A Stable-transmitted Transgenic Microalgae, Nannochloropsis oculata, Harbors a DNA Fragment Encoding a Viral Protein

• Main Authors: Ko-Ping Sun, 孫可蘋
• Other Authors: Rang Huang
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/22782484489721483836

碩士 === 臺灣大學 === 海洋研究所 === 95 === 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.