| 摘要: |
| yC16(cDNA,2.9kb)是果蝇yema基因的转录片段。为制备yC16编码蛋白作为免疫原,本实验用GST融合基因表达载体pGEX-2T在Escherichia coli中合成该基因的蛋白,并与谷胱苷肽-s-转移酶(GST)融合形成融合蛋白。结果由裂解细菌所得到的融合蛋白为水溶性,并且可以在非变性的条件下,通过亲和层析作用在固定化的谷胱苷肽上加以纯化,然后再利用具有识别特异位点的蛋白水解酶——凝血酶或凝血因子Xa的切割作用,将融合蛋白中的GST成分除掉。实验结果表明,使用pGEX载体合成克隆基因多肽,是一种有效而可靠的方法,步骤简单。 |
| 关键词: 载体pGEX 克隆基因yC16 融合蛋白 纯化 |
| DOI： |
| 投稿时间：1998-12-15修订日期：1999-02-05 |
| 基金项目: |
|
| Expression of Gene yC16 in Escherichia coli and Purification of the Gene Product |
|
Lan Guobao
|
| (Guangxi Institute of Oceanology, 92 East Changqinglu, Beihai, Guangxi, 536000) |
| Abstract: |
| yC16 (cDNA, 2.9kb), cloned in Bluescript ⅡSK, is a transcript of yemanuclein-α gene of Drosophila. For preparation of the protein encoded by yC16 as immunogen, pGEX-2T, the GST fusion gene expression vector[2] was used to synthesize the protein in E. coli as fusion with the C-terminus of Sj26, a26-kDa glutathione S-transferase(GST). The result showed that the fusion protein was soluble in aqueous solution and can be purified from crude bacterial lysate under non-denaturing conditions by affinity chromatogaphy on immobilized glutathione, and further purification of the protein of yC16 could be done by cleavage with site-specific protease, thrombine or blood coagulation factor Xa to remove the GST carrier. Experiment results suggests that using pGEX vectors to synthesize polypeptides from cloned genes is an efficient and reliable method with simple procedures. |
| Key words: vecter pGEX cloned gene yC16 fusion protein purification |
