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Anti EB virus resulted tumour polypeptide, and its use and preparing method

An Epstein-Barr virus, anti-tumor technology, applied in anti-tumor drugs, anti-viral agents, botanical equipment and methods, etc., can solve the problem of not many specific markers on the cell surface, and achieve the effect of specific targeting

Active Publication Date: 2005-07-20
畿晋庆堂国际生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The search for specific markers on the surface of tumor cells has been carried out for decades, but there are not many specific markers on the surface of cells available, so this difficulty has become an academic / technical bottleneck in the development of specific anti-tumor drugs

Method used

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  • Anti EB virus resulted tumour polypeptide, and its use and preparing method
  • Anti EB virus resulted tumour polypeptide, and its use and preparing method
  • Anti EB virus resulted tumour polypeptide, and its use and preparing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Construction of plasmids expressing anti-tumor polypeptides and preparation of recombinant anti-tumor polypeptides

[0020] The original plasmid is pSELECT loaded with colistin Ia and Immunity protein genes TM -1 commercial plasmid (plasmid size 8.3kb, donated by UCSF Dr.P.Gosh), double-stranded oligonucleotide point mutation technology (QuickChange TM Kit, Strategene Company) inserts the gene (the nucleotide sequence described in SEQ ID NO.2 and SEQ ID NO.4 in the sequence listing) encoding the induced polypeptide into the I626 site of the colicin Ia gene, and has prepared the anti- Two recombinant plasmids pCHCEB1 (such as figure 1 shown), pCHCEB2 (as shown figure 2 shown). The recombinant plasmid was transfected into E. coli TG1 engineering bacteria (AECOM, donated by Dr. K. Jakes) to prepare the polypeptide, and the anti-tumor polypeptide described in SEQ ID NO.7 and SEQ ID NO.9 in the sequence listing was obtained. Wherein, the anti-tumor polypeptide ...

Embodiment 2

[0076] Embodiment 2: Recombinant anti-tumor polypeptide is used for external killing of human malignant lymphosarcoma (Burkitt's lymphoma) caused by Epstein-Barr virus

[0077] Epstein-Barr virus positive and negative cell lines: American ATCC standard cell lines.

[0078] Cell culture: Take out 0.1ml of revived cultured Raji cell suspension, slowly add it into a petri dish containing 3ml 1640 liquid medium (plus 10% serum) (dilution ratio is 1:30), mix well, put in 37°C CO 2cultured in an incubator. A Epstein-Barr virus-positive cell line was used in the experiment: ATCC CCL-86 (the standard human Burkitt's malignant lymphosarcoma cells used in cell culture laboratories all over the world, Raji cells, isolated from a 12-year-old African male child in 1963). The test cells were divided into 4 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 (pH7.4)). The second ...

Embodiment 3

[0080] Example 3: Multiple fluorescent staining observation of the in vitro killing effect of recombinant anti-tumor polypeptide on Burkitt's human malignant lymphosarcoma cells and other tumor cells and normal cells caused by Epstein-Barr virus

[0081] Cell culture conditions are the same as in Example 2. A total of seven cell lines were used in the experiment, Epstein-Barr virus-positive cell lines: ATCC CCL-86 (Raji cells, Burkitt's malignant lymphosarcoma cells); ATCC CRL-2230, 46-year-old male AIDS patient (AIDS) malignant lymphosarcoma cell lines, Epstein-Barr virus and Kaposi sarcoma virus (HHV8) positive; Epstein-Barr virus negative cell lines: ATCC CRL-1648 (CA-46, cells isolated from ascites of American Burkitt's malignant lymphosarcoma patients); ATCCHepG2 human hepatoma cells; SMMC-7721 human liver cancer Cells, 3T3 mouse embryonic fibroblasts; ECV-304 human umbilical vein endothelial cells (the latter three cells are all from the Cell Center of Peking Union Medic...

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Abstract

The present invention provides one kind of gene, recombinant plasmid and polypeptide of EB virus resisting tumor polypeptide. The coding inducing polypeptide gene and coliform group gene are connected via operation to obtain polynucleotide sequence expressing recombinant antitumor polypeptide. The nucleotide sequence of the coding inducing polypeptide is inserted into coliform group gene by means of double strand oligomeric nucleotide point mutation technology to form the said recombinant plasmid. The obtained recombinant plasmid is transfected into engineering colibacillus TG1 to obtain engineering bacillus cell capable of generating recombinant antitumor polypeptide; and through mass proliferation, centrifugal precipitation and other steps, recombinant antitumor polypeptide is obtained. The antitumor polypeptide has specific targeting property, high efficiency of killing solid tumor, no attack to health cell, low toxicity and less negative reaction.

Description

technical field [0001] The invention relates to a gene, recombinant plasmid, polypeptide and application and preparation method of anti-Epstein-Barr virus-induced tumor polypeptide. Background technique [0002] Malignant tumors are a huge threat to human health. There are about 7 million patients who die from malignant tumors every year in the world, among which China accounts for one-sixth. Malignant tumors are the second cause of death in my country. Because its etiology, pathogenesis, and clinical manifestations have not yet been clarified, so the control effect is not ideal. 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 ...

Claims

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Application Information

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IPC IPC(8): A61K38/16A61K38/17A61K47/48A61P35/00C07K14/435C12N15/12C12N15/62C12N15/63C12P21/02
CPCC12N15/62C07K14/245C07K14/32A61K47/48246C07K16/085C07K2318/10C07K2319/00C07K2317/56C07K2319/01C07K2318/20A61K38/00C07K14/705C07K14/34C07K14/24C07K2317/565A61K38/164C07K14/212A61K47/64A61P31/12A61P35/00
Inventor 丘小庆
Owner 畿晋庆堂国际生物技术有限公司
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