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Fusion protein of tumor blood vessel targeted polypeptide and tissue factor and preparation method thereof

A technology for targeting peptides and fusion proteins, which can be used in peptide/protein components, anti-tumor drugs, drug combinations, etc., and can solve the problem that TF is not immunogenic.

Inactive Publication Date: 2011-08-17
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] 5) TF of human sequence is not immunogenic

Method used

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  • Fusion protein of tumor blood vessel targeted polypeptide and tissue factor and preparation method thereof
  • Fusion protein of tumor blood vessel targeted polypeptide and tissue factor and preparation method thereof
  • Fusion protein of tumor blood vessel targeted polypeptide and tissue factor and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1E

[0084] Embodiment 1 Construction of EG3287-tTF recombinant gene

[0085] 1) According to the amino acid sequence of tumor blood vessel targeting polypeptide EG3287 reported in literature (see literature: Haiyan Jia, Azadeh Bagherzadeh, Basil Hartzoulakis, Ashley Jarvis, Marianne Lo¨hr, Shaheda Shaikh, Rehan Aqil, LiliCheng, Michelle Tickner, Diego Esposito, Richard Harris , Paul C. Driscoll, David L. Selwood, and Ian C. Zachary. Characterization of a bicyclic peptide Neuropilin-1(NP-1) antagonist(EG3287) reveals importance of vascular endothelial growth factor exon8 for NP-1binding and role of NP- 1in KDR signaling. Journal of biological chemistry, 2006, 281(19): 13493-13502.), according to the principle of primer design, according to the degeneracy of codons and the codon preference principle of Escherichia coli, without affecting the amino acid sequence Under the premise, the nucleotide sequence of EG3287 was designed.

[0086] 2) Using P1 and P2 as upstream and downstream ...

Embodiment 2

[0106] Construction and Identification of Embodiment 2 Recombinant Expression Plasmid

[0107] 1) Digest the PCR product of the EG3287-tTF recombinant gene and the pET32b(+) plasmid with restriction endonucleases Nco I and Xho I, respectively, and recover the digested products with a conventional digestion purification kit (see figure 2 ).

[0108] 2) The EG3287-tTF gene clone was ligated into the plasmid pET32b(+) with T4 DNA ligase to construct the recombinant plasmid pET32b(+) / EG3287-tTF.

[0109] 3) Transform the recombinant plasmid pET32b(+) / EG3287-tTF into Escherichia coli BL21(DE3), select positive clones and send them to Shanghai Yingjun Company for sequencing.

Embodiment 3

[0110] Example 3 Expression of recombinant expression plasmid pET32b(+) / EG3287-tTF and purification of its expression product

[0111] 1) Pick a single colony of Escherichia coli BL21(DE3), which contains the recombinant plasmid pET32b(+) / EG3287-tTF) with correct sequencing, culture it overnight with shaking at 37°C, dilute it into LB culture medium at 1:100, and keep at 37°C Shake culture to OD 600 When the value is 0.6-0.8, add IPTG with a final concentration of 0.6mmol / L to induce expression for 6h.

[0112] 2) Purification of the fusion protein EG3287-tTF, the purification of the fusion protein EG3287-tTF was carried out referring to the nickel column protein purification operation manual provided by Amersham Pharmacia Biotech. The fusion protein EG3287-tTF mainly existed in the form of inclusion body, the inclusion body was washed and dissolved, and the supernatant was collected by centrifugation and purified by nickel column. The expression of the fusion protein EG3287...

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Abstract

The invention provides the expression and the purification of a recombinant fusion protein, particularly a fusion protein of the tumor blood vessel targeted polypeptide and tissue factor and a preparation method and application thereof. The preparation method of the fusion protein of the tumor blood vessel targeted polypeptide and tissue factor comprises the following steps: designing primers, carrying out PCR (polymerase chain reaction) to amplify an recombinant gene of the fusion protein EG3287-tTF, guiding the recombinant gene of the fusion protein EG3287-tTF into a carrier, guiding a recombinant carrier into a host cell, constructing a recombinant engineering bacteria for expressing the fusion protein EG3287-tTF, fermenting to culture, and separating and purifying fermentation liquor to obtain the fusion protein of the tumor blood vessel targeted polypeptide and tissue factor. The targeted antitumor fusion protein EG3287-tTF is constructed, a novel tTF derivative with the antitumor advantage of the tTF and the targeting effect is obtained, the antitumor effect of the tTF is improved, and a foundation is laid to the development of a novel medicament for the targeted therapy of the tumor blood vessel.

Description

technical field [0001] The present invention relates to the expression and purification of recombinant fusion protein, in particular to the fusion protein of tumor blood vessel targeting polypeptide and tissue factor and its preparation method. Background technique [0002] Neuropilin-1 (NRP1 for short) is a type I transmembrane glycoprotein on the cell surface, with a relative molecular mass of 130KDa, encoded by a gene located on chromosome 10p12. NRP1 is a vascular growth factor 165 (Vascularendothelial growth factor, VEGF 165 ), a co-receptor for VEGF 165 Plays an important role in angiogenesis and can induce the proliferation of blood vessels. This effect is mediated by vascular growth factor receptor-2 (VEGF receptors 2, VEGFR-2) and depends on the presence of NRP1 diploids, only diploids can make VEGF 165 Effectively binds to VEGFR-2 (1, WestDC, Rees CG, Duchesne L, Patey SJ, Terry CJ, Turnbull JE, Delehedde M, Heegaard CW, Allain F, Vanpouille C, Ron D, Fernig DG....

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/36A61K47/48A61P35/00A61K47/42
Inventor 颜江华何杰李响王生育
Owner XIAMEN UNIV
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