- 學位論文
黑角舞蛾核多角體病毒抑制細胞凋亡基因iap2 及iap3 之選殖、定序、表現及比較
The study of anti-apoptotic genes iap2 and iap3 of Lymantria xylina multiple nucleopolyhedrovirus, (LyxyMNPV): cloning, sequencing, expression and comparison
摘要
昆蟲桿狀病毒感染非寄主昆蟲細胞，即可以觀察到細胞凋亡的現象。昆蟲桿狀病毒為能夠順利在感染昆蟲細胞達到增殖的目的，其基因組內主要含有兩種抑制細胞凋亡之基因，即為p35 和iap 基因群(inhibitor of apoptosis gene family, iap1~iap5)，其中iap 基因較p35 基因廣泛地分布於桿狀病毒中。經PCR 增幅再利用南氏墨點法證實，黑角舞蛾核多角體病毒(Lymantria xylina multiple nucleopolyhedrovirus, LyxyMNPV) 之基因組內含有兩個iap 基因，分別為ly-iap2 (687 bp) 與ly-iap3 (450 bp)，而不具有加州苜蓿夜蛾核多角體病毒(Autographa californica MNPV, AcMNPV) 之iap1 及p35 同源基因。此外黑角舞蛾核多角體病毒之iap 基因與吉普賽舞蛾核多角體病毒(L.dispar MNPV) 之iap 基因皆呈高相似度，iap2 及iap3 分別為81% 及86%。利用北方雜合法證實黑角舞蛾核多角體病毒ly-iap2 在細胞感染72 小時後，即可偵測到一1.95-kb 的轉錄區；而ly-iap3 在感染48 小時後，可偵測到兩個轉錄區，分別為1.04-kb 及2.09-kb。黑角舞蛾核多角體病毒的兩個iap 基因和榕樹透翅毒蛾核多角體病毒(Perina nuda MNPV, PenuMNPV) 的相同iap 基因均已解序，據此序列與其他桿狀病毒之相同基因序列分析親緣關係，結 果顯示LyxyMNPV 之iap 基因與LdMNPV 關係密切，而與PenuMNPV 和 AcMNPV 皆處於不同群中。LY-IAP3 經表現後，並成功製造出抗血清，利 用間接螢光免疫偵測法(indirect fluorescen immunoassay, IFIA)，LY-IAP3 蛋白質在LyxyMNPV 感染之不接受細胞株(NTU-SL7B) 以及可接受細胞株(IPLB-LD 652Y-7) 後之表現，發現LY-IAP3 皆可在SL7B 或LD7 細胞中表現。因此LY-IAP3 在病毒感染過程中所扮演的角色，需更進一步釐清。
並列摘要
The anti-apoptosis genes of baculoviruses are essential genes for viral propagation in their susceptible host cells. There are two main groups of antiapoptotic genes in baculovirus genome, p35 and iap gene family (iap1~iap5), the latter is more wildly distributed in baculovirus. According to the results of PCR amplification and Southern blot hybridization, Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) contains only two iap genes ly-iap2 (687 bp) and ly-iap3 (450 bp). The sequences of iap2 and iap3 of LyxyMNPV had high identities with those of L. dispar MNPV (LdMNPV), about 81% and 86%, respectively. The results of Northern blot hybridization revealed that, a 1.95-kb transcript of ly-iap2 was first detected at 72 h postinfection (pi) and 1.04- and 2.09-kb transcripts of ly-iap3 were first detected at 48 h pi. The iap genes of Perina nuda MNPV (PenuMNPV) had been also cloned and sequenced. LyxyMNPV and PenuMNPV were compared with other known baculovirus iap genes for phylogenetic analysis. The trees showed that LyxyMNPV was closely related to LdMNPV, in addition, LyxyMNPV, PenuMNPV and Autographa californica MNPV (AcMNPV) were located seperately in three different clusters. LY-IAP3 polyclonal antibody had been prepared and used to detect the expression of LY-IAP3 in LyxyMNPV-infected IPLB-LD 652Y-7 cells (LyxyMNPV permissive cells) and NTU-SL7B cells (LyxyMNPV non-permissive cells) by indirdct fluorescence immunoassay (IFIA). LY-IAP3 could be expressed in the cytoplasm of both cells; therefore, the role of LY-IAP3 needs to be further elucidated.