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Antineoplastic dibasic polypeptide and application and preparation method thereof

An anti-tumor and antigen technology, applied in anti-tumor drugs, chemical instruments and methods, medical preparations containing active ingredients, etc., can solve problems such as limiting the application value of antibodies, achieve specific targeting, and prevent adverse prognosis effects

Active Publication Date: 2008-03-12
姜荣锡
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This greatly limits the application value of these antibodies, causing drugs that kill tumor cells to also kill some normal cells

Method used

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  • Antineoplastic dibasic polypeptide and application and preparation method thereof
  • Antineoplastic dibasic polypeptide and application and preparation method thereof
  • Antineoplastic dibasic polypeptide and application and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of plasmid expressing anti-tumor binary polypeptide and preparation of recombinant anti-tumor polypeptide

[0029] The original plasmid is the pET22b plasmid (7.6kb in size of the plasmid, provided by Harvard Univ.Dr.J.Collier) loaded with the wild-type Bacillus anthracis protein antigen gene SEQ ID NO.1, through the double-stranded oligonucleotide point mutation technique (QuickChange TM Kit, Strategene Company) after inserting the gene-CACCACCACCACCACCAC- of six histidines at the amino-terminus, the genes SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.8 encoding the mutant Bacillus anthracis protein antigen and the wild-type The Bacillus anthracis protein antigen gene is operably linked to obtain the nucleotide sequences SEQ ID NO.6 and SEQ ID NO.10 expressing the recombinant Bacillus anthracis protein antigen gene. After inserting the genes SEQ ID NO.12 and SEQ ID NO.18 encoding antibody mimics into the carboxy-terminal G734 site of the recombinant Bacil...

Embodiment 2

[0099] Example 2: Comparison of the toxic effects of the anti-tumor binary polypeptide of the present invention and the wild-type Bacillus anthracis protein antigen on mice

[0100] The tested mice were BALB / c immunodeficient nude mice inoculated with human non-small cell lung cancer cells (ATCC CCL-185) in the left armpit, and were divided into 5 experimental groups, each with 5 mice.

[0101] Experimental group 1: intraperitoneal injection of wild-type Bacillus anthracis protein antigen / wild-type necrosis factor group, the injection volume was 2 μg / 2 μg / day.

[0102] Experimental group 2: intraperitoneal injection of wild-type Bacillus anthracis protein antigen / wild-type necrosis factor group containing lung cancer antibody simulant, the injection amount was 2 μg / 2 μg / day.

[0103] Experimental group 3: intraperitoneal injection of mutant N682SD683K Bacillus anthracis protein antigen / wild-type necrosis factor group containing lung cancer antibody mimics, and the injection vo...

Embodiment 3

[0108] Example 3: Comparison of the anti-tumor binary polypeptide of the present invention and the wild-type Bacillus anthracis protein antigen on the killing effect of human non-small cell lung cancer cells in vitro

[0109] Human non-small cell lung cancer cells: American ATCC standard cell line CCL-185.

[0110] Cell culture: Take out 0.1ml of the revived cultured CCL-185 cell suspension, slowly add it into a petri dish containing 3ml of 1640 liquid medium (plus 10% serum) (the dilution ratio is 1:30), mix well, put in 37 ℃CO 2 cultured in an incubator. The test cells were divided into 3 groups. The first group was the blank group, which was added with recombinant anti-tumor polypeptide blank preservation solution (10mMPB+0.2M NaCI phosphate buffer (pH 8.0)). In the second group, 10 μg / ml of wild-type Bacillus anthracis protein antigen and 10 μg / ml of Bacillus anthracis wild-type necrosis factor were added. The third group was adding 10 μg / ml anti-tumor binary polypeptid...

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Abstract

The invention provides a gene, recombinant plasmid and polypeptide for an anti-tumor binary polypeptide. The gene of the recombinant anti-tumor binary polypeptide is obtained by connecting in an operable way the gene of a coding antibody simulator with a recombinant bacillus anthraci protein antigen gene. The recombinant plasmid of the invention is formed by inserting the gene of the coding antibody simulator by double-chain oligomeric nucleotide directed mutagenesis method into the recombinant bacillus anthraci protein antigen gene. The obtained recombinant plasmid is infected into engineering bacillus coli BL-21 to get engineering bacillus coli cell of anti-tumor binary polypeptide; the anti-tumor binary polypeptide can be obtained by expanding the bacillus coli, settling in centrifugal way the bacillus coli body, crushing in altrasonic way, settling and crushing bacillus coli body by hi-speed centrifuging and treating the upper clean solution. The anti-tumor binary polypeptide is of special targeting characteristic, higher efficiency in killing special physical tumor than prior anti-tumor medicine, and will not attack normal cells, and has much lower toxicity and poor-reaction than prior anti-tumor medicine.

Description

technical field [0001] The present invention relates to a gene of anti-tumor binary polypeptide, recombinant plasmid, polypeptide and its application and preparation method. The tumor is human non-small cell lung cancer, small cell lung cancer or ovarian cancer. Background technique [0002] Malignant tumors such as non-small cell lung cancer, small cell lung cancer or ovarian cancer are a huge threat to human health. There are about 7 million patients who die from malignant tumors every year in the world, of which China accounts for one-sixth. Existing anti-tumor drugs play an important role in the treatment of tumors. Although they have achieved certain curative effects on some tumors, they still have defects such as poor selectivity for tumor cells, many and serious immunosuppression and adverse reactions, and drug resistance. [0003] In recent years, great progress has been made in the basic and clinical research of tumors, and one of the most important efforts in the ...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00A61K38/16A61P35/00
Inventor 丘小庆
Owner 姜荣锡
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