Construction and screening of a cDNA library from the triactinomyxon spores of Myxobolus cerebralis, the causative agent of salmonid whirling diseases.
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 x 10(6) 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.
el-Matbouli
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Institute of Zoology, Fish Biology and Fish Diseases, Faculty of Veterinary Medicine, University of Munich, Germany. El-Matbouli@lmu.de
Soliman
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H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D016054:DNA, Protozoan / Q000737:chemistry; D056890:Eukaryota / Q000235:genetics*; D005393:Fish Diseases / Q000469:parasitology*; D015723:Gene Library; D007158:Immunologic Techniques / Q000662:veterinary; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009835:Oligochaeta; D017686:Oncorhynchus mykiss / Q000469:parasitology*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D016415:Sequence Alignment; D033761:Spores, Protozoan / Q000235:genetics
D017931:DNA Primers; D016054:DNA, Protozoan; C511510:L23 protein, Myxobolus cerebralis; C097584:MRPL23 protein, human; D024101:Mitochondrial Proteins; D015800:Protozoan Proteins; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins