Chimeric vector and preparation method and application thereof

A chimeric vector and expression vector technology, applied in the field of chimeric vector and its preparation, can solve the problems of disrupting the growth law, tumor, stimulating cell growth and the like

Active Publication Date: 2014-11-05
GUANGZHOU QIANYANG BIO-TECH PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The normal KRAS gene can inhibit the growth of tumor cells, but once a mutation occurs, it will continue to stimulate cell growth and disrupt the growth pattern, thus leading to the occurrence of tumors

Method used

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  • Chimeric vector and preparation method and application thereof
  • Chimeric vector and preparation method and application thereof
  • Chimeric vector and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction model of chimeric vector RBD-Vif-C

[0037] It is well known that the occurrence of pancreatic cancer, lung cancer, colon cancer and other tumors is closely related to the mutation of KRAS gene. A single point mutation of KRAS is enough to cause tumors, and the mutation of the 12th amino acid of KRAS accounts for 98% of the single point mutations of KRAS, and among the mutations of the 12th amino acid, G12V and G12D mutations are the most severe. Significantly, accounting for 30% and 51% respectively. We use Vif to induce the degradation mechanism of target protein to achieve specific degradation of mutant KRAS protein, so as to develop new anti-tumor drugs.

[0038] According to literature review, there is a binding domain RBD (KRAS binding domain) at the N-terminus of RAF-1 protein that specifically binds to mutant KRAS, which can specifically bind to mutant KRAS protein in vivo and in vitro, and its effect on GTP -Kras has a 1000-fold differe...

Embodiment 2

[0050] Example 2 Chimeric carrier RBD-Vif-C can degrade KRAS protein and inhibit its downstream phosphorylation signaling pathway

[0051] The mutant KRAS genes KRAS-G12D and KRAS-G12V were fused with the RFP gene and cloned into the pcDNA3.1 vector, so that the expression of KRAS could be reflected by observing the expression of RFP; at the same time, western blot was used to further verify Expression of KRAS.

[0052] (1) Co-transfect KRAS-G12D-RFP and RBD-Vif-C vectors or KRAS-G12V-RFP and RBD-Vif-C vectors in 293t cells in a 6-well plate. After 48 hours of transfection, the expression of RFP was observed; at the same time, the cells were collected and lysed for Western Blot to detect the expression of KRAS

[0053] This experiment illustrates that RBD-Vif-C can degrade mutant KRAS protein.

[0054] (2) KRAS-G12D and RBD-Vif-C plasmids were transfected in 293t cells in a 6-well plate, and the cells were collected and lysed after 48 hours for Western Blot detection o...

Embodiment 3

[0056] Example 3 PTD-RBD-Vif-C protein has good tumor cell killing effect in vitro

[0057] Considering factors such as the low transfection efficiency of the plasmid system in pancreatic cancer, lung cancer and colorectal cancer cells, as well as the subsequent druggability of the protein, we chose to use Escherichia coli to express the corresponding RBD-Vif-C protein and GFP-Vif -C is used as a negative control protein, trying to verify the inhibition of KRAS gene expression by RBD-Vif-C directly through the mode of action of the protein.

[0058] We first refer to the method of relevant literature and synthesize a short peptide PTD that can efficiently transmembrane, the sequence is shown in the figure. Then, we cloned the transmembrane peptide and RBD-Vif-C into the pET-32a prokaryotic expression vector, and expressed the RBD-Vif-C protein through the prokaryotic system. We used a nickel column to purify the PTD-RBD-Vif-C protein with His-tag, and obtained a relativel...

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Abstract

The invention provides a chimeric vector obtained by replacing a Vif protein N end with an Raf protein N end RAS binding domain which can be specifically combined with GTP-Kras. In the study, the RBD-Vif-C vector is designed and constructed, and then cell-level in-vitro experiments and naked mouse tumor model in-vivo experiments prove that the RBD-Vif-C vector has a good tumor cell killing effect, and a novel antitumor technology specifically aiming at a mutant KRAS is provided. The Vif-C vector is successfully applied in degradation of an KRAS protein, and is expected to become a novel gene knockout means. The effect is similar to siRNA, but is superior to siRNA functions. The Vif-C vector can specifically degrade targeted proteins, especially some proteins in some special modification or special states.

Description

technical field [0001] [0002] The present invention relates to protein anticancer drugs, and more specifically relates to a chimeric carrier and its preparation method and application. Background technique [0003] [0004] For a long time, changes in the cell genome that control expression or function in cell growth and differentiation have been considered to be the main cause of tumor induction. Molecular biology studies of tumors aim to identify those genes that are altered in various tumor types and to elucidate the role of these genes in tumorigenesis. Among them, one of the most commonly mutated gene families in human tumors is the RAS gene. [0005] Under normal physiological conditions, when cells are activated by external stimuli such as EGFR and other signaling pathways, wild-type KRAS is temporarily activated after being phosphorylated by active EGFR and other tyrosine kinases, and activated KRAS can activate downstream signaling proteins in this signaling pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/64A61K48/00A61P35/00
Inventor 张辉潘婷
Owner GUANGZHOU QIANYANG BIO-TECH PHARM CO LTD
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