Interfusion protein of antineoplastic genetic engineering composed of target short peptide of receptor of epidermal growth factor and Lidamycin

A fusion protein, lidamycin technology, applied in the direction of antitumor drugs, peptide/protein components, medical preparations containing active ingredients, etc., to achieve the effects of strong penetration, good stability and strong killing effect

Inactive Publication Date: 2007-12-05
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The high-efficiency and miniaturized enhanced fusion protein SG-LDP-AE constructed by the present invention uses lidamycin as the "warhead" drug and the EGFR targeting-specific binding short peptide SG as the "carrier", so far no relevant reports have been seen

Method used

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  • Interfusion protein of antineoplastic genetic engineering composed of target short peptide of receptor of epidermal growth factor and Lidamycin
  • Interfusion protein of antineoplastic genetic engineering composed of target short peptide of receptor of epidermal growth factor and Lidamycin
  • Interfusion protein of antineoplastic genetic engineering composed of target short peptide of receptor of epidermal growth factor and Lidamycin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1. Construction of the expression vector pET30SGLDP containing the short peptide SG / lidamycin prosthetic protein LDP gene for EGFR targeting binding

[0092] L1 upstream 5'GA CAT ATG AAC CCC GTG GTG GGC TAC ATC GGT GAA CGT CCT CAG

[0093] Nde I

[0094] TAT CGT GAC CTG GGT GGA GGC GGT TCA GCG CCC GCC TTC TCC GTC3’

[0095] 5'GTTA downstream of R1 CTC GAG GCC GAA GGT CAG AGC CAC GTG3'

[0096] Xho I

[0097] L2 upstream 5'GAATCCA CAT ATG AAA TAC CTG CTG CCG ACC GCT GCT GCT GGT CTG

[0098] Nde I

[0099] CTG CTC CTC GCT GCC CAG CCG GCG ATG GCC ATG GCC AAC CCC GTG

[0100] GTG GGC TAC3'

[0101] 5'GTTA downstream of R2 CTC GAG GCC GAA GGT CAG AGC CAC GTG3'

[0102] Xho I

[0103] Using the plasmid pIJ1027GRGDS (containing the LDP gene, constructed by our laboratory, which can be provided to the public and issue relevant certificates) as a template, the prime...

Embodiment 2

[0105] Embodiment 2. Fusion protein SG-LDP is in Escherichia coli BL21 (DE3) star TM Induced expression in

[0106] The expression vector used in the present invention is Escherichia coli expression system E.coli BL21(DE3)star TM , for Invitrogen products. Transform Escherichia coli BL21(DE3)star with the constructed recombinant plasmid TM To obtain recombinant transformed bacteria, pick a single clone colony from the LB plate and inoculate it into the LB medium containing 50 μg / ml kanamycin, and cultivate overnight at 37° C. with shaking at 180 rpm. On the next day, replant with 2-3% inoculum, shake culture at 37°C for 2-3 hours to OD 600 About 0.8-1.0, add isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.05mmol / L to the culture, and induce for 8 hours. The 15% SDS-PAGE analysis results (Fig. 3) show that there is an obvious exogenous protein expression band at about 14kDa in the induced Escherichia coli in the culture medium, and the expression am...

Embodiment 3

[0107] Example 3. Affinity chromatography purification of fusion protein SG-LDP and its separation and preparation

[0108] The supernatant of the bacterial cell fermentation broth induced to express was collected, and ammonium sulfate was added at a concentration of 113 g / L at 4° C. under the condition of slow stirring. Stand still at 4°C for 1 hour, centrifuge at 10,000 g for 20 minutes at 4°C, and collect the supernatant. Ammonium sulfate was added to the supernatant at 4°C under slow stirring to make the final concentration 390 g / L. Stand still at 4°C for half an hour, centrifuge at 10,000g for 20 minutes at 4°C, and collect the precipitate. Use 2ml 1×Ni-NTA Binding Buffer (50mMNaH 2 PO 4 , 300mM NaCl, 10mM imidazole, pH8.0) were dissolved and dialyzed with the same solution.

[0109] Ni-NTA His-Bind resin (Novagen) was loaded into the chromatographic column, equilibrated with 1×Ni-NTA Binding Buffer, and the dialyzed sample was filtered through a 0.45um filter membran...

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Abstract

The present invention relates to an intensified gene engineering fusion protein SG-LDP-AE. It is prepared by mainly utilizing two-step technical lines of gene engineering construction of fusion protein and molecular intensification. The described fusion protein has the strong action of killing tumor cells, and can be specifically combined with tumor cell expressing epidermal growth factor receptor, and can be used as a new target medicine for clinically-curing tumor.

Description

Technical field: [0001] The invention relates to a strengthened genetic engineering recombinant fusion protein, its preparation method and its application in anti-tumor targeted drugs. Background technique: [0002] Malignant tumor is a disease that seriously threatens human life and health. Among them, cancers of the digestive tract and respiratory tract have the highest incidence rates in my country, mainly including gastric cancer, liver cancer, lung cancer, esophageal cancer, and colorectal cancer. With the deteriorating human living environment, the incidence of such tumors is increasing year by year. [0003] For the above-mentioned tumors, the current clinical treatment options mainly include surgery, radiotherapy, chemotherapy, interventional radiology, immunology, and guided therapy. The so-called targeted therapy is to use antibodies or compounds with specific affinity for tumors; or to use magnetic materials with physical guidance; or to use lipiodol specifically...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/19C12N15/62A61K38/16A61P35/00
Inventor 陈红霞甄永苏师以康尚伯杨
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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