A recombinant mhcc97-1 hepatoma cell with high expression of mutant egfr and its construction
A technology for MHCC97-L and liver cancer cells, which is applied in the direction of tumor/cancer cells, genetically modified cells, cells modified by introducing foreign genetic material, etc. It can solve the problems of tumor drug resistance and affecting the effect of treatment, etc.
Active Publication Date: 2022-05-31
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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EGFR mutation not only causes tumor resistance to targeted drugs, but also off-targets EGFR-targeted specific drugs, seriously affecting the effect of treatment
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[0021] The full length of the EGFR (I645L) gene obtained will be recovered after double digestion with XbaI and NotI (as shown in Figure 2).
[0023]. After the restriction enzyme digestion, the plasmid and the PCR product are connected and transformed into the DH5α competent state. Single clones were picked and sequenced. get
[0029] Placed at 37°C, forced transfection in a 5% CO2 incubator for 6-8 hours. Then add 9ml containing 10% volume concentration
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The invention discloses a recombinant MHCC97-L liver cancer cell that stably and highly expresses mutant EGFR constructed on the basis of an exogenous recombinant plasmid pCDH-puro-EGFR (I645L). Specifically, after obtaining the full length of the EGFR-I645L gene, constructing the pCDH-puro-EGFR(I645L) plasmid, packaging the virus, and then performing puromycin selection on the virus-infected cells and passage, to establish stable expression of MHCC97-L cells. The establishment of this cell can provide a cell model for drug screening and drug resistance research related to the EGFR pathway of liver cancer tumors, and improve the specificity and sensitivity of the screening; it can also be applied to the comparative study of the differences in the related pathway proteins affected by EGFR.
Description
A recombinant MHCC97-L liver cancer cell with high expression of mutant EGFR and its construction technical field The invention belongs to biotechnology field, be specifically related to a kind of construction of mutant EGFR expression plasmid and mutant Construction of hepatoma cells with high EGFR expression. technical background Liver cancer is one of the malignant tumors that seriously threaten human health. Inherent drug resistance and other characteristics, ranking sixth in cancer incidence and third in mortality. Current global estimated annual new cases About 748,300 people, my country is the country with the highest incidence of liver cancer and the largest number of deaths in the world, and liver cancer patients account for the world's 55%. Recent studies have shown that EGFR is often highly expressed in liver cancer tissue and adjacent liver tissue of patients with primary liver cancer. Activation of EGFR and its downstream signal transduction pathway ...
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IPC IPC(8): C12N5/10C12N15/85C12N15/12C12R1/91
CPCC12N5/0693C07K14/71C12N2510/00
Inventor 刘扬尹雅蕾
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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