Functional genomic study of the parotoid secretion from Bufo gargarizans

  • Yiguo Huo

Student thesis: Masters ThesisMaster of Philosophy

Abstract

The parotoid secretion from Bufo gargarizans has been used as a traditional Chinese medicine for 2000 years in the treatment of inflammation, swelling and carbuncles. Recent studies have been focused on the small molecules, the bufadienolides and biogenic amines, the main medicinal parts in its parotoid secretion. However, the peptides and proteins, which were reported to have large quantities in parotoid secretion have not been widely studied.
Here, we focused on the proteins and peptides derived from the parotoid secretion of Bufo gargarizans. A comprehensive technology, RNA-Seq, was performed to obtain the whole transcriptome of which one of the most abundant proteins, CL4590, was investigated. Subsequent molecular cloning was carried out to validate the sequence of CL4590 using proper-designed primers and the Sanger sequencing method. In the meantime, the parotoid secretion was subjected to tryptic digestion to obtain the peptide fragments that were fractionated by RP-HPLC and analysed by LC-MS/MS to identify CL4590 at the proteomic level. The fractionated peptides were subjected to biological screening. Also, the six tryptic peptides with large quantities derived from CL4590 were identified and they were named by their molecular weight, QUB-1132, QUB-2617, QUB-802, QUB-826, QUB-1153 and QUB-1260. Then, further chemically-synthesised were employed to evaluate their biological activities as well.
The results showed that RNA sequencing induced a low degree of sequence bias by a validation using specific primers. It indicated that RNA-Seq is an authority technic for the acquisition of full-length sequence. On the other hand, none of these fractionated tryptic peptides had observable biological activities on three typical organisms, S. aureus, E.coil and C. albicans. In terms of synthesised peptides, QUB-826 is the only one who displayed antimicrobial activity against S. aureus and C. albicans. However, QUB-1132 and QUB- 2617 exhibited anti-proliferative activity against several human cancer cell lines, especially on prostate cancer cell lines. These two tryptic peptides were supposed to be a factor of TRPM8, which might have relationship with cancer cells. The results indicated that the discovered tryptic peptides can be potential drug candidates.
Date of AwardNov 2017
Original languageEnglish
Awarding Institution
  • Queen's University Belfast
SupervisorChris Shaw (Supervisor), Tianbao Chen (Supervisor), Lei Wang (Supervisor), Xinping Xi (Supervisor) & Chengbang Ma (Supervisor)

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