CN103834686A - Efficient clone selection expression vector, and preparation method and use thereof - Google Patents
Efficient clone selection expression vector, and preparation method and use thereof Download PDFInfo
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- CN103834686A CN103834686A CN201210483844.7A CN201210483844A CN103834686A CN 103834686 A CN103834686 A CN 103834686A CN 201210483844 A CN201210483844 A CN 201210483844A CN 103834686 A CN103834686 A CN 103834686A
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Abstract
The invention discloses an efficient clone selection expression vector, which is obtained by an ITRs sequence of a piggyBac transposition system inserted into a mammal expression vector. The invention also discloses a preparation method and use thereof. The efficient clone selection expression vector is built by using the ITRs sequence of the piggyBac transposition system, and meanwhile, a transcriptase gene which is optimized by a codon and can be efficiently expressed in mice is cloned to another eukaryotic expression vector. A selection marker and a target gene are integrated into a genome of a host cell in common by coexpression of an expression vector containing the ITRs sequence and the vector containing transcriptase. Thus, the transfection efficiency and the positive clone screening rate are greatly improved.
Description
Technical field
The present invention relates to field of biology, particularly relate to a kind of high-efficient cloning screening expression vector of the ITRs sequence construct with piggyBac transposon system, construction process of this carrier and uses thereof.
Background technology
Transposon is one section of DNA sequence dna movably in genome, can be by a series of processes such as cutting, reintegrate from a genomic position " jump " to another position.Transposon accounts for the more than 40% of people and mouse genome sequence (Nature, 2001,409,860 – 921; Nature, 2002,420,520 – 562).
Since McClintock finds first transposon from corn (Proc.Natl.Acad.Sci.1950, USA36,344 – 345), transposable element has become a lot of biological valuable genetic analysis instruments.In prokaryotic organism, the mutation research that utilizes transposon to carry out has been found the gene (Science, 1999,286,2165 – 2169 that in the time that harmful microorganism causes a disease, play an important role; J.Virol.2003,77,123 – 134).In eukaryote, the transgenosis of the utilization P factor (a kind of transposon) and insertion mutation technology have promoted the development of Drosophila genetics greatly.Many transposons including the P factor do not have activity in the organism beyond its natural host, illustrate that swivel base process relates to some host's factors (Arch.Insect Biochem.Physiol.1993,22,373 – 384).Tc1/Mariner family has been applied to mouse and zebra fish at interior several transposon systems.A kind of Tc1 class transposon Sleeping Beauty (SB) that utilizes comparison system auxology means synthetic is proved to be has activity (Cell 1997,91,501-510 in mouse and people's cell; Proc.Natl.Acad.Sci.1998, USA 95,10769 – 10773).Although the transposon such as SB and Minos has carried out insertion mutation test in Mice Body, but because new insertion point concentrates on around original site, swivel base inefficiency and carry the reasons such as DNA limited length, these transposons are not widely used (Genomics 2003,81,108-111; Mol.Cell.Biol.2003,23,9189-9207).
The PB factor is a kind of DNA transposon deriving from moth wild cabbage looper, 2472 bases of total length.The inverted terminal repeat (ITR) that 13 bases are contained at its two ends, and 594 the amino acid whose transposases of encoding.PB has been successfully used to the genetic analysis of drosophila melanogaster and other insect.It inserts four base TTAA sites specifically, and forms TTAA repetition (Virology 1989,172,156 – 169 in insertion point both sides; Virology 1995,211,397 – 407; Insect Mol.Biol.1996,5,141 – 151).Due to its distinctive transposase and TTAA target sequence, PB becomes a new DNA transposon family---the representative (In Mobile DNA II, 2002, pp.1093 – 1110) of piggyBac family.PB has been applied to four objects more than ten as reproductive tract transgenosis instrument and has planted insect (Insect Biochem.Mol.Biol.2003,33,449 – 458).As mutagenic compound, the swivel base efficiency of PB in drosophila melanogaster at least with quite (Nat.Genet.2004,36,283 – 287) of the P factor.In red flour beetle Tribolium castaneum, PB also can high-efficiency transposon (Insect Mol.Biol.2003,12,433 – 440) between nonhomologous chromosome.Mould, to the sequence that all finds that there is a lot of similar PB in the different species gene group of many Phylogenetics such as Mammals, has further indicated that the activity of PB may and not only be confined to (Mol.Genet.Genomics, 2003,270,173 – 180) in insect.In fact, recent findings PB also can have efficient transposition activity in turbellarian worm Girardiatigrina, Mammals and cell thereof, be widely applied (Proc.Natl.Acad.Sci.2003 in fields such as Animal genome functional study, transgenosis and induced multi-potent stem cells, USA 100,14046 – 14051).
Although PB system has host's property widely, and efficient transfection efficiency, and in mammalian cell, be widely used in gene therapy (Molecular Therapy, 2007,15,139-145), still current its studied in vitro and also do not had relevant report.
Summary of the invention
One of the technical problem to be solved in the present invention is to provide a kind of high-efficient cloning screening expression vector, and it can improve the screening rate of transfection efficiency and positive colony greatly.
For solving the problems of the technologies described above, high-efficient cloning screening expression vector of the present invention, is inserted in mammalian expression vector and is obtained by the ITRs sequence of piggyBac transposon system.
Two of the technical problem to be solved in the present invention is to provide the preparation method of above-mentioned high-efficient cloning screening expression vector.
For solving the problems of the technologies described above, the preparation method of high-efficient cloning screening expression vector of the present invention, comprises the following steps:
1) take pGH as cloning vector, the synthetic 5 ' ITR of full gene and 3 ' ITR DNA fragmentation, two of fragment adds the restriction enzyme site of EcoRV, obtains carrier pGH-ITR5 and pGH-ITR3;
2) take pGH as cloning vector, the transcriptase gene fragment that the synthetic GeneBank sequence number of full gene is EF587698, and two of fragment adds respectively the restriction enzyme site of EcoRI and HindIII, obtains carrier pGH-Transposase;
3) cut carrier pGH-ITR5 and pGH-ITR3 with EcoRV enzyme, EcoRI and HindIII enzyme are cut carrier pGH-Transposase;
4) NruI enzyme is cut mammalian expression vector, and EcoRI and HindIII enzyme are cut expression vector pTriEx2 simultaneously;
5) fragment ITR5 is connected to the mammalian expression vector of cutting through NruI enzyme, obtains the expression vector containing ITR5 fragment;
6) fragment Transposase is connected to the expression vector pTriEx2 cutting through EcoRI and HindIII enzyme simultaneously, obtains new expression vector pTriEx2-Transposase;
7) Bstz17I enzyme is cut the expression vector containing ITR5 fragment that step 5) obtains;
8) fragment ITR3 is connected to the expression vector that step 7) obtains.
Three of the technical problem to be solved in the present invention is to provide above-mentioned high-efficient cloning screening expression vector and expresses the application in target protein clone at transfection mammalian cell and screening.
The ITRs sequence construct high-efficient cloning screening expression vector of piggyBac transposon system for the present invention, codon is optimized simultaneously can be in mouse the transcriptase gene clone of high efficient expression to another one carrier for expression of eukaryon, by containing the expression vector of ITRs sequence with containing the coexpression of the carrier of transcriptase, selection markers and goal gene are together incorporated in the genome of host cell, thereby the efficiency of transfection and the screening rate of positive colony are greatly improved, also for the clone that surely turns that builds multiple subunit coexpressions provides possibility, for the function of Study of Exogenous gene in mammalian cell provides favourable condition.
Accompanying drawing explanation
Fig. 1 is the structural representation of expression vector pcDNA3.1 (+)-ITRs.
Fig. 2 is the structural representation of expression vector pcDNA4/TO-ITRs.
Embodiment
Understand for technology contents of the present invention, feature and effect being had more specifically, existing in conjunction with illustrated embodiment, details are as follows:
The preparation of embodiment 1 pcDNA3.1 (+)-ITRs carrier
The structure of pcDNA3.1 (+)-ITRs expression vector as shown in Figure 1, take pc DNA3.1 (+) as carrier, the direction (see figure 1) correct according to DNA sequence dna is inserted into ITRs sequence on carrier, obtains carrier for expression of eukaryon pcDNA3.1 (+)-ITRs containing ITRs sequence.This expression vector pcDNA3.1 (+)-ITRs can carry goal gene and the screening-gene (Neomycin) that mammalian promoter (CMV), multiple clone site (MCS) locate and be incorporated into together in the genome of host cell in the time of swivel base.
Concrete preparation method is as follows:
(1), from commercial pXL-BacII plasmid vector, find out the concrete sequence of its 5 ' ITR and 3 ' ITR; From the document (Nucleic Acids Research, 2007,35, e87) of having reported, find that codon optimizes can be in mouse transcriptase (Transposase) gene order (GeneBank sequence number: EF587698) of high efficient expression.Wherein,
5 ' ITR sequence (SEQ ID No:1) is:
TATAACAAGAAAATATATATATAATAAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCATTTTGACTCACGCGGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGGGAGCTCCAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTAGCTGCATCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGCTGGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGAAGCGGCATGGAAAGAGTTTGC
3 ' ITR sequence (SEQ ID No:2) is:
TAAAACGACGGCCAGTGAGCGCGCCTCGTTCATTCACGTTTTTGAACCCGTGGAGGACGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGATGCTCGACACGCTGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATTGACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATCGATATCGTC
(2), take pGH as cloning vector, synthetic required inverted terminal repeat 5 ' ITR and 3 ' the ITR DNA fragmentation of gene, and two of fragment entirely all adds the restriction enzyme site (GATATC) of EcoRV, obtains respectively carrier pGH-ITR5 and pGH-ITR3.
Meanwhile, take pGH as cloning vector, the synthetic required transcriptase gene fragment of full gene, and two of fragment adds respectively EcoRI(5 ' GAATTC) and HindIII(3 ' AAGCTT) restriction enzyme site, obtain carrier pGH-Transposase.
(3) cut carrier pGH-ITR5 and pGH-ITR3 with EcoRV enzyme, EcoRI and HindIII enzyme are cut carrier pGH-Transposase.
(4) NruI enzyme is cut expression vector pcDNA3.1 (+), and EcoRI and HindIII enzyme are cut expression vector pTriEx2 simultaneously.
(5) fragment ITR5 is connected to the expression vector pcDNA3.1 (+) cutting through NruI enzyme, obtains new expression vector ITR5-pcDNA3.1 (+).
(6) fragment Transposase is connected to the expression vector pTriEx2 cutting through EcoRI and HindIII enzyme simultaneously, obtains new expression vector pTriEx2-Transposase.
(7) Bstz17I enzyme is cut expression vector ITR5-pcDNA3.1 (+).
(8) fragment ITR3 is connected to the expression vector ITR5-pcDNA3.1 (+) cutting through Bstz17I enzyme, obtains novel vector ITR5-pcDNA3.1 (+)-ITR3.
The sequence (SEQ ID No:3) of carrier ITR5-pcDNA3.1 (+)-ITR3 is:
GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCG
ATCTATAACAAGAAAATATATATATAAT AAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTAAAAGTCACGTAAAAGATAAT CATGCGTCATTTTGACTCACGCGGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGGGA GCTCCAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGA ATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTAGCTGCATCAGGATCATATCGTCGGGTCTTTTTTC CGGCTCAGTCATCGCCCAAGCTGGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGAAGC GGCATGGAAAGAGTTTGCGATCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTA
ATCGATAAAAGTTTTGTTACTTTATAGAAGAAATTTTGAGTTTTTGTTTTTTTTTAATAAATAAATAAACA TAAATAAATTGTTTGTTGAATTTATTATTAGTATGTAAGTGTAAATATAATAAAACTTAATATCTATTCAAATTAATAAA TAAACCTCGATATACAGACCGATAAAACACATGCGTCAATTTTACGCATGATTATCTTTAACGTACGTCACAATATGATT ATCTTTCTAGGGTTAATCTAGCTGCGTGTTCTGCAGCGTGTCGAGCATCTTCATCTGCTCCATCACGCTGTAAAACACAT TTGCACCGCGAGTCTGCCCGTCCTCCACGGGTTCAAAAACGTGAATGAACGAGGCGCGCTCACTGGCCGTCGTTTTAGATTACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC
The preparation of embodiment 2 pcDNA4/TO-ITRs carriers
As shown in Figure 2, take pc DNA4/TO as carrier, the direction (see figure 2) correct according to DNA sequence dna is inserted into ITRs sequence on carrier the construction process of pcDNA4/TO-ITRs carrier, obtains the carrier for expression of eukaryon pcDNA4/TO-ITRs containing ITRs sequence.This expression vector pcDNA4/TO-ITRs can carry goal gene and the screening-gene (Zeocin) that mammalian promoter (CMV), multiple clone site (MCS) locate and be incorporated into together in the genome of host cell in the time of swivel base.
Concrete preparation method is as follows:
(1)～(3) are with embodiment 1.
(4) NruI enzyme is cut expression vector pcDNA4/TO, and EcoRI and HindIII enzyme are cut expression vector pTriEx2 simultaneously.
(5) fragment ITR5 is connected to the expression vector pcDNA4/TO cutting through NruI enzyme, obtains new expression vector ITR5-pcDNA4/TO.
(6) fragment Transposase is connected to the expression vector pTriEx2 cutting through EcoRI and HindIII enzyme simultaneously, obtains new expression vector pTriEx2-Transposase.
(7) Bstz17I enzyme is cut expression vector ITR5-pcDNA4/TO.
(8) fragment ITR3 is connected to the expression vector ITR5-pcDNA4/TO cutting through Bstz17I enzyme, obtains novel vector ITR5-pcDNA4/TO-ITR3.
The sequence (SEQ ID No:4) of carrier ITR5-pcDNA4/TO-ITR3 is:
GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCG
ATCTATAACAAGAAAATATATATATAAT AAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTAAAAGTCACGTAAAAGATAAT CATGCGTCATTTTGACTCACGCGGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGGGA GCTCCAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGA ATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTAGCTGCATCAGGATCATATCGTCGGGTCTTTTTTC CGGCTCAGTCATCGCCCAAGCTGGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGAAGC GGCATGGAAAGAGTTTGCGATCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGAACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGATCGTCGACGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTA
ATCGATAAAAGTTTTGTTACT TTATAGAAGAAATTTTGAGTTTTTGTTTTTTTTTAATAAATAAATAAACATAAATAAATTGTTTGTTGAATTTATTATTA GTATGTAAGTGTAAATATAATAAAACTTAATATCTATTCAAATTAATAAATAAACCTCGATATACAGACCGATAAAACAC ATGCGTCAATTTTACGCATGATTATCTTTAACGTACGTCACAATATGATTATCTTTCTAGGGTTAATCTAGCTGCGTGTT CTGCAGCGTGTCGAGCATCTTCATCTGCTCCATCACGCTGTAAAACACATTTGCACCGCGAGTCTGCCCGTCCTCCACGG GTTCAAAAACGTGAATGAACGAGGCGCGCTCACTGGCCGTCGTTTTAGATTACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC
Embodiment 3 application of pcDNA3.1 (+)-ITRs carrier in Hela cell
1. experimental technique:
(1) recovery Hela(human cervical carcinoma cell) cell, for transfection, substratum is: RM1640+10% foetal calf serum+1% suis (Pen Strep).
(2) transfection the day before yesterday, with the quantity in 4 × 105/ holes, Hela cell kind is entered to six orifice plates.In the time that cell fraction of coverage is 80%-90%, change into without any antibiotic substratum.
(3) prepare two 1.5ml EP pipe (
3810 type Eppendorf tubes), add respectively following two kinds of combination plasmids:
Combination 1:2 μ g pcDNA3.1 (+)-EGFP
Combination 2:1.5 μ g pcDNA3.1 (+)-ITRs-EGFP and 0.5 μ g transcriptase
Use respectively again OPTI-MEM substratum polishing to 100 μ l volume.
(4) in above-mentioned two EP pipes, carefully add 5 μ l Fugene HD transfection reagents respectively, carefully blow and beat and mix with rifle head, room temperature leaves standstill 15 minutes.
(5) mixing solutions is dropwise joined in six orifice plates that have been covered with cell equably, mix gently.37 ℃ of baking ovens are cultivated.
(6) after transfection 48 hours, with 6 × 10
3cell is laid on 96 orifice plates that black wall is transparent by the quantity in/hole, four multiple holes of each combination, and remaining cell is layered in 6cm culture dish again by identical quantity, and adding final concentration is 400 μ g/ml G418 screenings.
(7) next day, after transfection 72 hours, carry out Array Scan(array scanning), detect the signal value of its fluorescence.
(8) screening is after 2 days, with 6 × 10
3cell is laid on 96 orifice plates that black wall is transparent by the quantity in/hole, four multiple holes of each combination, carry out Array Scan detection, remaining cell is layered in 6cm culture dish again in initiating cell is identical separately ratio (70%), and adding final concentration is 400 μ g/ml G418 screenings.
(9) screen after 6 days, after 11 days, carry out same processing according to step (8) respectively.
2. experimental result:
Experimental result is as shown in table 1, as can be seen from Table 1, and along with the increase of screening number of days, cell number in combination 1 reduces gradually, has arrived while screening latter 11 days, and the number of cell reaches minimum, for 3.1E+05, the number (1.8E+05) of this and cellular control unit is suitable; And the cell number combining in 2 presents the trend increasing progressively, it is maximum that the cell number while screening latter 11 days reaches, and is 5.7E+06, is 18 times of combination 1 cell number, is 32 times of cellular control unit number.
The quantity of different time cell after table 1 transfection
3. conclusion:
Can find out by this experiment, cell (combination 2) expression rate of Green fluorescin (GFP) and the survival rate (as table 1) of cell that contain PB swivel base sequence (ITRs) carrier are significantly higher than the cell of transfection without transformation carrier, show that this carrier can improve the screening rate of positive colony greatly, simultaneously this system also successfully built monogenic ionophorous protein TRPV3 and three subunit coexpressions calcium channel PROTEIN C av1.2 surely turn clone, specific experiment scheme is not just repeating here.
Claims (10)
1. a high-efficient cloning screening expression vector, is characterized in that, this carrier is inserted in mammalian expression vector and is obtained by the ITRs sequence of piggyBac transposon system.
2. carrier according to claim 1, is characterized in that, described mammalian expression vector is pcDNA3.1 (+).
3. carrier according to claim 2, is characterized in that, this carrier has the sequence as shown in SEQ ID No:3.
4. carrier according to claim 1, is characterized in that, described mammalian expression vector is pcDNA4/TO.
5. carrier according to claim 4, is characterized in that, this carrier has the sequence as shown in SEQ ID No:4.
6. the preparation method of carrier described in claim 1 to 5 any one, is characterized in that, comprises the following steps:
1) take pGH as cloning vector, the synthetic 5 ' ITR of full gene and 3 ' ITR DNA fragmentation, two of fragment adds the restriction enzyme site of EcoRV, obtains carrier pGH-ITR5 and pGH-ITR3;
2) take pGH as cloning vector, the transcriptase gene fragment that the synthetic GeneBank sequence number of full gene is EF587698, and two of fragment adds respectively the restriction enzyme site of EcoRI and HindIII, obtains carrier pGH-Transposase;
3) cut carrier pGH-ITR5 and pGH-ITR3 with EcoRV enzyme, EcoRI and HindIII enzyme are cut carrier pGH-Transposase;
4) NruI enzyme is cut mammalian expression vector, and EcoRI and HindIII enzyme are cut expression vector pTriEx2 simultaneously;
5) fragment ITR5 is connected to the mammalian expression vector of cutting through NruI enzyme, obtains the expression vector containing ITR5 fragment;
6) fragment Transposase is connected to the expression vector pTriEx2 cutting through EcoRI and HindIII enzyme simultaneously, obtains new expression vector pTriEx2-Transposase;
7) Bstz17I enzyme is cut the expression vector containing ITR5 fragment that step 5) obtains;
8) fragment ITR3 is connected to the expression vector that step 7) obtains.
7. method according to claim 6, is characterized in that, described mammalian expression vector comprises pcDNA3.1 (+) or pcDNA4/TO.
8. described in claim 1 to 5 any one, carrier is expressed the application in target protein clone in screening.
9. the application of carrier in transfection mammalian cell described in claim 1 to 5 any one.
10. application according to claim 9, is characterized in that, described mammalian cell comprises HEKC HEK293 or human cervical carcinoma cell Hela.
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| CN106480081A (en) * | 2016-09-22 | 2017-03-08 | 苏州泓迅生物科技有限公司 | A kind of CRISPRi carrier of single pUC pUC and preparation method thereof |
| CN106755096A (en) * | 2016-12-20 | 2017-05-31 | 上海药明生物技术有限公司 | The method for obtaining the stable cell mass of expression target protein in Chinese hamster ovary celI using piggyBac transposon |
| CN109628488A (en) * | 2018-12-28 | 2019-04-16 | 赛业（苏州）生物科技有限公司 | The method for constructing gene overexpression based on piggyBAC system or interfering stable cell strain |
| EP3433366A4 (en) * | 2016-03-25 | 2019-08-28 | Genomefrontier Therapeutics, Inc. | Kit for constructing transposon and use thereof |
| WO2020034097A1 (en) * | 2018-08-14 | 2020-02-20 | Wuxi Biologics (Shanghai) Co., Ltd. | Transcriptional regulatory element and its use in enhancing the expression of exogenous protein |
| CN111471714A (en) * | 2020-05-07 | 2020-07-31 | 西南大学 | Eukaryotic transgenic cell line mediated by Minos transposon system and construction method |
| CN116144705A (en) * | 2022-12-28 | 2023-05-23 | 昭衍（苏州）新药研究中心有限公司 | Method for efficiently preparing voltage-dependent calcium ion channel cell model |
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| EP3433366A4 (en) * | 2016-03-25 | 2019-08-28 | Genomefrontier Therapeutics, Inc. | Kit for constructing transposon and use thereof |
| CN106480081A (en) * | 2016-09-22 | 2017-03-08 | 苏州泓迅生物科技有限公司 | A kind of CRISPRi carrier of single pUC pUC and preparation method thereof |
| CN106755096A (en) * | 2016-12-20 | 2017-05-31 | 上海药明生物技术有限公司 | The method for obtaining the stable cell mass of expression target protein in Chinese hamster ovary celI using piggyBac transposon |
| WO2020034097A1 (en) * | 2018-08-14 | 2020-02-20 | Wuxi Biologics (Shanghai) Co., Ltd. | Transcriptional regulatory element and its use in enhancing the expression of exogenous protein |
| US11254952B2 (en) | 2018-08-14 | 2022-02-22 | Wuxi Biologies Ireland Limited | Transcriptional regulatory element and its use in enhancing the expression of heterologous protein |
| CN109628488A (en) * | 2018-12-28 | 2019-04-16 | 赛业（苏州）生物科技有限公司 | The method for constructing gene overexpression based on piggyBAC system or interfering stable cell strain |
| CN111471714A (en) * | 2020-05-07 | 2020-07-31 | 西南大学 | Eukaryotic transgenic cell line mediated by Minos transposon system and construction method |
| CN116144705A (en) * | 2022-12-28 | 2023-05-23 | 昭衍（苏州）新药研究中心有限公司 | Method for efficiently preparing voltage-dependent calcium ion channel cell model |
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