人组织型纤溶酶原激活剂真核表达质粒的构建及生物学活性

Construction and biological activities of human tPA eukaryotic expression plasmid.

作者信息

Wu Zhong-Jun, Yang Su-Fen, Zheng Shu-Sen, Shi De, Wu Wei-Wei, Li De-Wei

机构信息

Organ Transplant Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China.

出版信息

Hepatobiliary Pancreat Dis Int. 2004 Nov;3(4):511-5.

PMID:15567735

Abstract

BACKGROUND

Tissue-type plasminogen activator(tPA), which is highly efficient and specific in resolution of thrombus, could significantly improve the survival rate and life-quality of the patients with thrombosis. This study was designed to clone human tPA gene, construct eukaryotic expression plasmid pcDNA3.1(+)/tPA, and evaluate their biological activities.

METHODS

The tPA gene was obtained from dead human heart tissue with reverse transcriptase-polymerase chain reaction (RT-PCR). It was cloned to eukaryotic expression plasmid pcDNA3.1(+) by recombination strategy. The eukaryotic expression plasmid pcDNA3.1(+)/tPA was identified by restriction enzyme digestion and sequenced. The pcDNA3.1(+)/tPA was transfected into vascular smooth muscle cell (VSMC) by using lipofection. The tPA expression level was detected by Northern blot and dot blot, and the protein biological activities of tPA were detected by the fibrin plate technique.

RESULTS

The tPA gene was cloned and pcDNA3.1(+)/tPA was constructed. The tPA expression levels of mRNA and protein were highly increased after pcDNA3.1(+)/tPA transfected into VSMC, and the expression of tPA protein showed evident biological activities.

CONCLUSIONS

The present study has laid a foundation for further animal experiment in treating thrombus in transplanted organ by tPA gene transfection.

摘要

背景

组织型纤溶酶原激活剂（tPA）在血栓溶解方面具有高效性和特异性，可显著提高血栓形成患者的生存率和生活质量。本研究旨在克隆人tPA基因，构建真核表达质粒pcDNA3.1(+)/tPA，并评估其生物学活性。

方法

采用逆转录聚合酶链反应（RT-PCR）从人死亡心脏组织中获取tPA基因。通过重组策略将其克隆至真核表达质粒pcDNA3.1(+)。通过限制性内切酶消化和测序鉴定真核表达质粒pcDNA3.1(+)/tPA。利用脂质体转染法将pcDNA3.1(+)/tPA转染至血管平滑肌细胞（VSMC）。通过Northern印迹和斑点印迹检测tPA表达水平，采用纤维蛋白平板技术检测tPA的蛋白生物学活性。