS, resuspended after digestion, detect luciferase expression situation, calculation formula: the per-cent of virus titer (TU/mL)=cell quantity x fluorescencepositive cell/virus liquid volume (ml) through flow cytometer.Cells were tested by flow cytometry shows that the virus titer of Le-P53-15T is 8.75 * 10
9tU/mL, and the virus titer of Le-P53 is 1.37 * 10
8tU/mL, the virus titer of Le-P53-15T is 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 subject to the slow virus of miR-15a regulation and control to express the level determination of P53 in tumour cell
Hepatoma cell strain Hep3B, lung cancer cell line A549, breast carcinoma cell strain MCF7 is purchased from ATCC, and miR-15a expresses all negative.Above-mentioned tumour cell is pressed to 5 * 10
4cells/well is layered in the 24-orifice plate, at 37 ℃ of incubator 5%CO
2overnight incubation.Second day adds respectively recombinant slow virus Le-P53, Le-P53-15T, and the MOI value is 10.At the 1st, 2,3 days, extract total RNA respectively, apply 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 without significant difference.The insertion that shows miR-15a target site nucleotide sequence does not cause that the expression amount of external source P53 in 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 affect the expression level of foreign gene in target cell.
reference:
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Berezikov?E，Cuppen?E，Plasterk?R.Approaches?to?microRNAdiscovery.Nat?Genet.2006，38(6)：2-6
2.LandgrafP，RusuM，SheridanR，SewerA，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，Müller?RU，Schermer?B，Bissels?U，Inman?J，PhanQ，ChienM，Weir?DB，Choksi?R，De?Vita?G，Frezzetti?D，TrompeterHI，Hornung?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，ZavolanM，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，Aider?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 > recombinant viral vector of a kind of foreign gene-carrying High-efficient Production in packing cell, its construction process and uses thereof
<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+SV40polyA 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 (4)
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| CN106591361A (en) * | 2015-10-20 | 2017-04-26 | 钱文斌 | Recombinant pox oncolytic virus, and construction method and application thereof |
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| CN106591361A (en) * | 2015-10-20 | 2017-04-26 | 钱文斌 | Recombinant pox oncolytic virus, and construction method and application thereof |
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