Anti-tumor fusion protein as well as encoding gene and application thereof

A technology of fusion protein and coding gene, which is applied in the direction of antineoplastic drugs, peptide/protein components, hybrid peptides, etc., can solve the problems of difficult acquisition and purification of the main product, small molecular weight, and no binding ability of tumor cells, so as to improve the anticancer Tumor curative effect, better anti-tumor effect, good anti-tumor effect

Inactive Publication Date: 2014-11-19
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in chemical synthesis, there are too many by-products, the main product is not easy to obtain and purify, and it is difficult to produce in large quantities
In addition, it is known that the molecular weight of the Tuftsin molecule is too small to bind tumor cells, and it has certain toxicity when used in large quantities in animal experiments.

Method used

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  • Anti-tumor fusion protein as well as encoding gene and application thereof
  • Anti-tumor fusion protein as well as encoding gene and application thereof
  • Anti-tumor fusion protein as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Construction of recombinant expression vector pET30-ET-tuftsin

[0054] The recombinant plasmid pEL used in this example contains the EGFR oligopeptide ligand Ec gene and LDP gene. For the construction method and its map, please refer to Guo Xiaofang, "Construction of Oligopeptide Targeting Epidermal Growth Factor Receptor and Lidamycin Enhanced Fusion Protein and its antitumor activity", "Cancer", 2009, 28(6):561-568). Escherichia coli competent DH5α was a product of Beijing Quanshijin Biotechnology Co., Ltd., pET30a(+) was a product of Novagen (69909-3), and PCR primers were synthesized by Invigorate Shanghai Company.

[0055] Three primers are required to construct the recombinant vector pET30-ET-tuftsin:

[0056] E1 (SEQ ID NO:8): 5'-gc cat atg aaa tac ctg ctg ccg acc-3' (NdeI restriction site is underlined)

[0057] P2 (SEQ ID NO:9): 5'-gcc gaa ggt cag agc cac gtg-3'

[0058] T2 (SEQ ID NO: 10): 5'-gc ctc gag acg cgg ctt ggt tga acc gcc tcc acc...

Embodiment 2

[0069] Example 2 Expression and Purification of Fusion Protein ET-tuftsin in Escherichia coli

[0070] Escherichia coli bacterial strain BL21star that the present invention uses TM (DE3) is a product of Novagen.

[0071] The recombinant expression vector pET30-ET-tuftsin constructed in Example 1 was transformed into Escherichia coli BL21, and a positive single clone was picked and inoculated in LB medium (kanamycin 50 μg / ml), and cultured overnight at 37°C. Sent for sequencing, and selected positive strains with correct sequencing and enzyme digestion results were stored at -80°C.

[0072]The positive single clone was inoculated into 5ml LB medium, cultured overnight at 37°C, when OD=1.0-2.0, added 1mmol / L IPTG, induced for 8h, and the culture supernatant, Periplasmic lumen, cytoplasmic soluble and insoluble fractions were analyzed. The fusion protein ET-tuftsin has a molecular weight of 16.3KD. It was found that ET-tuftsin protein was mainly localized in the periplasmic...

Embodiment 3

[0075] Example 3 Determination of cell binding activity of fusion protein ET-tuftsin in vitro

[0076] experiment one):

[0077] Human skin squamous cell carcinoma cell A431 and mouse macrophage J774A.1 in the logarithmic growth phase were washed twice with pre-cooled PBS, digested with trypsin, washed twice with PBS, and an appropriate amount of cell lysate (Beijing Baosai Biotechnology Company), lysed on ice for 10 minutes. Afterwards, centrifuge at 10,000 rpm for 30 minutes at 4°C, and collect the supernatant.

[0078] The fusion protein ET-tuftsin was quantified with the BCA kit, and 100 μg of the protein was mixed with an appropriate amount of 5× loading buffer, denatured in a boiling water bath for 5 minutes, and stored at -80°C for later use.

[0079] The expression level of EGFR in tumor cells was detected by Western Blot, and the results showed that A431 was a cell line with high expression of EGFR, while J774A.1 could not detect the expression of EGFR ( image 3...

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Abstract

The invention provides an anti-tumor fusion protein. The fusion protein has the following first-grade amino acid sequence structure: EGFR specifically targeted oligopeptide-linking peptide 1-lidamycin apoprotein-linking peptide 2-(tuftsin)n, wherein the EGFR specifically targeted oligopeptide-linking peptide has an amino acid sequence as shown in SEQ ID No.1, the lidamycin apoprotein has an amino acid sequence as shown in SEQ ID No.2, the tuftsin has an amino acid sequence as shown in SEQ ID No.3, and n is equal to 1-3. The fusion protein is a novel protein targeting EGFR and containing LDP and Tuftsin, not only retains the phagocytosis promoting effect of the Tuftsin, but also has targeting property and has a favorable anti-tumor effect in an animal body. The invention also provides an encoding gene of the fusion protein, a strengthened fusion protein assembled with a lidamycin chromophore and application of the anti-tumor fusion protein and the strengthened fusion protein in preparation of medicines.

Description

technical field [0001] The invention belongs to the technical field of medicinal protein. Specifically, the present invention relates to an anti-tumor fusion protein, its coding gene, a drug with the fusion protein as an active ingredient, and its pharmaceutical application. Background technique [0002] Abnormal expression of epidermal growth factor receptor (EGFR) is an important characteristic of tumor cells of various epithelial origins, so EGFR has become an ideal target for tumor-targeted therapy. However, anti-EGFR antibodies have large molecular weight and strong immunogenicity, and it is difficult to penetrate into the interior of solid tumors. Although small molecule tyrosine inhibitors of EGFR have strong tissue diffusion and penetration and low immunogenicity, they are generally compatible with used with cytotoxic drugs. [0003] Tuftsin was first discovered by Najjar et al. at Tufts University in the United States in 1970, and hence its name. It is a naturall...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K38/17A61P35/00
Inventor 刘文娟甄永苏刘秀均李毅张胜华
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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