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Archive > Volume 23 Issue 2 > 2016,23(2):161-167. DOI:10.3872/j.issn.1007-385X.2016.02.002 Prev Next
Prompt Report
Construction of PSMA targeting system with fusion protein of evolutionary conserved district of Beclin1-streptavidin coupling biotin-aptamer A10 and inhibition effect of the targeting system on prostatic cancer cells
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Abstract:
Objective:To construct a fusion protein prokaryotic expression vector of streptavidin (SA) and evolutionary conserved district (ECD) of Beclin1, to prepare PSMA targeting system the fusion protein coupling specific aptamer A10 of biotin-prostate specific membrane antigen (PSMA) and to identify its promotion effect on apoptosis and autophagy activities of PSMA+ prostatic cancer cells. Methods: Fusion SA-ECD gene was obtained by gene synthesis technology and then cloned into vector PUC57; after confirmation of correct synthesis, the SA-ECD gene was directly inserted into the prokaryotic expression vector PET30a; the fusion protein was expressed with inducement of IPTG, purified with Ni affinity gel chromatography column and identified with Western blotting. PSMA specific conjugate of PSMA, namely the targeting system, was prepared by mixing the biotin-A10 and the SA-ECD at the molar ratio of 4∶1; Combination of the biotin-A10 and the SA-ECD was confirmed by electrophoretic mobility shift assay (EMSA). Effects of the targeting system on apoptosis, expression of autophagy related proteins and viability of the prostatic cancer cells were detected by flow cytometry, Western blotting and trypan blue assays, respectively. Results:Double restriction enzyme digestion and gene sequencing assays verified that the recombinant plasmid PET30a-SA-ECD was successfully constructed; the SA-ECD fusion protein was efficiently expressed in E.coli with induction of IPTG and successfully purified by affinity gel chromatography. EMSA confirmed that SA-ECD could effectively bind biotin-A10. The targeting system could promote apoptosis and autophagy of the prostatic cancer cells, and inhibit its viability.Conclusion: The SA-ECD fusion protein was successfully expressed and purified, and the targeting system of biotin-A10 and SA-ECD could kill the PSMA+ prostatic cancer cells.
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Project Supported:
Project supported by the National Natural Science Foundation of China (No.30973463)
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