Part:BBa_K3981013
pET28a-vgb-pelb-linker-his-linker-her2-linker-ISZ-sTRAIL
This is a hypoxia-inducible promoter plasmid modified by pET28a-his-linker-her2-linker-ISZ-sTRAIL, in order to enable our modified probiotics to further enhance the killing of cancer cells. In the hypoxic microenvironment of cancer cells, probiotics are affected by hypoxic conditions and secrete a fusion protein with her2, which binds to cancer cell receptors to further enhance the killing ability.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 189
Illegal NgoMIV site found at 199 - 1000COMPATIBLE WITH RFC[1000]
Based on the result that Her2-ISZ-sTRAIL protein could efficiently inhibit the growth of breast cancer cells, in order to enhance the anti-tumor effect of Her2-ISZ-sTRAIL protein, E.coli Nissle 1917 (EcN 1917), an intestinal probiotic with higher tumor-targeting ability to proliferate in hypoxic regions of tumors, was utilized as a targeted transport vector to deliver Her2-ISZ-sTRAIL protein to tumor hypoxic regions. Therefore, a new part (BBa_K3981013) of pET28a(+)-Vgb-pelB-linker-His-linker-Her2-linker-ISZ-Strail (Vgb-pelB-Her2-ISZ-sTRAIL for short) was constructed, in which Her2-linker-ISZ-Strail was placed under the hypoxia promoter Vgb and signal peptide pelB was added to increase the secretory expression of Her2-linker-ISZ-Strail protein (Fig. 1).
1. Construction of Vgb-pelB-Her2-ISZ-sTRAIL expression plasmid
The sequence of Vgb-pelB-Her2-ISZ-sTRAIL was synthesized and inserted into ApaI and XhoI sites of pET28a(+) expression vectors by company to obtain the hypoxia expression vector -- pET28a(+)-Vgb-pelB-Her2-ISZ-sTRAIL recombinant plasmid (Fig. 2 and Fig. 3).
2. Construction of an engineered EcN 1917 probiotic strain with Vgb-pelB-Her2-ISZ-sTRAIL
The Vgb-pelB-Her2-ISZ-sTRAIL plasmid was electrotransformed into EcN 1917, and the positive colonies were screened by patching onto kanamycin LB agar plates. An antitumor engineered strain EcN 1917 (Her2-ISZ-sTRAIL) was successfully obtained (Fig. 4-1). Also the control engineered EcN 1917 (28a) was constructed (Fig. 4-2).
3. Her2-ISZ-sTRAIL protein was successfully expressed in tumor-targeting bacteria EcN 1917
EcN(Her2-ISZ-sTRAIL) and EcN(28a) were cultured in LB medium for 24 h. Then, the proteins in LB medium were concentrated, and subjected to Western blot analysis which was used to confirm the secretory expression of Her2-ISZ-sTRAIL by using Anti-6 × His antibody. It was shown in Fig. 5 that Her2-ISZ-sTRAIL could be efficiently expressed under the control of the hypoxia promoter Vgb and successfully secreted in the medium to exert its anti-tumor activity.
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