自從蝦白點症爆發迄今，蝦白點症病毒疫情在全球肆虐嚴重，造成蝦養殖經濟產業受到極大重創。白點症病毒的宿主範圍涵遢撳M其他水產甲殼類動物，其病毒的形態與基因體序列不同於已知的大型雙股 DNA 病毒，在分類上被歸屬在一新成立的病毒科 Nimaviridae (Whispovirus 屬)。
本論文研究主題是針對蝦白點症病毒臺灣分離株的新穎基因 – wssv 162，進行基因體學與蛋白質體學的探討，分別在原核大腸桿菌系統與真核昆蟲細胞系統進行基因表現。在原核表現系統中以 pET28b 載體構築重組質體，轉型入大腸桿菌 BL-21(DE3)，以 IPTG 誘導表現含有 6x His 標記的 His6-WSSV162 融合性蛋白質；並以 Ni-NTA 樹脂純化蛋白質，目前已順利製成免疫抗體，可供後續相關實驗應用。在真核表現系統部分則以 pIZ/V5-EGFP 載體構築重組質體，利用微脂粒包裹方式，轉染送入昆蟲細胞內，表現具有綠螢光的 EGFP-WSSV162 融合性蛋白質，透過螢光顯微鏡可快速檢測表現狀況。本研究發現pIZ/V5-EGFP-WSSV162在Sf9細胞轉染量極低，因此只有極少量的Sf9細胞有綠螢光表現，但螢光表現強度不弱於正控制組(轉染pIZ/V5-EGFP)，且其螢光主要呈現於細胞質區域，並有類似包涵體構造表現出強螢光。
另為探就 WSSV162 蛋白質與寄主蝦體組織內蛋白質的交互作用關係，設計以 Pull-down assay 方式，將大腸桿菌生產的 His6-WSSV162 融合性蛋白質與蝦組織萃取出的蛋白質混合反應，以分析可能的交互作用關係。 White spot syndrome virus (WSSV) is a widespread viral agent in shrimps and other crustaceans population. It has caused severe mortalities and huge enconomic losses to the shrimp farming industry, not only in Asia but also globally. Sequence analysis revealed a low level of homology between most WSSV ORFs and known genes from GeneBank, and this virus has been a member of the genus Whispovirus within a new virus family called Nimaviridae.
This study is to investigate the function of one open reading frame (designated wssv162) from the WSSV genome of Taiwan isolate. We try to construct the recombinant plasmids containing the ORFs, and to express them by transformation into Escherichia coli, BL-21(DE3). Until now, the 6x His-tagged fusion proteins containing the WSSV162 were expressed successfully and used to prepare a specific antibody for western blotting. On the other hand, we transfected EGFP-WSSV162 construct into insect cells, and examined the cellular localization of the WSSV162 protein. The numbers of transfected insect cells, which expressed EGFP-WSSV162 fusion proteins, are low. But, it is interesting that EGFP-WSSV162 fusion proteins are mainly expressed in the cytoplasma of transfected insect cells. Then we also found that some EGFP-WSSV162 fusion proteins seem to be expressed as inclusion bodies.
In order to study the interactions between WSSV162 protein and host shrimp proteins. We used the pull-down assay to analyze the possibilities of ineteractions between His6-WSSV162 fusion proteins from E. coli and shrimp proteins.
