人源抗菌肽LL-37表达载体的构建及其在毕赤酵母中的表达  

Construction of human antimicrobial peptide LL-37 vector and its expression in Pichia pastoris

作　　者：陆建荣[1] 王惠民[2] 吴萍[1] 黄松平[1] 常秋月 凌勇武[1] 倪晓蓉[1] 

机构地区：[1]南通市第三人民医院感染病实验室,南通226006 [2]南通大学附属医院检验科,南通226001 [3]南通市中心血站,南通226006

出　　处：《第二军医大学学报》2007年第8期833-837,共5页Academic Journal of Second Military Medical University

基　　金：江苏省南通市科学技术委员会资助项目(No.S40032)~~

摘　　要：目的:构建人源抗菌肽LL-37的表达载体pPIC9-LL-37,将其转化毕赤酵母GS115,诱导LL-37表达。方法:根据抗菌肽LL-37氨基酸序列及毕赤酵母偏爱密码子,应用互补延伸PCR技术扩增抗菌肽LL-37基因,定向克隆到毕赤酵母表达载体pPIC9上,转化E.coli DH5a.构建重组分泌型酵母表达载体pPIC9-LL-37.PCR鉴定并测序;原生质球法转化毕赤酵母GS115.PCR扩增鉴定:甲醇诱导LL-37表达,筛选最高表达株.检测表达产物对大肠杆菌的抑菌活性,并对其进行聚丙烯酰胺凝胶电泳和Western印迹分析。结果:pPIC9-LL-37载体构建成功:转化毕赤酵母后PCR鉴定出LL-37基因:0.5%甲醇能诱导LL-37高表达,筛选出最高表达株,发酵上清约含LL-37 0.5μg/ml,对E.coli具有较强的抑菌活性,电泳及Western印迹分析证实表达产物为LL-37。结论:成功构建pPIC9-LL-37载体.转化毕赤酵母后.经甲醇诱导能高表达分泌LL-37,表达的LL-37蛋白具有较强的抑菌活性。Objective： To construct a eukaryotic expression vector of human antimicrobial peptide LL-37 （ pPIC9 LL-37） and to express it in P. pastoris. Methods： The full-length gene encoding antimicrobial peptide LL-37 was synthesized by overlap extension-PCR method using the sequence of LL37 and P. pastoris biased codon. The full-length gene was cloned into pPIC9 vector and the product was transformed into E. coli DHS to construct expression vector pPICg-LL-37. After identification by PCR and sequencing, pPICg-LL-37 was used to transfect P. pastoris. The expression of LL-37 was induced by methanol and the highest expressing strain was screened. The concentrated fermentation product was analyzed by Tricine-SDS-PAGE and Western-blot, and the antibacterial activity of the expression product on E. coli. DHSα was tested. Results： The eukaryotic expression vector pPICg-LL-37 was successfully constructed. The fusion of LL-37 gene into P. pastoris was confirmed by PCR. High expression of LL-37 was expressed by 0.5%. methanol and the highest expressing strain was screened out. The fermentation supernatant contained 0. 5 μg/ml LL-37 and had strong antibacterial activity against E. coli. Tricine-SDS-PAGE and Western-blot analysis confirmed that the product was LL-37. Conclusion： We have successfully constructed pPIC9-LL-37 for transfecting P. pastori Methanol can induce the high expression of LL-37 and the expressed LL-37 has strong antimicrobial activity.

关 键 词：LL-37 毕赤酵母 遗传载体 基因表达 

分 类 号：Q78[生物学—分子生物学]