Human and mammal cell expression vector and application thereof
An expression vector and mammalian technology, applied in the field of genetic engineering, can solve problems such as unsatisfactory expression levels of transgenes, and achieve the effect of increasing expression and increasing expression levels
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[0022] Example 1 Construction of expression vector pCAD
[0023] 1.1 Construction of expression vector pCATG containing neomycin eukaryotic resistance gene selection complex
[0024] 1.1.1 PCR amplification of neomycin eukaryotic resistance gene selection complex
[0025] According to the sequence of the neomycin eukaryotic resistance gene selection complex (GenBank accession number is EF437957.1), the primers P1 and P2 were designed, and the 5′ ends of the primers were introduced into Sal I restriction sites respectively. The primer sequences are as follows (the underline is the restriction site Point): P1: 5′-GTC GTCGAC CTGTGGAATGTGTGTCAGTTAGGGTGTGGA-3′; P2: 5′-AGC GTCGAC CAGACATGATAAGATACATTGATGAGA-3'. Using the extracted pCDNA3.1 plasmid as a template, primers P1 and P2 were used to amplify the sequence of the neomycin eukaryotic resistance gene selection complex, and the conventional PCR method was used for amplification. The PCR system is shown in Table 1.
[0026] Table 1 P...
Example Embodiment
[0036] Example 2 Construction of expression vectors pCAC1 and pCAC2
[0037] 1.2.1 PCR amplification of DNA fragments of different sizes
[0038] Refer to the enhanced green fluorescent protein (EGFP) gene fragment sequence of pEGFP-C1 vector (GenBank accession number is U55763.1) to design the following primers: P5:5′-CCGGCTAGCGCTACCGGTCGCCA-3′;P6:5′-CGCAGATCTCTGAGTGCGGACTTG-3′;P7 : 5'-AATAGATCTCGGCGCGGGTCTTGTA-3'. P5 and P6 are used to amplify 750 bp fragments, P5 and P7 are used to amplify 350 bp fragments, while introducing Bgl II / Nhe Ⅰ restriction sites, using the extracted pEGFP-C1 plasmid as a template, the PCR reaction system is the same as before, PCR The reaction conditions are as follows: 95°C for 3min; 94°C for 40s, 60-56°C for 30s, 72°C for 40s, 4 cycles per annealing temperature, and finally 55°C for 30 cycles; 72°C for 3min. The amplified products were recovered by agarose gel electrophoresis and sent to the biological company for sequencing verification. The resu...
Example Embodiment
[0041] Example 3 Expression of VEGF gene in Chinese hamster ovary (CHO) cells using the expression vector of the present invention
[0042] (1) Construction of pCAD-VEGF vector containing vascular endothelial growth factor (VEGF)
[0043] The PCR primers P8 and P9 were designed according to the human VEGF gene cDNA sequence (GenBank accession number is AF022375.1). In order to achieve directional cloning, NcoI / XbaI restriction sites were introduced at the 5'end of the primers. The primer sequence is as follows: P8: 5′-GGC CCATGG ATGAACTTTCTGCTGTCTTGGGTG-3′; P9: 5′-GCG AAGCTT TCACCGCCTCGGCTTGTCACATCTG-3'. Take fetal myocardial tissue, extract total RNA by Trizol method, and verify its integrity by 1% formaldehyde denaturation agarose gel electrophoresis. In a 25μl reaction system, add RNA 1μg, dNTP 1μl (10mmol / L), P8 and P9 primers at a concentration of 20μmol / L 1μl each, AMV-Taq DNA polymerase 1μl (5U / μl) and AMV reverse transcriptase 1μl (5U / μl), follow the One Step PCR Kit (...
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