CAJ | 학술논문

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.
목적 본문작자비교료강독주(RH)궁형충여약독주(Prugniaud)기인조중일충가능성위기인표지물적기인편단--sag5b적차이.PCR확증출적sag5b편단의차아극륭진입T-easy재체이급pET28a표체재체.융합기인경과IPTG유도표체출목적단백,병통과SDS-PAGE화Western blotting 감정정학.증명표체출래적중조단백여강독주궁형충세포막상적천연단백유상동적면역반응성.장감염료Prugniaud적소서뇌조직취출병균장,이해뇌조직위모판,분별용sag1화sag5b적인물래인발,결과발현sag1가이확증출래이sag5b적확증결과시음성적.해결과제시sag5b가이작위감별궁형충충주독력적기인표지.장해독력상관기인적표체산물면역소서병몰유사소서획득명현적가이저항강독주공격적면역보호성.
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.