Recombinant immunotoxin based on pseudomonas exotoxin containing (Arg) 9 peptide segment

An immunotoxin and pseudomonas technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, peptides, etc., to achieve the effect of increasing the scope of application, good practicability, and improving internalization efficiency

Inactive Publication Date: 2005-10-05
BEIJING ABT GENETIC ENG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, only the charge effect is not enough. The same positively charged amino acid residues, peptides composed of histidine, lysine and ornithine, have much lower transmembrane transport efficiency than Tat 48-67 ; On the contrary, the transmembrane transport efficiency of a peptide (R9) composed of 9 L-arginine residues was Tat 48-67 20 times of that; the transmembrane transport efficiency of the peptide (r9) composed of 9 D-arginine residues is Tat 48-67 More than 100 times that of (Wender et al., 2000)

Method used

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  • Recombinant immunotoxin based on pseudomonas exotoxin containing (Arg) 9 peptide segment
  • Recombinant immunotoxin based on pseudomonas exotoxin containing (Arg) 9 peptide segment
  • Recombinant immunotoxin based on pseudomonas exotoxin containing (Arg) 9 peptide segment

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, the construction of expression vector pCIT35:

[0051] PE35KDEL is a truncated and modified form of PE molecule. Its gene fragment is based on the PE38 gene (from the vector pMS8-38f+T, donated by Dr. NIH Brinkmann) as a template, and the following primers are designed: (1) Nde35:5 '-gACATATgTgggAACAACTggAgCAgTgC-3' and

[0052] (2) EcoR: 5'-CAggAATTCATATTCgATTgggCTg-3' amplified and transformed.

[0053] After the nucleotide sequence site encoding amino acid 607 of PE35KDEL, the gene fragment of anti-CEA single-chain antibody was inserted into the gene fragment of the anti-CEA single-chain antibody with the enzyme cutting sites of XhoI and SacI to obtain the gene fragment of PE35 / CEA(Fv)KDEL. pCIT35 is constructed by inserting the PE35 / CEA(Fv) / KDEL gene fragment into the NdeI and EcoRI double restriction sites on the basis of the general expression vector pET21a(+) (Novagen) (Figure 2.A).

Embodiment 2

[0054] Embodiment 2, the construction of expression vector pCIT35a:

[0055] This expression vector will contain (Gly 4 Ser) 2 The primers of the connecting peptide nucleotide sequence were amplified with the pCIT35 plasmid as a template containing (Gly 4 Ser) 2 And the nucleotide fragment containing SacII at the 5' end and XhoI at the 3' end was double digested and inserted into the pCIT35 vector digested with the same restriction endonuclease to construct the expression vector pCIT35g (Fig. 2.B). will contain (Arg) 9 (Gly 4 Ser) 2 The primers of the connecting peptide nucleotide sequence use the pCIT35 plasmid as a template to amplify (Arg) 9 (Gly 4 Ser) 2And the nucleotide fragment containing SacII at the 5' end and XhoI at the 3' end was double digested and inserted into the pCIT35 vector digested with the same restriction endonuclease to construct the expression vector pCIT35a (Fig. 2.C).

[0056] Design and synthesize 4 oligonucleotide fragments,

[0057] SacFo...

Embodiment 3

[0082] Example 3, Expression of anti-CEA immunotoxins containing different linking peptides

[0083] 1. Expression of Immunotoxin

[0084] The expression vector plasmids pCIT35a and pCIT35g were transformed into Escherichia coli E. coli strain BL21(DE3) star (Novagen Company) and a single clone was picked and cultured overnight at 30°C in LB (containing 100 μg / ml Amp) medium. Transplant at 10% and grow to OD at 37°C 600 About 0.6-0.8, add IPTG to the final concentration of 0.4mmol / ml, continue to cultivate for 4 hours, and collect the bacteria by centrifugation. The bacteria were resuspended in ultrasonic buffer, frozen and thawed three times, and centrifuged at 12000 rpm for 30 minutes. The pellet was resuspended in sonication buffer and sonicated in an ice bath. The supernatant and precipitate were collected by centrifugation and analyzed by 12% SDS-PAGE.

[0085] The preparation of polyacrylamide is shown in Table 1 below:

[0086] Stacking gel 5...

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Abstract

This invention belongs to anti-carcinoe mbryonic antigen recombination immuno toxin. It is concretely relates to said antigen immuno toxic with (Arg)9 based on pseudomonic extotoxin. Nucleuotide sequence code-connects ectotoxin and the antigen connecting peptide, contains the expressing carrier of the sequence and the xeno-cell of the sequence and the utilization of the prevention and cure of tumor.

Description

technical field [0001] The invention belongs to a recombinant immunotoxin against carcinoembryonic antigen. Specifically, a peptide containing 9 arginines (Arg) 9 The anti-carcinoembryonic antigen recombinant immunotoxin based on pseudomonas exotoxin, the nucleotide sequence encoding the connecting exotoxin and anti-carcinoembryonic antigen linking peptide, the expression vector containing said sequence and the host containing this expression vector Cells and their application in the preparation of drugs for treating tumors. Background technique [0002] Toxins produced by certain bacteria and plants linked to growth factors, antibodies, or other cell-targeting molecules can be used to selectively kill target cells carrying the corresponding receptors or antigens (Pastan et al., 1986; Vitetta et al., 1987;) . In this class of toxins, pseudomonas exotoxin A (PE) is a monomeric protein with extremely high activity. Immunotoxins based on PE have attractive application prospe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395A61P35/00C07K19/00C12N15/62C12N15/70
Inventor 何丹黄华樑杨慧林晴尹长城张众
Owner BEIJING ABT GENETIC ENG TECH
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