Unlock instant, AI-driven research and patent intelligence for your innovation.

New lung-oncocyte-apoptosis inducing fusion protein and application thereof

A fusion protein and tumor cell technology, applied in peptide/protein components, medical preparations containing active ingredients, hybrid peptides, etc., can solve the problems of lung tumor cell apoptosis

Active Publication Date: 2014-04-23
中海峡福建细胞生物科技有限公司
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to overcome the deficiencies of the prior art, the present invention provides a novel fusion protein that induces apoptosis of lung tumor cells, which solves the potential drug resistance risk problem existing in current targeted drugs

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • New lung-oncocyte-apoptosis inducing fusion protein and application thereof
  • New lung-oncocyte-apoptosis inducing fusion protein and application thereof
  • New lung-oncocyte-apoptosis inducing fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Preparation of EGFR (L858R) mutant-specific mouse monoclonal antibody.

[0046] Artificial chemical synthesis of EGFR (L858R) mutant-specific short peptide, the short peptide sequence is TDFGRAKKLLGGGC. The EGFR (L858R) short peptide was covalently cross-linked with the KLH carrier protein using the C amino acid residue at the end of the short peptide (Kit from Thermo Scientific, product number #77605). The preparation method of the hybridoma secreting monoclonal antibody in the present invention is carried out according to Harlow, E & Lane, D: Antibodies: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 1988, unless otherwise specified. The cross-linked product was used to immunize Balb / c mice with Freund's complete adjuvant and Freund's incomplete adjuvant, and the antigen dose for each immunization was 0.5 mg. After three immunizations, when the EGFR (L858R) specific antibody titer in the serum reached 1:10000, the mouse spleen cells were fused w...

Embodiment 2

[0047] Example 2: Preparation of heavy chain and light chain variable regions of humanized EGFR (L858R) mutant mouse monoclonal antibody

[0048]In order to clone the cDNA encoding antibody VH and VL from hybridoma cells (4G8) that secrete specific monoclonal antibodies that recognize human EGFR (L858R) mutants, first follow the method published by Zhou et al. (Zhou, H. et al: Nucleic Acids Res22:888-9, 1994), synthesized a set of universal (universal) oligonucleotide primers for cloning mouse VH and VL cDNA. Total RNA was extracted from 5×106 hybridoma (4G8) cells as a template, and oligo (dT) was used as a primer to catalyze the synthesis of cDNA under the action of ligase. The cDNA synthesis reaction mediated by reverse transcriptase is as follows:

[0049]

[0050] Reaction environment: 30°C, 10 minutes; 50°C, 30 minutes; 95°C, 5 minutes; store at 4°C for later use.

[0051] Further, the cDNA synthesized by the reverse transcription reaction was used as a template, an...

Embodiment 3

[0055] Example 3: Expression and preparation of VH-GGG-Apaf-1 (1-97aa)-9R fusion protein (SBC-426) in Escherichia coli

[0056] According to the VH-GGG-Apaf-1 (1-97aa)-9R amino acid sequence and the codon preference of Escherichia coli, the optimized DNA sequence encoding the fusion protein suitable for expression in Escherichia coli was artificially synthesized. When synthesizing the gene, a KpnI restriction site GGTACC was introduced at its 5' end, and a stop codon TAA and a SalI restriction site GTCGAC were introduced at its 3' end. The following molecular cloning operation methods, unless otherwise specified, refer to the literature: Green.M.R: Molecular cloning, A Laboratory Manual (4th), Cold Spring Harbor Press, 2012. The synthetic DNA sequence was first cloned into pBluescript (+ ) plasmid to hold the synthetic DNA sequence. Further, the gene encoding the fusion protein was excised from pBluescript (+) plasmid with KpnI and SalI and cloned into pET32a (+) expression v...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a new lung-oncocyte-apoptosis inducing fusion protein which is composed of four part sequences: a protein fragment I capable of specific binding with a human EGF receptor (epidermal growth factor receptor) ( (EGFR) (L858R) ) mutation sequence; a human Apaf (antipernicious anemia factor)-1 (1-97) protein fragment II; a protein fragment III of 9R-tag sequence formed by series of nine arginine residues; and a linker sequence comprising three Glycine residues (3G) for connection between the protein fragment I and the protein fragment II. The fusion protein can be expressed in quantity by a genetic engineering method, uby the mediation of 9R-tag, the purified fusion protein can enter lung oncocyte and bind with EGFR dimers or polymers containing (EGFR) (L858R) mutation so as to mediate the formation of secondary Apaf-1 dimers or polymers; the Apaf-1 dimers or polymers can induce Caspase-9 and-3 protease activation so as to activate a Caspase mediated apoptosis cell signal transduction pathway to cause oncocyte apoptosis, and the new lung-oncocyte-apoptosis inducing fusion protein can be used as a drug for the treatment of lung cancer.

Description

technical field [0001] The invention relates to the technical fields of biopharmaceuticals and genetic engineering, in particular to a fusion protein containing human EGFR (L858R) mutant protein sequence binding protein, Apaf-1 (1-97aa) and 9R-tag and an expression vector thereof. Background technique [0002] Lung cancer is the most common and deadly type of lung malignancy. In many countries and regions, the incidence of lung cancer ranks first among malignant tumors. In the past few decades, although the diagnosis and treatment technology of lung cancer has made great progress, the survival rate of lung cancer patients has not improved significantly. The main reasons for this result are, on the one hand, due to the complex pathobiological characteristics of lung cancer and the high rate of severe disease, most patients have advanced or metastasized when they are first diagnosed; on the other hand, there is a lack of effective lung cancer treatment. [0003] At present, ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K19/00A61K38/16A61P35/00
Inventor 吴炯
Owner 中海峡福建细胞生物科技有限公司