Preparation method and application of conjugate of doxorubicin and double-targeting elastin-like polypeptide/anti-EGFR nanometer antibody/iRGD fusion protein

An elastin-like and fusion protein technology, applied in the field of medicine, can solve the problems of cumbersome humanization transformation steps, size-limited antibody penetration, complex preparation process, etc., and achieve the effects of improving precise control of molecular weight, improving water solubility, and simple preparation method.

Active Publication Date: 2018-06-22
NANJING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, its large size limits the penetration of antibodies in tumors and achieves better therapeutic effects
At

Method used

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  • Preparation method and application of conjugate of doxorubicin and double-targeting elastin-like polypeptide/anti-EGFR nanometer antibody/iRGD fusion protein
  • Preparation method and application of conjugate of doxorubicin and double-targeting elastin-like polypeptide/anti-EGFR nanometer antibody/iRGD fusion protein
  • Preparation method and application of conjugate of doxorubicin and double-targeting elastin-like polypeptide/anti-EGFR nanometer antibody/iRGD fusion protein

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Embodiment 1: Construction of elastin-like gene

[0050] The recursive directional linking method is used to link the elastin-like monomer genes synthesized by genes together to construct the gene sequence of the target size. The pUC19 plasmid containing the elastin-like monomer gene was digested with BglI and PflM I and BglI respectively, and purified with PCR purification kits. The single- and double-digested products were ligated at a ratio of 3:7 with T4 ligase overnight at 16°C. Take all the connection solution and heat shock it into TOP10 competent medium, spread it on X-gal ampicillin plate, culture it overnight, carry out blue-white screening, pick the white colonies of positive clones and culture them in 5mL shaking tube at 37℃, 210rpm for 12h, use plasmid extraction The kit extracts the plasmid, and performs nucleic acid electrophoresis verification and DNA sequencing verification. Repeat the single and double digestion steps above until the desired sequence...

Embodiment 2

[0054] Embodiment 2: Construction of pET-25b plasmid containing anti-EGFR-iRGD gene

[0055] The pET-28a plasmid containing the anti-EGFR-iRGD gene was subjected to PCR reaction using the synthetic primer pair P1 and P2 containing Hind III and EcoR I restriction sites, and the anti-EGFR-iRGD gene was amplified. PCR system is 50μL (ddH 2 O 22 μL, template 1 μL, P1 1 μL, P2 1 μL, Extaq enzyme 25 μL). The PCR reaction conditions are: 95°C for 5min, (95°C for 30s; 55°C for 30s; 72°C for 1min)*30 cycles; 72°C for 5min; 4°C for maintenance. The reaction mixture was purified with a PCR purification kit to obtain an anti-EGFR-iRGD gene fragment. The resulting anti-EGFR-iRGD gene and blank pET-25b plasmids were double digested with Hind III and EcoR I enzymes, respectively. Then, they were purified separately with a PCR purification kit, and then ligated overnight at 16° C. under the action of T4 ligase to obtain the pET-25b plasmid containing the anti-EGFR-iRGD gene.

[0056] The ...

Embodiment 3

[0058] Example 3: Construction and amplification of elastin-like anti-EGFR-iRGD recombinant gene

[0059] First, the pET-25b plasmid containing the anti-EGFR-iRGD gene was digested with SfiI, and purified using a PCR purification kit. Next, 5 μL of the elastin-like gene digested with Bgl I and PflM I and 5 μL of the pET-25b plasmid containing the anti-EGFR-iRGD gene digested with Sfi I were ligated overnight at 16°C under the action of T4 ligase. 10 μL of the connection solution was mixed with TOP10 competent cells for heat shock transformation, spread on ampicillin-resistant TB plates, and grown overnight. Pick a single colony of positive clones and place them in a 5 mL ampicillin-resistant TB shaker tube, and culture them at 37°C and 210 rpm for 12 hours. Plasmid extraction was performed using a plasmid extraction kit to obtain the pET-25b plasmid containing the elastin-like anti-EGFR-iRGD recombinant gene.

[0060] The sequence of the pET-25b plasmid containing the elasti...

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Abstract

The invention discloses a double-targeting fusion protein based on elastin-like polypeptide (ELP), i.e., a double-targeting ELP/anti-EGFR nanometer antibody/iRGD fusion protein. The double-targeting fusion protein is prepared by constructing a recombinant plasmid containing ELP, an anti-EGFR nanometer antibody and an iRGD based on gene recombination technology and expressing and purifying the recombinant plasmid in Escherichia coli. The double-targeting fusion protein can be conjugated with the drug doxorubicin so as to form the ELP/anti-EGFR/iRGD double-targeting fusion protein doxorubicin conjugate. The ELP/anti-EGFR/iRGD double-targeting fusion protein doxorubicin conjugate has a hydration particle size of about 5 nm, mainly exists in the state of molecules and presents a good doxorubicin slow-release function and a good tumor cell-targeting function. The invention discloses a preparation method for the ELP/anti-EGFR/iRGD double-targeting fusion protein doxorubicin conjugate.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to the preparation of an acid-sensitive dual-targeting antibody drug conjugate used for tumor treatment. Background technique [0002] In recent years, the number of cancer patients in my country has been increasing. The diversification, malignancy and refractory nature of cancer have put forward high requirements for the development of treatment technology. Chemotherapy is still the most effective cancer treatment at present, but chemotherapy drugs are generally small molecule drugs, and the circulation time in the body is relatively short. In order to obtain a good therapeutic effect, multiple times of chemotherapy are often required, accompanied by relatively large toxic and side effects. Therefore, chemotherapy is more harmful to patients. Among them, doxorubicin (DOX) is a broad-spectrum antitumor drug. It has a strong cytotoxic effect, and the mechanism of action is mainly...

Claims

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Application Information

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IPC IPC(8): A61K47/68A61K31/704C07K19/00C07K1/34C12N15/70C12N15/66A61P35/00
CPCA61K31/704C07K14/00C07K16/2863C07K2319/00C12N15/66C12N15/70
Inventor 蒋锡群陈伟芝
Owner NANJING UNIV
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