CN103834686B - High-efficient cloning screening expression vector, Preparation Method And The Use - Google Patents
High-efficient cloning screening expression vector, Preparation Method And The Use Download PDFInfo
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Abstract
The invention discloses a kind of high-efficient cloning screening expression vector, this carrier is inserted in mammalian expression vector and is obtained by the ITRs sequence of piggyBac transposon system. The invention also discloses the preparation method and its usage of above-mentioned carrier. The present invention utilizes the ITRs sequence construct high-efficient cloning screening expression vector of piggyBac transposon system, codon is optimized simultaneously can be in mouse the transcriptase Gene cloning 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 genes of interest are together incorporated in the genome of host cell, thereby have greatly improved the efficiency of transfection and the screening rate of positive colony.
Description
Technical Field
The invention relates to the field of biology, in particular to an efficient cloning and screening expression vector constructed by using an ITRs sequence of a piggyBac transposition system, a construction method of the vector and application of the vector.
Background
Transposons are moveable DNA sequences in a genome that can "jump" from one location to another in the genome through a series of processes, such as cutting, reintegration, and the like. Transposons account for over 40% of the human and mouse genomic sequences (Nature, 2001,409, 860-562; Nature,2002,420, 520-562).
Since the first transposon was discovered in maize by McClintock (Proc. Natl. Acad. Sci.1950, USA36, 344-. In prokaryotes, mutation studies using transposons have found genes that play an important role in the pathogenesis of harmful microorganisms (Science, 1999,286, 2165-2169; J.Virol.2003,77, 123-134). In eukaryotes, transgenic and insertional mutagenesis techniques using P-elements, a transposon, have driven the development of Drosophila genetics considerably. Many transposons, including P elements, are not active in organisms other than their native host, suggesting that some host elements are involved in the transposition process (Arch. InsectBiochem. Physiol.1993,22, 373-384). Several transposition systems including the Tc1/Mariner family have been applied to mice and zebrafish. A Tc 1-like transposon SleepingBeauty (SB) artificially synthesized using the comparative phylogenetic approach was shown to be active in mouse and human cells (Cell 1997,91, 501-510; Proc. Natl. Acad. Sci.1998, USA95, 10769-10773). Although transposons such as SB and Minos have been tested for insertion mutation in mice, these transposons have not been widely used due to the concentration of new insertion sites around the original site, low transposition efficiency and limited length of the carrying DNA (Genomics 2003,81, 108-.
The PB factor is a DNA transposon derived from moth and cabbage looper, and has 2472 bases in the total length. It contains 13 base Inverted Terminal Repeats (ITRs) at both ends and encodes a 594 amino acid transposase. PB has been successfully used for genetic analysis of Drosophila melanogaster and other insects. It specifically inserts into the four-base TTAA site and forms TTAA repeats flanking the insertion site (Virology1989, 172, 156-169; Virology1995,211, 397-407; InsectMol. biol.1996,5, 141-151). PB is a representative of a new DNA transposon family, piggyBac family, due to its unique transposase and TTAA target sequences (InMobileDNAII, 2002, pp.1093-1110). PB has been used as a germline transgenic tool for over ten insects of four purposes (InsectBiochem. mol. biol.2003,33, 449-458). As a mutagen, PB transposes in Drosophila melanogaster at least as efficiently as P-element (nat. Genet.2004,36, 283-287). In Triboliumcastaneum, PB is also efficiently transposable between non-homologous chromosomes (InsectMol. biol.2003,12, 433-440). The PB-like sequences found in the genomes of a number of phylogenetically different species, from moulds to mammals, further predict that PB activity may not be restricted to insects (mol. Genet. genomics,2003,270, 173-180). In fact, PB has recently been found to have high-efficiency transposition activity in Giraradiatigrina, mammals and their cells, and has been widely used in the fields of animal genome function research, gene transfer and induced pluripotent stem cells (Proc. Natl. Acad. Sci.2003, USA100, 14046-.
Although the PB system has wide host range and high transfection efficiency, and has been widely used for gene therapy in mammalian cells (molecular therapy,2007,15, 139-145), no relevant report has been reported in vitro studies.
Disclosure of Invention
One of the technical problems to be solved by the invention is to provide an expression vector for efficient cloning and screening, which can greatly improve the transfection efficiency and the screening rate of positive clones.
In order to solve the technical problems, the efficient cloning and screening expression vector is obtained by inserting an ITRs sequence of a piggyBac transposition system into a mammalian expression vector.
The second technical problem to be solved by the present invention is to provide a method for preparing the above efficient cloning and screening expression vector.
In order to solve the technical problems, the preparation method of the high-efficiency cloning and screening expression vector comprises the following steps:
1) taking pGH as a cloning vector, synthesizing 5 'ITR and 3' ITRDNA fragments through a whole gene, and adding enzyme cutting sites of EcoRV at two ends of the fragments to obtain vectors pGH-ITR5 and pGH-ITR 3;
2) taking pGH as a cloning vector, synthesizing a transcriptive enzyme gene fragment with the sequence number of EF587698 of a GeneBank through a whole gene, and respectively adding enzyme cutting sites of EcoRI and HindIII into two ends of the fragment to obtain a vector pGH-Transposase;
3) cutting the vector pGH-ITR5 and pGH-ITR3 by EcoRV enzyme, cutting the vector pGH-Transposase by EcoRI and HindIII enzyme;
4) NruI enzyme-cutting the mammalian expression vector, and simultaneously EcoRI and HindIII enzyme-cutting the expression vector pTriEx 2;
5) connecting the fragment ITR5 to a mammal expression vector subjected to NruI enzyme digestion to obtain an expression vector containing an ITR5 fragment;
6) meanwhile, the fragment Transposase is connected to an expression vector pTriEx2 which is cut by EcoRI and HindIII to obtain a new expression vector pTriEx 2-Transposase;
7) bstz17I enzyme-cutting the expression vector containing the ITR5 fragment obtained in the step 5);
8) the fragment ITR3 was ligated to the expression vector obtained in step 7).
The invention also provides the application of the efficient cloning and screening expression vector in transfecting mammalian cells and screening and expressing target protein clones.
The invention constructs an efficient cloning and screening expression vector by using an ITRs sequence of a piggyBac transposition system, clones a transcription enzyme gene which is optimized by a codon and can be efficiently expressed in a mouse to another eukaryotic expression vector, and integrates a screening marker and a target gene into a genome of a host cell together through the co-expression of the expression vector containing the ITRs sequence and the vector containing the transcription enzyme, thereby greatly improving the transfection efficiency and the screening rate of positive cloning, providing possibility for constructing a stable transfer cell line co-expressed by a plurality of subunits and providing favorable conditions for researching the functions of exogenous genes in mammalian cells.
Drawings
FIG. 1 is a schematic diagram of the structure of expression vector pcDNA3.1(+) -ITRs.
FIG. 2 is a schematic diagram of the structure of expression vector pcDNA 4/TO-ITRs.
Detailed Description
For a more detailed understanding of the technical content, features and effects of the present invention, reference is now made to the following detailed description taken in conjunction with the accompanying drawings, in which:
EXAMPLE 1 preparation of pcDNA3.1(+) -ITRs vector
The construction of the pcDNA3.1(+) -ITRs expression vector is shown in FIG. 1, and the ITRs sequence is inserted into the vector in the correct direction of the DNA sequence (see FIG. 1) with pcDNA3.1(+) as the vector to obtain the eukaryotic expression vector pcDNA3.1(+) -ITRs containing the ITRs sequence. The expression vector pcDNA3.1(+) -ITRs can carry the target gene at the mammalian promoter (CMV), Multiple Cloning Site (MCS) and the selection gene (Neomycin) to be integrated into the genome of the host cell when being transposed.
The preparation method comprises the following steps:
(1) finding out specific sequences of 5 'ITR and 3' ITR from a commercial pXL-BacII plasmid vector; a codon-optimized transcription enzyme (Transposase) gene sequence (GeneBank sequence number: EF 587698) which can be efficiently expressed in mice is found in reported literature (nucleic acids research,2007,35, e 87). Wherein,
the 5' ITR sequence (SEQ ID NO: 1) is:
TATAACAAGAAAATATATATATAATAAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCATTTTGACTCACGCGGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGGGAGCTCCAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTAGCTGCATCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGCTGGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGAAGCGGCATGGAAAGAGTTTGC
the 3' ITR sequence (SEQ ID NO: 2) is:
TAAAACGACGGCCAGTGAGCGCGCCTCGTTCATTCACGTTTTTGAACCCGTGGAGGACGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGATGCTCGACACGCTGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATTGACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATCGATATCGTC
(2) pGH is taken as a cloning vector, inverted terminal repetitive sequences 5 'ITR and 3' ITRDNA fragments required by whole-gene synthesis are synthesized, enzyme cutting sites (GATATC) of EcoRV are added to two ends of the fragments, and the vectors pGH-ITR5 and pGH-ITR3 are respectively obtained.
Meanwhile, pGH is used as a cloning vector, a required transcriptive enzyme gene fragment is synthesized by the whole gene, and enzyme cutting sites of EcoRI (5 'GAATTC) and HindIII (3' AAGCTT) are respectively added to two ends of the fragment to obtain the vector pGH-Transposase.
(3) The vector pGH-ITR5 and pGH-ITR3 were cut with EcoRV, EcoRI and HindIII, and pGH-Transposase was cut with EcoRV.
(4) The NruI restriction enzyme digestion expression vector pcDNA3.1(+), and the EcoRI and HindIII restriction enzyme digestion expression vector pTriEx 2.
(5) The fragment ITR5 was ligated to NruI digested expression vector pcDNA3.1(+) to give the new expression vector ITR5-pcDNA3.1 (+).
(6) Meanwhile, the fragment Transposase was ligated to EcoRI and HindIII digested expression vector pTriEx2 to obtain a novel expression vector pTriEx 2-Transposase.
(7) Bstz17I digested expression vector ITR5-pcDNA3.1 (+).
(8) The fragment ITR3 was ligated to Bstz17I digested expression vector ITR5-pcDNA3.1(+), to obtain the new vector ITR5-pcDNA3.1(+) -ITR 3.
The sequence of the vector ITR5-pcDNA3.1(+) -ITR3 (SEQ ID NO: 3) is:
GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGATCTATAACAAGAAAATATATATATAAT AAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTAAAAGTCACGTAAAAGATAAT CATGCGTCATTTTGACTCACGCGGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGGGA GCTCCAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGA ATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTAGCTGCATCAGGATCATATCGTCGGGTCTTTTTTC CGGCTCAGTCATCGCCCAAGCTGGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGAAGC GGCATGGAAAGAGTTTGCGATCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTAATCGATAAAAGTTTTGTTACTTTATAGAAGAAATTTTGAGTTTTTGTTTTTTTTTAATAAATAAATAAACA TAAATAAATTGTTTGTTGAATTTATTATTAGTATGTAAGTGTAAATATAATAAAACTTAATATCTATTCAAATTAATAAA TAAACCTCGATATACAGACCGATAAAACACATGCGTCAATTTTACGCATGATTATCTTTAACGTACGTCACAATATGATT ATCTTTCTAGGGTTAATCTAGCTGCGTGTTCTGCAGCGTGTCGAGCATCTTCATCTGCTCCATCACGCTGTAAAACACAT TTGCACCGCGAGTCTGCCCGTCCTCCACGGGTTCAAAAACGTGAATGAACGAGGCGCGCTCACTGGCCGTCGTTTTAGATTACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC
EXAMPLE 2 preparation of pcDNA4/TO-ITRs vector
The construction method of pcDNA4/TO-ITRs vector is shown in FIG. 2, and the ITRs sequence is inserted into the vector with pcDNA4/TO as the vector according TO the correct direction of DNA sequence (see FIG. 2), so as TO obtain eukaryotic expression vector pcDNA4/TO-ITRs containing ITRs sequence. The expression vector pcDNA4/TO-ITRs, when transposed, can carry the mammalian promoter (CMV), the gene of interest at the Multiple Cloning Site (MCS), and the selection gene (Zeocin) together into the genome of the host cell.
The preparation method comprises the following steps:
(1) examples (3) and (3) are the same as example 1.
(4) The expression vector pcDNA4/TO was digested with NruI, while the expression vector pTriEx2 was digested with EcoRI and HindIII.
(5) The fragment ITR5 was ligated TO NruI digested expression vector pcDNA4/TO TO obtain the new expression vector ITR5-pcDNA 4/TO.
(6) Meanwhile, the fragment Transposase was ligated to EcoRI and HindIII digested expression vector pTriEx2 to obtain a novel expression vector pTriEx 2-Transposase.
(7) Bstz17I digested the expression vector ITR5-pcDNA 4/TO.
(8) The fragment ITR3 is connected TO an expression vector ITR5-pcDNA4/TO cut by Bstz17I TO obtain a new vector ITR5-pcDNA4/TO-ITR 3.
The sequence of the vector ITR5-pcDNA4/TO-ITR3 (SEQ ID NO: 4) is as follows:
GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGATCTATAACAAGAAAATATATATATAAT AAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTAAAAGTCACGTAAAAGATAAT CATGCGTCATTTTGACTCACGCGGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGGGA GCTCCAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGA ATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTAGCTGCATCAGGATCATATCGTCGGGTCTTTTTTC CGGCTCAGTCATCGCCCAAGCTGGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGAAGC GGCATGGAAAGAGTTTGCGATCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGAACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGATCGTCGACGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTAATCGATAAAAGTTTTGTTACT TTATAGAAGAAATTTTGAGTTTTTGTTTTTTTTTAATAAATAAATAAACATAAATAAATTGTTTGTTGAATTTATTATTA GTATGTAAGTGTAAATATAATAAAACTTAATATCTATTCAAATTAATAAATAAACCTCGATATACAGACCGATAAAACAC ATGCGTCAATTTTACGCATGATTATCTTTAACGTACGTCACAATATGATTATCTTTCTAGGGTTAATCTAGCTGCGTGTT CTGCAGCGTGTCGAGCATCTTCATCTGCTCCATCACGCTGTAAAACACATTTGCACCGCGAGTCTGCCCGTCCTCCACGG GTTCAAAAACGTGAATGAACGAGGCGCGCTCACTGGCCGTCGTTTTAGATTACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC
example 3 application of pcDNA3.1(+) -ITRs vector in Hela cell
1. The experimental method comprises the following steps:
(1) resuscitating Hela (human cervical cancer cell) cells for transfection in the following culture media: RM1640+10% fetal bovine serum +1% Streptococcus mutans (PenStrep).
(2) Hela cells were seeded into six-well plates at 4X 105/well the day before transfection. When the cell coverage rate is 80% -90%, the culture medium is changed to a culture medium without any antibiotics.
(3) Two 1.5ml EP tubes (b) were preparedModel 3810 microcentrifuge tubes) were added the following two combined plasmids:
combination 1: 2 μ g of pcDNA3.1(+) -EGFP
And (3) combination 2: 1.5. mu.g of pcDNA3.1(+) -ITRs-EGFP and 0.5. mu.g of transcriptase
Then, the volume was filled up to 100. mu.l with OPTI-MEM medium.
(4) Carefully add 5 μ l fugenehd transfection reagent to each of the two EP tubes, gently blow the mixture with a tip, and allow the mixture to stand at room temperature for 15 minutes.
(5) The mixed solution was added dropwise to a six-well plate on which cells had been spread, and gently mixed. Oven culture at 37 ℃.
(6) 48 hours after transfection, at 6 × 103Number of wells cells were plated in a black-walled clear 96-well plate,four replicate wells were combined for each, and the remaining cells were replated in equal numbers in 6cm dishes and screened by adding 400. mu.g/ml G418 final concentration.
(7) The next day, 72 hours after transfection, ArrayScan (array scan) was performed, and the signal value of fluorescence was measured.
(8) After 2 days of screening, 6 × 10 days3Number of wells cells were plated in a black-walled clear 96-well plate, four wells each were combined, and the ArrayScan assay was performed, and the remaining cells were plated in 6cm dishes at the same ratio (i.e., 70%) as the respective original cells, and screened by adding 400. mu.g/ml G418 at the final concentration.
(9) After 6 days and 11 days of the screening, the same treatment as in step (8) was carried out, respectively.
2. The experimental results are as follows:
the results are shown in Table 1, and it can be seen from Table 1 that the number of cells in combination 1 gradually decreased with the increase of the number of screening days, and by 11 days after screening, the number of cells reached a minimum of 3.1E +05, which is equivalent to the number of cells in the control group (1.8E + 05); the number of cells in combination 2 showed an increasing trend, with the number of cells at 11 days after screening reaching a maximum of 5.7E +06, 18 times the number of cells in combination 1 and 32 times the number of cells in the control group.
TABLE 1 number of cells at different times after transfection
3. And (4) conclusion:
it can be seen from the experiment that the expression rate of Green Fluorescent Protein (GFP) and the survival rate of cells (as shown in table 1) in cells (combination 2) containing PB Transposition Sequence (ITRs) vectors are significantly higher than those of cells transfected with unmodified vectors, indicating that the vectors can greatly improve the screening rate of positive clones, and meanwhile, the system has also successfully constructed a stable transgenic cell line of a monogenic ion channel protein TRPV3 and a calcium ion channel protein cav1.2 co-expressed by three subunits, and the specific experimental scheme is not repeated here.
Claims (3)
1. The efficient cloning and screening expression vector is characterized in that the vector is obtained by inserting an ITRs sequence of a piggyBac transposition system into a mammalian expression vector, the mammalian expression vector is pcDNA3.1(+) or pcDNA4/TO, the vector obtained by inserting the ITRs sequence into the pcDNA3.1(+) vector has a sequence shown as SEQIDNo:3, and the vector obtained by inserting the ITRs sequence into the pcDNA4/TO vector has a sequence shown as SEQIDNo: 4.
2. A method for preparing the carrier of claim 1, comprising the steps of:
1) taking pGH as a cloning vector, synthesizing 5 'ITR and 3' ITRDNA fragments through a whole gene, and adding enzyme cutting sites of EcoRV at two ends of the fragments to obtain vectors pGH-ITR5 and pGH-ITR 3; the sequence of the 5 'ITR is shown as SEQIDNo:1, and the sequence of the 3' ITR is shown as SEQIDNo: 2;
2) cutting the vector pGH-ITR5 and pGH-ITR3 by EcoRV enzyme;
3) NruI enzyme-digested mammalian expression vector;
4) connecting the fragment ITR5 to a mammal expression vector subjected to NruI enzyme digestion to obtain an expression vector containing an ITR5 fragment;
5) bstz17I enzyme-cutting the expression vector containing the ITR5 fragment obtained in the step 4);
6) the fragment ITR3 was ligated to the expression vector obtained in step 5).
3. The method of claim 2, wherein the mammalian expression vector comprises pcDNA3.1(+) or pcDNA 4/TO.
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