FAK and EGFR targeted RNA interference plasmid-lipidosome antineoplastic complex
A technology of liposome complex and RNA interference, which is applied in the direction of DNA / RNA fragments, antineoplastic drugs, liposome delivery, etc., can solve the problems of poor quality of life of patients, large amount of siRNA, not very ideal, etc., to achieve Long-lasting action time in the body, significant curative effect, good effect
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Embodiment 1
[0041] Example 1 Construction of an shRNA expression plasmid targeting FAK and EGFR double genes
[0042] Preliminary screening obtained siRNA sequences with obvious function of inhibiting FAK gene expression:
[0043] 5'-AACCACCTGGGCCAGTATTAT-3'(SEQ ID NO.1)(human FAK: GenBank accession no:NM_153831) and the siRNA sequence that significantly inhibited the expression of EGFR gene: 5'-CACAGTGGAGCGAATTCCT-3'(SEQ ID NO.2)(human EGFR: GenBank accession no: x00588).
[0044] The construction process of the shRNA plasmid expression vector expressing the double-gene RNA interference sequence is as follows:
[0045] 1. Design and synthesize the RNA interference expression framework according to the following structure
[0046] Express frame structure: (Where LOOP refers to the connection sequence).
[0047] (1), RNA interference target sequence of FAK gene (SEQ ID NO.1)
[0048] 5'-AACCACCTGGGCCAGTATTAT-3'
[0049] Synthesize the RNA interference expression framework of the fol...
Embodiment 2
[0078] Embodiment two, the preparation of plasmid DNA / DOTAP-Chol cationic liposome complex
[0079] 1. Preparation of DOTAP-Chol cationic liposomes
[0080] Cationic liposome DOTAP and neutral liposome cholesterol (Chol) were mixed at a molar ratio of 1:1, and the mixture was dissolved with HPLC grade chloroform, and placed on a rotary evaporator at 30°C for 45 minutes. After forming a film, vacuum dry overnight after film formation. The formed film was taken out and dissolved in 5% glucose solution, then shaken for 45 minutes in a water bath at 50°C and 10 minutes in a water bath at 35°C, the mixture was ultrasonically broken at 30°C for 5 minutes at 400w, and the mixture was transferred to a test tube and heated at 50°C for 10min, and finally squeezed through 450nm, 220nm, and 100nm polycarbonate membranes in turn, and finally dissolved the mixture in an appropriate amount of 5% glucose solution to obtain 5mg / ml DOTAP-Chol cationic lipid body suspension.
[0081] 2. Prepa...
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