人源多肽抗生素LL-37真核表达载体的构建

Construction of eukaryotic expression vector of human antimicrobial peptide LL-37

目的构建抗菌肽LL-37的表达载体pPIC9-LL-37,转化毕赤酵母GS115以获得表达LL-37的工程菌。方法通过重叠区扩增法人工合成抗菌肽LL-37的基因,定向克隆到毕赤酵母表达载体pPIC9上,转化大肠杆菌DH5α,提取质粒,PCR鉴定及序列分析后转化毕赤酵母GS115。MM、MD平板筛选His+Mut+、His+Mut-表型,PCR扩增鉴定基因的插入。结果PCR鉴定及序列分析表明抗菌肽LL-37基因正确插入载体pPIC9,MM、MD平板筛选及PCR鉴定获得10个His+Mut+、9个His+Mut-克隆。结论获得含LL-37基因的工程菌。

Objective To construct eukaryotic expression vector of human antimicrobial peptide LL-37 pPIC9-LL-37, and transform the plasmid pPIC9-LL-37 into P. pastoris GS115 to obtain the recombinant P. pastoris strains. Methods The full-length of antimicrobial peptide LL-37 gene was artificially synthesized by overlap extension method and was fused to pPIC9 and then the fused plasmid was transformed into E. coli DH5α. After analysis by PCR and sequencing,the plasmid pPIC9-LL-37 was transformed into P. pastoris. The colonies exhibiting the phenotype of His ＋ Mut ＋ or His ＋ Mut - were screened by means of MM and MD plates and the insertion was confirmed by PCR. Results The results of PCR and sequencing confirmed that the LL-37 gene was correctly inserted into pPIC9. The colonies of 10 His ＋ Mut ＋ and 9 His ＋ Mut- were obtained by means of MM and MD plates screening and were confirmed by PCR. Conclsion The recombinant P. pastoris strains containing LL-37 were successfully obtained.