Cationic liposome compound of cancer suppressor gene LKB1 eukaryotic expression plasmid as well as preparation method and anti-tumor effect thereof
A liposome complex and liposome technology, which is applied in the field of genetic engineering to achieve the effects of inhibiting invasion and metastasis, prolonging survival period and improving transfection rate
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0035] Example 1 Construction of human LKB1 gene eukaryotic expression vector
[0036] Primers were designed with the sequence NM000455 of LKB1 in GenBank, and the LKB1 gene fragment of the gene was amplified by nested PCR reaction after reverse transcription from the total RNA of human lung fibroblast MRC-5 cells. Then clone the LKB1 gene fragment into the pVAX vector to obtain the pVAX-LKB1 expression vector. For the construction process, see Figure 10 .
[0037] Upstream primer (SEQ ID No.3): 5'- CCAGCATGGAGGTGTGGAC-3'.
[0038] Downstream primer (SEQ ID No.4): 5'- TCACTGCTGCTTGCAGG'CCGA-3'.
[0039] The parts marked in boldface are EcoRI at the 5' end and the XbaI restriction site at the 3' end respectively, and the parts marked in italics are protected bases. An EcoRI restriction site was added upstream of the LKB1 gene fragment, and an XbaI site was added downstream. The sequence of the amplified human LKB1 gene is SEQ ID No.1.
[0040] 2): Construct the above ...
Embodiment 2
[0043] Embodiment two The preparation of the liposome complex of human LKB1 gene eukaryotic expression vector
[0044] PVAX-LKB1(P-L) and PVAX(P) vector plasmids were extracted and quantified using QIAGEN's endotoxin-free large-scale plasmid extraction kit.
[0045] The present invention has undergone a large number of pre-tests in the early stage to determine that DOTAP ([1-(2,3-dioleoyl)]-N,N,N-trimethylamine propyl methylsulfate, 1,2-Dioleoyl-3-Trimethyllammonium- Liposomes made of Propane, C43H83NO4, molecular weight, 698.55) and cholesterol Chol are more suitable for preparing complexes with PVAX-LKB1, and the ratio of the two is mol / mol=1:0.9~1.1. Preferably the molar ratio of DOTAP and CHOL is 1:1.
[0046] The double-gene cationic liposome complex was obtained as follows.
[0047] 1. Preparation of cationic liposomes:
[0048] Accurately weigh DOTAP 58mg, cholesterol Chol 32mg (mol / mol=1:1), put in a 100ml rotary evaporating bottle, add 20ml of chloroform to dissolv...
experiment example 3
[0056] Experimental example 3. Expression and antitumor activity of human LKB1 gene eukaryotic expression plasmid in vitro
[0057] 1. Expression of LKB1 protein in transfected recombinant plasmid cells
[0058] The recombinant plasmid was transfected into A549 cells, and untransfected A549 cells and cells transfected with empty vector pVAX were used as negative controls. Western blot detection results after 24 hours showed that LKB1 was expressed in A549 cells transfected with PVAX-LKB1, and the relative molecular The mass is about 54kDa, all consistent with the expected size ( image 3 ).
[0059] 2. Detection of anti-tumor activity of LKB1 gene expression plasmid in vitro
[0060] Cells were treated with pVAX-LKB1 plasmid, and untransfected A549 cells and cells transfected with empty vector pVTRO2 were used as negative controls. Cell growth was observed to be inhibited 24 hours after transfection. In order to further explore the type of cell death, we carried out Hoechst3...
PUM
Property | Measurement | Unit |
---|---|---|
molecular weight | aaaaa | aaaaa |
molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com