A human and other mammalian cell attachment expression vector, expression system, preparation method and application
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An expression vector and mammalian technology, applied in the field of genetic engineering and gene therapy, can solve problems such as low expression level, low copy number of transgene expression, and increased gap
Active Publication Date: 2019-05-17
XINXIANG MEDICAL UNIV
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Publication number CN102703503A discloses a human and mammalian episomal expression vector, which is inserted with a synthetic β-interferon characteristic MAR sequence (387bp, specifically composed of human β-interferon MAR sequence 2200bp and human β-bead The 2150bp characteristic elements of the protein MAR sequence are cut and spliced), which can mediate the expression of foreign genes in mammalian cells, and because the foreign genes are attached rather than integrated into the host chromosome, it can also overcome the integration effect of traditional vectors However, the study also found that the transgene expression driven by the expression vector had defects such as low copy number, unstable expression and low expression level, which led to a widening gap between the research itself and clinical application
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Embodiment 1
[0041] The construction of the expression vector pMAR-InE, the specific steps are as follows:
[0042] 1) using human peripheral blood genomic DNA as a template, using primers to perform PCR amplification on E1 / E2 to obtain MAR fragments;
[0046] The amplification reaction system is: 10×PCR buffer 2.5μL, 25μmol / L dNTP 2.0μL, 5U / μL Taq enzyme 0.5μL, 100ng / μL template DNA 1.0μL, 10μmol / L primer E1 / E2 1.0μL each, ddH 2 O 17.0 μL, a total of 25 μL;
[0047] The reaction program of the amplification is: 95°C for 3min, 94°C for 40s, 56°C for 30s, 72°C for 40s, 30 cycles, 72°C for 3min;
[0048] 2) Digest the above MAR fragment and pEGFP-C1 plasmid DNA with KpnⅠ and BamHI enzymes, recover the digested MAR fragment and pEGFP-C1 linear plasmid DNA from the gel, transform E.coli JM109 strain competent cells after ligation, and identify , to o...
Embodiment 2
[0053] The construction of the expression vector pMAR-InD, except that the primer pair D1 / D2 is used for PCR amplification, other operations are basically the same as in Example 1;
[0054] Primer pair D1 / D2 is:
[0055] D1: 5′-ATC GGTACC TACCCCATGGGCTTCCTCCC-3′;
[0056] D2: 5′-TGA GGATCC TCTCAATTTTGCTATGAACC-3'.
Embodiment 3
[0058] The construction of the expression vector pMAR-InC, except that the primer pair C1 / C2 is used for PCR amplification, other operations are basically the same as in Example 1;
[0059] Primer pair C1 / C2 is:
[0060] C1: 5′-ATC GGTACC CATGCCATCATGACTTCAGT-3′;
[0061] C2: 5′-TGA GGATCC TATTTTTGATGACCTTGACA-3'.
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Abstract
The invention discloses a human and other mammalian cell attachment expression vector, expression system, preparation method and application, and belongs to the technical field of genetic engineering and gene therapy. The attachment expression vector is inserted with the segmented β-interferon MAR sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 (or nucleotides more than 95% homologous to the sequence sequence), compared with expression vectors containing full-length MAR sequences or 387bp MAR sequences, this vector can efficiently, continuously and stably express exogenous target genes, especially vectors containing E-segment MAR sequences have the best expression and can be used for gene therapy . In this vector, the segmented MAR sequence is inserted into the multiple cloning site of the vector, that is, downstream of eGFP. On the one hand, it can overcome transgene silencing, and on the other hand, it can improve the expression level of the target protein and the effectiveness of subsequent monoclonal cell line screening.
Description
technical field [0001] The invention relates to a human and other mammalian cell attachment expression vector, and also relates to the expression system, preparation method and application of the attachment expression vector, belonging to the technical field of genetic engineering and gene therapy. Background technique [0002] Gene therapy is to introduce normal and functional genes into target cells to correct or compensate for the defects produced by disease-causing genes, so as to achieve the purpose of treating diseases. Effective gene therapy depends on the efficient and stable expression of foreign genes in recipients, and depends to a large extent on the vector system used. Vectors are divided into viral vectors and plasmid vectors due to different ways of entering host cells. At present, viral vectors with high transfection efficiency are the most used, such as adenovirus and retroviral vectors. However, after the viral vector is transfected into the host cell, it ...
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