抗菌肽Cecropin B—肿瘤血管生长抑制因子Kringle5融合蛋白表达载体的构建及其表达  

Construction and Expression of the Recombinant Expression Plasmid for A Novel Fusion Protein CecropinB-Kringle5

作　　者：樊钰虎[1] 冯瑄[1] 杨晓燕[1] 边六交[1] 

机构地区：[1]西北大学生命科学院基因工程中心,陕西西安710069

出　　处：《药物生物技术》2005年第5期281-284,290,共5页Pharmaceutical Biotechnology

基　　金：陕西省科技攻关项目(项目号:2003K10-G58)

摘　　要：通过重叠区扩增法人工合成抗菌肽Cecropin B(CB)基因,从ThioA/L15-7上卸下肿瘤血管生长抑制因子Kringle5(K5)基因,将两者按正确的阅读框架融合并定向克隆至大肠杆菌高效表达载体pET32a上,构建成pET32a/CB-K5重组载体。重组载体转化BL21(DE3)细胞后,提取质粒进行PCR鉴定和序列分析,证明重组质粒pET32a/CB-K5阅读框架和融合基因序列均与预期相符。实验结果显示,成功地构建了抗菌肽CecropinB(CB)和肿瘤血管生长抑制因子Kringle5(K5)融合蛋白的表达载体pET32a/CB-K5。转化E.coliBL21(DE3),SDS-PAGE分析显示,在IPTG诱导下,融合蛋白以包涵体的形式在重组转化菌株中获得高效表达,表达量达到菌体总蛋白的50%。After Kringle 5（K5） gene was isolated from plasmid ThioA/L15-7, the full-length antibacterial peptide cecropin B gene （CB） was artificially synthesized using gene splicing by overlap extension （gene SOEing） method and was genetically fused to the 5＇ end of K5 gene, and then the fusion gene CB- K5 was subcloned into expression plasmid pET32a and the termination codon was designed at the 3＇ end of K5 gene, the recombinant expression plasmid pET32a/CB-K5 could be obtained. By using PCR and sequence analysis, it was proved that all the tested clones contained the recombinant plasmid pET32a/ CB-KS. The novel fusion expression plasmid pET32a/CB-K5 was successfully constructed. The fusion protein is efficiently expressed after IPTG induction as a 37kD band on SDS-PAGE.

关 键 词：抗菌肽CECROPIN B 肿瘤血管生长抑制因子Kringle5 重叠区扩增法 融合表达载体 

分 类 号：Q786[生物学—分子生物学]