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Construction and screening of a cDNA library from the triactinomyxon spores of Myxobolus cerebralis, the causative agent of salmonid Whirling Diseases

Published online by Cambridge University Press:  03 January 2006

M. EL-MATBOULI  and
H. SOLIMAN
M. EL-MATBOULI
Affiliation:
Institute of Zoology, Fish Biology and Fish Diseases, Faculty of Veterinary Medicine, University of Munich, Germany
H. SOLIMAN
Affiliation:
Institute of Zoology, Fish Biology and Fish Diseases, Faculty of Veterinary Medicine, University of Munich, Germany
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Abstract

The ZAP Express cDNA library was constructed using mRNA extracted from the triactinomyxon spores of Myxobolus cerebralis. First-strand cDNA was synthesized using Moloney Murine leukaemia virus reverse transcriptase. Following second-strand cDNA synthesis, the double-stranded cDNA was digested with Xho I restriction enzyme, cDNA fragments less than 400 bp were removed and the remaining cDNA was ligated with the lambda ZAP Express vector. The recombinants were packaged in vitro using Gigapack III gold packaging extract. The primary cDNA library titre contained 0·5×106 clones, with 97% recombinant and only 3% non-recombinant clones. The cDNA library was then screened using the anti-triactinomyxon antibodies. Positive clones were selected and re-screened twice more to give a final selection of 526 clones. One clone (46-5) was selected randomly and subjected to in vivo excision of the pBK-CMV phagemid from the ZAP express vector. The sequence of the entire clone was obtained using rapid amplification of the cDNA ends. A search of the clone sequence against GenBank revealed that it related to ribosomal protein L23 and it had a high percentage similarity to this protein from different species. A conserved domain for ribosomal protein L23 was also identified in the clone sequence.

Keywords

triactinomyxon sporesMyxobolus cerebraliscDNA library constructioncDNA library screeningZAP express vectorchemiluminescence
Type
Research Article
Information
Parasitology , Volume 132 , Issue 4 , April 2006 , pp. 467 - 477
Copyright
2006 Cambridge University Press

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