Antitumor and thrombolytic double-effect chimeric protein, preparation method thereof and use thereof

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

Inactive Publication Date: 2010-08-18
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 d...

Method used

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  • Antitumor and thrombolytic double-effect chimeric protein, preparation method thereof and use thereof
  • Antitumor and thrombolytic double-effect chimeric protein, preparation method thereof and use thereof
  • Antitumor and thrombolytic double-effect chimeric protein, preparation method thereof and use thereof

Examples

Experimental program
Comparison scheme
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 antitumor and thrombolytic double-effect chimeric protein, a preparation method thereof and the use thereof. The amino acid sequence of the chimeric protein is represented by an amino acid sequence SEQ ID No.4 or an amino acid sequence SEQ ID No.5. The preparation method of the chimeric protein comprises: constructing a chimeric gene; inserting the chimeric gene subjected to enzyme digestion into the EcoRI/XhoI locus of a carrier plasma Pet28a(+) to construct a recombinant plasma; transferring the recombinant plasma into the BL 21 competent cells of Escherichia coli to obtain recombinant Escherichia coli; culturing the recombinant Escherichia coli in a shake flask, disrupting the recombinant Escherichia coli by ultrasonic waves, passing collected supernate through a Ni-NTA affinity chromatographic column, performing gradient elution, condensation and desalination to obtain the chimeric protein. The chimeric protein combines antitumor and antithrombotic effects.

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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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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