臺灣博碩士論文加值系統

假性狂犬病毒（Pseudorabies virus, PrV）被歸類為阿爾法疱疹病毒亞科（alphaherpesvirinae）中的Varicellovirus屬，為Aujeszky’s disease （AD）之病原。至今假性狂犬病毒已被證實在封套上具有10種醣蛋白，且皆與病毒的侵入及在細胞間的散佈有關。假性狂犬病毒若缺損醣蛋白B、D、H與L其中的一種，就喪失其感染能力，所以這四種醣蛋白被認為是參與膜融合的必要因子。為了進一步研究醣蛋白H（glycoprotein H, gH）的功能及其在病毒感染時所扮演的角色，我們首先將假性狂犬病毒的gH基因以PCR及限制酵素切割等步驟選殖到原核表現載體－pET32a (＋)，命名為pETgH。再將所構築的重組原核表現質體pETgH利用大腸桿菌（E. coli）BL21（DE3）之表現系統進行大量表現。於IPTG誘導後，經由SDS-PAGE及Western blotting的分析結果顯示有一分子量約為93 kDa的蛋白質被表現且分子量大小與預期相符。進一步將此表現蛋白純化後免疫小白鼠，所收集之免疫血清在immunoperoxidase staining試驗中可特異性辨識假性狂犬病毒所感染LM細胞，證實此表現蛋白具有正確之抗原性。接著利用短暫轉染分析配合CAT報導質體，以評估適用於LM細胞內有效活化gH基因表現之啟動子，由CAT assay的結果決定選擇含有可被dexamethasone激活的MMTV-LTR啟動子之真核表現載體－pMAMneo在LM細胞內進行表現gH蛋白。將含有gH基因之真核表現質體pMAMgH轉染到LM細胞中，利用G418篩選出單株化的細胞後，再分別以PCR、Southern blot analysis、RT-PCR、Northern blot analysis、immunoperoxidase staining及immunoprecipitation等試驗進一步進行篩選與驗證，最後所得LMgH7細胞株其染色體內確實嵌有假性狂犬病毒gH基因且具有表現gH mRNA及gH蛋白的能力。此外，若將gH－PrV缺陷病毒株分別感染LM及LMgH7細胞後發現唯有在LMgH7細胞內此缺陷病毒才具有散佈到鄰近細胞的能力。此外，將此可受dexamethasone刺激而持續穩定表現假性狂犬病毒gH蛋白之LMgH7細胞，經由MTT assay與細胞生長速率分析證實細胞表現此外源性蛋白並不會影響LM細胞的生長速率，且藉由偵測LMgH7與LM細胞對假性狂犬病毒的感染病毒力價試驗所得之結果亦顯示可表現病毒gH蛋白的LMgH7細胞並不會影響PrV的複製能力。綜合以上結果可知此穩定表現gH之細胞株確實能產生具有功能性的醣蛋白H以提供gH缺陷病毒在感染細胞時所需之醣蛋白H。

Pseudorabies virus (PrV), a double-stranded DNA virus, belongs to the subfamily Alphaherpesvirinae of neurotropic herpesviruses and is the causative agent of Aujeszky’s disease in swine. PrV encodes information for at least 11 glycoproteins, 10 of which are found in the virion envelope as well as on the surfaces of infected mammalian cells. Glycoprotein H (gH), gL, gB and gD of PrV are not only essential for virus entry at a post-binding stage, but are also sufficient for cell-to-cell spread via fusion with the cellular membranes. Virions that lack glycoprotein H, L, B or D fail to infect cells, thus the PrV glycoprotein H plays an essential role in the fusion of virus envelopes with cellular membranes and in the fusion of an infected cell membranes with the uninfected neighbours. For a detailed analysis of the function of gH, this gene was cloned into prokaryotic and eukaryotic expression systems in this study. The recombinant expression plasmid pETgH was constructed by cloning the gH gene located in the BamH I fragments 11 and 17 of PrV genomic DNA into pET32a(+) vector. The gH protein expressed in E. coli was 76 kDa in size, and the purified product was used as an antigen to immunize mice for generating specific antibodies against viral gH protein. The specificity of immunoserum was identified by recognizing the gH protein in the PrV infected cells in immunoperoxidase staining. Using a transient transfection assay, we have chosen the MMTV-LTR promoter to induce the expression of viral gH gene in mouse-L (LM) cells from several viral promoters, including the CMV and SV40 promoters using chloramphenicol acetyltransferase (CAT) reporter gene assay. In addition, the recombinant eukaryotic expression plasmid pMAMgH that contains the intact gH gene under the control of MMTV-LTR promoter and a neomycin-resistant gene was transfected into LM cells to establish the stable gH-expressing LMgH7 cells. The LMgH7 cells were demonstrated to contain gH gene and stably express gH protein by several analytical methods including PCR, Southern blotting, RT-PCR, Northern blotting and immunoprecipitation. The LMgH7 cells were found to grow at a similar rate as the parental and pMAMneo vector-transfected LM cells. Furthermore, a gH-deletion PrV (gH－PrV) was shown to be capable of penetrating and infecting adjacent LMgH7 cells in contrast to LM cells. These results clearly indicate that gH expressing in cells can provide the gH－PrV as viral glycoprotein H. Thus, the gH protein produced from LMgH7 cells are functional that could displace viral gH protein during infection.

中文摘要………………………………………………………………………………I英文摘要……………………………………………………………..…………........III
第一章 前言……………………………………………………………….…...…...1
第二章 文獻探討…………………………………...……………………...……….2
第三章 材料與方法.………...………………………………………………….…10
第四章 結果…...…………………………………………………………………..30
第五章 討論……...………………………………………………………………..63
參考文獻…………………………………………………………………………..…69

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