J Integr Plant Biol. ›› 2001, Vol. 43 ›› Issue (1): 46-50.

• Research Articles • Previous Articles     Next Articles

Expression of TNF-α Gene in Anabaena sp. PCC 7120 and Purification of Its Product by Affinity Chromatography

SHI Ding-Ji, YE Xin, ZHONG Hui, CAI Yi-Ying, SHAO Ning, PENG Guo-Hong, DONG Xiao-Jun, WANG Chun-Mei, OUYANG Ye-Xin, LI Zhen-Jia   

Abstract:

The effects of N (NaNO3) and C (NaAc) source in medium on the expression of tumor necrosis factor-α (TNF-α) gene in transgenic Anabaena sp. PCC 7120 were compared. The data showed that N source stabilized the expression of foreign protein and C source altered the synthesis of cell walls. Comparing several methods for breaking the cells, supersonic was able to extract TNF-α better than others. For purification of TNF-α, transgenic Anabaena cells were broken, the extracts were precipitated with ammonia sulfate, and the impure TNF-α was eluted from DEAE ion exchange chromatography. Electrophoresis (PAGE-SDS) showed a single band at 17 kD position.

TNF-a基因在鱼腥藻7120中的表达及其产物的亲和层析纯化
施定基1*  叶 欣1,2 钟 晖 蔡以滢1 邵 宁1彭国宏4 董晓军3 王春梅1 欧阳叶新1 李振甲3

(1. 中国科学院植物研究所,北京100093;2. 中国科技大学生命科学学院,合肥230027;
3. 中国人民解放军#&! 医院,北京10085;2. 中国科学院海洋研究所,青岛266071)

摘要: 用转入TNF-a基因并稳定表达了4 年多的丝状体蓝藻———鱼腥藻7120(Anabaena sp.PCC7120),研究比较了在培养液中氮源(NaNO3)和碳源(NaAc)对转基因藻生长和TNF-a基因的不同影响。氮源能稳定外源蛋白的表达,而碳源则影响细胞壁的合成。通过比较几种不同的破碎细胞的方法,发现超声破碎得到的可溶性总蛋白含量和TNF-a含量均要比冻融法高出1 倍左右,经过细胞破碎、硫酸铵分级沉淀、常压离子交换液相层析后,除去杂蛋白及大量容易堵柱的糖类等物质,最后通过亲和柱层析,分离出了纯的目标蛋白———重组肿瘤坏死因子(TNF-a)。经凝胶电泳鉴定为单一的谱带,分子量为17 kD.

关键词: 肿瘤坏死因子a(TNF-a);转基因蓝藻(鱼腥藻7120);亲和层析

Key words: tumor necrosis factor-α(TNF-α), transgenic cyanobacterium (Anabaena sp. PCC 7120), affinity chromatography

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