Hepatic targeting peptide and angiogenesis inhibitor fusion protein as well as preparation method and application thereof

An angiogenesis inhibition and fusion protein technology, which is applied in the field of medicine and biology, can solve the problems of increasing drug costs, adverse effects of angiogenesis, and side effects, and achieve the effects of reducing systemic dosage, improving specificity, and treating liver cancer

Active Publication Date: 2013-09-25
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these angiogenesis inhibitors are easy to diffuse and degrade in the body during treatment, resulting in a relatively uniform tissue distribution and failing to form an effective anti-tumor concentration at the lesion site. In order to achieve clinical effects, o

Method used

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  • Hepatic targeting peptide and angiogenesis inhibitor fusion protein as well as preparation method and application thereof
  • Hepatic targeting peptide and angiogenesis inhibitor fusion protein as well as preparation method and application thereof
  • Hepatic targeting peptide and angiogenesis inhibitor fusion protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of Fusion Protein of Liver Targeting Peptide and Endostatin

[0037] For the construction process of liver-targeting peptide and ES fusion gene, see figure 1

[0038] S1. Cloning of the ES gene

[0039] Cell culture and total RNA extraction: Embryonic liver cells L-02 were cultured in 1640 cell culture medium containing double antibodies (100U / ml penicillin, 100ug / ml streptomycin) and 10% fetal bovine serum until the cells were confluent. TIANGEN TRNzol Total RNA Extraction Kit Instructions Operation, every 10cm 2 Add 1ml TRNzol to the area to extract total cellular RNA.

[0040] Detection of RNA: Take 2 μL of RNA sample, add sample buffer, and electrophoresis on 1.0% agarose gel at 80V for 20 min. EB staining, gel imager to detect RNA integrity; use RNase-free ultrapure water to dilute the total RNA product at 1:50, read with a nucleic acid and protein analyzer, and analyze the concentration and purity of the RNA sample. 1.8≤OD 260 / OD ...

Embodiment 2

[0076] Example 2 Preparation of Liver Targeting Peptide and Angiostatin Fusion Protein (AS-CSP)

[0077] Design primers, use the SOE-PCR method to add the CSP I-plus gene sequence at the amino or carboxyl end of the angiostatin gene; when the amino end is added, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 2, and its corresponding nucleotide The sequence is shown in SEQ ID NO: 13; the amino acid sequence of the fusion protein when the carboxyl terminal is added is shown in SEQ ID NO: 3, and the corresponding nucleotide sequence is shown in SEQ ID NO: 14. The linked fusion protein gene was eukaryotically expressed and purified to obtain the fusion protein. The specific method was the same as in Example 1.

Embodiment 3

[0078] Example 3 Preparation of fusion protein of liver targeting peptide and human plasminogen kringles5

[0079] Primers were designed, and the CSP I-plus gene sequence was added to the amino or carboxyl terminus of the human plasminogen kringles5 gene using the SOE-PCR method; when the amino terminus was added, the amino acid sequence of the fusion protein was shown in SEQ ID NO: 4, and its corresponding The nucleotide sequence is shown in SEQ ID NO: 15; the amino acid sequence of the fusion protein when the carboxyl terminal is added is shown in SEQ ID NO: 5, and the corresponding nucleotide sequence is shown in SEQ ID NO: 16. The linked fusion protein gene was eukaryotically expressed and purified to obtain the fusion protein. The specific method was the same as in Example 1.

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Abstract

The invention discloses a hepatic targeting peptide and angiogenesis inhibitor fusion protein as well as a preparation method and an application thereof. According to the invention, 19 amino acids, which can be specifically combined with an acceptor on the hepatocyte surface (namely heparin sulfate proteoglycan), in a circumsporozoite protein (CSP) N-terminal conserved block I (CSPI-plus) are adopted, and are fused on the amino or carboxyl terminal of the angiogenesis inhibitor by a genetic engineering process to prepare the hepatic targeting peptide and angiogenesis inhibitor fusion protein. The fusion protein can specifically target the liver to inhibit the neovascularization, improve the local concentration of focus part, reduce the dosage of a whole body and reduce the toxic and side effects.

Description

technical field [0001] The invention belongs to the technical field of medicine and biology, and more specifically relates to a fusion protein of liver targeting peptide and angiogenesis inhibitory factor, its preparation method and application. Background technique [0002] Primary hepatocellular carcinoma (HCC) is a serious disease with the fifth incidence rate and the second fatality rate among all cancers, and the incidence rate is increasing year by year without effective treatment. Research on innovative drugs and new therapeutic targets is of great significance for improving the current status of liver cancer treatment. The theory that the growth and metastasis of solid tumors depend on angiogenesis has opened up a new way for tumor treatment. At present, targeting tumor angiogenesis, using tumor angiogenesis inhibitors (tumor angiogenesis inhibitors, TAI) to inhibit or destroy tumor angiogenesis, cutting off the source of tumor nutrition and "starving" tumor new can...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70A61K38/18A61K47/48A61P35/00
Inventor 朱家勇马艳金小宝卢雪梅
Owner GUANGDONG PHARMA UNIV
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