Construction and characterization of a cDNA library from head kidney of Japanese sea bass (Lateolabrax japonicus)
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In this paper, a cDNA expression library from head kidney of Japanese sea bass (Lateolabrax japonicus) was constructed for the first time. The first-strand cDNA was synthesized with Moloney Murine Leukaemia virus reverse transcriptase and the double-stranded cDNA was digested by Xho I enzyme. Size fractionation was performed on CHROMA SPIN-400 columns. cDNA fragments longer than 500 bps were ligated into the λZAPExpress vector. The recombinant DNA was packaged in vitro with Gigapack III gold packaging extract. The titers of the primary and amplified library were 1.0 × 105 and 5.0 × 109 pfu/ml, respectively. To characterize the constructed cDNA library, 15 phage plaques were selected randomly to test the inserted fragments. The results showed that the inserts were mostly longer than 500 bps. To test the utility, the library was screened with primers designed for three immune-related genes of, Myxovirus resistant (Mx), tumor necrosis factor-alpha (TNF-α) and Toll-like receptor (TLR). Results of Blastn and alignment showed that they are members of Mx, TNF-α and TLR gene families, respectively, which meets our anticipates for this cDNA library as an immune-related one. These results confirmed that the cDNA library constructed will provide a useful tool for gene cloning and expression analysis in immune system of Japanese sea bass.
KeywordsJapanese sea bass Head kidney cDNA library construction λZAPExpress vector PCR amplification Immunology
This work was supported by National Scientific Foundation of China (30070586), Specialized Research Fund for the Doctoral Program of Higher Education of China (20060445003) and Shandong Provincial Natural Foundation (Y2007D37) of China.
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