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Cloning and expression of a gene encoding NTPase of Toxoplasma gondii (practical use for diagnosis)

弓形虫 NTPase 基因的克隆和表达（诊断实用）

基本信息

批准号：
05670238
负责人：
ASAI Takashi
金额：
$ 1.15万
依托单位：
Keio University, School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05670238/
关键词：
Parasite Protozoa Toxoplasma gondii NTPase cDNA Gene expression Serodiagnosis cDNA シグナルペプタイド

项目摘要

In this project, we defined that there were two NTPase isozymes (NTPase-I and NTPase-II) in the RH strain of Toxoplasma gondii and two genes encoding NTPase-I and NTPase-II of the RH strain and a gene encoding NTPase-II of the Beverly strain were cloned and the nucleotide sequences of these genes were determined. It was found that all the strains as tested have NTPase-II universally and only virulent strains have both isozymes by the enzyme kinetic studies and PCR gene amplification. These results will be published in May issue of Journal of Biological Chemistry, 1995. Above results suggested that a target isozyme to express in E.coli cells was NTPase-II which was present universally in all strains. Thus we constructed an expression plasmid including a gene encoding NTPase-II and tested gene expression in E.coli cell. Isopropyl beta-D-thiogalactopyranoside (IPTG) -inducible pGEMEX-1 and heat-inducible pPL-lambda were used as the expression vectors. It was confirmed that the E.coli cells treated with IPTG produced NTPase-II protein by SDS-polyacrylamide gel electrophoresis and following western blot analysis using anti-NTPase antibody and peroxidase conjugated antibody. Heat induction was a similar result. Although we succeeded to express NTPase-II protein in E.coli cells using IPTG and heat inducible plasmids, the efficiencies of both expression systems were insufficient because of a possibility that NTPase protein was toxic to E.coli cell. It is possible to use these E.coli cell lysates for a diagnostic ELISA method using a monoclonal antibody, however, more efficient expression system will be necessary to simplify the ELISA method. In this project, it was also found that NTPase gene was coding extra 25 aminoacids with some properties of a signal peptide. The data suggested that NTPase was a secretory enzyme. We found that NTPase was secreted form parasite to host cell actually. These results were published in Experimental Parasitology (79,301-311,1994) .

本研究确定了弓形虫RH株存在两种NTPase-I和NTPase-II同工酶，并克隆了RH株NTPase-I和NTPase-II的编码基因和Beverly株NTPase-II的编码基因，测定了它们的核苷酸序列。酶动力学研究和PCR基因扩增结果表明，供试菌株普遍具有NTPase-II，只有强毒株具有两种同工酶。这些结果将发表在1995年5月出版的《生物化学杂志》上。以上结果表明，在大肠杆菌细胞中表达的目标同工酶是NTPase-II，该同工酶在所有菌株中普遍存在。因此，我们构建了包含编码NTPase-II的基因的表达质粒，并在大肠杆菌细胞中测试了基因表达。将异丙基β-D-硫代半乳糖苷（IPTG）诱导型pGEMEX-1和热诱导型pPL-λ用作表达载体。通过SDS-聚丙烯酰胺凝胶电泳和使用抗NTPase-II抗体和过氧化物酶缀合抗体的蛋白质印迹分析，证实用IPTG处理的大肠杆菌细胞产生NTPase-II蛋白。热感应是类似的结果。虽然我们成功地使用IPTG和热诱导质粒在大肠杆菌细胞中表达NTPase-II蛋白，但是由于NTPase-II蛋白对大肠杆菌细胞有毒性的可能性，两种表达系统的效率都是不足的。可以将这些大肠杆菌细胞裂解物用于使用单克隆抗体的诊断ELISA方法，然而，需要更有效的表达系统来简化ELISA方法。在本研究中，还发现NTR基因编码额外的25个氨基酸，具有信号肽的某些特性。提示NTR是一种分泌型酶。我们发现NTR实际上是由寄生虫分泌到宿主细胞中的。这些结果发表在《实验寄生虫学》（79，301 - 311，1994）上。