Title page for etd-0327113-142030


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URN etd-0327113-142030
Author Shuo-Fu Chang
Author's Email Address m992010015@student.nsysu.edu.tw
Statistics This thesis had been viewed 5578 times. Download 110 times.
Department Biological Sciences
Year 2012
Semester 2
Degree Master
Type of Document
Language zh-TW.Big5 Chinese
Title Production and characterization of polyclonal antibody against Epinephelus coioides retinoic acid inducible gene-I
Date of Defense 2013-03-02
Page Count 82
Keyword
  • Epinephelus coioides
  • RIG-I
  • poly(I:C)
  • antiserum
  • fusion protein
  • Abstract Orange-spotted Grouper (Epinephelus coioides) is one of the important economically farmed fish in Taiwan. However, grouper aquaculture in Taiwan has a serious problem of infection, especially in grouper larvae breeding stage. The infection resulted in very high mortality, which causes massive economic loss. Therefore, early detection of pathogen is critical to prevent epidemic outbreak. Retinoic acid inducible gene-I (RIG-I) is an intracellular receptor recognizing dsRNA virus. Upon dsRNA virus infection, RIG-I is induced and over-expressed in protein level that mediating type I Interferon (IFNs) antiviral signaling. In this study, total RNA extracted from fertilized eggs of Epinephelus coioides was extracted for the reverse transcription-polymerase chain reaction (RT-PCR) amplification of 432 bp cDNA encoding regulatory domain (RD) of RIG-I protein. The amplified cDNA fragment was then cloned into pQE32 for the prokaryotic expression of 17 kDa His-tagged RIG-I (His-RIG-I-RD) fusion protein. His-RIG-I-RD fusion protein was purified for immunization of New Zealand white rabbit to produce antiserum against RIG-I protein. Our Western blot result confirmed the specificity of antiserum for recognizing 0.01 μg His-RIG-I-RD fusion protein at 1:5000 dilution. In addition, 0.02 μg of immunogen His-RIG-I-RD fusion protein can be readily detected at 1:25000 dilution. Furthermore, this antiserum could detect the increase of a 72 kDa protein in lysate prepared from head kidney of dsRNA viral mimic poly(I:C) injected Epinephelus coioides, but not in uninjected control fish. The immunostaining results of frozen head kidney sections also revealed the RIG-I immunoreactivity using this antiserum. RIG-I protein immunoreactivity was prominently increased in melanomacrophage center (MMC) of head kidney from poly(I:C) injected fish compared to that of uninjected control fish. In summary, we have generated a specific antiserum against RIG-I protein of Epinephelus coioides, which could be a useful reagent for further study of Grouper immune response and signal transduction upon virus infection.
    Advisory Committee
  • Tsung-Chou Chang - chair
  • Chung-Da Yang - co-chair
  • Jiin-Tsuey Cheng - advisor
  • Files
  • etd-0327113-142030.pdf
  • Indicate in-campus at 5 year and off-campus access at 5 year.
    Date of Submission 2013-03-27

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