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1.Proteomic Analysis of the Venom from Chinese Tarantula Chilobrachys Jingzhao 2.Preparation of Antiserum Against Latrodectus Tredecimguttatus Venom

Author: ZuoXiaoJun
Tutor: WangXianChun；LiangSongPing
School: Hunan Normal University
Course: Biochemistry and Molecular Biology
Keywords: Chilobrachys jingzhao proteomics crude venom HPLC two-dimensional gel electrophoresis N-terminal sequencing MS/MS Latrodectus tredecimguttatus antiserum preparation purification neutralization of venom
CLC: Q51
Type: Master's thesis
Year: 2006
Downloads: 10
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Abstract

Part I: Proteomic analysis of the venom from Chinese tarantulas Chilobrachys jingzhaoChinese tarantulas Chilobrachys jingzhao are primarily distributed in hainan province. The venom of this spider contains a mixture of peptides and proteins with different biological activities. The venom was analyzed via proteomic strategies to gain an overview of the venom composition. After gel filtration chromatography, the venom peptides with molecular mass of less than 10 kDa were applied to ion-exchange and reversed-phase high performance liquid chromatography (RP-HPLC) separation, followed by peptide identification using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and Edman degradation sequencing. The venom proteins of grater than 10 kDa were separated and visualized by 2-dimensional gel electrophoresis (2~DE). Protein identification was performed by liquid-chromatography -quadrupole time-of-flight (LC-ESI-Q-TOF) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry.About 150 peptides were detected by MALDI-TOF MS, of which 22 peptides’ full sequences were identified, and another 20 peptides’ N-teminal sequences were determined. About 250 protein dots were visualized on the 2-DE gel, of which 19 protein spots were directly identified by mass spectrometry with conventional stringent Mascot searching. Another 55 protein spots were identified by sequence similarity search using Mass Spectrum driven BLAST (MS BLAST).This study provides a global view of peptides and proteins composition in the venom from the Chinese tarantula C. jingzhao for the first time, and the results demonstrate the diversity and complexity of the spider venom, which provides a better understanding of the mechanism of the envenomation of this spider. PartⅡ: Preparation of antiserum against Latrodectus tredecimguttatus venomThe spider Latrodedtus tredecimguttatus is one of the most venomous spiders in the world and distributed worldwide. Envenomation by this spider could cause serious clinical symptoms, and the most effective therapy method is using the antivenom. But there is no antivenom specific for the envenomation by L. tredecimguttatus bite. In fact, the venom of this spider contains many kinds of proteins which are rather suitable for preparing antiserum.In this study we prepared the antiserum against L. tredecimguttatus venom with guinea pigs. The filtered crude venom was mixed with Freund’s complete adjuvant or Freund’s incomplete adjuvant, and then injected subcutaneously into the back of guinea pigs every 8-10days. From the second reimmunization on, the titre of antiserum against the crude venom were determined through double immunodiffusion detection. Then the eligible serum was separated from the blood and was purified by ammonium sulphate fractionation and anion-exchange chromatography. To confirm the antivenom, all of the eluted peaks were detected by western blot. The neutralization of the toxic effect of L. tredecimguttatus venom by the antivenom was assessed in vitro with an isolated rat nerve-spermaduct preparation and in vivo with a lethality experiments in mice.After the third reimmunization, the titre of antiserum reached 1:8. rhe fourth reimmunization was performed and then the experimental animals were bled. Lots of non-specific proteins were removed from the serum by ammonium sulphate fractionation, and the antivenom was effectively purified by anion-exchange chromatography further. Both in vivro and in vivo experiments showed the obvious neutralization of toxic effects of the spider venom.The results demonstrated that we prepared a novel, specific antivenom against Latrodectus tredecimguttatus venom successfully, and this made a foundation for the specific treatment of envenomation by the spider L. tredecimguttatus.

1.Proteomic Analysis of the Venom from Chinese Tarantula Chilobrachys Jingzhao 2.Preparation of Antiserum Against Latrodectus Tredecimguttatus Venom

Abstract

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