| Summary: | 碩士 === 中山醫學院 === 生物化學研究所 === 87 === The major capsid protein VP1 of human plyomavirus, JC virus, has been expressed in E. coli and found to self-assembled into a capsid-like structure. The capsid-like particles have been purified by sucrose cushion, CsCl density gradient centrifugation and sucrose gradient centrifugation from E. coli lysate. The purified capsid-like particles were capable of packaging exogeneous DNA by osmotic shock and delivering the DNA into human kidney cells(293 cell line). Osmotic shock for 90 min was the optimum period for DNA packaging. The capsid-like particles could protect exogeneous DNA from DNase I digestion at the biological osmotic condition. One kilobase-pair DNA in length was the maximun for accommodation in the particles. Capsid-like particles could be dissociated into pentameric capsomeres by treating EDTA and DTT. Capsid-like particles could be re-assembled from capsomeres in the presence of calcium ions. The exogeneous DNA was packaged in the re-assembled capsid and delivered into 293 cells. In addition, VP1 protein expressed in E. coli was able to form capsid-like particles and package host DNA and RNA molecules. Based on this finding, a reporter plasmid, pEGFP, was introduced into the E. coli carrying JCV VP1 expressing plasmid, ΔpFJCV1. The capsid-like particles purified from E. coli with dual plasmids were found to package both plasmids as demonstrated by PCR and E. coli transformation. Furthermore, the capsid-like particles packaging reporter plasmid, pEGFP, in vivo were able to deliver the plasmid DNA into human kidney 293 cells for expression. These findings indicate that human JC virus capsid-like particles self-assembled in E. coli were potentially to be developed as a gene transfer vecter for human gene therapy in the future.
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