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

Inactive Publication Date: 2013-04-17
SICHUAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although some achievements have been made in local tumor therapy by adenovirus-mediated gene, the adenovirus-encoded protein can stimulate the body's immune response, so that the viral vector can be cleared quickly; at the same time, systemic administration of the adenoviral vector may cause fatal allergic reactions. , can only be used for local injection therapy of tumors, and there are obviously some limitations in using this carrier for the treatment of systemically spread tumor diseases such as lung cancer, breast cancer, and colon cancer.

Method used

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  • Cationic liposome compound of cancer suppressor gene LKB1 eukaryotic expression plasmid as well as preparation method and anti-tumor effect thereof
  • Cationic liposome compound of cancer suppressor gene LKB1 eukaryotic expression plasmid as well as preparation method and anti-tumor effect thereof
  • Cationic liposome compound of cancer suppressor gene LKB1 eukaryotic expression plasmid as well as preparation method and anti-tumor effect thereof

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Experimental program
Comparison scheme
Effect test

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...

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Abstract

The invention belongs to the field of gene treatment, provides a new gene therapy product, and specifically relates to a cationic liposome compound of cancer suppressor gene LKB1 eukaryotic expression plasmid as well as a preparation method and an anti-tumor effect thereof. The gene expression carrier contains genes encoding human LKB1 protein and can express the human LKB1 protein in eukaryotic cells. Based on experiments, the cationic liposome compound of cancer suppressor gene LKB1 eukaryotic expression plasmid, disclosed by the invention, has excellent functions of resisting tumor growth and invasion and metastasis so as to provide a new selection for the tumor treatment.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a cationic liposome complex of a tumor suppressor gene LKB1 eukaryotic expression plasmid, a preparation method thereof and an antitumor effect. Background technique [0002] Lung cancer is a common malignant tumor in all countries in the world today, and it is also the malignant tumor with the fastest growing morbidity and mortality worldwide and the greatest threat to human health and life. According to the latest statistics from WHO, it is estimated that there are more than 2 million new lung cancer patients and about 1.8 million deaths worldwide each year [1]. The morbidity and mortality of lung cancer in my country are rapidly rising, and lung cancer has become the first cause of death from malignant tumors in urban population in my country. Governments of various countries have invested a lot of manpower and material resources in order to make breakthroughs i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N15/54C12N9/12A61K48/00A61K9/127A61P35/00
Inventor 朱文魏于全陈俐娟
Owner SICHUAN UNIV
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