Eukaryon recombinant plasmid, preparation process and applications thereof
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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
Inactive Publication Date: 2012-08-22
SICHUAN UNIV
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Human laryngeal squamous cell carcinoma is a common malignant tumor, and now some oncogenes (such as survivin, c-myc, Bcl-2, etc.) and tumor suppressor genes (such as p53, DCC, nm23, APC, PTEN, DPC4, etc.) have been discovered , the oncogenes and tumor suppressor genes are closely related to the occurrence and development of human laryngeal squamous cell carcinoma, but so far there are still many oncogenes and tumor suppressor genes that have not yet been discovered
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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;
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Abstract
A eukaryon recombinant plasmid is constructed by a eukaryotic vector and a CY-FBXL20 segment in the nucleotide sequence of human FBXL20 gene shown in SEQ ID NO.1 in a sequence table, and the CY-FBXL20 segment is a nucleotide sequence from 10045bp to 10063bp in initiator codon downstream. A preparation process of the eukaryon recombinant plasmid comprises the following steps of firstly, obtaining an RNAi segment of the CY-FBXL20 segment in the human FBXL20 gene by means of a ribonucleic acid (RNA) solid phase synthesis method; secondly, inserting the RNAi segment with respective restriction enzyme cutting sites of BamI and BbsI of the CY-FBXL20 segment into the eukaryotic vector to construct the eukaryon recombinant plasmid, transforming the eukaryon recombinant plasmid into competence bacteria for culturing, and screening out clones containing correct inserted segments; and thirdly, extracting the constructed eukaryon recombinant plasmid by means of an alkaline lysis method, analyzing the constructed eukaryon recombinant plasmid by means of a restriction enzyme restriction enzyme map, and performing deoxyribonucleic acid (DNA) sequence analysis. The eukaryon recombinant plasmid can be applicable to the preparation of drugs for curing laryngeal squamous cell carcinoma.
Description
technical field [0001] The invention belongs to the field of genetic engineering, and particularly relates to a eukaryotic recombination plasmid, a preparation method thereof and an application in preparation of medicine for treating laryngeal squamous cell carcinoma. Background technique [0002] In recent years, the threat of malignant tumors to humans has become increasingly prominent, and tumors have become one of the common causes of death, seriously affecting human health. Human laryngeal squamous cell carcinoma is a common malignant tumor, and now some oncogenes (such as survivin, c-myc, Bcl-2, etc.) and tumor suppressor genes (such as p53, DCC, nm23, APC, PTEN, DPC4, etc.) have been discovered , the oncogenes and tumor suppressor genes are closely related to the occurrence and development of human laryngeal squamous cell carcinoma, but there are still many oncogenes and tumor suppressor genes that have not been discovered so far. [0003] The human FBXL20 gene has b...
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