Dissertation > Excellent graduate degree dissertation topics show

Screening Diagnostic Antigen of Toxoplasmosis Based on Proteomics Technology

Author: LiuLiLi
Tutor: ShenJiLong
School: Anhui Medical University,
Course: Pathogen Biology
Keywords: Proteomics diagnosis two dimensional electrophoresis ROP2 toxoplasmosis
CLC: R446.6
Type: Master's thesis
Year: 2011
Downloads: 40
Quote: 0
Read: Download Dissertation

Abstract


ObjectiveToxoplasma gondii （T. gondii） is an obligatory intracellular parasite that infects almost one third of people worldwide. Since the vagaries associated with lack of specificity, sensitivity, and not reliability of many commercial serologic test kits on the market, there is no reliable evidence in clinical diagnosis and treatment. In an attempt to discover useful candidates for the diagnosis of toxoplasmosis, proteomics in combination with western blotting were employed in this study. Furthermore, assess its role in the diagnosis of toxoplasmosis.MethodsFollowing two dimensional electrophoresis （2-DE）, the tachyzoite lysates of T. gondii RH strain were transfered onto NC membranes for Western-blot analysis. The developed films were aligned and then matched with the homologous Coomassie brilliant blue （CBB）-stained gels by PDQuest software and artificial recognition. The matched spots were excised, digested by trypsin and further analyzed by LC/MS-MS. The potential biomarkers of the identified protein was cloned and expressed from the transformed E. coli. The recombinant was purified, coated in micro-well plates and evaluated their performance in diagnosis of toxoplasmosis using enzyme linked immunosorbent assay （ELISA）. Besides, the BALB/c mice were orally infected with cysts of PRU strain of T. gondii and bled to obtain sera at different time points for specific IgG and IgM detection.ResultsThis serological proteome assay yielded more than 50 immunodominant spots. 21 of these spots in Toxo-IgM–-IgG+ group were precisely matched with a homologous two-dimensional electrophoresis （2-DE） gel and successfully identified by LC/MS-MS as corresponding to 13 different proteins. However, pooled sera with Toxo-IgM+-IgG– showed just 3 spots could be matched to 2-DE gel perfectly, and only one of them could be identified and characterized by LC/MS-MS: the rhpotry protein 2 （ROP2）. Of these proteins, rROP2186–533 was successfully expressed and further applied in the diagnosis of human toxoplasmosis using ELISA. The ELISA results revealed sensitivities of 100% and 0% for anti-Toxo IgM and IgG antibodies, respectively. Furthermore, the assays showed a specificity of 100% for IgM antibody test. There was a significant difference of OD values of Toxo-IgM between the sera of Toxo-IgM+-IgG– and control （P﹤0.0001）.Blood samples from the mice infected with the PRU strain were detected by rROP2186–533, data showed that The Toxo-IgM antibodies appeared on day 7 post-infection （PI）, peaked on 14 PI and rapidly decreased on 28 PI when tested with rROP2186–533 antigen. The data showed that the kinetics of Toxo-IgM antibodies response was consistent with that generally accepted. But, the titer of specific IgG antibodies had no significant change before and after infection.ConclusionProtein ROP2 was successfully screened and recombined from the tachyzoite lysates of T. gondii RH strain. Our present study confirmed that rROP2186–533 was an useful diagnostic antigen for detecting Toxo-IgM antibody and could clearly distinguish acute and chronic infection. And the immunoproteomic approach has promising potential and offers a powerful tool in the screening of candidates for the diagnosis of toxoplasmosis. Our primary investigation laid the foundation for the development of laboratory reagent with high specificity and sensitivity for the diagnosis of toxoplasmosis.

Related Dissertations

  1. Design and Development of Application Programme for Fault Analyzer Based on Wince Platform,TP311.52
  2. Research on the 6-Dof Fault Tolerant Control of the Vibration Isolation Platform with Eight Actuators,TB535.1
  3. Tongue Feature Extraction and Research of Fusion Classification,TP391.41
  4. Research on Feature Extraction and Classification of Tongue Shape and Tooth-Marked Tongue in TCM Tongue Diagnosis,TP391.41
  5. Research on Classification Method of Tongue Substance Color and Tongue Coating Color Based on SVM,TP391.41
  6. Fault Diagnosis Method Based on Support Vector Machine,TP18
  7. Fault Diagnosis Research on Three-Tank System,TP277
  8. The Value of the Heart-Type Fatty Acid Binding Protein in Early Diagnosis of Acute Myocardial Infarction,R542.22
  9. The Means of Clinical Screening in Make Differential Diagnosis between L4-L5 and L5-S1,R681.53
  10. Research on the Key Technology of Waterborne Transport Security System,U698
  11. Research on Intellectualized Diagnostic Device Applied to Distribution Lines,TM755
  12. Cellular and Proteomic Analyses of Endocytosis Mechanism Involved in Pollen-tube Growth in Nicotiana Tabacum,Q942
  13. Proteomic Analysis of Vibrio Alginolyticus in Response to Four Types of Antibiotics,S941
  14. Quantifying the Responses of Weak Gluten Wheat Leaf Photosynthesis and Chlorophyll Fluorescence Parameters to Weak Light at Filling Stage,S512.1
  15. Responses of the Double-Cropping Rice Population Growth and Nitrogen Uptake to Density and Nitrogen Management and the Diagnosis Based on NDVI,S511
  16. Proteomic Research of Rice Plasma Membrane Protein under Blast Stress,S511
  17. Salt-Tolerance Evaluation of Brassica Napus Germplasm in the Bud and Seeding and Establishment of the Two-Dimensional Electrophoresis System,S565.4
  18. Isollation and Identification of Haemophilus Parasuis and Its Proteomic Analysis under in Vitro Growth Conditions Mimicking the in Vivo Environment,S858.28
  19. The mRNA Expression of PAPPA, PLAC8 and PAG in Pregnant Diary Cows,S858.23
  20. The Establishment and Application of cPL Double Anti-Body Sandwich Elisa Method for the Diagnosis of Canine Acute Pancreatisis,S858.292
  21. Clinical Research of Nonpalpable Breast Lesions (Diameter≤1.0cm) in 1040 Cases,R737.9

CLC: > Medicine, health > Clinical > Diagnostics > Laboratory diagnosis > Immunology Examination
© 2012 www.DissertationTopic.Net  Mobile