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

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%。

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

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-K5.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.