Abstract
BackgroundRNA interference (RNAi) is an important tool to determine the role of genes. RNAi has been widely used to downregulate target molecules, resulting in the reduction of mRNA for protein expression. Matrix metalloprotease 12A (MMP-12) is known to have important roles during embryonic development, organ morphogenesis and pathological processes in animals. However, MMP-12 from Haemonchus contortus has not been characterized.MethodsHaemonchus contortus MMP-12 gene was cloned and recombinant protein of MMP-12 (rHc-MMP-12) was expressed. Binding activities of rHc-MMP-12 to goat peripheral blood mononuclear cells (PBMCs) were assessed by immunofluorescence assay (IFA) and the immuno-regulatory effects of rHc-MMP-12 on cell proliferation and nitric oxide production were observed by co-incubation of rHc-MMP-12 with goat PBMCs. Furthermore, a soaking method was used to knockdown the expression of Hc-MMP12 gene using three siRNA, targeting different regions of the gene and infectivity of effective siRNA on the development of H. contortus was evaluated in goat.ResultsrHc-MMP-12 was successfully expressed in an expression vector as well as the tissues of the cuticle of adult H. contortus worms and a successful binding with PBMCs surface were observed. Increased cellular proliferation and nitric oxide production by goat PBMCs was observed in a dose-dependent manner. Quantitative real time PCR (qRT-PCR) results confirmed the successful silencing of Hc-MMP-12 gene in siRNA of 1, 2 and 3 treated third-stage larvae (L3) of H. contortus in vitro. The most efficient qRT-PCR-identified siRNA template was siRNA-2, with a 69% suppression rate compared to the control groups. Moreover, in an in vivo study, silencing of the Hc-MMP-12 gene by siRNA-2 reduced the number of eggs (54.02%), hatchability (16.84%) and worm burden (51.47%) as compared to snRNA-treated control group. In addition, a shorter length of worms in siRNA-2-treated group was observed as compared to control groups.ConclusionsOur results indicate that siRNA-mediated silencing of Hc-MMP-12 gene in H. contortus significantly reduce the egg counts, larval hatchability, and adult worm counts and sizes. The findings of the present study demonstrate important roles of Hc-MMP-12 in the development of H. contortus.
Highlights
RNA interference (RNAi) is an important tool to determine the role of genes
Sequence analysis and western blotting of Hc‐MMP‐12 The amplified PCR products of the Hc-Matrix metallopro‐ tease 12A (MMP-12) gene were obtained from cDNA of H. contortus using a specific pair of primers and a correct fragment size of 1275 bp encoding a protein of 424 amino acids was detected (Fig. 1a)
The recombinant plasmid of Hc-MMP-12 induced with IPTG and expressed in Escherichia coli BL21 cells showed a His6-tagged HcMMP-12 protein on SDS-PAGE after Coomassie blue staining (Fig. 1b) that was purified with a denaturation procedure followed by N i2+ affinity chromatography and a single band was detected with a calculated molecular weight of c.64 kDa (Fig. 1c)
Summary
RNA interference (RNAi) is an important tool to determine the role of genes. RNAi has been widely used to downregulate target molecules, resulting in the reduction of mRNA for protein expression. Haemonchus contortus is an important trichostrongylid nematode that affects small ruminants and causes significant economic losses due to high mortality and morbidity worldwide [1,2,3]. Bloodfeeding adult H. contortus can lead to severe hemorrhagic gastritis, anemia, hypoproteinemia, edema, and even death in acute infections [5,6,7] Control of this parasitic nematode is based on the application of anthelmintics, but excessive usage of these drugs has led to the development of anthelmintic resistance [8,9,10,11]. Development of more effective control approaches is urgently needed
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