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

本项目确定弓形虫RH株中存在两个NTPase同工酶（NTPase- i和NTPase- ii），克隆了RH株的两个编码NTPase- i和NTPase- ii的基因，以及Beverly株的一个编码NTPase- ii的基因，并测定了这些基因的核苷酸序列。酶动力学研究和PCR基因扩增结果表明，所有菌株普遍具有NTPase-II同工酶，只有强毒株同时具有两种同工酶。这些结果将发表在1995年5月号的《生物化学杂志》上。上述结果表明，在大肠杆菌细胞中表达的靶同工酶是NTPase-II，该酶在所有菌株中普遍存在。因此，我们构建了包含编码NTPase-II基因的表达质粒，并在大肠杆菌细胞中进行了基因表达测试。以异丙基β - d -硫代半乳糖苷（IPTG）诱导的pGEMEX-1和热诱导的pPL-lambda为表达载体。经sds -聚丙烯酰胺凝胶电泳和抗ntpase抗体和过氧化物酶偶联抗体western blot分析，证实IPTG处理的大肠杆菌细胞产生NTPase-II蛋白。热感应也有类似的结果。虽然我们成功地利用IPTG和热诱导质粒在大肠杆菌细胞中表达了NTPase- ii蛋白，但由于NTPase蛋白可能对大肠杆菌细胞有毒性，这两种表达系统的效率都不足。这些大肠杆菌细胞裂解物可用于单克隆抗体的诊断ELISA方法，但需要更高效的表达系统来简化ELISA方法。在本项目中，还发现NTPase基因编码额外的25个氨基酸，具有信号肽的一些特性。这些数据表明NTPase是一种分泌酶。我们发现NTPase实际上是由寄生虫向宿主细胞分泌的。这些结果发表在《实验寄生虫学》（79,301-311,1994）。

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) .