【摘要】 通过逆转录-聚合酶链式反应,从中国三黄鸡脾组织细胞中扩增得到459bp的鸡可溶性B淋巴细胞刺激因子(CycsBAFF)基因片段,将其克隆入原核表达载体pET-28a,转化大肠杆菌BL21(DE3)后高效表达了带有His6标签的包涵体形式重组蛋白,通过聚丙烯酰胺凝胶电泳及免疫转印检测鉴定。表达产物经Ni2+-NTA纯化后,经透析复性得到活性目的蛋白,通过单独刺激以及与PMA共刺激体外培养的鸡法氏囊B细胞,均有明显的促法氏囊B细胞存活的效应。更多还原

【Abstract】 459 bp Chinese 3-Yellow chicken soluble BAFF（CycsBAFF） cDNA was amplified by reverse transcription polymerase chain reaction（RT-PCR） method using total RNA isolated from chicken spleen as template, and then inserted into pET-28a vector. After the recombinant vector was transformed into E.coli BL21（DE3） which was then induced with Isopropyl-β-D-thiogalactopyanoside（IPTG）, the recombinant protein with polyhistidine tag（His₆-Tag） was expressed with high efficiency as inclusion bodies and identified by SodiumDedecylSulfate-Polyacrylamide Gel Electrophoresis（SDS-PAGE） and Western blot. After the target protein was purified by Ni 2+ -nitrilotriacetic acid（Ni 2+ -NTA） and refolded by dialysis, the bioactivity of recombinant CycsBAFF was analyzed by stimulating and co-stimulating chicken bursal B cells with Phorbol 12-Myristate 13-Acetate（PMA）, and both suggest obvious effects of promoting chicken bursal B cell survival.更多还原