Eukaryon recombinant plasmid, preparation process and applications thereof
A recombinant plasmid and eukaryotic technology, applied in the field of genetic engineering, can solve the problem of undiscovered oncogenes and tumor suppressor genes, and achieve the effect of promoting development and benefiting rehabilitation
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Embodiment 1
[0034] Example 1: Preparation of eukaryotic recombinant plasmid pGPU6 / GFP / Neo / CY-FBXL20
[0035] 1. Synthesis of oligonucleotides
[0036] The loop structure in the shRNA template uses TTCAAGAGA to avoid the formation of a termination signal, and the shRNA transcription termination sequence adopts the T6 structure. CACC is added to the 5' end of the sense strand template, which is complementary to the sticky end formed after digestion with BbsI; GATC is added to the 5' end of the antisense strand template, which is complementary to the sticky end formed after digestion with BamHI; if the first siRNA If a base is not G, add a G after CACC.
[0037] The target sequence is 5-CCAAATGCTTAGCCAATCT-3, the complementary strand is 3-AGATTGGCTAAGCATTTGG-5; the sense strand of the target sequence is 5- CACCG CCAAATGCTTAGCCAATCT TTCAAGAGA AGATTGGCTAAGCATTTGG TTTTTTG -3,
[0038] The target sequence antisense strand is 5- GATCCAAAAAA CCAAATGCTTAGCCAATCT TCTCTTGAA AGATTGGCTAAGCA...
Embodiment 2
[0060] Example 2: Activity determination of eukaryotic recombinant recombinant plasmid pGPU6 / GFP / Neo / CY-FBXL20
[0061] 1. Cell culture
[0062] DMEM high-glucose medium (purchased from Gbico) with a volume concentration of fetal bovine serum of 10% was used at 37° C., 5% CO 2 The laryngeal squamous cell carcinoma Hep2 cell line (purchased from ATCC, USA) was cultured in a cell culture incubator until the culture flask was confluent.
[0063] 2. Transfection of Hep2 cells
[0064] 2.1 Inoculate 1 / 24 laryngeal squamous cell carcinoma Hep2 cells in each well of a 24-well cell culture plate;
[0065] 2. After 224 hours, discard the original medium, and add 0.4ml of serum-free and antibiotic-free DMEM high-glucose medium;
[0066] 2.3A solution preparation: 1μg eukaryotic recombinant plasmid pGPU6 / GFP / Neo / CY-FBXL20+50μl DMEM culture medium;
[0067] Solution B preparation: 2.5 μl Lipo2000 (SIGMA company) + 50 μl DMEM culture solution;
[0068] Preparation of liquid C: After s...
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