The aim of this study was to construct the eukaryotic expression vector of pCMV-Myc-NOVA1 based on NOVA1 gene, and to screen the optimum expression condition after transfecting to PC12 cells, and further to explore the distribution of NOVA1 protein in PC12 cells using cell immunohistochemistry, and to identifyits anti-hypoxia activity. According to the NOVA1 gene sequence of NCBI database, we designed the upstream and downstream primers, and performed polymerase chain reaction (PCR) to amplify the full length cDNA coding sequence using pCR4-TOPO-NOVA1 as a template. The products were digested by restriction endonuclease SalⅠand XhoⅠ, and conjugated to the eukaryotic expression vector ofpCMV-Myc followed by validating by digestion and direct sequencing. Subsequently, the validated pCMV-Myc-NOVA1 was transfected to PC12 cells followed by optimizing of transfection ratio and transfection time, and identified by qPCR, Western blotting and cell immunohistochemistry respectively. After validation by digestion and direct sequencing, the eukaryotic expression vector of pCMV-Myc-NOVA1 was correctly constructed. The optimum transfection ratio of plasmid to Lipo 2000 was 1:2.5, and the optimum transfection time was 72 h. At the optimum transfection condition, the expression level of NOVA1 mRNA and protein significantly increased, and after transfection of pCMV-Myc-NOVA1, NOVA1 protein mainly distributed in cell nucleus and cytoplasm. After 6 h hypoxia, the cell proliferation activity was significantly increased compared to that of the control and pCMV-Myc group. Our findings provided a reference for exploring the mechanism of NOVA1, and also a technical support for potential drug development of NOVA1.
构建人NOVA1 基因的真核表达载体pCMV-Myc-NOVA1，转染PC12 细胞后筛选最佳转染条件，进而结合细胞免疫组织化学研究NOVA1 蛋白在PC12 细胞中的表达分布，并探究其抗低氧活性。根据NCBI 数据库NOVA1 基因序列设计上下游引物，以pCR4-TOPO-NOVA1 载体为模板采用聚合酶链式反应扩增获得NOVA1基因的全长cDNA 编码序列，限制性内切酶SalⅠ和XhoⅠ双酶切后插入pCMV-Myc 真核表达载体，酶切及直接测序验证后采用脂质体转染法转染入PC12 细胞，针对转染比例和转染时间进行优化，进而采用实时定量PCR和Western blotting 检测NOVA1 蛋白的表达，最后采用细胞免疫组织化学检测NOVA1 蛋白在PC12 细胞中的表达定位及其抗低氧活性。通过酶切和直接测序验证，成功构建了真核表达载体pCMV-Myc-NOVA1；质粒和Lipo2000 最佳转染比例为1:2.5，最佳转染时间为72 h；最佳转染条件下NOVA1 基因和蛋白的表达水平显著增加，转染pCMV-Myc-NOVA1 质粒后，NOVA1 蛋白主要分布于细胞核和细胞质；过表达NOVA1 的PC12 细胞增殖活性明显增加。本文采用分子克隆的方法成功构建了NOVA1 基因的真核表达载体，通过条件优化实现了高效表达并测定过表达NOVA1 蛋白具有明显的抗低氧活性，不仅为深入揭示NOVA1 蛋白的作用机理提供了重要参考，而且为NOVA1 蛋白潜在的药物开发提供了重要技术支撑。.
Keywords: anti-hypoxia; distribution; eukaryotic expression vector; human NOVA1 protein; transfection.