斑点叉尾NK-lysin成熟肽在毕赤酵母中的表达

Expression of Ictalurus punctatus NK- lysin Mature Peptide in Pichia pastoris

NK-lysin是毒性T淋巴细胞和自然杀伤细胞产生的具有抗菌作用的多肽,在机体免疫系统中发挥重要作用,被认为是抗生素的理想替代品。NK-lysin的生物学功能主要由其成熟肽(m NK-lysin)决定。本文在已有斑点叉尾(Ictalurus punctatus)NK-lysin成熟肽原核重组表达载体p ET-32a(+)-m NK-lysin的基础上,通过PCR在m NK-lysin的5’端和3’端分别添加EcoRI和Xba I酶切位点,并在3’端添加6×His标签以便于纯化;扩增到的片段与表达载体p PICZαA连接构建重组表达载体p PICZαA-m NK-lysin,转化至毕赤酵母X-33细胞中,通过博来霉素筛选以及对酵母转化子基因组的PCR鉴定得到高拷贝酵母转化子;在29℃,250 rpm,采用0.5%甲醇进行诱导表达;表达产物经固化金属离子亲和层析(IMAC)获得纯化的重组体m NK-lysin。经Tricine-SDS-PAGE分析,其分子量约为11.5 k D;经Western blot分析,证明m NK-lysin在毕赤酵母中成功表达。

NK- lysins are antimicrobial peptides that are produced in cytotoxic T lymphocytes（ CTLs） and natural killer（ NK） cells and play an important role in immune response against microbial invasion. Thus,they are considered to be good substitutes for traditional antibiotics. In the present study,the prokaryotic recombinant expression vector ＂p ET- 32a（ ＋）- m NK- lysin＂for Ictalurus punctatus NK- lysin mature peptide,which was constructed in previous study,was used as the template of PCR. EcoRⅠand XbaⅠrestriction sites were added to 5＇and 3＇ends of the m NK- lysin fragment respectively,and 6 x His tag was also added to 3＇end to purify the target protein easily. The m NK- lysin fragment was ligated to p PICZαA vector to construct a recombinant expression vector p PICZαA- m NK- lysin,which then was transformed into competent Pichia pastoris X- 33. The yeast transformants containing multicopy gene insertions were selected by using Zeocin and PCR identification for genomic DNA from yeast transformants. Recombinant m NK- lysin was induced with 0. 5%methanol at 29 ℃ and 250 rpm. The expression product was purified by immobilized metal affinity chromatography（ IMAC）.Tricine- SDS- PAGE analysis indicated that molecular mass of the purified recombinant m NK- lysin was11. 5k D. Furthermore,Western blot analysis demonstrated that the recombinant m NK- lysin was expressed successfully in P. pastoris.