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Construction and Screening of an Expression Cdna Library from the Triactinomyxon Spores of Myxobolus Cerebralis, the Causative Agent of Salmonid Whirling Diseases*

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Construction and Screening of an Expression Cdna Library from the Triactinomyxon Spores of Myxobolus Cerebralis, the Causative Agent of Salmonid Whirling Diseases* Institute of Zoology, Fish biology and Fish Diseases Ludwig-Maximilians-University Munich Chair: Prof. Dr. Dr. R.W. Hoffmann Under Supervision of Prof. Dr. Dr. M. El-Matbouli Construction and Screening of an Expression cDNA Library from the Triactinomyxon Spores of Myxobolus cerebralis, the causative agent of Salmonid Whirling Diseases* A Thesis Submitted for the Doctor Degree in Veterinary Medicine Faculty of Veterinary Medicine Ludwig- Maximilians- University Munich From Hatem Mohamed Toughan Soliman Sohag- Egypt Munich 2005 *This study was supported by the Egyptian government as a full term mission and in part by the Whirling Disease Foundation and U.S. Fish and wildlife Service. Gedruckt mit Genehmigung der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Dekan: Univ.-Prof. Dr. A. Stolle Referent: Prof. Dr. M. El-Matbouli Referent: Univ.-Prof. Dr. R. Schulz Tag der Promotion: 15. Juli 2005 Construction of cDNA Library from the Triactinomyxon spores Dedication This thesis is dedicated to My Parents, my wife and my kids; Ahmed, Mohamed and Reem Construction of cDNA Library from the Triactinomyxon spores Table of Contents Page I Table of Contents Page 1. Introduction ................................................................................................. 1 2. Literature Review........................................................................................ 3 2.1 Myxozoa...................................................................................................................... 3 2.1.1 Taxonomy of Myxobolus cerebralis ................................................................... 3 2.1.2 Development of Myxobolus cerebralis in its hosts............................................ 4 2.2 Whirling disease......................................................................................................... 6 2.2.1 Symptoms and pathogenicity............................................................................. 7 2.2.2 Host susceptibility............................................................................................... 8 2.2.3 Diagnosis.............................................................................................................. 9 2.2.4 Factors influencing infection and disease....................................................... 12 2.2.5 Vectors for the spread of whirling disease ..................................................... 15 2.2.6 Fish immune response to Myxobolus cerebralis ............................................. 15 2.3 Control and eradication of whirling disease ......................................................... 16 2.4 Polyacrylamide gel electrophoresis (PAGE) and western blot (Electroblotting) ......................................................................................................................................... 17 2.5 Complementary DNA (cDNA) library................................................................... 18 3. MATERIALS AND METHODS .............................................................20 3.1 MATERIALS......................................................................................................... 20 3.1.1 Specimens .......................................................................................................... 20 3.1.2 Media ................................................................................................................. 20 3.1.3 Bacterial strains................................................................................................ 21 3.1.4 Antiserum.......................................................................................................... 21 3.1.5 Primer Sequences ............................................................................................. 21 3.1.6 Reagents used.................................................................................................... 22 3.2 METHODS............................................................................................................... 26 3.2.1 Experimental production of triactinomyxon spores ................................... 26 3.2.2 Sodium Dodecyl Sulphate Polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting of triactinomyxon spores....................................................... 27 3.2.3 RNA extraction ................................................................................................. 29 3.2.4 Construction of the cDNA library................................................................... 30 3.2.5 Antibody screening of the cDNA library using ECL™ chemiluminescent detection...................................................................................................................... 34 3.2.6 In vivo excision of the pBK-CMV phagemid vector containing insert from the ZAP Express vector............................................................................................. 36 Construction of cDNA Library from the Triactinomyxon spores Table of Contents Page II 3.2.7 Isolation of plasmid DNA and estimation of the cDNA insert size .............. 38 3.2.8 DNA sequencing and sequence analysis:........................................................ 38 3.2.9 Primer construction from the selected sequence ........................................... 39 3.2.10 Triactinomyxon spores and Tubifex tubifex cDNA synthesis..................... 39 3.2.11 A verification PCR test for the cDNA sequence .......................................... 40 4. Results.........................................................................................................44 4.1 Experimental production and collection of triactinomyxon spores.................... 44 4.2 SDS-PAGE and western blotting of triactinomyxons .......................................... 44 4.3 RNA from triactinomyxons, non-infected and highly-infected T. tubifex ......... 47 4.4 Construction of the cDNA library.......................................................................... 47 4.5 Immunological screening for the triactinomyxon positive plaques .................... 47 4.6 Purification of clones of interest and characterisation of the library inserts..... 49 4.7 In vivo excision, plasmid isolation and size estimation of the selected clone ..... 49 4.8 Synthesis of cDNA from triactinomyxon spores and non-infected oligochaetes ......................................................................................................................................... 50 4.9 Determination of the specificity of clone 46-5 to triactinomyxon spores............ 51 4.10 RACE amplification of the cDNA ends ............................................................... 52 4.11 Sequence analysis of the full-length cDNA clone 46-5........................................ 52 5. Discussion...................................................................................................55 6. Summary ....................................................................................................62 7. Zusammenfassung....................................................................................64 8. References ..................................................................................................66 Acknowledgment ...........................................................................................81 Curriculum Vitae ..........................................................................................82 Construction of cDNA Library from the Triactinomyxon spores List of Figures Page III List of Figures Page Figure 1: Mature waterborne triactinomyxon spores released from infected T. tubifex. .................................................................................................................. 44 Figure 2: SDS-PAGE analysis of soluble proteins from non-infected T. tubifex stained with coomassie brilliant blue. .................................................................. 45 Figure 3: PAGE electropherogram of triactinomyxon soluble proteins stained with silver stain. ................................................................................................... 46 Figure 4: Detection of triactinomyxon spore antigens by western blot analysis using anti-triactinomyxon antiserum, and ECL detection reagents...................... 46 Figure 5: Total RNA extracted from triactinomyxon spores (1)......................................... 47 Figure 6: ECL-Hyperfilm showing primary screening for triactinomyxon positive plaques visible as black spots on the plate............................................ 48 Figure 7: ECL-Hyperfilm showing the result of secondary screening................................ 48 Figure 8: ECL-Hyperfilm showing the result of tertiary screening. ................................... 49 Figure 9: Gel showing results of PCR of random samples of positive plaques. ................. 49 Figure 10: Gel showing amplified cDNA insert fragment. ................................................. 50 Figure 11: Gel showing effect of varying PCR cycles number; the double- stranded cDNA appears as a moderately strong smear from 500- 6000bp. ................................................................................................................. 51 Figure 12: Gel showing that the clone is specific to triactinomyxon spores....................... 51 Figure 13: Gel showing RACE amplification products....................................................... 52 Figure 14: Nucleotide sequence of clone 46-5. ..................................................................
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