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Recombinant human endostatin fusion protein as well as preparation method and application thereof

A technology of endostatin and fusion protein, which is applied in the field of medicine and biology, can solve the problems of easy decomposition of nanobody monomers, lack of organ targeting or targeting, and low stability, so as to inhibit the formation of new vascular endothelial cells , Inhibit tumor cell growth and improve stability

Active Publication Date: 2017-10-10
SUZHOU SIXTH PHARMA PLANT OF JIANGSU WUZHONG PHARMA GROUP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Therefore, the first technical problem to be solved by the present invention is to overcome the defects in the prior art that nanobody monomers are easy to decompose after entering the human body, have low stability during storage, and are easy to degrade with prolonged storage time, thereby providing a A Her2 nanobody dimer that can be stored and transported stably for a long time
[0007] The second technical problem to be solved by the present invention is to overcome the problem that the recombinant human endostatin in the prior art cannot be effectively transported to the lesion to cause diffusion and degradation after entering the human body due to lack of organ targeting or poor targeting, thereby Provide a recombinant human endostatin fusion protein capable of targeting Her2-positive breast cancer lesion sites and effectively inhibiting the proliferation of breast cancer cells, as well as its preparation method and application

Method used

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  • Recombinant human endostatin fusion protein as well as preparation method and application thereof
  • Recombinant human endostatin fusion protein as well as preparation method and application thereof
  • Recombinant human endostatin fusion protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 (N)- Preparation of Her2 Nanobody Dimer-Recombinant Human Endostatin Protein-(C)

[0081] 1. Construction of TOP10-pET28a-Dim-endo(m) recombinant expression vector

[0082] The Her2 nanobody dimer includes two Her2 nanobody monomers, a connecting peptide and a Her2 binding peptide, the Her2 binding peptide sequence is shown in SEQ ID NO.2, and the connecting peptide sequence is shown in SEQ ID NO.4 As shown, the amino acid sequence of the Her2 nanobody dimer is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.3.

[0083] 1) The Her2 nanobody dimer gene was synthesized by Nanjing GenScript Company according to the nucleotide sequence shown in SEQ ID NO.3, using primer 1 (5'-CAAGTCAAACTGGTGGAATCG-3') and primer 2 (5'-CACGTCCATGTAACACGCATAG -3') Add a BamHI restriction site and an EcoRI restriction site to the carboxyl end of the Her2 nanobody dimer gene by PCR, and clone it into the BamHI and EcoRI restriction sites of the pET28a(+) vecto...

Embodiment 2

[0104] Example 2 (N)- Preparation of Recombinant Human Endostatin Protein-Her2 Nanobody Dimer-(C)

[0105] 1. Construction of TOP10-pET28a-endo(m)-Dim recombinant expression vector

[0106] 1) The sequence synthesized in Example 1 is the Her2 nanobody dimer gene shown in SEQ ID NO.3, using primer 3 (5'-CAAGTCAAACTGGTGGAATCG-3') and primer 4 (5'-TAACACGTCCATGTAACACGCATAG-3' ) Add an EcoRI restriction site to the amino-terminus and an XhoI restriction site to the carboxy-terminus by PCR, and clone into the EcoRI and XhoI restriction sites of the pET28a(+) vector to obtain pET-28a-Dim-2 recombinant carrier;

[0107] 2) Design primers Endo-F' and Endo-R', Endo-F'(5'-ATGCGCCGCCGCCGCCGCCGCCGCCGCCGC-3'), Endo-R'(5'-CTTGGAGGCAGTCATGAAGCTG-3'), using pET28a-m-endostatin as DNA Template, using the above two primers to PCR amplify the recombinant human endostatin fusion protein gene, the PCR amplification system is based on the PCR amplification system described in Example 1;

[0108]...

Embodiment 3

[0112] Example 3 (N)- Preparation of Her2 Nanobody Dimer-Connecting Peptide-Recombinant Human Endostatin Protein-(C)

[0113] 1. Construction of TOP10-pET28a-Dim-linker-endo(m) recombinant expression vector

[0114] 1) The sequence synthesized in Example 1 is the Her2 nanobody dimer gene shown in SEQ ID NO.3, using primer 1 and primer 2 to pass PCR at the amino-terminal BamHI restriction site, and at the carboxyl-terminal along the protein coding Direction to add linking peptide sequence and EcoRI restriction site; the amino acid sequence of the linking peptide is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.13; the above-mentioned Her2 nanobody dimer gene is cloned Enter the BamHI and EcoRI restriction sites of the pET28a(+) vector to obtain the pET-28a-Dim-3 recombinant vector;

[0115] 2) Amplify the recombinant human endostatin fusion protein gene with the primers and PCR methods described in Example 1, and connect it to the pET-28a-Dim-3 recomb...

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Abstract

The invention discloses fusion protein composed of Her2 nano antibody dipolymer and recombinant human endostatin as well as a preparation method and application thereof. The recombinant human endostatin fusion protein comprises the Her2 nanobody dipolymer and recombinant human endostatin protein, and the fusion protein is easy to purify, can specifically target to breast cancer cells with high expression of Her2, has good transmembrane effect and can effectively inhibit the growth of tumor cells. The invention discloses the application of the recombinant human endostatin fusion protein in preparing a medicine for detecting or treating breast cancer in a targeting mode clinically, the concentration of the medicine in the location of tumor lesion can be increased, and correspondingly the dose-effect ratio is increased. The invention also discloses the recombinant expression vectors, transgenic cell lines or transgenic recombinant bacteria of the encoding gene of the recombinant human endostatin fusion protein as well as a gene engineering preparation method to be applied in clinical production and preparation of the fusion protein.

Description

technical field [0001] The invention belongs to the field of medical biotechnology, and in particular relates to a recombinant human endostatin fusion protein and its preparation method and application. Background technique [0002] Tumors require a functional vascular network to provide oxygen, nourishment and remove metabolites. In addition to obtaining some blood vessels through fusion with host blood vessels, tumors must also build their own blood vessel system by forming a new blood vessel network, so that they can continue to grow and develop. Solid tumors do not grow more than 1mm without the vasculature to provide oxygen and nutrients 3 . The theory that the growth and metastasis of solid tumors depend on angiogenesis has opened up a new way for tumor treatment. [0003] In 1997, Harvard University O'Reilly et al. found that the culture medium of the mouse hemangioendothelial tumor (EOMA) cell line can inhibit the proliferation of vascular endothelial cells. A ne...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/13C12N15/62C07K19/00G01N33/68G01N33/574A61K38/39A61K47/42A61P35/00
CPCA61K38/00C07K14/78C07K16/32C07K16/46C07K2319/00G01N33/57415G01N33/68
Inventor 徐根兴华子春谷鹏荣志刚朱浩文黄佩华
Owner SUZHOU SIXTH PHARMA PLANT OF JIANGSU WUZHONG PHARMA GROUP
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