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

A technology of fusion protein and targeting polypeptide, which is applied in the direction of peptide/protein components, antineoplastic drugs, drug combinations, etc., and can solve the problem that TF has no immunogenicity, etc.

Inactive Publication Date: 2012-08-15
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] Example 1 Construction of EG3287-tTF recombinant gene

[0085] 1) According to the amino acid sequence of tumor blood vessel targeting polypeptide EG3287 reported in the literature (refer to the literature: Haiyan Jia, Azadeh Bagherzadeh, Basil Hartzoulakis, Ashley Jarvis, Marianne Lo¨hr, Shaheda Shaikh, Rehan Aqil, Lili Cheng, 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-1 binding 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 principle of codon preference 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 the up...

Embodiment 2

[0106] Example 2 Construction and identification of recombinant expression plasmids

[0107] 1) The PCR product of the EG3287-tTF recombinant gene and the pET32b(+) plasmid were digested with restriction endonuclease Nco I and Xho I respectively, and the digested product was recovered with a conventional enzyme 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) The recombinant plasmid pET32b(+) / EG3287-tTF was transformed into Escherichia coli BL21(DE3), and positive clones were selected and sent 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 correctly sequenced recombinant plasmid pET32b(+) / EG3287-tTF), shake and culture at 37°C overnight, and dilute it into LB medium at 1:100, at 37°C Shake to OD 600 When the value was 0.6-0.8, IPTG with a final concentration of 0.6 mmol / L was added to induce expression for 6 h.

[0112] 2) Purification of fusion protein EG3287-tTF, the purification of fusion protein EG3287-tTF was carried out according to the nickel column protein purification operation manual provided by Amersham PharmaciaBiotech Company. The fusion protein EG3287-tTF mainly existed in the form of inclusion bodies. The inclusion bodies were washed and dissolved, and the supernatant was collected by centrifugation and purified by nickel column. The expression of fusion protein EG3287-tTF accounted for mor...

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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 a preparation method thereof. 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 an vascular growth factor 165 (Vascularendothelial growth factor, VEGF 165 ) co-receptor, VEGF 165 It plays an important role in angiogenesis and can induce the proliferation of blood vessels. This effect is mediated by VEGF receptors 2 (VEGFR-2) and is dependent on the presence of NRP1 diploids, only diploids capable of activating VEGF 165 Efficient binding 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. Interactions ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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