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標題: 犬小病毒重組蛋白VP2的表現及純化
Expression and Purification of recombinant VP2 protein of Canine Parvovirus
作者: 伍子善
Wu, Tzu-San
關鍵字: canine parvovirus
VP2 protein
出版社: 獸醫學系暨研究所
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摘要: 犬小病毒 (canine parvovirus, CPV) 屬於小病毒科 (Parvoviridae),具有正20面體直徑約25 nm的蛋白鞘 (capsid)、不具外套膜 (non-enveloped) 。病毒為線性單股DNA (single-stranded DNA) 基因體長度約5.2 kb。CPV主要感染犬科動物,造成新生幼犬心肌炎與出血性腸炎。依學者研究指出,CPV的結構蛋白鞘是由VP1、VP2以及VP3所組成,VP2為其主要蛋白,並且和宿主的免疫反應有重要相關。本實驗材料為來自中興大學獸醫教學醫院的臨床病犬糞材,抽取糞材內CPV的DNA所建構的重組pGEM-VP1/VP2質體,並參考實驗室在GenBank登錄的犬小病毒VP2全長基因序列 (編號AY869724),設計一對引子,利用聚合酶連鎖反應 (PCR) 增幅病毒VP2的核甘酸片段。此片段經適當的限制酵素剪接後,轉接到pET-32b (Novagen) 原核表現載體上,成為pET32b-VP2質體,藉由大腸桿菌BL21 (DE3) 進行VP2全長基因片段的蛋白質表現。經SDS-PAGE分析,可以觀察到如預期大小的VP2蛋白質之表現,並利用西方墨點法 (Western blot) 與質譜儀分析鑑定重組蛋白,結果與預期符合。此外,我們進一步建構pET32b-VP2-1225與pET32b-VP2-1000二個重組質體,以表現C端缺損 (deletion) 的VP2重組蛋白。與全長VP2蛋白相較,此缺損VP2重組蛋白有較佳的表現量。最後利用親合性樹脂管柱,我們已順利地純化全長以及C端缺損的VP2蛋白質。
Canine parvovirus (CPV) is a non-enveloped virus with a single-stranded DNA genome and causes severe enteritis in dog of all ages and myocarditis in puppies. The CPV capsid contains two overlapping proteins, VP1 (82 kDa) and VP2 (65 kDa),. VP3 (63 kDa) is produced by proteolytic processing of VP2. VP2, the most abundant protein of the CPV capsid, seemed to contain all the neutralization sites for host immunity reaction. In this study, a full-length gene of VP2 was cloned into the plasmid pET-32b, expressed in E. coli system, and the recombinant VP2 was purified by affinity chromatography. The recombinant VP2 protein was identified with Western blotting and mass spectrometry analysis. In addition, two recombinant VP2 proteins with C terminal deletions were constructed and expressed. The characterization of these two deletion mutant proteins was performed with techniques used for the full-length VP2. Our results showed that the protein expression level of the VP2 deletion mutant is higher than that of full-length VP2.
其他識別: U0005-2408200619065500
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