title: Molecular cloning, bioinformatics analysis and functional characterization of B-cell activating factor in goat (Capra hircus).
abstract: B-cell activating factor of the TNF family (BAFF) induces B cell survival, proliferation, immunoglobulin secretion and has a role in enhancing immune responses. In the present study, we amplified the cDNA of goat (Capra hircus) BAFF (designated gBAFF) from spleen by reverse transcription-PCR (RT-PCR). The open reading frame (ORF) of gBAFF covers 843 bp encoding 280 amino acids, with a 152-aa mature peptide. Sequence comparison indicated that the amino acid of gBAFF possessed the TNF signature, a transmembrane domain, a putative furin protease cleavage site and three cysteine residues. The predicted three-dimensional (3D) structure of the soluble part of gBAFF (gsBAFF) analyzed by "comparative protein modelling" revealed that it was very similar to its counterparts. Real-time quantitative PCR (qPCR) analysis indicated that gBAFF mRNA was predominantly expressed in goat lymphoid tissue spleen. Recombinant gsBAFF was fused with a small ubiquitin-related modifier (SUMO) gene to enhance the soluble expression level in Escherichia coli BL21 (DE3). The resulting fused protein SUMO-gsBAFF was efficiently expressed and purified using metal chelate affinity chromatography (Ni-NTA), then confirmed by SDS-PAGE and Western blotting analysis. In vitro, the MTT assays and flow cytometric analysis indicated that SUMO-gsBAFF as well as gsBAFF could promote the survival/proliferation of goat splenic B cells or mouse splenic B cells. Therefore, BAFF may be a potential immunologic factor for enhancing immunological efficacy in goat.
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
authors.forename: Yuefen
authors.initials: Y
authors.lastname: Shen
authors.forename: Fengtao
authors.initials: F
authors.lastname: You
authors.forename: Chunlan
authors.initials: C
authors.lastname: Li
authors.forename: Zhijuan
authors.initials: Z
authors.lastname: He
authors.forename: Zhenning
authors.initials: Z
authors.lastname: Liang
authors.forename: Hongxin
authors.initials: H
authors.lastname: Ai
authors.forename: Shule
authors.initials: S
authors.lastname: Wang
authors.forename: Shuangquan
authors.initials: S
authors.lastname: Zhang
mesh_terms: D000818:Animals; D053264:B-Cell Activating Factor / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D001483:Base Sequence; D049109:Cell Proliferation; D002469:Cell Separation; D003001:Cloning, Molecular; D019295:Computational Biology; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006041:Goats; D008213:Lymphocyte Activation; D008297:Male; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D019076:Transgenes / Q000235:genetics
chemical_list: D053264:B-Cell Activating Factor