Prof. Dr. Hans Jürgen Prömel
Präsident in Vertretung der Humboldt-Universität zu Berlin

• 1 Introduction

  • 1.1  Structure and function of chitinases

  • 1.2 Filarial chitinases are potential targets for interventions

  • 1.3  A. viteae as a model to investigate parasitological parameters of filarial diseases

  • 1.4 Aims and objectives of the study

• 2 Results

  • 2.1  Characterisation of A. viteae chitinase genes

    • 2.1.1  Determination of the gene structure of A. viteae chitinase genes

      • 2.1.1.1 Isolation and characterisation of A. viteae chitinase sequences from a genomic phage library

      • 2.1.1.2 Restriction analysis of genomic inserts

      • 2.1.1.3 Sequencing of A. viteae genomic chitinase and determination of gene structure

  • 2.2 Comparative analysis of A. viteae genomic chitinase sequences with genomic data bases of nematodes

  • 2.3  A. viteae chitinase transcripts

    • 2.3.1  Screening of A. viteae L3 and adult female cDNA libraries for chitinase transcripts

    • 2.3.2 Identification of A. viteae chitinase transcripts

  • 2.4 Identification of homologous chitinase sequences from nematode databases

  • 2.5 Cloning, expression and purification of recombinant A. viteae chitinase

    • 2.5.1  Cloning, periplasmatic/ cytoplasmatic expression and purification of N-terminal portion of A. viteae chitinase in the expression vector pET 22 b (+) in E. coli

    • 2.5.2 Activity of N-terminal chitinases

  • 2.6 Immunisation studies with A. viteae chitinase in Meriones unguiculatus

    • 2.6.1  Immunisation studies with A. viteae N-terminal chitinase fragment

    • 2.6.2 Immunisation studies with synthetic peptides from the active site of A. viteae chitinase

• 3 Discussion

  • 3.1  Analysis of A. viteae chitinase genes and transcripts

  • 3.2 Expression, purification and activity of N-terminal chitinase

  • 3.3 Immunological control

  • 3.4 Outlook

• 4 METHODS

  • 4.1  Identification of recombinant cDNA and genomic clones from phage libraries by plaque hybridisation

    • 4.1.1  Preparation of plating bacteria

    • 4.1.2 Plating of lambda phage

    • 4.1.3 Titration of phage libraries

    • 4.1.4 Amplification and cryopreservation of phage stocks

  • 4.2 Screening of libraries

    • 4.2.1  Screening of libraries with a DIG-labelled A. viteae chitinase probe

      • 4.2.1.1  Screening of libraries by plaque hybridisation

      • 4.2.1.2 In vivo excision

  • 4.3 Purification and manipulation of DNA

    • 4.3.1  Isolation of lambda DNA and sub-cloning of genomic inserts

      • 4.3.1.1 Preparation of crude phage lysates

      • 4.3.1.2 Isolation of phage DNA

      • 4.3.1.3  Release and sub-cloning of genomic inserts into pBluescript II SK

    • 4.3.2 Small scale Plasmid isolation from E. coli

    • 4.3.3  Electrophoresis and detection of DNA on agarose gels

    • 4.3.4  Isolation of DNA from agarose gels

    • 4.3.5 Isolation and concentration of DNA from aqueous solutions

      • 4.3.5.1  Extraction with Nucleospin kit

      • 4.3.5.2 Phenol chloroform extraction

      • 4.3.5.3 Precipitation of DNA

    • 4.3.6  Isolation of high molecular weight genomic DNA from A. viteae

    • 4.3.7 Determination of DNA Concentration

    • 4.3.8 Restriction digestion of DNA

    • 4.3.9 5' Dephosphorylation of digested DNA

    • 4.3.10 Ligation of DNA fragments

      • 4.3.10.1  Ligation of PCR fragments into T-overhang vectors

      • 4.3.10.2 Ligation of DNA fragments with sticky ends

    • 4.3.11 mRNA isolation, reverse transcription and RT-PCR

    • 4.3.12 Polymerase chain reaction (PCR)

      • 4.3.12.1 Standard PCR

      • 4.3.12.2 Mid-range PCR

  • 4.4 Southern blotting

    • 4.4.1  Digestion, electrophoresis, and blotting of genomic DNA

    • 4.4.2 Radioactive labeling of A. viteae chitinase probe

    • 4.4.3 Hybridisation and detection

  • 4.5 Microbiological methods

    • 4.5.1  Preparation of competent E. coli

    • 4.5.2 Transformation of competent E. coli

    • 4.5.3 Screening of bacterial colonies for plasmids/ recombinant plasmids

      • 4.5.3.1 Blue-white screening

      • 4.5.3.2 Restriction analysis of isolated plasmids

    • 4.5.4 Bacteria cultures and long-term storage of bacterial stocks

    • 4.5.5 Expression and purification of recombinant proteins from E. coli

  • 4.6 Protein analytical methods

    • 4.6.1  Determination of protein concentration

    • 4.6.2 Sodium dodecly sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

    • 4.6.3 Chitinase activity assays

  • 4.7 Immunochemical and immunological methods

    • 4.7.1  Western blot

      • 4.7.1.1 Transfer of proteins onto nitrocellulose membranes

      • 4.7.1.2 Immunodetection of immobilised proteins

    • 4.7.2 Coupling of peptides to KLH

    • 4.7.3 Immunisation studies

      • 4.7.3.1 Bleeding of animals for production of sera

  • 4.8 Parasitological methods

    • 4.8.1  Maintenance of the life cycle of A. viteae

    • 4.8.2 Quantification of microfilarial load in blood of jirds

    • 4.8.3 Isolation of filariae

      • 4.8.3.1 Isolation of adult A. viteae

      • 4.8.3.2  Isolation of L3 from the vector Ornithodoros moubata

      • 4.8.3.3 Isolation of uterine microfilariae from gravid female worms

  • 4.9 Computer analysis and statistical methods

    • 4.9.1  Analysis of DNA sequences

    • 4.9.2 Statistical analysis

• 5 Materials

  • 5.1  Commercial Kits and Enzymes

  • 5.2 Laboratory Equipment and consumables

  • 5.3 Synthetic oligonuceotides

    • 5.3.1  PCR Primers

      • 5.3.1.1 Primers used in long range PCR

      • 5.3.1.2 Primers used for standard PCR

    • 5.3.2 Sequencing primers

      • 5.3.2.1 Universal sequencing primers

        • Primers on pGEM-T vector

      • 5.3.2.2 Primers on pBluescript SK (-) vector

    • 5.3.3  Primers used for sequencing A. viteae chitinase genomic clone 1

    • 5.3.4 Primers used for sequencing A. viteae chitinase genomic clone 9

    • 5.3.5 Primers used for sequencing A. viteae chitinase genomic clone 12

  • 5.4 Plasmids

    • 5.4.1  Cloning plasmids

      • 5.4.1.1 Cloning plasmid for genomic inserts: pBluescript (Stratagen)

      • 5.4.1.2 TA- overhang vector for cloning PCR products

    • 5.4.2 Plasmids harbouring genomic and cDNA inserts: λ Dash II and λ Zap, respectively

  • 5.5 Buffers and solutions

    • 5.5.1  Molecular biology

      • 5.5.1.1 Buffers for separation, purification and storage of DNA

      • 5.5.1.2 Buffers for purification of plasmids: CTAB miniprep

      • 5.5.1.3 Buffers for purification of lambda DNA

      • 5.5.1.4 Buffers and reagents used for screening of libraries

      • 5.5.1.5 Buffers and reagents used for Southern blotting

  • 5.6 Media and buffers for E. coli

  • 5.7 Protein and immuno- chemistry

    • 5.7.1  SDS-PAGE

    • 5.7.2 Western blot

    • 5.7.3 Buffers for native purification of N-terminal chitinase with Ni²⁺-NTA

    • 5.7.4 Buffers for denaturing purification of N-terminal chitinase with Ni²⁺-NTA

    • 5.7.5 Synthetic peptides

    • 5.7.6 Buffers and solutions for chitinase enzyme activity assay

    • 5.7.7 Stock solutions

  • 5.8 Antibiotic stock solutions

  • 5.9  E. coli host strains and plasmids

  • 5.10 Databanks, softwares and online services

  • 5.11 Softwares

• Acknowledgements

• Abbreviations

• References

• Appendix

• Eidesstattliche Erklärung

• Tab. 1: Comparison of exon/intron sizes and identities of Acanthocheilonema viteae genomic chitinase sequences I and III.

• Tab. 2: Comparison of exon/intron sizes and identities of Acanthocheilonema viteae genomic chitinase sequences I and II.

• Tab. 3: Comparison of exon/intron sizes and identities of Acanthocheilonema viteae genomic chitinase sequences II and III.

• Tab. 4: Comparison of exons/ intron sizes and identities between Acanthocheilonema viteae L3 (Gene I) and Brugia malayi MF 1 chitinases

• Tab. 5: Nucleotide differences between published A. viteae chitinase and the chitinase sequence used in this study

• Tab. 6: Orthologous filarial transcripts to A. viteae gene sequence I

• Tab. 7: Brugia malayi ESTs

• Tab. 8: C. elegans homologues of A. viteae chitinase gene sequence I

• Tab. 9: Specific activity of purified A. viteae and O. volvulus chitinases

• Tab. 10: Summary of biometric data from immunization studies with N-terminal Acanthocheilonema viteae chitinase

• Tab. 11: Summary of biometric data from immunization studies with synthetic peptides from Acanthocheilonema viteae chitinase

• Tab. 12: Pipetting scheme for restriction digestion of DNA

• Tab. 13: Pipetting scheme for ligation of PCR fragments into T-overhang vectors

• Tab. 14: Pipetting scheme for ligation of DNA fragments with sticky ends

• Tab. 15: Pipetting scheme for standard PCR

• Tab. 16: Thermal profile for standard PCR

• Tab. 17: Pipetting scheme for Mid range PCR

• Tab. 18: Thermal profile for mid range PCR

• Tab. 19: Pipetting scheme for radioactive labeling of probes

• Fig. 1 : Structure of chitinase-1 from Coccidiodes immitis .

• Fig. 2: Structural model of the active sites of Acanthocheilonema viteae (light green) and Onchocerca volvulus (blue) chitinases.

• Fig. 3: Life cycle of Acanthocheilonema viteae

• Fig. 4: Sizes of genomic inserts cloned into pBluescript

• Fig. 5: Restriction analysis of Acanthocheilonema viteae genomic chitinase/ pBluescript recombinant plasmids.

• Fig. 6: Long-range PCR fragments used in sequencing of genomic insert 12.

• Fig. 7: Southern blot analysis of Acanthocheilonema viteae genomic DNA.

• Fig. 8: Schematic diagram showing Acanthocheilonema viteae gene sequences.

• Fig. 9: Structure and organisation of Acanthocheilonema viteae L3 chitinase gene.

• Fig. 10: Comparison of genomic organisation between Acanthocheilonema viteae and Caenorhabditis elegans chitinase genes.

• Fig. 11: Transcripts of A. viteae genomic sequences in L3 and microfilariae (mf)

• Fig. 12: Amplification of 3’ end of cDNA chitinase from different stages using Rapid amplification of cDNA ends (RACE)

• Fig. 13: Amino acid sequence comparison of different filarial chitinases.

• Fig. 14: Phylogenetic relationships of filarial chitinases with C. elegans chitinases

• Fig. 15: Amino acid sequence comparison of Acanthocheilonema viteae chitinase with Caenorhabditis elegans sequence C04F6.3.

• Fig. 16: Expression and purification of A. viteae chitinase from the periplasmatic space

• Fig. 17: Expression and purification of A. viteae chitinase under denaturing conditions

• Fig. 18: Activity assay of purified cytoplasmic N-terminal chitinase

• Fig. 19: Immunisation scheme of Meriones unguiculatus with experimental vaccines

• Fig. 20: Immunisation of Meriones unguiculatus with N-terminal Acanthocheilonema viteae chitinase

• Fig. 21: Recognition of native Acanthocheilonema viteae L3 chitinase by sera of mice immunized with peptides.

• Fig. 22: Vaccination of Meriones with KLH-coupled chitinase peptides 1 and 2

• Fig. 23: Microfilaria burden from Meriones vaccinated with KLH-coupled chitinase peptides