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
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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
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Application Information
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