Abstract
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.
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References
Torrecillas S, Makol A, Caballero MJ et al (2007) Immune stimulation and improved infection resistance in European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides. Fish Shellfish Immunol 23(5):969–981. doi:10.1016/j.fsi.2007.03.007
Ai Q, Mai K, Li H et al (2004) Effects of dietary protein to energy ratios on growth and body composition of juvenile Japanese sea bass, Lateolabrax japonicus. Aquaculture 230:507–516. doi:10.1016/j.aquaculture.2003.09.040
Zhang C, Mai K, Ai Q et al (2006) Dietary phosphorus requirement of juvenile Japanese seabass, Lateolabrax japonicus. Aquaculture 255:201–209. doi:10.1016/j.aquaculture.2005.11.040
Xue M, Luo L, Wu X et al (2006) Effects of six alternative lipid sources on growth and tissue fatty acid composition in Japanese sea bass (Lateolabrax japonicus). Aquaculture 260:206–214. doi:10.1016/j.aquaculture.2006.05.054
Mai K, Zhang L, Ai Q et al (2006) Dietary lysine requirement of juvenile Japanese seabass, Lateolabrax japonicus. Aquaculture 258:535–542. doi:10.1016/j.aquaculture.2006.04.043
Ye H-Q, Chen S-L, Xu J-Y (2007) Molecular cloning and characterization of the Myf5 gene in sea perch (Lateolabrax japonicus). Comp Biochem Physiol B 147(3):452–459. doi:10.1016/j.cbpb.2007.02.008
Xie ZY, Hu CQ, Zhang LP et al (2007) Identification and pathogenicity of Vibrio ponticus affecting cultured Japanese sea bass, Lateolabrax japonicus (Cuvier in Cuvier and Valenciennes). Lett Appl Microbiol 45(1):62–67. doi:10.1111/j.1472-765X.2007.02141.x
Li-hua QIU, Lin-sheng Song, Zhong-hua CAI et al (2003) Cloning and expression analysis of tumor necrosis factor α from Japanese sea perch (Lateolabrax japonicus). Fish Sci China 10:370–375
Li-hua QIU, Shi-gui JIANG, Han-hua ZHANG et al (2007) Molecular cloning and characterization of interleukin 8 from Japanese sea perch (Lateolabrax japonicus). Fish Sci China 14:201–207
Ren H-L, Wang K-J, Zhou H-L et al (2006) Cloning and organisation analysis of a hepcidin-like gene and cDNA from Japan sea bass, Lateolabrax japonicus. Fish Shellfish Immunol 21(3):221–227. doi:10.1016/j.fsi.2005.10.011
Savan R, Sakai M (2002) Analysis of expressed sequence tags (EST) obtained from commom carp, Cyprinus carpio L., head kidney cells after stimulation by two mitogens, lipopolysaccharide and concanavalin-A. Comp Biochem Physiol B 131(1):71–82. doi:10.1016/S1096-4959(01)00488-2
Dos Santos NMS, Hermsen T, Rombout JHWM et al (2001) Ig light chain variability in DNP494-KLH immunised sea bass (Dicentrarchus labrax L.): evidence for intra-molecular induced suppression. Dev Comp Immunol 25:387–401. doi:10.1016/S0145-305X(01)00002-7
Wang H-J, Shao J-Z, Xiang L-X et al (2006) Molecular cloning, characterization and expression analysis of an ILF2 homologue from Tetraodon nigroviridis. Mol Biol 39(6):686–695
Piyaviriyakul P, Kondo H, Hirono I, Aoki T et al (2007) A novel immune-type receptor of Japanese flounder (Paralichthys olivaceus) is expressed in both T and B lymphocytes. Fish Shellfish Immunol 22(5):467–476. doi:10.1016/j.fsi.2006.05.007
Ying ShaoYao (2004) Complementary DNA libraries: an overview. Mol Biotechnol 27(3):245–252. doi:10.1385/MB:27:3:245
Rebl A, Siegl E, Köllner B et al (2007) Characterization of twin toll-like receptors from rainbow trout (Oncorhynchus mykiss): evolutionary relationship and induced expression by Aeromonas salmonicida salmonicida. Dev Comp Immunol 31(5):499–510. doi:10.1016/j.dci.2006.08.007
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Lab Press, Cold Spring Harbor, Chapter 11.8
Larade K, Storey KB (2008) Constructing and screening a cDNA library. Methods for identification and characterization of novel genes expressed under conditions of environmental stress. Methods Mol Biol 410:55–80. doi:10.1007/978-1-59745-548-0_5
Fülle H-J (2003) Quality assessment of cDNA libraries. Methods Mol Biol 221:145–153
Stanfford JL, Ellestad KK, Magor KE et al (2003) A toll-like receptor (TLR) gene that is up-regulated in activated goldfish macrophages. Dev Comp Immunol 27:685–698. doi:10.1016/S0145-305X(03)00041-7
Lee J-Y, Hirono I, Aoki T (2000) Cloning and analysis of expression of Mx cDNA in Japanese flounder, Paralichthys olivaceus. Dev Comp Immunol 24(4):407–415. doi:10.1016/S0145-305X(99)00060-9
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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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Shao, Zt., Cong, X., Yuan, Jd. et al. Construction and characterization of a cDNA library from head kidney of Japanese sea bass (Lateolabrax japonicus). Mol Biol Rep 36, 2031–2037 (2009). https://doi.org/10.1007/s11033-009-9536-0
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DOI: https://doi.org/10.1007/s11033-009-9536-0