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Anti-tumor thrombolytic double-effect chimeric protein and its preparation method and application

A chimeric protein, anti-tumor technology, applied in the field of microbiology, can solve problems such as single drugs

Inactive Publication Date: 2011-12-14
LIAONING UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] At present, the drugs for the treatment of tumors and thrombosis are single-function drugs, and there are no reports of drugs with anti-tumor and thrombolytic functions at the same time.

Method used

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  • Anti-tumor thrombolytic double-effect chimeric protein and its preparation method and application
  • Anti-tumor thrombolytic double-effect chimeric protein and its preparation method and application
  • Anti-tumor thrombolytic double-effect chimeric protein and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 Preparation of anti-tumor thrombolytic double-effect chimeric protein SAK-linker-SEC2

[0034] (1) Construction of expression plasmid with chimeric gene sak-linker-sec2

[0035] In order to ensure that the structure and function of SAK and SEC2 will not be affected in the chimeric protein, the present invention uses a flexible linker composed of 9 alanines to connect SAK and SEC2. The chimeric gene was constructed by overlapping extension splicing technology, connected to the expression vector pET28a(+), and transformed into Escherichia coli BL21(DE3).

[0036] 1. Construction of chimeric gene sak-linker-sec2:

[0037] The gene sak (its DNA sequence as shown in SEQ ID NO.6) and the gene sec2 (its DNA sequence as shown in SEQ ID NO.7) encoding SEC2 are respectively cloned to obtain encoding SAK through PCR method, and at the 3' of sak The gene linker (the DNA sequence of which is shown in SEQ ID NO.8) encoding the connecting peptide (linker) was added to the 5'...

Embodiment 2

[0076] Embodiment 2 Preparation of chimeric protein SEC2-linker-SAK

[0077] (1) Construction of expression plasmid with chimeric gene sec2-linker-sak

[0078] 1. Construction of chimeric gene sec2-linker-sak:

[0079] The gene sak (its DNA sequence as shown in SEQ ID NO.10) and the gene sec2 (its DNA sequence as shown in SEQ ID NO.11) encoding SEC2 were respectively cloned by PCR method, and at the 3' of sec2 The gene linker (the DNA sequence of which is shown in SEQ ID NO.8) encoding the connecting peptide (linker) was added to the 5' end of the end and sak respectively, and then the complementary end of the linker was amplified by splicing by overlap extension (SOE-PCR) The chimeric gene sec2-linker-sak (its DNA sequence is shown in SEQ ID NO.12) was obtained by adding. At the same time, in order to connect the chimeric gene sec2-linker-sak to the expression plasmid pET-28a(+) and express it successfully, EcoRI was added to the 5' end of the chimeric gene sec2-linker-sak ...

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Abstract

The invention relates to an anti-tumor thrombolytic double-effect chimeric protein and a preparation method and application thereof. The amino acid sequence of the chimeric protein of the present invention is shown in SEQ ID NO.4 or as shown in SEQ ID NO.5. The preparation method of the chimeric protein is as follows: construct a chimeric gene; after the chimeric gene is digested with enzyme, insert it into the EcoRI / XhoI site of the vector plasmid pET28a(+) to construct a recombinant plasmid; transfer the recombinant plasmid into Escherichia coli BL21 to sense The recombinant Escherichia coli was obtained by culturing the recombinant Escherichia coli in a shake flask, and ultrasonically disrupted, and the collected supernatant was passed through a Ni-NTA affinity chromatography column, followed by gradient elution and concentration and desalting to obtain a chimeric protein. The chimeric protein of the present invention can simultaneously manifest two effects of anti-tumor and anti-thrombosis.

Description

technical field [0001] The invention relates to an anti-tumor thrombolytic double-effect chimeric protein and its preparation method and application, belonging to the fields of microbiology, molecular biology and genetic engineering. Background technique [0002] In 2009, the chairman of ASCO in the United States mentioned in the introduction of tumor progress that the first cause of death from cancer is the cancer itself and cancer recurrence, and the second cause of death is blood clots. The relationship between cancer and thrombus is very close. Paying attention to the prevention and treatment of cancer thrombus has become one of the key points of clinicians' work in the future. [0003] In 1865, French doctor Armand Trousseau reported for the first time that gastric cancer patients were prone to venous thrombosis. Soon, Billroth discovered the presence of cancer cells in thrombus in 1878, and further proposed that canceration can cause coagulation disorders. Clinically,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/085A61P35/00A61P7/02A61K38/45
Inventor 胡风庆戴有金胡静
Owner LIAONING UNIVERSITY
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