荧光标记人透明带融合蛋白质粒的构建及其在中国仓鼠卵巢细胞内的表达

Construction of fluorescent human zona pellucida fusion protein expression vectors and expression in CHO cells

目的 :分别构建人透明带蛋白(zona pellucida, ZP)基因ZP1、ZP2、ZP3、ZP4与荧光蛋白基因的融合蛋白表达载体,并在中国仓鼠卵巢(Chinese hamster ovary, CHO)细胞和卵母细胞中表达这些荧光标记的重组融合蛋白。方法:设计合适的PCR引物,采用GeneArt Gibson Assembly克隆技术,将荧光蛋白基因与人透明带基因形成融合基因片段,用特定的双酶分别酶切中间载体pENTR1A(no ccdB)和融合基因片段后,用T4 DNA连接酶将融合基因片段连入中间载体,再通过Gateway克隆技术将中间载体上的融合基因转入表达载体pInducer20,并对产物进行DNA测序。验证融合基因构建成功后,将含透明带融合基因的质粒进行转化,筛选阳性克隆并扩增、抽提质粒。用脂质体转染法将含融合基因的表达载体转染入CHO细胞,采用逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction, RT-PCR)检测透明带融合基因mRNA的表达,用蛋白印迹法检测透明带融合蛋白的表达,并在激光共聚焦显微镜下观察透明带融合蛋白在CHO细胞内的表达及分布。结果:成功构建所需载体并转染CHO细胞;经RTPCR检测证实人透明带ZP1、ZP2、ZP3、ZP4 mRNA在CHO细胞中表达;经蛋白印迹法检测证实CHO细胞中有重组融合蛋白质的表达;在激光共聚焦显微镜下观察到重组融合蛋白在CHO细胞内表达和定位。结论:本研究成功构建了人类透明带4个基因的荧光融合蛋白表达质粒,以此为实验工具,今后可用于诊断卵子异常导致不孕的病因机制研究。

Objective: To construct expression vectors of the fluorescent fusion proteins of all four human zona pellucida(ZP) genes respectively, and explore their expression in Chinese hamster ovary(CHO) cells and oocytes in vitro.Methods: The desired fluorescent protein sequence was inserted into a specific site of the zona pellucida gene sequence using the Gibson Assembly multi-fragment one-step splicing method to form the desired fusion gene fragment, and both ends of the fragment contained a specific sticky cleavage site of the entry vector. The entry vector pENTR 1 A(no ccdB)and the fusion gene fragment were digested with a specific double enzyme, and the target gene fragment was ligated into the entry vector by T4 DNA ligase. Transfer genes from entry vector into destination vector via the LR reaction of the Gateway cloning technology. The plasmids validated by DNA sequencing were amplified and transfected into CHO cells.The expression of desired fusion protein was proved by RT-PCR and Western Blot. The distribution of target proteins in cytoplasm of CHO cells was visualized by laser confocal microscopy. Results: The expression vectors were successfully constructed, and the expression of each ZP protein in CHO cells was detected by RT-PCR and Western blot. The laser confocal microscopy revealed the distribution of ZP proteins. Conclusions: In this study, we successfully constructed fluorescent fusion protein expression plasmids of human zona pellucida genes, enabling the diagnosis of infertility caused by oocyte abnormalities.