人β-NGF融合蛋白真核表达载体的构建及其表达研究

Construction of eukaryotic expression vector of human beta-NGF fusion protein and its expression

研究构建一种能高效、稳定表达且易分离纯化人神经生长因子的pcDNA3.1-TM-FactorXa-β-NGF真核表达载体,此载体能在CHO细胞中表达具有高活性的rh-β-NGF。实验利用Trizol溶液从人胎盘中提取总RNA,利用分光光度计测定总RNA浓度后经RT-PCR克隆β-NGF基因,然后添加TM-FactorXa跨膜元件,实验成功构建了pcDNA3.1-TM-FactorXa-β-NGF真核表达载体,用LipofectamineTM 2000把此载体转染CHO细胞进行表达,收集表达酶切液,透析后经DEAE-Sepharose F F和Sephadex G-75层析,经Bradford法蛋白定量后,利用Western-blot和ELISA等方法对目标蛋白进行质量检测,研究结果表明:人β-NGF融合蛋白能高效表达,细胞总蛋白中β-NGF含量进行ELISA检测结果为602μg/mL,对照组<1.15E-03μg/mL。SDS-PAGE电泳结果在13KD有明显的目标蛋白条带。此载体是一种能高效、稳定表达重组人神经生长因子,且易分离纯化的真核表达载体。实验添加TM-FactorXa跨膜元件达到可控酶切,以便更有利于目的蛋白的分离收集纯化,为下一步开展rh-β-NGF活性鉴定奠定基础。

Our experiment aims to construct a eukaryotic expression vector of pcDNA3.1-TM-FactorXa-beta-NGF,which can express efficiently,stably and purify human nerve growth factor easily.This vector can express rh-beta-NGF with high activity in CHO cells.Total RNA was extracted from human placenta by Trizol solution.The concentration of total RNA was determined by spectrophotometer.Then the gene of beta-NGF was cloned by RT-PCR.The eukaryotic expression vector of pcDNA3.1-TMFactorXa-beta-NGF was successfully constructed by adding TM-FactorXa transmembrane element.The vector was transfected into CHO cells for expression by LipofectamineTM 2000.DEAE-Sepharose F and Sephadex G-75 chromatography were performed after dialysis of the collected digestion fluid.After quantifying the target protein by Bradford method,we detected the quality of the target protein by Western-blot and ELISA.The results showed that human beta-NGF fusion protein was highly expressed.The content of beta-NGF in total cell protein was 602μg/mL by ELISA,with control group<1.15E-03μg/mL.SDS-PAGE electrophoresis showed that there were obvious target protein bands at 13KD.This vector is a eukaryotic expression vector which can express recombinant human nerve growth factor efficiently and stably,and is easy to isolate and purify.TM-FactorXa transmembrane element was added to achieve controlled digestion in order to facilitate the separation,collection and purification of the target protein.It lays a foundation for further identification of rh-beta-NGF activity.