請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/25046
標題: | 表現病毒外套膜蛋白的擬球藻轉殖品系 A Stable-transmitted Transgenic Microalgae, Nannochloropsis oculata, Harbors a DNA Fragment Encoding a Viral Protein |
作者: | Ko-Ping Sun 孫可蘋 |
指導教授: | 黃穰(Rang Huang) |
關鍵字: | 白點病毒,電破,草蝦,口服疫苗,擬球藻, WSSV,electroporation,Penaeus monodon,oral vaccine,Nannochloropsis oculata, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | 擬球藻 (Nannochloropsis oculata)是目前養殖魚蝦貝類幼苗生產中重要的餌料生物之一,它是一種單細胞藻類,具有生活週期短、繁殖快速、容易培養、繼代方便、可以短時間內大量生產、成本低廉等優勢,是一種具潛力的生物反應器(bioreactor)。白點病毒(white spot syndrome virus, WSSV)一直是水產養殖上相當具有威脅性的致病病原,在典型感染中常可發現100%感染率及九成以上死亡率,通常會造成很嚴重的經濟損失。VP28是白點病毒最主要的外鞘膜蛋白,而且已被證實能使草蝦產生對WSSV的抗病能力。因此,我們希望利用擬球藻表現VP28並生產以口服投餵的疫苗,將VP28的cDNA片段構築到包含衣藻 (Chlamydomonas reinhartii)的heat shock 70A (hsp70A)與ribulose bisphosphate carboxylase small subunit 2 (rbcs2)基因之雙啟動子的質體pCB740中,利用電穿孔(electroporation)方式將外源性的去氧核醣核酸 (Deoxyribonucleic acid, DNA) 導入擬球藻的基因體中。經過多次繼代培養與PCR (polymerase chain reaction) 檢測後,得到23株轉殖藻株。轉殖後一年半內,都可以在藻內檢測出DNA的存在,其轉殖率為4.6% (497株中挑選到23株穩定轉殖株)。我們從中選擇蛋白質誘導表現最高的轉殖株D5來量產VP28膜蛋白。將此轉殖藻株經過熱處理誘發後,利用反轉錄聚合酶鏈反應,可以偵測到VP28基因大小約650個鹼基的片段,證實轉殖藻株可產生VP28 mRNA。抽取藻類蛋白質經由蛋白質電泳分析後證實轉殖藻株能表現一野生組所沒有的分子量在28kDa附近之蛋白質,經熱處理誘導的轉殖藻株在28kDa的蛋白質表現量比沒有熱處理誘導所表現的basal level蛋白質量還多,表示VP28膜蛋白有被熱處理誘導成功。我們以抗VP28的多株抗體(mouse polyclonal anti-VP28 IgG),藉由西方浸漬法 (Western blotting)證實VP28膜蛋白確實存在於轉殖藻株中。而用口服疫苗VP28轉殖藻株的方式進行草蝦的生物活性測試,也證實能夠增加草蝦對白點病毒的抵抗能力。 Microalga, Nannochloropsis oculata, is the commonly useful foodstuff for finfish and shellfish larvae in aquaculture. In this study, we attempt to generate transgenic N. oculata strain which can enhance the quality of larvae. White spot syndrome virus (WSSV) causes severe mortality in shrimp industry. VP28 is a major envelope protein of WSSV and enables to stimulate disease-resistant ability of shrimp. We constructed a plasmid, pCB740-VP28, in which the VP28 cDNA is driven by a heat shock 70A (hsp70A) promoter combined with a ribulose bisphospate carboxylase small subunit 2 (rbcs2) promoter of Chlamydomonas reinhartii, and transferred into N. oculata by electroporation. The transformants containing pCB740-VP28 were screened, and 23 stable-transmitted transgenic algae were selected, and the transferred pCB740-VP28 is present over one-and-a-half-year passage. The success rate of stable-transmitted transgenic algal clones was 4.6% of the examined transgenic algae (n=497). We selected a stable transgenic clone-D5, in which VP28 protein is highly expressed after induction. After we cultured this transgenic alga and induced with heat shock treatment, its mRNA was extracted and detected by reverse transcription-polymerase chain reaction (RT-PCR). Results showed that a RT-PCR-product with a 650-bp was generated, which was corresponding to that of RT-PCR-product amplified from VP28 mRNA template. Furthermore, when we extracted total proteins from this recombinant microalga and analyzed the protein profile by SDS-PAGE, we found that a band near 28-kDa was observed in the transgenic alga, but not present in the wild-type alga. And, compared to the transgenic alge before induction, the intensity of this band greatly increased after induction, indicating that the exogenous VP28 protein was significantly heat-induced from a basal leaky. The result of Western blotting analysis with polyclonal antibody VP28-IgG also showed that transgenic alga could produce VP28 envelope protein by heat shock induction. According to biological assay of vaccination experiment result, we demonstrated that feeding transgenic algae containing an exogenous viral protein enables to protect shrimps from WSSV infection in shrimp cultivation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/25046 |
全文授權: | 未授權 |
顯示於系所單位: | 海洋研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-96-1.pdf 目前未授權公開取用 | 686.93 kB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。