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Targeted anti-tumor recombinant protein and preparation method thereof

A recombinant protein and anti-tumor technology, applied in anti-tumor drugs, peptide/protein components, recombinant DNA technology, etc., can solve the problems of low protein renaturation rate, low expression, low activity, etc., to overcome low expression or inclusion The effects of body expression, improved affinity, and increased activity

Active Publication Date: 2011-08-17
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The invention provides a targeted anti-tumor recombinant protein and its preparation method to solve the problems of low protein renaturation rate, low activity after renaturation, and low expression level

Method used

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  • Targeted anti-tumor recombinant protein and preparation method thereof
  • Targeted anti-tumor recombinant protein and preparation method thereof
  • Targeted anti-tumor recombinant protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The following gene was artificially synthesized, its nucleotide sequence is SEQ No.3, and the recombinant targeting toxin hIL6(T23)-PE38KDEL of the following structure was expressed in E. coli cytoplasm by using the multiple cloning sites Nco I and Xhol I on pET28a, recombinantly expressed vector construction see Figure 4 . The recombinant plasmid was transferred into Rosstabule host bacteria, and induced with LB medium, 100uM IPTG, 28°C for 5 hours, and the expression amount accounted for about 14%-20% of the supernatant protein. After ultrafiltration with a molecular weight of 30,000MW and dialysis with 10mM PBS (pH7.4), the amino acid sequence is SEQ No.1.

[0031] U266 cells and 293T cells were used to detect cell killing activity, and U266 had obvious killing activity, while 293T cells had no killing activity.

Embodiment 2

[0033] The following gene was artificially synthesized, its nucleotide sequence is SEQ No.3, using pET22b to secrete and express the recombinant targeting toxin hIL6(T23)-PE38KDEL with the following structure in Escherichia coli in the periplasmic space, see for recombinant expression vector construction Figure 5 . The recombinant plasmid was transferred into the Rosstabule host bacteria, induced with LB medium, 100uM IPTG, 28°C for 5 hours, and the periplasmic protein was extracted by the osmotic shock method, and the expression amount accounted for about 20%-25% of the supernatant protein. After ultrafiltration with a molecular weight of 30,000MW and dialysis with 10mM PBS (pH7.4), the amino acid sequence is SEQ No.1.

[0034] U266 cells and 293T cells were used to detect cell killing activity, and U266 had obvious killing activity, while 293T cells had no killing activity.

Embodiment 3

[0035] Embodiment 3: The following genes are artificially synthesized. The optimized recombinant toxin gene, whose nucleotide sequence is SEQ No.4, is inserted between the pGAPZa expression vector EcoRI and Kpn I. For the construction of the recombinant expression vector, see Figure 6 . The recombinant targeting toxin hIL6(T23)-PE38KDEL secreted and expressed in Pichia pastoris SMD1168 with the following structure was induced by YPD medium at 30°C for 72 hours, and the expression amount accounted for about 16% of the supernatant protein in the medium. After ultrafiltration with a molecular weight of 30,000MW and dialysis with 10mM PBS (pH7.4), the amino acid sequence is SEQ No.2.

[0036] U266 cells and 293T cells were used to detect cell killing activity, and U266 had obvious killing activity, while 293T cells had no killing activity.

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Abstract

The invention relates to a targeted tumor-resistant recombinant protein and a preparation method thereof, and belongs to a targeted tumor-resistant recombinant protein medicament. A truncated cell factor hIL6 is connected to a derivative PE38KDEL of pseudomonas aeruginosa exotoxin A through a joint, and an amino acid mutation derivative of hIL6 serving as a guide vector comprises 1-28 amino acidsof truncated hIL6 amino ends. The invention further relates to application of the recombinant targeted protein to tumor resistance. The targeted tumor-resistant recombinant protein hIL6 (T23)-PE38KDEL is constructed by gene modification and codon optimization, the vacant site resistance between two ligands is reduced, and the activity is improved; and efficient soluble expression is realized in escherichia coli, the bottleneck of low expression of natural genes is broken, and large-scale preparation of the recombinant toxin becomes possible.

Description

technical field [0001] The invention relates to a targeted anti-tumor recombinant protein drug and a preparation method thereof through genetic engineering. Background technique [0002] The most important thing for tumor treatment is to be able to distinguish tumor cells from normal cells relatively accurately and kill tumor cells efficiently and specifically. This ability to accurately distinguish is also one of the hotspots in cancer treatment research, which is the so-called targeted therapy. strategies to kill tumors. The strategy of targeting and killing tumors is based on special markers on the surface of tumor cells or overexpressed specific receptors as targets, so that cytotoxic drugs are relatively concentrated around tumor cells and killed under the guidance of targeted substances. It does not harm normal cells. Using the ability of specific binding between cytokines and receptors, according to the fact that some cytokine receptors are overexpressed on the surf...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N15/81A61K38/16A61K47/48A61P35/00A61K47/64
Inventor 柳增善郭德军任洪林卢士英周玉李岩松
Owner JILIN UNIV
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