Anti-tumor and thrombolytic dual-effect chimeric protein with low immunogenicity, its preparation method and application

A chimeric protein, immunogenic technology, applied in the field of microbiology, can solve the problems of single, tumor complicated by thrombus without specific drugs, etc., and achieve the effect of reducing toxic side effects, low immunogenicity, and small molecular weight

Active Publication Date: 2013-09-18
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 tumor and thrombus are single-function drugs, and there is no specific drug for tumor complicated with thrombus

Method used

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  • Anti-tumor and thrombolytic dual-effect chimeric protein with low immunogenicity, its preparation method and application
  • Anti-tumor and thrombolytic dual-effect chimeric protein with low immunogenicity, its preparation method and application
  • Anti-tumor and thrombolytic dual-effect chimeric protein with low immunogenicity, its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Preparation of low immunogenicity anti-tumor thrombolytic dual-effect chimeric protein ΔSak-ΔSEC2

[0036] (1) With chimeric genes Δsak-Δsec2 Expression plasmid construction

[0037] 1) Δsak PCR amplification, Δsak The gene sequence is shown in SEQ ID NO.1, Δsak by EcoR I / Xho I was connected to pET28a to construct plasmid pET28a- Δsak , With plasmid pET28a- Δsak As a template, by designing specific primers:

[0038] Upstream: 5’-ATTAATCATATGGACGACGACGACAAAAAAGGCGATGACGCGAGT-3’

[0039] Downstream: 5’-CGGGCCGAATTCTTTCTTTTCTATAACAAC-3’

[0040] Amplify using PCR Δsak And make Δsak The upstream of the encoded amino acid has enterokinase recognition as a point, Δsak Restriction enzyme sites on both ends Nde I and Eco RI.

[0041] 2) pET28a- Δsak-linker-Δsec2 The build: Δsec2 The gene sequence is shown in SEQ ID NO.2, Δsec2 by EcoR I / Xho I was connected to pET 28a to construct plasmid pET28a- Δsec2 , Δsak through Nde I and Eco Insert pET28a after RI d...

Embodiment 2

[0082] Example 2 Preparation of anti-tumor thrombolytic dual-effect chimeric protein ΔSEC2-ΔSak

[0083] (1) With chimeric genes Δsec2-Δsak Expression plasmid construction

[0084] 1) Δsec2 PCR amplification, Δsec2 The gene sequence is shown in SEQ ID NO.2, Δsec2 by EcoR I / Xho I was connected to pET28a to construct plasmid pET28a- Δsec2 , With plasmid pET28a- Δsec2 As a template, by designing specific primers:

[0085] Upstream: 5’-TTATCGCATATGGACGACGACGACAAAACTGGTACGATGGGTAATAT-3’

[0086] Downstream: 5’-CGCGAATTCACCACCTGTAACTTTAC-3’

[0087] Amplify using PCR Δsec2 And make Δsec2 The upstream of the encoded amino acid has enterokinase recognition as a point, Δsec2 Restriction enzyme sites on both ends Nde I and Eco RI.

[0088] 2) pET28a- Δsec2-linker-Δsak The build, Δsak The gene sequence is shown in SEQ ID NO.1 , Δsak by EcoR I / Xho I was connected to pET28a to construct plasmid pET28a- Δsak , Δsec2 through Nde I and Eco Insert pET28a after RI double digestion...

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Abstract

The invention relates to an anti-tumor and thrombolytic dual-effect chimeric protein with low immunogenicity, its preparation method and application. The amino acid sequence of the chimeric protein is as shown in SEQ ID NO.11 or SEQ ID NO.12. The chimeric protein of the invention makes staphylokinase delta Sak with 10 amino acids deleted on the N end and staphylococcal enterotoxins delta SEC2 with 10 and 132 amino acids deleted respectively on the N and the C end jogged together, and keeps no connecting peptide between the two. The constructed chimeric proteins delta SEC2-delta Sak and delta Sak-delta SEC2 have substantially reduced molecular weights, and amino acid sequences with high immunogenicity are removed, so that the problems of large molecular weight and high immunogenicity of chimeric proteins are solved. Meanwhile, the anti-tumor and thrombolytic functions are retained.

Description

technical field [0001] The invention relates to low immunogenic 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 existence of cancer cells in thrombus in 1878, and further proposed that canceration can cause coagulation disorders...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/70A61K38/45A61K38/16A61P35/00A61P7/02
Inventor 胡风庆崔小进林家帅
Owner LIAONING UNIVERSITY
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