利用人類多瘤性病毒,JC病毒,殼體作為基因輸送載體之研究

人類多瘤性病毒，JC病毒，屬乳多瘤病毒科。結構蛋白VP1是主要殼體蛋白，約占病毒總蛋白量75 ％。將JCV VP1基因選殖至大腸桿菌進行表達，其蛋白質表達後會自行組裝成類病毒顆粒。此類病毒顆粒可以滲透壓震盪法（osmotic shock）體外包裝運送外源性核酸，進入哺乳類細胞，表達或是抑制特定蛋白質，以影響細胞生理。此結果顯示JC病毒顆粒有可能可發展為基因輸送載體。我們發展出在大腸桿菌體內，表達組裝病毒殼體的同時，進行外源性的DNA包裝。這種細胞內包裝DNA的表達系統可以對包裝入的DNA有更好的保護性，也相對增加了輸送基因的效率。以此系統所生產的JCV類病毒顆粒經測試，至少可感染腎臟、腦部、肺、卵巢、大腸、骨頭、皮膚及B淋巴球等13種人類的癌細胞株，並可表達轉殖基因之蛋白產物。這些結果暗示著此基因載體可能可輸送治療性基因以治療特定的腫瘤。為增加此基因載體的應用性，我們進一步分析在大腸桿菌表達系統中，JCV類病毒顆粒可以包裹的DNA長度。實驗結果顯示，約10 kb左右的DNA長度可以被包入殼體顆粒內。這結果顯示利用JC類病毒殼體作為基因載體，在治療性基因或組織專一性的啟動子的選擇有更大的彈性。大腸桿菌是一種相當廉價的蛋白質表達系統，在此系統下表達出攜帶有外源性基因的JCV類病毒顆粒，只要以蔗糖或氯化銫梯度進行超高速離心，即可被純化出，製備過程簡單且經濟。綜合以上各點，我們認為JCV類病毒殼體可能可發展成為基因輸送載體。

關鍵字

JC病毒主要結構蛋白VP1 ；滲透壓震盪法；細胞內包裝系統

並列摘要

JC virus (JCV), a human polyomavirus, belongs to the polyomaviridae. The JC virion conatins three capsid proteins（VP1, VP2 and VP3）and a viral minichromosome. VP1 is the major capsid protein constituting approximately 75 ％ of the total proteins. We generated JCV virus-like particles（VLPs）when the major capsid protein VP1 was expressed in E. coli. The recombinant VLPs were demonstrated to be able to package and deliver exogenous DNA into mammalian cell. These findings indicate that the recombinant JCV VLP potentially could be used as a human gene transfer vector for gene therapy. A reporter plasmid, pEGFP, was introduced into the E. coli carrying JCV VP1 expressing plasmid. The VLPs purified from the E. coli with dual plasmids were found to package both plasmids as demonstrated by PCR and E. coli transformation. Furthermore, the VLPs which purified from in vivo packaging system were able to deliver the reporter plasmid, pEGFP, into 13 different mammalian cell lines and transduce the gene for expression. In this study, we also tried to determint the limitation of DNA size that could be packaged and protected in the VLP from in vivo packaging system. The maximum size of DNA molecule could be packaged in the VLP was approximately 10 Kbp. The findings demonstrate that the JCV VLP may provide more flexible gene delivery vector in terms of accommodation of large size of DNA molecule. Furthermore, it is easy to purify the VLPs just by using sucrose or CsCl gradient centrifugation. The findings in the thesis indicate that the human JCV VLPs generated in E. coli are potentially to be developed as a gene delivery vector for human gene therapy in the future.

並列關鍵字

JCV major capsid protein VP1 ； osmotic shock ； in vivo packaging system

參考文獻

1. Arthur, R.R., Shan, K.V., Charache, P., and Saral, R. 1988. BK and JC virus infections in recipients of bone marrow transplants. J. Infect. Dis. 158:563-569.

2. Atwood, W.J., Amemiya, K., Traub, R., Harms, J., and Major, E.O. 1992. Interaction of the human polyomavirus, JCV, with human B-Lymphocytes. Virology.190:716-723.

4. Auricchio, A., Rivera, V.M., Clackson, T., O'Connor, E.E., Maguire, A.M., Tolentino, M.J., Bennett, J., and Wilson, J.M. 2002. Pharmacological regulation of protein expression from adeno-associated viral vectors in the eye. Mol. Ther. 6, 238–242

5. Berger, J.R., Kaszovitz, B., Donovan Post, M.J., and Dickinson, G. 1987. Progressive leukoencephalopathy associated with human immunodeficiency virus infection. A review of the literature with a report of sixteen cases. Ann. Intern. Med. 107:78-87.

7. Bonini, C., Ferrari, G., Verzeletti, S., Servida, P., Zappone, E., Ruggieri, L., Ponzoni, M., Rossini, S., Mavilio, F., Traversari, C., and Bordignon, C. 1997. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science 276, 1719–1724

國際替代計量

利用人類多瘤性病毒,JC病毒,殼體作為基因輸送載體之研究

未授權

主題瀏覽