摘要
球虫子孢子表面抗原TA4(25kD)是通过二硫键连接8和17kD 2条多肤形成的,在孢子化后期合成。E.tenella BJ株的TA4基因同国外株的同源性为99％。本试验进行了TA4基因的原核融合表达,并对表达蛋白的SDS-PAGE电泳特性进行探究。试验发现,以pGEX-KG为载体,用IPTG可诱导TA4基因在E.coli BL21(DE3)中的表达。SDS-PAGE表明,融合蛋白大小约43 kD,而非推测的51 kD,诱导6 h的蛋白表达量即可达到较高水平,表达量约为31％。采用抗GST抗体进行Western blotting,成功检测到了特异性条带。说明SDS-PAGE前的样品煮沸处理可使连接TA4 2条多肽的二硫键断裂,只有17 kD的多肽可与26 kD GST蛋白结合。样品采用反复冻融法处理,加入还原型谷胱苷肽后,采用GST凝胶回收柱Sepharose 4B纯化的融合蛋白主要为51 kD的单一条带,煮沸变性后电泳,则43 kD带含量增加。说明纯化过程可影响GST-TA4融合蛋白的特性。
The antigen TA4 is a 25 kD protein that is composed of 2 polypeptides of 17 and 8 kD respectively, linked by a disulfide bond. It is located on the surface of sporozoites and synthesized throughout the latter half period of sporulation. The TA4 gene of Eimeria tenella BJ strain has a 99% homology with the published data. In this study, the gene TA4 of E. tenella BJ strain was ligated to the prokaryotic expression vector pGEX-KG and induced to express in E.coli BL21 (DE3). The results indicated that the fusion GST-TA4 protein is about 43 kD instead of 51 kD shown from SDS-PAGE result. This means that the disulfide bond linking the 17 and 8 kD polypeptides would be broken during the boiling pre-treatment of samples before SDS-PAGE, and only the 17 kD polypeptide was linked to the 26 kD GST protein. In the large-scale purification of the fusion protein, the cells were lysed with repeated freeze-melt; the inclusion bodies were denatured and dissolved in 8mol·L-1 urine solution containing reduced glutathione. The recombinant protein purified with glutathione Sepharose 4B showed 2 bands of 51 kD (primary) and 43 kD (secondary) respectively in SDS-PAGE. This means that the disulfide bond would not break completely in purifying conditions of this experiment. In the further investigation, when the purified protein was treated boiledly, the ratio of 51 kD protein decreased while that of 43 kD protein increased simultaneously. This means that the purifying conditions v/ill affect the structure of GST-TA4 protein. Further investigation should be done to compare the immune-protective effects of different kinds of fusion proteins.
出处
《中国农业科学》
CAS
CSCD
北大核心
2004年第8期1217-1221,共5页
Scientia Agricultura Sinica
基金
国家自然科学基金资助项目(30170695)
国家"863"计划资助项目(2002AA241331)
