The Yangtze Finless Porpoise And Bats B Lymphocyte Stimulation Factor (baff) Research Cloning, Expression, And Biological Function

This thesis contains four chapters:1. Some advances in the studies of BAFF (B cell activating factor of the TNF family) were reviewed.2. The research progress of the finless porpoises and bat.3. The present study, the full-length cDNA of BAFF (designated NpBAFF) from the finless porpoise was cloned using RT-PCR and rapid amplification of cDNA ends (RACE) techniques, and its biological activities have been characterized. To our knowledge, this is the first report of any BAFF gene being cloned from an aquatic mammal. The full-length cDNA of NpBAFF consists of1502bases including an852bp open reading frame encoding283amino acids. This protein was found to contain a predicted transmembrane domain, a putative furin protease cleavage site, and a typical TNF homology domain corresponding to other, known BAFF homologues. Sequence comparison indicated that the amino acid sequence of NpBAFF was very similar to its bovine (87.68%), porcine (76.33%), hircine (87.68%) and canine (82.19%) counterparts. The predicted three-dimensional (3D) structure of the NpsBAFF monomer, analyzed by comparative protein modeling, revealed that it was very similar to its human counterpart. The SUMO-NpsBAFF was efficiently expressed in Escherichia coli BL21(DE3) and confirmed by SDS-PAGE and Western blot analysis. Laser scanning confocal microscopy analysis showed that NpsBAFF could bind to its receptors on mouse lymphocytes. In vitro, MTT assays indicated that SUMO-NpsBAFF could promote the survival of mouse splenic lymphocytes grown with anti-mouse IgM. These findings indicate that NpBAFF plays an important role in the survival of lymphocytes and has functional cross-reactivity among cetaceans and other mammals. The present findings may provide valuable information for research into the immune system of the finless porpoise.4. In the present study, the full-length cDNA of BAFF (designated bBAFF) from the bat was cloned using RT-PCR and rapid amplification of cDNA ends (RACE) techniques, and its biological activities have been characterized. The full-length cDNA of bBAFF consists of986bases including an873bp open reading frame encoding290amino acids. Sequence comparison indicated that the amino acid of bBAFF possessed the TNF signature, a transmembrane domain, a putative furin protease cleavage site and three cysteine residues. Real-time quantitative PCR (qPCR) analysis indicated that bBAFF mRNA was predominantly expressed in bat lymphoid tissue spleen. The SUMO-bsBAFF was efficiently expressed in Escherichia coli BL21(DE3) and confirmed by SDS-PAGE and Western blotting analysis. In vitro, the MTT assays indicated that SUMO-bsBAFF was able to stimulate survival of mouse lymphocytes grown with anti-mouse IgM. These findings indicate that bsBAFF plays an important role in the survival of lymphocytes and has functional cross-reactivity among mammalians.