抗菌肽Defensin真核表达载体的构建及其在中国仓鼠卵巢细胞中的表达  

Construction of antibacterial peptide Defensin eukaryotic expression vector and its expression in Chinese hamster ovary cells

作　　者：许琴英[1] 朱家勇[2] 金小宝[2] 徐建华[2] 

机构地区：[1]广东医学院寄生虫学与临床寄生虫检验学教研室,广东省湛江市524023 [2]广东药学院寄生虫学教研室

出　　处：《中华生物医学工程杂志》2011年第6期509-512,共4页Chinese Journal of Biomedical Engineering

基　　金：湛江市科技攻关计划项目（2007C07004）

摘　　要：目的 构建家蝇幼虫抗菌肽的真核表达载体,并检测它在中国仓鼠卵巢（CHO）细胞中的表达情况.方法 以pUCm-T/Defensin为模板,设计带His标签特异引物,扩增C-末端融合6×His标签的Defensin基因的cDNA全长编码区序列,将其克隆至pcDNA3.1（＋）真核表达载体.经酶切及测序鉴定后,以阳离子脂质体Lipofect AmineTM 2000转染CHO细胞,G418抗性筛选稳定转染的阳性克隆细胞,以RT-PCR分析其mRNA的转录情况,通过Ni-NTA琼脂糖柱层析纯化目的蛋白并以Western免疫印迹鉴定蛋白质表达情况.结果 构建了pcDNA3.1 （＋）-Defensin-His表达质粒,建立了稳定转染的CHO细胞系.RT- PCR从阳性克隆CHO细胞系中扩增出320 bp大小的目的片段,从转录水平证实pcDNA3.1（＋）-Defensin-His重组细胞株表达了Defensin基因.Western免疫印迹检测可见相对分子质量为10000的阳性条带,表明Defensin-His在该细胞系中成功表达.结论 成功构建了质粒pcDNA3.1（＋）-Defensin-His,并获得了稳定表达的CHO-Defensin细胞株,有利于进一步研究家蝇幼虫抗菌肽Defensin基因的生物学功能.Objective To construct eukaryotic vector expressing antibacterial Defensin （pcDNA3.1 （＋）-Defensin-His） in housefly larva and detect its expression in Chinese hamster ovary （CHO） cells.Methods The whole coding sequence of Defensin gene cDNA with 6×His fusion tag at C-terminal was amplified using the Histagged specific primer with pUCm-T/Defensin as a model and was cloned into eukaryotic expression vectors pcDNA3.1 （＋）.After identification using restriction enzyme and sequencing technique,the recombinant pcDNA3.1 （＋）-Defensin-His was transfected into CHO cells via Lipofect AmineTM 2000.The stably transfected positive cloning cells were screened with G418 site.Transcription of mRNA was analyzed using RT-PCR,and the protein expressed was purified adopting Ni-NAT agar column and detected by Western blotting.Results The eukaryotic expression plasmid of pcDNA3.1 （＋） -Defensin-His was constructed and the CHO cell lines with stable transfection were established.The targeted 320 bp cDNA fragment was amplified from positive CHO cloning cells.Expression of Defensin gene in recombinant pcDNA3.1 （＋）-Defensin-His cell lines was proved by RT-PCR at the level of transcription.Western blotting showed a 10 000 positive band,indicating successful expression in Defensin-His cell lines.Conclusion Recombinant pcDAN3.1 （＋）-Defensin-His expression plasmid is successfully constructed and CHO-Defensin cell lines with stable expression are obtained,which benefit further researches on the biological activities of Defensin gene in housefly larva.

关 键 词：抗菌肽类 基因表达 载体 真核细胞 中国仓鼠卵巢细胞 

分 类 号：Q78[生物学—分子生物学]