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Recombinant immunotoxin and preparation method and application thereof

An immunotoxin and toxicity technology, which is applied in the field of recombinant immunotoxin and its preparation, can solve the problem of multiple varieties, and achieve the effects of enhanced specificity, reduced toxicity, and reduced usage

Pending Publication Date: 2018-10-19
FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many varieties of type I RIP, and the activity of killing cells after internalization is also quite different.

Method used

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  • Recombinant immunotoxin and preparation method and application thereof
  • Recombinant immunotoxin and preparation method and application thereof
  • Recombinant immunotoxin and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Preparation of recombinant immunotoxin 7D12-CUS and determination of its antitumor activity

[0031] 1. Construction of recombinant nanobody 7D12 expression vector pET32a-7D12 According to the literature (Laeremans T, et al. Nanobodies and polypeptides against EGFR and ICF-IR: U.S. Patent 2009 / 0252681A1 [P]. 2009-10-8.) to obtain the nanobody According to the amino acid sequence (Seq.ID no2) of 7D12, a gene sequence (Seq.ID no3) suitable for expression in Escherichia coli containing NdeI / XhoI restriction sites at both ends was synthesized accordingly, amplified by PCR, using NdeI and Recover the gene and the pET32a plasmid after double digestion with XhoI, and connect the two fragments with T4 DNA ligase, and transform the ligated product into E. Positive clones were screened out by XhoI double digestion and PCR, and identified by sequencing. The results showed that the 7D12 gene was amplified ( figure 1 ) and successfully constructed the expression vector ...

Embodiment 2

[0038] Example 2: Preparation of recombinant immunotoxin 7D12-9G8 / CUS and determination of its antitumor activity

[0039] The pumpkin protein gene was linked with the EGFR-targeting bispecific nanobody 7D12-9G8 gene through a genetic engineering method, and the immunotoxin recombinant immunotoxin 7D12-9G8 / CUS was prepared through a prokaryotic expression system for determining its effect on EGFR positive tumor cell killing effect.

[0040] 1. Construction of recombinant bispecific nanobody 7D12-9G8 expression vector According to the literature (Laeremans T, etal.Nanobodies and polypeptides against EGFR and ICF-IR: U.S.Patent2009 / 0252681A1[P].2009-10-8.) to obtain the nanobody The amino acid sequence of 9G8 (Seq.ID no7), based on which a gene sequence suitable for expression in Escherichia coli was synthesized, spliced ​​with the 7D12 gene by the overlap extension PCR method through the Linker sequence, and the two ends of the gene contain NdeI / XhoI restriction sites The sequ...

Embodiment 3

[0047] Example 3: Preparation of recombinant immunotoxin HER2-ScFv-CUS and determination of its antitumor activity

[0048] The pumpkin protein gene was linked with the single-chain antibody gene targeting HER2 through a genetic engineering method, and the immunotoxin HER2-ScFv-CUS was prepared through a prokaryotic expression system, which was used to determine its killing effect on HER2-positive tumor cells. effect.

[0049] 1. Construction of recombinant single-chain antibody HER2-ScFv expression vector pET32a-HER2-ScFv Cloning Herceptin variable region V with Linker connection H and V L The single-chain antibody plasmid pTT21-Her2scFv of the gene was purchased from Hongye Xinchuang Antibody Technology Co., Ltd., and primers containing NdeI / XhoI restriction sites were designed to amplify the Her2-scFv gene by PCR, and NdeI and XhoI double enzymes were used to amplify the Her2-scFv gene. The gene and the pET32a plasmid were cut and recovered respectively, and the two fragm...

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Abstract

The invention relates to a recombinant immunotoxin and a preparation method and application thereof, the recombinant immunotoxin is a fusion protein of a toxic molecule and a carrier molecule, the toxic molecule is pumpkin protein or a pumpkin protein mutant, the recombinant immunotoxin is the fusion protein expressed by connection of the pumpkin protein or a pumpkin protein mutant gene with an antibody or a cytokine gene capable of binding to target cells (including tumor cells, pathogenic cells, and the like), the anti-tumor activity of the recombinant immunotoxin and the anti-tumor activityof the recombinant immunotoxin in treatment of rejection reaction after organ transplant, autoimmune diseases, and possible indications such as infectious diseases caused by pathogens are tested to prove that the recombinant immunotoxin is superior to the toxic molecule and the carrier molecule.

Description

technical field [0001] The invention relates to a recombinant immunotoxin and its preparation method and application. Background technique [0002] Immunotoxin is a hybrid molecule with specific cell killing ability, which is composed of carrier molecules with targeting ability (such as antibodies, cytokines, etc.) and toxic molecules with cytotoxicity (such as plant toxins, bacterial toxins, etc.). Through the guiding effect of the carrier, it makes the toxic molecules carried by it reach the target cells, and then inhibits protein synthesis in the cells to cause cell death, so as to achieve the effect of specifically killing target cells without damaging normal tissues. Immunotoxins can be used to treat malignant tumors, rejection after organ transplantation, autoimmune diseases and viral infections, and have broad application prospects especially in the treatment of malignant tumors. [0003] Toxic molecules of immunotoxins refer to protein toxins, mainly including bacte...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K47/42A61K38/16A61P35/00A61P37/02A61P37/06A61P31/00
CPCA61K38/168A61K47/42A61P31/00A61P35/00A61P37/02A61P37/06C07K14/415C07K16/00C07K2319/33C07K2319/55
Inventor 谢捷明邓翠敏顾晓帆蔡玉梅
Owner FUJIAN MEDICAL UNIV
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