摘要
HIV I反式激活蛋白TAT中的一段富含碱性氨基酸的短序列具有独特的跨膜转运能力 ,被称为TAT蛋白转导结构域 (proteintransductiondomain ,PTD)。从人肺cDNA文库中PCR扩增hCu ,Zn SOD基因 ,插入pUC19测序 ;以测序质粒为模板 ,再用含TATPTD编码序列的 3’引物PCR扩增得hCu ,Zn SOD TATPTD融合基因 ,并插入 pUC19。将SOD基因和SOD TATPTD融合基因分别亚克隆至温控表达载体 pJW 2 ,转化大肠杆菌DH5α。SDS PAGE和Westernblot结果表明 ,热击诱导 4h后 ,分别在 19ku和 2 2ku处出现SOD和SOD TATPTD目标表达条带 ,表达蛋白以包含体形式存在。分别提取SOD和SOD TATPTD包含体 ,复性纯化后SOD和SOD PTD重组蛋白均具有特异的SOD酶活性 ,其比活性分别为 12× 10 3 NU/mg和 9.9× 10 3 NU/mg。细胞试验结果表明 ,与SOD相比较 ,SOD TATPTD融合蛋白能显著提高细胞内SOD酶活力。
Trans-activating protein (TAT) encoded by HIV-I has been accorded the unusual ability to translocate across cell membranes in a receptor-independent and temperature-independent manner, and this activity has been mapped to a highly basic series of residues currently termed a protein transduction domain (PTD).Human gene was amplified by PCR from human lung cDNA library, inserted into pUC19 and identified by sequencing. The plasmid was used as the template for PCR amplification of Cu,Zn-SOD-TAT PTD fusion gene with a primer which contained TAT PTD coding frame. The Cu,Zn-SOD gene and Cu,Zn-SOD-TAT PTD fusion gene were sub-cloned into temperature-inducing expression vector pJW2 and the recombinant plasmids were transformed into E. coli DH5α,respectively. The results of SDS-PAGE and western blot showed the nascent band of recombinant protein was obviously appeared with a molecular weight of 19 ku and 22 ku,respectively, and recombinant proteins could be specifically recognized by monoclonal antibody against human Cu,Zn-SOD. Biossay in vitro showed that specific activity of purified rhCu,Zn-SOD and rhCu,Zn-SOD-TAT PTD were 12×10 3 NU/mg and 9.9×10 3 NU/mg respectively. Comparied with rhCu, Zn-SOD,the fusion protein rhCu,Zn-SOD-TAT PTD might translocate through cell membrane and enter cultured Wish cells.
出处
《药物生物技术》
CAS
CSCD
2004年第1期11-15,共5页
Pharmaceutical Biotechnology
基金
安徽省 2 0 0 1年度优秀青年科技基金 (2 0 0 1 2 5)
