人類多瘤性病毒JC病毒主要結構蛋白VP1可在E. coli系統中表達，並能自行組裝成殼體。利用蔗糖cushion、CsCl密度梯度、10-30%蔗糖梯度離心，可分別純化出virion-like particle和capsid-like particle。利用純化的VP1空殼體發展成新的人類基因治療新載體。將純化的空殼體可利用osmotic shock方式包裝外來DNA，並可運送螢光質體到人類腎臟細胞(293細胞)中表達。結果也發現osmotic shock時病毒殼體處於低張狀態90分鐘有最大DNA包裝量，而在低張之後恢復等張狀態可防止DNase 進入殼體分解DNA。VP1殼體利用osmotic shock只能包裝1 Kbp以下的DNA片段。純化出來的VP1殼體也可利用EDTA移除鈣離子和DTT還原雙硫鍵的方式將殼體瓦解成為次殼體(capsomere)，之後再加入CaCl2使其再組裝回來成為殼體(capsid)，在此過程可包裝螢光質體。並運送到293細胞中表達以再組裝方式只能包裝1 Kbp以下的直線DNA片段。由先前的研究得知JCV VP1殼體在宿主細胞組裝時會包入DNA片段，因此我們將螢光質體直接送入能表達VP1的E. coli中，分析在in vivo狀態下JCV殼體是否可以包裝螢光質體。由PCR及E. coli transformation的結果得知JCV殼體能在E. coli中包裝VP1質體及螢光質體。此外，從含兩個質體的E. coli中純化出來的virion-like particle可感染293細胞並表達螢光蛋白。
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.
