抗菌肽Cecropin B-人溶菌酶融合蛋白表达载体的构建

Construction of the Recombinant Plasmid Expressing A Novel Fusion Protein Cecropin B-human Lysozyme

目的是构建抗菌肽B (CecropinB)和人溶菌酶 (hLyso)的融合蛋白表达载体。从pUC118-hLyso上卸下人溶菌酶基因后 ,通过重叠区扩增法人工合成抗菌肽B基因 ,并将其融合到人溶菌酶基因的 5’端。将抗菌肽B基因和人溶菌酶基因按正确的阅读框架定向克隆至大肠杆菌高效表达载体pET32a,终止子位于人溶菌酶基因的 3’端。PCR鉴定及序列分析表明 ,所转化的BL2 1(DE3)菌落中含有插入CecropinB -hLyso基因的重组质粒pET32a -CB -hLyso。

The objective of this paper is to construct the recombinant plasmid expressing a novel fusion protein antibacterial peptide cecropin B - human lysozyme.Human lysozyme gene (hLyso) was isolated from plasmid pUC118-hLyso,the full-length antibacterial peptide cecropin B gene (Cecropin B) was artificially synthesized through using gene splicing by overlap extension (gene SOEing) method and then was genetically fused to the 5' end of hLyso gene.By subcloning cecropin B gene and hLyso gene into expression plasmid pET-32a,the recombinant expression plasmid pET32a-CB-hLyso can be obtained,and the termination codon was designed at the 3' end of hLyso gene.By using PCR and sequence analyses,it can be proved that all the tested clones contain the recombinant plasmid pET32a-CB-hLyso.And thus,it can be concluded that the novel fusion expression plasmid pET32a-CB-hLyso is successfully constructed.