人血管内皮细胞生长因子基因的克隆与表达及其在原核系统中反义抑制。

Cloning and expression of human vascular endothelial growth factor gene and inhibition of its expression by antisense in prokaryotic system.

机构信息

Islamic Azad University, Research and Sciences Campus.

出版信息

Daru. 2010;18(4):281-5.

PMID:22615629

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3304348/

Abstract

BACKGROUND AND THE PURPOSE OF THE STUDY

Angiogenesis is an important process in physiology and disease pathogenesis and is controlled in a healthy body by a number of stimulatory and inhibitory factors. The aim of this study was to determine the effect of antisense transcript on the sense transcript of the endothelial growth factor (EGF) gene in bacterial system as an approach for the gene regulation in tumors.

METHODS

The hepatoma cell line (HepG2) was stimulated by PMA. VEGF mRNA was used for RT-PCR. VEGF cDNA was synthesised and cloned into T-vector pTZ57R, then sense fragment of VEGF subcloned into pACYC Duet-1 expression vector and antisense VEGF subcloned into pCDNA3 expression vector. Recombinant plasmids were transforemed into BL21 bacterial cells. Expression of recombinant plasmid was analysed by western blot technique.

RESULTS

The recombinant pCDNA3-VEGF (pYZantiVEGF) was successfully expressed in BL21 cells. Western blot analysis showed that the expression of VEGF decreased significantly in the cells transfected with VEGF antisense RNA compared with the pACYCDUET-1-VEGF (pYZsenseVEGF) transfected and control.

MAJOR CONCLUSIONS

The expression of VEGF in BL21 cells was strong. In vitro, antisense of VEGF inhibited VEGF expression significantly in BL21 cells.

摘要

背景与研究目的

血管生成是生理和疾病发病机制中的一个重要过程，在健康的体内受多种刺激和抑制因子的控制。本研究旨在探讨反义转录本在细菌系统中对内皮生长因子（EGF）基因的正义转录本的影响，作为肿瘤基因调控的一种方法。

方法

用 PMA 刺激肝癌细胞系（HepG2）。用 RT-PCR 检测 VEGF mRNA。合成 VEGF cDNA 并克隆到 T 载体 pTZ57R 中，然后将 VEGF 的正义片段亚克隆到 pACYC Duet-1 表达载体中，将反义 VEGF 亚克隆到 pCDNA3 表达载体中。将重组质粒转化到 BL21 细菌细胞中。通过 Western blot 技术分析重组质粒的表达。