FGFR single-stranded antibody fusion protein and application thereof in preparing targeting tumor cells medicines

A fusion protein and single-chain antibody technology, applied in the fields of genetic engineering and biopharmaceuticals, can solve the problem of not being able to fundamentally eradicate tumors, and achieve the effects of increasing protein expression and soluble protein content.

Inactive Publication Date: 2013-01-16
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although targeted inhibition of FGFR3-mediated signal transduction can inhibit tumor cell proliferation and tumor tissue growth, it still cannot fundamentally eradicate ...

Method used

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  • FGFR single-stranded antibody fusion protein and application thereof in preparing targeting tumor cells medicines
  • FGFR single-stranded antibody fusion protein and application thereof in preparing targeting tumor cells medicines
  • FGFR single-stranded antibody fusion protein and application thereof in preparing targeting tumor cells medicines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 PCR synthesis of FGFR3 ScFV-basic polypeptide fusion gene

[0042] According to the gene sequence encoding FGFR3 single-chain antibody reported in the literature (Martínez-Torrecuadrada J et al., 2005), Shanghai Jierui Bioengineering Co., Ltd. was entrusted to synthesize the fusion gene of FGFR3ScFV and basic polypeptide. Among them, the FGFR3 single-chain antibody-fish The nucleotide sequences of the protamine fusion gene and the FGFR3 single-chain antibody-polyarginine 9R fusion gene are Seq ID No4 and No. 6, respectively.

Embodiment 2

[0043] Example 2 Construction of pET-20b-Sumo-FGFR3 ScFV-basic polypeptide expression vector

[0044] Refer to the vector construction method described in ZL200810102542.4. In order to fuse the synthetic FGFR3 ScFV-basic polypeptide fusion gene with the nucleotide sequence encoding the Sumo molecular chaperone, the synthetic primer P1 was designed according to the N-terminal of the Sumo sequence, and the primer P2 was designed according to the Sumo C-terminal and the ScFV N-terminal. P2 contained 20bp and ScFV-basic polypeptide gene complementary sequence. Using P1 and P2 as primers (see Table 1 for primer sequences), the Sumo-S sequence was synthesized by PCR.

[0045] PCR reaction system one:

[0046]

[0047] Reaction program: denaturation at 94°C for 5 min; denaturation at 94°C for 1 min; annealing at 55°C for 0.5 min; extension at 72°C for 0.5 min, 30 cycles, and finally extension at 72°C for 10 min. After no non-specific bands were detected by 2% agarose gel electr...

Embodiment 3

[0056] Example 3 Obtaining of FGFR3 ScFV-Basic Polypeptide Fusion Protein

[0057] 1. Induced expression

[0058] The correctly sequenced pET-20b-Sumo-FGFR3 ScFV-basic polypeptide plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, and positive transformants were screened on an ampicillin resistance plate. A single colony of the screened positive recombinant bacteria BL21(DE3) / pET-20b-Sumo-ScFV-basic polypeptide was inoculated into LB medium and cultured with shaking at 37°C for 16h. Then it was transferred to fresh LB medium at a ratio of 5%, and the expression conditions such as temperature, IPTG concentration and induction time were optimized. The results showed that when the OD value was 0.6, the bacteria were induced by 1mM IPTG at 37°C for 3h, ultrasonic (100w, ultrasonic 2s, interval 2s) broke the bacteria, and the soluble expressed target protein could account for more than 90% of the total bacterial target protein.

[0059] 2. Protein Purificat...

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Abstract

The invention provides a single-stranded antibody fusion protein in growth factor receptors of human fibroblast cells. The single-stranded antibody fusion protein is an FGFR single-stranded antibody-alkaline polypeptide fusion protein, wherein the FGFR single-stranded antibody is the single-stranded antibody of FGFR1, 2, 3 or 4; and alkaline polypeptide is a polypeptide which comprises 9 to 30 alkaline amino acids. The invention further provides the application of the FGFR single-stranded antibody-alkaline polypeptide fusion protein in preparing targeting tumor cells medicines. In the application, the fusion protein is used as nucleic acid carriers to carry micro-molecules to disturb RNA (such as siRNA) to attack various tumor cells and tissues (such as leukemia stem cell, bladder cancer cell, breast cancer cell and the like) with high expression of FGFR correspondingly; and by inducing the tumor cells to die, restraining the growth and the division of the tumor cells and the like, the growth, the attack and the transference of the tumor cells are restrained at last.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and biopharmaceuticals, and in particular relates to an FGFR single-chain antibody fusion protein and its application in preparing drugs targeting tumor cells. Background technique [0002] Fibroblast growth factor (FGF) is a class of structurally related proteins encoded by members of the FGF gene family. At present, 23 members have been found, and their central regions all contain a segment with a homology of 30%-70%. amino acid sequence. The members of the large FGF family, as intercellular multifunctional signaling molecules, regulate various physiological functions of organisms. [0003] FGFs mainly exert regulatory functions through FGFR receptors on the cell membrane. FGFR belongs to tyrosine kinase receptors and is a single transmembrane protein, including the extracellular part, transmembrane part and intracellular tyrosine kinase structure combined with FGFs The domain has 3 parts. ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/15C12N1/19C12N1/21A61K48/00A61K47/42A61P35/00
Inventor 肖业臣李校堃
Owner JILIN UNIV
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