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Yuan T.,Sichuan University | Li C.,Sichuan University | Gu J.-R.,Tongwei Technology Center | Fu Y.-J.,Sichuan University | Xu H.,Sichuan University
Fish Physiology and Biochemistry | Year: 2013

An expressed sequence tag of grass carp leukocyte cell-derived chemotaxin 2 (LECT2) gene was screened from an established intestinal cDNA library. Rapid amplification of cDNA ends gave rise to a full-length LECT2 cDNA (gcLECT2) with a complete open-reading frame of 474 bp, encoding 158 amino acids about 17.9 kDa. Homology search and sequence alignment showed that this deduced protein sequence shared a high identity with LECT2 from other vertebrates. Western blotting indicated immunological cross-reactivity occurs between grass carp and human LECT2 protein. This gcLECT2 genomic sequence is 1,868 bp in size, which consists of five exons and four introns. Real-time quantitative PCR analysis revealed that gcLECT2 gene is ubiquitously expressed in different tissues of healthy grass carp including brain, gut, liver, spleen, kidney, muscle and heart, while the expression levels were significantly increased in liver and spleen followed by Aeromonas salmonicida infection. 992 bp 5′-flanking region sequence was cloned and analyzed, where one CAAT box and one GC island were found. Our results showed that the LECT2 is suggested to be most possibly involved in the grass carp's immune response. © 2012 Springer Science+Business Media Dordrecht.

Yuan T.,Sichuan University | Gu J.-R.,Tongwei Technology Center | Gu W.-B.,Sichuan University | Wu J.,Tongwei Technology Center | And 2 more authors.
Molecular Biology Reports | Year: 2011

Adenylosuccinate lyase (ADSL) is a bifunctional enzyme acting in de novo purine synthesis and purine nucleotide recycling. In the present study, we have constructed a grass carp (Ctenopharyngodon idella) intestinal cDNA library that has over 2.3 × 105 primary clones. An expressed sequence tag (EST) of grass carp adenylosuccinate lyase (gcADSL) gene was screened from this library. Both 5′-RACE and 3′-RACE were carried out in order to obtain the complete cDNA sequence, which contains a 1,446 bp open reading frame encoding 482 amino acids about 54.552 kDa. The deduced amino acid sequence shares high homology with its vertebrate counterparts, which shares 94% similarity with zebrafish, 81% with African clawed frog as well as chicken, 77% with human and 76% with mouse. This gcADSL genomic sequence, consisted of 13 exons and 12 introns, is 8,557 bp in size. Real-time quantitative PCR analysis revealed that the highest expression level of gcADSL was detected in muscle and the lowest in gill. In western blotting analysis, His6-tagged gcADSL protein expressed in Escherichia coli could be recognized not only by an anti-His6-tag monoclonal antibody but also by an anti-human ADSL polyclonal antibody, indicating immunological crossreactivity occurs between grass carp and human ADSL protein. 1,082 bp 5′-flanking region sequence was cloned and analyzed. © 2010 Springer Science+Business Media B.V.

Fu Y.J.,Sichuan University | Fu Y.J.,Tongwei Technology Center | Huang F.G.,Sichuan University | Yuan T.,Sichuan University | And 4 more authors.
Journal of Fish Biology | Year: 2012

An expressed sequence tag (EST) of B cell translocation gene (BTG) 1 (gcbtg1) was obtained from a grass carp Ctenopharyngodon idellus intestinal complementary (c)DNA library and the full-length cDNA sequence was acquired by rapid amplification of cDNA ends (RACE) technology. The predicted Gcbtg1 protein contains the box A and box B motifs which characterized the BTG and transducer of ERBB2 (TOB) family. Multiple alignment analysis reveals that Gcbtg1 shares an overall identity of 65-94% with Gcbtg1s of other vertebrates. Real-time quantitative PCR analysis reveals that the highest expression level of gcbtg1 was detected in liver and the lowest in muscle. Western blotting analysis indicates that the immunological cross-reactivity occurs between C. idella and human Homo sapiens BTG1 protein. A 1008 bp 5′-flanking region sequence was cloned and analysed. © 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.

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