Analysis of proteome in clonorchis sinensis adult worms and identification of antigenic protein by immuno-proteomics

다국어 초록 (Multilingual Abstract)

Clonorchis sinensis, the causative agent of clonorchiasis, is widespread in East and Southeast Asia, including China, Vietnam and the Republic of Korea. I identified antigenic proteins from adult C. sinensis liver flukes using immunoproteomic analysis. In this study, I found 23 candidate antigenic proteins with a pI in the range of 5.4-6.2 in total lysates of C. sinensis. The antigenic protein spots reacted against sera from clonorchiasis patients and were identified as cysteine proteases, glutathione transferases, gelsolin, propionyl-CoA carboxylase (PCC), prohibitin and 14-3-3 protein (14-3-3) using LC-coupled ESI-MS/MS and an EST database for C. sinensis. PCC and 14-3-3 were identified for the first time as serological antigens for the diagnosis of C. sinensis. To validate the antigenicity of PCC and 14-3-3, recombinant proteins were immunoblotted with sera from clonorchiasis patients. The structural, functional and immunological characteristics of the putative amino acid sequence were predicted by bioinformatics analysis. These proteins will contribute to the development of diagnostics for clonorchiasis. These results suggest that immunoproteomic approaches are valuable tools to identify antigens that could be used as targets for effective parasitic infection control strategies.

목차 (Table of Contents)

• Introduction of Clonorchis sinensis
• 1. Overview 2
• 2. Life cycle and infection 3
• 3. Epidemiology 7
• 4. Pathology with C. sinensis 9
• 5. Diagnosis 11
• 6. Treatment and control 11
• Research Paper
• Abstract 14
• Ⅰ. Introduction 16
• Ⅱ. Materials and Methods 19
• 2.1 Sample preparation 19
• 2.2 Extraction of total lysates 19
• 2.3 IEF and 2DE 20
• 2.4 SDS-PAGE 20
• 2.5 Fractionation based on IEF and 2DE 21
• 2.6 Western blot 21
• 2.7 Silver Staining of 2DE Gel 22
• 2.8 In-Gel protein Digestion 23
• 2.9 Identification of Proteins by LC-MS/MS 23
• 2.10 Identification of Proteins by MALDI-TOF 24
• 2.11 Protein Expression Constructs and Purification 25
• 2.12 Bioinformatic analysis 28
• 2.13 ELISA 28
• Ⅲ. Results 30
• 3.1 Pattern of C. sinensis development stages 30
• 3.2 2-DE map of C. sinensis adult worms 32
• 3.3 Identification on C. sinensis adult worms 34
• 3.4 2-DE and immunoblot 37
• 3.5 Fractionation of C. sinensis total lysates 39
• 3.6 Soluble fractionation and 2-DE 41
• 3.7 Identification of antigenic proteins by MS/MS analysis 44
• 3.8 Expression of antigenic proteins and immunoblot analysis 46
• 3.9 Bioinformatic analysis of antigenic recombinant proteins 51
• 3.10 Western blot analysis of 14-3-3 protein at the development stages of C. sinensis 56
• Ⅳ. Conclusions and Discussion 53
• Ⅴ. References 56
• Ⅵ. Abstract in Korean 74