Human Baff Gene Cloning, Expression And Antibody Preparation

BAFF(B cell activating factor belonging to the TNF family), a new member of thetumor necrosis factor superfamily, which is mainly expressed on monocytes, maerophagesand dendritic cells. The human BAFF gene codes for a typeⅡtransmembrance proteinwith full lengh of 285 amino acids. BAFF signals through three different receptors TACI,BCMA and BAFF-R. The expression of BAFF receptors is restricted to B cells.Since 1999, BAFF has been identified to stimulate proliferation, differentiation andproduction of immunoglobulin of B cells. Elevated amounts of BAFF can be found in theserum of autoimmune patients such as SLE and RA. Moreover, BAFF has been proposedto contribute to the development of B cell malignancies. Therefore, the more we knowabout BAFF, the more we know about these dieases.In our study, we extracted total RNA from human PBMCs to amplify the cDNAfragment encoding amino acid 83-285 of BAFF by RT-PCR. The sequence was insertedinto pET-30a(+) plasmid and transformed into E.coli BL21(DE3). BALB/c mice wereimmunized by injection of recombinant hBAFF_83-285-His fusion protein. Semi-solidmedium containing methycellulose was used in hybridoma production to get McAbs.Indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showedthat the purified McAbs had a good antigen specificity. At the same time, we amplified thefull length cDNA of human soluble BAFF by PT-PCR from total RNA of human PBMCs.The sequence was inserted into pGEX-4T-1, a prokaryotie expressing vector. Therecombinant plasmid containing hsBAFF was transformed into DH5αto express a solublefusion protein tagged with GST. The fusion protein was then purified by GST affinitychromatography. Then we produced the rabbit anti-hsBAFF polyclonal serum with thispurified soluble fusion protein. Indirect enzyme-linked immunosorbent assay (ELISA) andWestern blot analysis demonstrated that the recombinant protein was recognized by rabbitpolyclonal serum.In conclusion, we produced McAbs and polyclonal serum against hsBAFF. Theseantibodies may offer a good tool for future basic and clinical research.