人類疱疹病毒第八型Kaposin 蛋白與神經細胞因子Sept4 之交互作用

Interaction of human herpesvirus 8 kaposin protein with neural cell factor sept4

塗炳峰(T)

指導教授 ： 林振文 ； 張清堯 ； 蕭乃文

亞洲大學/健康學院/生物科技學系碩士班/碩士(2006年)

摘要

人類疱疹病毒第八型（Human Herpesvirus8,HHV-8）又稱為卡波西氏肉瘤疱疹病毒(KSHV) ， 為引起卡波西氏肉瘤(Kaposi＇s sarcoma,KS）之病因，HHV-8 DNA在KS病患的背部根神經節與HIV陽性病患不明腦炎的大腦活檢樣本中被發現。HHV-8包含的89個ORFs中有若干ORFs與細胞基因同源，能編碼干擾免疫系統蛋白，參與核酸代謝的酶和細胞生長調節因數等。ORF K12可編譯成Kaposin蛋白，可能促進細胞增生。為了進一步了解人類疱疹病毒Kaposin蛋白與腦部病變之相關性，以噬菌體表達人腦cDNA基因庫的技術，找尋與人類疱疹病毒第八型Kaposin蛋白作用之神經因子。結果表現人腦細胞septin4 cDNA之噬菌體被篩選出來。Kaposin蛋白與septin4重組蛋白間專一性結合經免疫沉澱法證實兩者確實有交互作用的關係。藉共軛焦顯微鏡的觀察Kaposin 與septin4 共同轉染之SK-N-SH 與TE671 細胞確認kaposin與septin4在細胞內有同位化現象。深入作用機轉探討發現kaposin有anti-septin4抑制caspase3活性及active caspase3生成。 此外NFκB與Ap-1啟動子結合冷光報導基因之細胞內訊息傳遞偵測系統推測kaposin可能藉活化AP-1因子干擾septin4傳達細胞凋亡途徑有關。本論文將有助於深入探討人類疱疹病毒第八型Kaposin蛋白可能的致病機制。

關鍵字

HASH(0x8881c24) ； HASH(0x8881c9c) ； HASH(0x8881d14)

並列摘要

Human herpesvirus 8 (HHV-8) referred to as Kaposi’s sarcomaassociated
herpesvirus (KSHV) is forms of Kaposi’s sarcoma. HHV-8 DNA was detected in dorsal root ganglia of KS patients, cerebrospinal fluid of human immunodeficiency virus-positive patients, and brain biopsy specimens from patients with unexplained encephalitis. The genome of HHV-8 contains 89 ORFs, some ORFs with homologous to cellular genes encode to interfere with host immune system, enzymes of nucleotide metabolism, and growth factors. The kaposin protein derived from ORF K12 has been reported to stimulate cell proliferation. In the study, we investigate protein-protein interaction of HHV-8 ORF K12 with neural cell factors using phage display human brain cDNA library.
After six-run biopanning, septin4 that interacted with the kaposin protein has been identified. Co-immunoprecipitation showed the binding
interaction of recombinant kaposin protein with septin4 protein. In
addition, conforcol immunofluorescence assay demonstrated co-locolization of HHV-8 kaposin protein with septin4 protein in SK-N-SH and TE671 cells. Moreover, caspase3 assay showed that the kaposin protein inhibited septin4-induced apoptosis. NFκB-luciferase and Ap-1-luciferase cis-acting reporters revealed that the kaposin protein
activated Ap-1 promoter, being associated with the inhibitory affect on septin4-mediated apoptosis. This study will be helpful for insights into the role of the kaposin protein in the HHV-8 pathogenesis.

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