使用比色β-内酰胺酶测定法对杜氏利什曼原虫临床分离株的无鞭毛体进行高通量药物筛选。
High-throughput screening of amastigotes of Leishmania donovani clinical isolates against drugs using a colorimetric beta-lactamase assay.
作者信息
Mandal Swati, Maharjan Mahendra, Ganguly Sudipto, Chatterjee Mitali, Singh Sarman, Buckner Frederick S, Madhubala Rentala
机构信息
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India.
出版信息
Indian J Exp Biol. 2009 Jun;47(6):475-9.
Abstract
A simple colorimetric beta-lactamase assay for quantifying Leishmania amastigotes in macrophages grown in microtiter plates has been reported. The beta-lactamase gene was integrated into the rRNA region of the genome, thereby allowing for high-level stable expression of the enzyme. Both visceral leishmaniasis (VL) and post-kala azar dermal leishmaniasis (PKDL) isolates were transfected with beta-Lactamase gene. These beta-lactamase-expressing promastigotes were used for infecting intracellular J774A.1 macrophages in vitro. Quantification was done by a colorimetric readout with CENTA beta-lactamase as substrate and with an optical density plate reader. The assay was carried out in 96-well plates. Results obtained demonstrate that this methodology could be a valuable high-throughput screening assay for checking efficacy of anti-leishmanial drugs in the clinical isolates.
摘要
据报道，有一种简单的比色法β-内酰胺酶检测方法，可用于定量在微量滴定板中生长的巨噬细胞内的利什曼原虫无鞭毛体。β-内酰胺酶基因被整合到基因组的rRNA区域，从而使该酶能够高水平稳定表达。内脏利什曼病（VL）和黑热病后皮肤利什曼病（PKDL）的分离株均用β-内酰胺酶基因进行了转染。这些表达β-内酰胺酶的前鞭毛体用于体外感染细胞内的J774A.1巨噬细胞。通过以CENTAβ-内酰胺酶为底物并使用光密度酶标仪进行比色读数来进行定量。该检测在96孔板中进行。获得的结果表明，该方法可能是一种有价值的高通量筛选检测方法，可用于检查临床分离株中抗利什曼原虫药物的疗效。
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