Molecular biological research for correlation between isozyme type of Toxoplasma specific enzyme and virulence

我们以前曾报道在弓形虫快速繁殖的速殖子形式中存在一种新的核苷三磷酸水解酶(NTPase)。进一步检查发现速殖子含有两种形式的NTPase(NTPase-I和NTPase-II)。这些基因的核苷酸序列也已经确定。通过对许多弓形虫菌株这些同工酶的动力学研究，推测NTPase-II基本上存在于所有的弓形虫菌株中，NTPase-I只存在于强毒株中。在本研究中发现，许多菌株中同工酶的存在与这些同工酶基因的存在有关，即编码NTPase-I的基因在无毒菌株中不存在。也有人推测所有的菌株都有一个不产生RNA的假基因。然而，在本研究中也发现了一株没有假基因的无毒菌株。我们从弓形虫粘粒文库中克隆了含有NTPase基因启动子区域的基因。随后，我们研究了利用部分聚合酶链式反应产物重建功能基因的条件，并将构建的基因插入到抗环磷霉素的质粒中。然后将该质粒导入弓形虫无毒株。不幸的是，我们没有成功地在无毒菌株中生产NTPase-I。假基因可能在NTPase-I基因的表达中起关键作用。一株没有本研究中识别的假基因的无毒菌株可能对下一步的研究有用。

We have previously reported the presence of a novel nucleoside triphosphate hydrolase (NTPase) in the rapidly multiplying tachyzoite form of Toxoplasma gondii. On further examination, it was found that tachyzoites contain two forms of NTPase (NTPase-I and NTPase-II). The nucleotide sequences of these genes have also been determined. With kinetic studies for these isozymes of many Toxoplasma strains, it has been presumed that NTPase-II is essentially present in all the strains and NTPase-I is only present in virulent strains. In the present study, it was found that the presence of isozymes in many strains correlated to the presence of genes which corresponded to these isozyme, that is to say, the gene encoding NTPase-I was not present in avirulent strains. It has also been presumed that all the strains have a pseudogene which dose not produce mRNA.However, an avirulent strain without the pseudogene was also recognized in the present study. We have cloned the gene which contains a promoter area for NTPase gene from a cosmid library of Toxoplasma. Subsequently, we studied a condition for reconstruction of functional gene by using some PCR products, and the constructed gene was inserted to a Phlomycin resistant plasmid. This plasmid was then transfected to avirulent strain of Toxoplasma. Unfortunately, we did not succeed to produce NTPase-I in avirulent strain. Probably, the pseudogene may play a critical role for expression of NTPase-I gene. An avirulent strain without the pseudogene recognized in the present study may be useful for next study.