Anti-LMP1scFv/tP融合蛋白基因递送载体在大肠杆菌中的表达与纯化  

Expression and Purification of a Foreign Gene Delivery Fusion Protein anti-LMP1scFv/tP in Escherichia coli

作　　者：黄秀兰[1] 唐旭东[2] 林观平[2] 李祥勇[2] 崔国辉[2] 周克元[2] 

机构地区：[1]广东医学院附属医院儿科研究室,湛江524001 [2]广东医学院生物化学与分子生物学研究所,湛江524023

出　　处：《中国细胞生物学学报》2010年第3期377-386,共10页Chinese Journal of Cell Biology

基　　金：supported by Youth Foundation of Guangdong MedicalCollege(No.Q2007042)~~

摘　　要：潜伏膜蛋白1(LMP1)是一种EB病毒编码的膜蛋白,组成型表达于各种肿瘤细胞表面。通过体外基因拼装技术,构建抗LMP1单链抗体/鱼精蛋白截短体融合蛋白基因(anti-LMP1scFv/tP)。在抗LMP1单链抗体基因的3'末端连接上编码鱼精蛋白截短体的基因序列,设计anti-LMP1scFv/tP融合蛋白基因。采用PCR为基础的基因拼接获得anti-LMP1scFv/tP。在大肠杆菌Rosseta中诱导表达anti-LMP1scFv/tP融合蛋白并利用其His标签在变性条件下通过Ni^(2+)亲合层析介质进行纯化,纯化产物通过尿素浓度梯度透析的方法进行复性。间接免疫荧光染色检测anti-LMP1scFv/tP融合蛋白的抗原结合活性,DNA凝胶迁移阻滞实验检测anti-LMP1scFv/tP的DNA结合活性,结果显示anti-LMP1scFv/tP融合蛋白能够与LMP1阳性表达的CNE1-GL细胞结合,而不能与LMP1阴性表达的CNE1细胞结合;同时复性anti-LMP1scFv/tP融合蛋白也具有与DNA结合的活性。本研究为该单链抗体用于靶向输送外源核苷酸的研究奠定了基础。Epstein-B arr virus （EB V）-encoded latent membrane protein- 1 （LMP 1） constitutively expresses on cell surface of various cancer ceils. In this study, we aim to construct a fusion protein that contains a domain of the single chain of the human variable fragment （scFv） against LMP1 and a nucleotide-binding domain of truncated protamine （tP）. The fusion protein anti-LMP1 scFv/tP was designed with the tP coding sequence linked to the 3＇- terminus of the scFv against LMP1, which will not only target LMP1 but also maintain nucleotide binding activity. The anti-LMP1 scFv/tP gene was obtained by PCR-based gene assembly. The fusion protein was expressed in inclusion bodies in Escherichia coli Rosseta and was purified efficiently by immobilized metal （Ni2.） affinity chromatography with His-tag under denaturation condition, and then the purified product was refolded by dialysis against urea concentration gradient. Indirect cellular immunofluorescence staining confirmed that refolded anti- LMP1 scFv/tP maintained antigen binding activity, which could bind to CNE1-GL cells expressing LMP1 and couldn＇t bind to CNE1 cells not expressing LMP1. Gel shift assay demonstrated that refolded anti-LMPlscFv/tP had DNA binding activity. Thus, the fusion protein provides a basis for further application for targeting gene delivery to LMP1 expressing tumor cells.

关 键 词：潜伏膜蛋白1 单链抗体 鱼精蛋白截短体 融合蛋白 基因递送 

分 类 号：Q78[生物学—分子生物学]