Cloning and Characterization of BAFF Gene from Whitespotted Catshark and Crucian Crap
|School||Nanjing Normal University|
|Course||Biochemistry and Molecular Biology|
|Keywords||Chilosoyllium plagiosum Carassius auratus BAFF clone invitro expression biological information|
This thesis contains four chapters:1. Some advances in the studies of BAFF (B cell activating factor of the TNF family) were reviewed.2. Discussion about the meaning of BAFF gene researches in fishes.3. In this study, a CpBAFF was amplified from Chiloscyllium plagiosum by RT-PCR and RACE strategies. The open reading frame of CpBAFF cDNA consists of 819 bases encoding a protein of 272 amino acids. Like other reported BAFF molecues, CpBAFF includes a typically transmembrane domain and a furin protease cleavage site. Sequence alignment shows that CpBAFF shares 36%-57% identity with BAFF sequences reported in other vertebrate animals, and it shares identical structural features. Real-time quantitative RT-PCR demonstrated high expression of CpBAFF in the main immune organ spleen, moderate expression in kidney, brankia and liver, and low expression in intestine, heart and pancreas. The predicted 3D structural analysis of the soluble mature part of CpBAFF (CpsBAFF) analyzed by comparative protein modeling revealed that it is very similar to the 3D structure of human BAFF. Recombinant CpsBAFF fused with NusA-His6 tag was efficiently expressed in Escherichia coli BL21 (DE3) and its molecular weight of-83 KDa was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In vitro MTT assay indicated that the purified pET43.1a (+)-CpsBAFF protein was able to promote splenic B cells survival in a dose-dependent manner. The present findings will provide valuable information for the shark immunity and the evolution of vertebrate adaptive immunity.4. In the present study, a CaBAFF gene was amplified from Carassius auratus by RT-PCR and RACE strategies. The full-length cDNA of CaBAFF was 1639 bp, consisiting of a 407 bp 5’-terminal untanslated region (UTR), and a 795 bp open reading frame (ORF) which encods a protein containing 264-amino acids bearing characteristics of the TNF family. Like other reported BAFF molecules, CaBAFF contains a typically transmembrane domain and a TNF homology domain. Sequence alignment shows that CaBAFF shares 47%-77% identity at the amino acid level with BAFF sequences reported in other bony fish, and it shares identical structural features. In addition, we analysed the fundamental property of CaBAFF protein usiong biological software. This study provided the basis for further investigation of its biological fuction in Carassius auratus and its relationship with imumune.