Bm-TFF2在毕赤酵母中的表达及鉴定  

Expression and identification of Bm-TFF2 in Pichia pastoris

作　　者：余果宇[1,2] 张红芸[3] 张勇[1] 江萍[1] 李文辉[1] 张云[1] 

机构地区：[1]中国科学院昆明动物研究所动物模型与人类疾病机理重点实验室,云南昆明650223 [2]昆明医学院生物化学教研室,云南昆明650500 [3]昆明医学院第一附属医院妇产科,云南昆明650032

出　　处：《Zoological Research》2010年第6期565-569,共5页动物学研究（英文）

基　　金："973"项目(2010CB529805);国家基金委重点项目(30630014);国家基金委面上项目(30570359);中国科学院重要方向项目(KSCX2-YW-R-088)

摘　　要：Bm-TFF2,一种从大蹼铃蟾皮肤分泌物中分离得到的两栖类三叶因子,具有比人三叶因子更强的生物学活性。该研究以Bm-TFF2的cDNA为模板,利用PCR方法扩增Bm-TFF2基因,然后插到含有AOX1启动子和α-因子信号肽序列的表达载体pPIC9K中,采用毕赤酵母表达系统进行分泌表达,并用G418筛选高拷贝整合转化子。SDS-PAGE和Western blotting都检测到Bm-TFF2被分泌性表达于酵母上清。在1%的甲醇诱导表达不同时间后,重组蛋白在72h的表达量最大,可达50mg/L;而不同浓度的饱和硫酸铵沉淀菌液上清时,80%的饱和硫酸铵沉淀量最大。这些结果表明,重组质粒Bm-TFF2-pPIC9K成功构建并在真核细胞中高效表达,这为进一步研究Bm-TFF2的生物学活性及其结构与功能关系奠定了基础。Bm-TFF2, an amphibian trefoil factor, which is isolated from skin secretions of frog Bombina maxima, has much stronger biological activities than human TFFs. In the present study, Bm-TFF2 gene was amplified by polymerase chain reaction （PCR） from its cDNA and cloned into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and α-factor leader sequence. Multi-copies insertion transformants were screened on G418 plates. After the induction by 1% methanol for 72 hours, the expression of Bm-TFF2 came up to the best quantity which was about 50 mg in 1L medium, and 80% saturation ammonium sulfate was suitable to collect the Bm-TFF2 protein, as identified by SDS-PAGE and Western blotting assay. The results showed that the plasmid of Bm-TFF2-pPIC9K was constructed successfully and expressed abundantly in eukaryotic expression system, which lies basis for researching further the biological activities and the relationship of structure and functions of Bm-TFF2.

关 键 词：Bm-TFF2 毕赤酵母 表达 鉴定 

分 类 号：Q51[生物学—生物化学]