Three Yellow Chicken Soluble B Lymphocyte Stimulator Cloning, Expression And Activity Of Research

B cell activating factor of the TNF family is a co-stimulating factor of B cells. Chicken BAFF cDNA has been cloned by Schneider et al. (2004 ) , while there has no reports about Chinese cBAFF till now. Chinese 3-Yellow chicken is a common and local chicken breed of China. In this paper, we designed primers according to the sequence of chicken (Gallus gallus) BAFF (GenBank accession no. AF506010). Then 864 bp chicken BAFF (CycBAFF) cDNA and 459 bp chicken soluble BAFF(CycsBAFF) cDNA were firstly amplified by reverse transcription polymerase chain reaction(RT-PCR) method using total RNA isolated from Chinese 3-Yellow chicken spleen as template. After that, on one hand, CycsBAFF was expressed in E.Coli BL21(DE3). The recombinant protein was then purified, analyzed by Western blot, refolded, and the bioactivity was tested. On the other hand, CycsBAFF was inserted in frame into pPIC9 expression vector and was ready to be transferred into Pichia pastoris GS115. Besides, rabbit polyclonal antibody(pAb) to CycsBAFF was prepared and the titer was calculated by indirect ELISA, and the Binding activity with antigen was also detected by Western blot.1 Expression and activities assay of recombinant CycsBAFF protein expressed in E. Coli BL21(DE3)The fragment encoding CycsBAFF was inserted into pET-28a vector. After the recombinant vector was transformed into E.Coli BL21(DE3) which was then induced with IPTG, the recombinant protein with His₆-Tag was expressed with high efficiency as inclusion bodies and identified by SDS-PAGE and Western blot. After the target protein was purified by Ni²⁺-NTA and refolded by dialysis, the bioactivity of recombinant CycsBAFF was analyzed by stimulating and co-stimulating chicken bursal B cells with PMA, and both suggest obvious effect of promoting chicken bursal B cells survival. 2 Construction of pPIC9-CycsBAFFThe fragment encoding CycsBAFF was inserted in frame into pPIC9 expression vector. After digested, the recombinant plasmid pPIC9-CycsBAFF was ready to be transferred into Pichiapastoris GS115.3 Preparation and identification of polyclonal antibody to CycsBAFFPolyclonal antibody to CycsBAFF was prepared by endermic injection of recombinant CycsBAFF, and the titer was 20,000 which had been calculated by indirect ELISA , and the specificity was analyzed by Western blot. Our study paved the way for the establishment of a sandwich ELISA method.