人類多瘤性病毒JC病毒主要結構蛋白VP1基因被選殖到昆蟲細胞桿狀病毒系統表達.此VP1蛋白質在昆蟲細胞內可以形成殼體(capsid).利用殼體密度為1.28 ~ 1.30 g/ml的特性,藉CsCl密度梯度離心可以純化出VP1蛋白質.其密度約為1.29 g/ml,不含DNA,在電子顯微鏡下為emptycapsid的構造.此VP1蛋白質具有結合DNA能力.VP1在昆蟲細胞表達後可被送入核內.
以2D-PAGE分析,共有6 species, 以EDTA及DTT一起處理純化的VP1殼體,會使殼體解離成次殼體( capsomere ).加入二價金屬離子Zn及Cd會使capsomere部份重組回殼體.若加入EDTA於再重組的殼體,不需DTT即可使殼
體再解離成capsomere.由此推測二價金屬離子在殼體組合扮演決定性角色,而且雙硫鍵則在殼體穩定上具有保護二價金屬離子的功能.在pH值方面,殼體在中性,鹼性下較穩定( pH 6.5 ~ 10.5 ). 相對的在酸性下(pH5.3以下),殼體較不易解離成capsomere.此外,以不同溫度測其穩定性得知,殼體在37及42度C較穩定,65及72度C則使殼體迅速解離.
The gene of the major capsid protein VP1 of human polyomavirus JC virus has been cloned into baculovirus and expressed in insect cells. The VP1 protein was able to self-assemble into a capsid structure in insect cells. The VP1 protein was purified by CsCl density gradient centrifugation to near homogeneity.The density of this purified capsid was 1.29 g/ml. Electron microscopy demostratedthat the VP1 protein was expressed in the cytoplasm and then transported into the nucleus. Two-dimensional gel analysis revealed that the VP1 proteins were composedof six distinct species. EDTA and DTT together were able to disrupt the capsid into capsomeres. Zn and Cd couldreassembled capsomeres into capsid. In additions the reassembled capsid could bedisrupted into capsomerew by EDTA alone. These findings indicated that the divalentcations played a major role in assembling the capsid structure and the disulphidebond protected the cations from chelation. Capsid appeared to be more stable inneutral or alkaline conditions (pH 6.5~ 10.5). Futhermore, capsid was found to be more stable at 37C and 42C, but not at above 65C.
