sag5b:一种用于鉴别弓形虫虫株毒力的新基因(英文)

目的本文作者比较了强毒株(RH)弓形虫与弱毒株(Prugniaud)基因组中一种可能成为基因标志物的基因片断——sag5b的差异。PCR扩增出的sag5b片断依次亚克隆进入T-easy载体以及pET28a表达载体。融合基因经过IPTG诱导表达出目的蛋白,并通过SDS-PAGE和Western blotting鉴定正确。证明表达出来的重组蛋白与强毒株弓形虫细胞膜上的天然蛋白有相同的免疫反应性。将感染了Prugniaud的小鼠脑组织取出并匀浆,以该脑组织为模板,分别用sag1和sag5b的引物来引发,结果发现sag1可以扩增出来而sag5b的扩增结果是阴性的。该结果提示sag5b可以作为鉴别弓形虫虫株毒力的基因标志。将该毒力相关基因的表达产物免疫小鼠并没有使小鼠获得明显的可以抵抗强毒株攻击的免疫保护性。

Sag5b:a novel gene for differentiation of strain virulence of Toxoplasma gondii

A promising genetic marker, sag5b, was cloned and expressed and the difference of the genes between highly virulent strain (RH) and less virulent strain(Prugniaud) of Toxoplasma gondii was compared. The PCR-generated product of sag5b was subcloned into T easy vector and plasmid pET28a consecutively. The fusion expression was induced by IPTG and identified by SDS-PAGE and Western blotting. The immunoreactivity of recombinant SAG5B was identical to that of native SAG5B on the membrane of tachyzoites of RH strain. The brains of mice infected with Prugniaud strain of T. gondii were homogenated. Sag1 was successully cloned by PCR from both RH strain tachyzoites and the homogenized brain tissues of mice infected with low virulent strain of Prugniaud,whereas sag5b was only detected in RH strain but not in Prugniaud strain, indicating that sag5b could be used as a genetic marker for differentiation of strain virulence. Expression and vaccination of the virulence-associated gene into mice failed to induce obvious protective immunity against the challenge of RH strain.