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Genet engineering recombinant protein capable of specifically killing tumor cell

A genetic engineering and protein technology, applied in the field of genetically engineered recombinant proteins that can specifically kill tumor cells, can solve the problems of low potency, large side effects, and high biological activity

Inactive Publication Date: 2005-01-19
BEIJING NORTHLAND BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are reports of biological targeting drugs including interleukin-2-diphtheria toxin, epidermal growth factor-diphtheria toxin, LHRH-PE40, specific antibody-linked cytotoxin, etc., but the clinical dosage of this toxoid is relatively large, resulting in relatively large side effects , so up to now, only interleukin-2-diphtheria toxin has been listed in the United States as a biologically targeted drug, while other types of drugs are still in the clinical or preclinical stage. This patent is aimed at biologically targeted drugs with low potency and large dosage A series of biologically oriented fusion proteins have been studied, which have the characteristics of high biological activity and small dosage

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0052] Firstly, the gene fragments of LHRH and PE40 were obtained from the cDNA library by PCR method. Then, PCR and gene recombination were used to mutate the 5 amino acids at the C-terminal part of the fusion protein PE40 into KDEL, and the recombinant gene vector pLHRH-PE40(KDEL) was obtained, and the sequence of the gene fragment LHRH-PE40(KDEL) inserted into the vector was determined. After verifying the correctness of its gene sequence, amplify and save, cut out the target gene fragment from the vector, insert it into the expression vector, and then introduce the recombinant expression vector into the expression host bacteria, express through culture, hydrophobic chromatography and ion exchange layer The target protein LHRH-PE40 (KDEL) was analyzed, and the activity of killing tumor cells of LHRH-PE40 (KDEL) was measured by MTT cell viability assay.

[0053] This sequence is LHRH-PE40(KDEL) (Sequence 1)

[0054] Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Al...

example 2

[0072] From the pLHRH-PE40(KDEL) vector, the 4 amino acids at the C-terminal part of the fusion protein PE40 were mutated into RQEL by PCR method, and the target gene fragment LHRH-PE40(RQEL) was obtained for sequence determination. After verifying the correctness of the gene sequence, amplify and store it, cut out the target gene fragment from the vector, insert it into the expression vector, and then introduce the recombinant expression vector into the expression host bacteria, cultivate and express, and purify to obtain the target protein LHRH-PE40 (RQEL), the activity of killing tumor cells of LHRH-PE40 (RQEL) was determined by MTT cell viability assay.

[0073] This sequence is LHRH-PE40(RQEL) (Sequence 2)

[0074] Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-

[0075] Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln

[0076] -Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg...

example 3

[0092] From the pLHRH-PE40(RQEL) vector, the 4 amino acids at the C-terminal part of the fusion protein PE40 were mutated into KEEL by PCR method, and the target gene fragment LHRH-PE40(KEEL) was obtained for sequence determination. After verifying the correctness of the gene sequence, amplify and store it, cut out the target gene fragment from the vector, insert it into the expression vector, and then introduce the recombinant expression vector into the expression host bacteria, cultivate and express, and purify to obtain the target protein LHRH-PE40 (KEEL), the activity of killing tumor cells of LHRH-PE40 (KEEL) was measured by MTT cell viability assay.

[0093] This sequence is LHRH-PE40(KEEL) (Sequence 3)

[0094] Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-

[0095] Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln

[0096] -Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-L...

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PUM

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Abstract

The invention discloses a genetic engineering recombinant protein capable of specifically killing tumor cell, wherein the genetic engineering reconstruction can substantially enhance the activity of the lethality tumor cells, thus has great medicinal potentiality in treating tumor.

Description

1. Technical field [0001] Cancer is a disease that seriously threatens people's health. According to reports, there are about 10 million new cancer patients in the world every year, and nearly 1 million new cancer patients and about 1.5 million deaths in my country every year. At present, there is no very effective method for cancer treatment. If it is found in the early stage, surgery can be used, but most of them are found in the later stage and can only be treated by radiotherapy or chemotherapy. Radiotherapy generally uses drugs such as doxorubicin, which has great side effects, mainly including various blood phase reductions caused by bone marrow suppression, gastrointestinal reactions, hair loss, liver function damage, and nervous system damage. The side effects of radiotherapy are even greater, which brings great pain to patients. And the effective rate of radiotherapy and chemotherapy is not high, about 30%. [0002] In view of this, it is urgent to find a method wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62
Inventor 陈瑞雪卢娟赵欣欣
Owner BEIJING NORTHLAND BIOTECH
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