Construction Of Recombinant Vectors Streptavidin-beclin1and Prokaryotic Expression And Purification Of Fusion Protein

In generally, Prostate cancer (prostate cancer, PCa) is a urinary system disease inolder men. As living standards improvement and the increase of Chinese aging population,the prevalence of prostate cancer has increased year by year. However, by the currentmedical standards, prostate cancer is basically diagnosed in advanced stage. And thecurrent conventional treatment effects, including surgery, hormone therapy, chemotherapy,radiotherapy and other methods is not ideal. The chemotherapy and radiotherapy are lackof specificity of cell target selection. They can play a role in tumor but also have serioussystemic toxic side effects which greatly limit its clinical application. Therefore, how toeffectively overcome the systemic toxicity of conventional chemotherapy drugs to killprostate cancer cells and to enhance its specificity have become one of research hotspots.The deficiency of autophagy and the ability to enhance the anti-apoptotic may be thereason of tumor cell proliferation of hormone-independent prostate cancer.Autophagy-related genes beclin1are present newly discovered tumor suppressor gene. Peptide NuBCP-9derived orphan receptors can affect apoptosis.Prostate-specificmembrane antigen (PSMA) aptamers can combine specifically with prostate cancer. Thispaper aims to use streptavidin-biotin technology to connect beclin1, PSMA ptamer andpeptide NuBCP-9together to increase targeting treatment for prostate cancer, Trying tofind an effective new treatment way for prostate cancer.1、Construction of streptavidin-beclin1recombinant vectorCore SA and Beclin1gene DNA sequence were retrieved from GenBank. beclin1wasconstructed into the prokaryotic expression vector pQE80L. Previously using in-Fusionprimer software to design SA specific primers including BamHⅠrestriction site. WithpQE60-SA as template, SA was amplified by PCR,381bp PCR products was obtained.SA and pQE80L-beclin1were connected by in-Fusion reagents. The recombinantplasmids was digested by restriction endonuclease and was sequenced. The alignment ofthe result of sequence was the same with designed. The experimental results showed thatthe recombinant plasmid was successfully constructed and named pQE80L-SA-beclin1.2、Prokaryotic expression and purification of fusion protein SA-beclin1The fusion vector SA-beclin1was induced by0.5mmol/L IPTG in E. coli rosettagami prokaryotic expression strain at37℃, the fusion protein was purified by nickelaffinity chromatography and was identified by Western blotting. The result showed thatSA-beclin1fusion protein (with His tag) was highly expressed in E. coli. SDS-PAGEanalysis showed that most of the fusion protein existed in inclusion body. The relativemolecular weight of fusion protein was about72,000identified by Western blotting, whichwas consistent with that of predictionThe peptide NuBCP-9was designed and synthesized andPeptide NuBCP-9it was labeledwith biotin and/or FITC. High pure biotin-labeled peptide NuBCP-9was obtained.These results indicate that the recombinant plasmid was successfully constructed andfusion protein SA-beclin1was expressed and purified, which laid a foundation for furtherresearch and clinical application functions of SA-beclin1.