A human and mammalian cell attachment expression vector, construction method and application
An expression vector and mammalian technology, applied in the field of genetic engineering and gene therapy, can solve the problems of unstable gene expression, low copy number, low gene expression level, etc., and achieve the effect of stable expression
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Embodiment 1
[0031] A method for constructing expression vectors for human and mammalian cell attachments, comprising:
[0032] 1. Synthesis of MAR sequence
[0033] The characteristic element (AT-rich sequence) of human β-interferon MAR sequence (GenBank accession number: M83137.1) and human β-globin MAR sequence (GenBank accession number: L22754.1) was cut and spliced to obtain the nucleus The characteristic sequence of the matrix binding region (MAR, shown as SEQ ID NO: 1).
[0034] 2. Synthesis of EF-1α promoter sequence
[0035] The EF-1α promoter sequence (shown as SEQ ID NO: 2) was synthesized by General Biosystems (Anhui) Co., Ltd. In order to realize directional cloning, Ase I restriction sites (5′-ATTAAT -3') and Nhe I restriction site (5'-GCTAGC-3').
[0036] 3. Construction of pEGFP-C1-MAR vector
[0037] 1) Enzyme digestion reaction
[0038] Use Kpn Ⅰ and BamH Ⅰ double enzymes to digest the characteristic sequence of the nuclear matrix binding region and the pEGFP-C1 pl...
Embodiment 2
[0091] The construction of the pEME-NGF expression vector containing nerve growth factor (NGF), including:
[0092] 1. PCR amplification of NGF target fragment
[0093] According to the human NGF gene cDNA sequence (GenBank accession number: AF150960.1), PCR primers P7 and P8 (as shown in SEQ ID NO: 9-10) were designed. In order to realize directional cloning, the 5' ends of the primers were respectively introduced into HindⅢ, Kpn Ⅰ Restriction site (see underline), the primer sequence of NGF gene is:
[0094] P7: 5′-GCC AAGCTT ATGTCCATGTTGTTCTACACTCT-3';
[0095] P8: 5′-TTA GGTACC TCAGGCTCTTTCCACAGCCTTCCT-3'.
[0096] Take human peripheral blood, use a DNA extraction kit to extract human peripheral blood genomic DNA as a template, and use a conventional PCR amplification method for amplification. The PCR system is as follows: ddH 2 O 17.0 μL, P7, P8 (10 μmol / L) each 1.0 μL, 10×PCR buffer 2.5 μL, dNTP (25 μmol / L) 2.0 μL, DNA template (100ng / μL) 1.0 template DNA, Taq enz...
Embodiment 3
[0105] Establishment of pEME-NGF vector transfection CHO cell expression system, including:
[0106] CHO cells at 37°C, 5% CO 2 Under the condition, culture in DMEM medium containing 10% inactivated fetal bovine serum, the transfection method is carried out according to the instructions of the liposome 2000 transfection kit (the operation is the same as the above-mentioned test example), and after 24 hours of transfection, use 800 μg / mL The G418 culture medium was pressurized and screened for 2 weeks. After the stable transformed cell colony was formed, it was digested with 0.25% trypsin, and each group of transfected cells was limitedly diluted and monoclonalized. They were divided into 96-well culture plates and continued to culture for 7 days. Afterwards, transfer to a culture bottle to continue culturing, and when the cell density reaches 85% (80%-90% is acceptable), the cells can be collected.
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