ABSTRACT
The RNA interference (RNAi) has the ability to turn off individual gene expression. So, it affords a remarkably specific tool for studying the effects of genes. It is regarded as a direct approach for determining such gene/genes functions and offers a valuable tool for modern drug discovery. The study aimed to develop in vitro RNAil in Brugia malayi with particular interest to study the function of Brugia malayi avr-14 (Bm-avr-14) and Brugia malayi f-tubulin (Bm-fi-tubulin) genes. Bm-avr-14 is a gene encoding glutamate gated chloride channel (GluCl) which binds ivermectin and Bm-ß-tubulin is a gene encoding ß-tubulin which binds albendazole. Adult female worms were soaked in heterogeneous short interfering RNA (hsiRNA) with interest to study the role of two genes Bm-avr-14 and Bm-ß-tubulin. Then, we assessed the knock down effects of target genes using worminator system and real time PCR. We found that worms treated with hsiRNA of Bm-avr-14 had a significant reduction in microfilariae (mf) production in comparison with untreated worms or worms treated with hsiRNA of green fluorescent protein (GFP). No significant reduction in mf production with Bm-ß-tubulin gene was obtained. There were no changes in the movement of adults treated with either Bm-avr-14 or Bm- ß-tubulin hsiRNAs. Inconsistent RNAi mediated suppression was achieved with Bm-avr-14 and Bm-ß- tubulin using real time PCR. The data may be helpful in assessment of drug target potential of genes.
Subject(s)
Brugia malayi/genetics , Brugia malayi/metabolism , Gene Expression Regulation/physiology , Helminth Proteins/metabolism , RNA Interference , Animals , Helminth Proteins/genetics , Polymerase Chain Reaction , RNA, Helminth/geneticsABSTRACT
Levamisole is an anthelmintic drug that acts by activating nicotinic acetylcholine receptors at the nematode neuromuscular junction and causing paralysis. We measured the in vitro effects of levamisole on the motility of Brugia malayi microfilariae; after 2 h incubation the apparent IC50 was 2.68 mM. Lower drug concentrations, such as 1 mM, caused an immediate total paralysis that lasted for up to 1 h, but was completely reversed by 2 h of incubation. The 'recovered' parasites were still completely susceptible to application of a second nicotinic agonist, pyrantel.
