A human and other mammalian cell attachment expression vector, expression system, preparation method and application
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
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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;
[0043] The primer pair E1 / E2 is:
[0044] E1: 5′-ATC GGTACC TATCCATAGCTGATTGGTCT-3′;
[0045] E2: 5′-TGA GGATCC CTATCAAGATATTTAAAGAAA-3';
[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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