| Toxocara canis is an intestinal nematode of canids with a worldwide distribution.T.canis can infect human beings as well as a wide range of other mammals causing serious parasitic zoonosis,toxocariasis.In the definitive host,the infective larvae usually migrate through viscera,and eventually develop into adult in the intestinal tract.While in paratenic hosts,development into the adult does not occur,but rather migrate throughout the viscera,muscle and neural system,and persist in tissues as a developmentally arrested infective third-stage larvae(L3).Animals as well as human beings can be infected by accidental ingestions of infective larvae in undercooked meat/viscera,infective eggs from contaminated soil or raw vegetables,causing various forms of diseases characterized by ocular larva migrans(OLM),visceral larva migrans(VLM),covert toxocariasis/common toxocariasis(CT),or nerve larvae migration(NLM).Aquaporins(AQPs)are a family of major intrinsic protein(MIP)superfamily that facilitate the transport of water and other small solutes such as glycerol,gas,urea,ammonia and ions across cell membranes.In addition,aquaporins are also involved in many biological processes such as cell migration,brain edema,neuroexcitation,adipocyte metabolism and so on.According to the permeability properties,AQPs were divided into three major subtypes: orthodox aquaporins,aquaglyceroporins and superaquaporins.Orthodox aquaporins are water-selective channels that allow only water to permeate the channel.Aquaglyceroporins are non-selective water channels that permeable to small neutral solutes such as glycerol or urea besides water.Superaquaporins,their functions remain undetermined.Aquaporins is the main molecular basis of water transmembrane transport and widely distributed in a variety of organisms such as mammalians,insects,plants,yeast even bacteria.Aquaporins are vital for parasite survival,they can involve not only in the transport of water but also in the regulation of osmotic pressure,nutrient absorption and excretion of metabolic waste,etc.AQP1 was the first discovered water channel protein,which can promote migration of endothelial cells and angiogenesis,mediate transmembrane transport of water,affect tumor spread,and regulate nutritional metabolism and vascular function of dilatation and contraction.Researches on AQP1 from Caenorhabditis elegans,Schistosoma mansoni,Fasciola gigantica,Opisthorchis viverrini,Leishmania major,Trypanosoma brucei and Toxoplasma gondii have been reported,but there was a lack of information about AQP1 of parasitic nematodes.In this study,the complete coding region sequence of Tc-aqp-1 was cloned,the tissue distribution of TcAQP1 was investigated,and the biological function of Tc-aqp-1 gene was studied by RNA mediated interference(RNAi)assay.The results were summarized as follows:1.Molecular cloning and sequence analysis of Tc-aqp-1Polymerase chain reaction(PCR)was performed to generate the full-length cDNA of Tc-aqp-1.The obtained Tc-aqp-1 coding sequence was 933 bp in length,which predicted to encode 310 amino acids,and the predicted molecular weight was 34.35 kDa.Two conserved NPA motifs were identified in the multiple alignments analysis.Phylogenetic analysis revealed the closest relationship among T.canis,C.elegans and B.malayi based on aquaporin-1 amino acid sequence.A structure was predicted with ligand binding sites predicted at N95,N226,L47,L183,I79 and I98 and with active sites predicted at I256 and G207.Gene Ontology(GO)annotations predicted its cellular component term of integral component of plasma membrane(GO: 0005887),molecular function term of channel activity(GO: 0015250),and biological process term of water transport(GO: 0006833).2.Prokaryotic expression of TcAQP1 and preparation of polyclonal antibodyThe C-terminal hydrophilic region sequence of Tc-aqp-1 was cloned and used for the construction of Tc-aqp-1c/pET32 a expression plasmid,the recombinant TcAQP1 c was expressed via Escherichia coli BL21(DE3)prokaryotic expression system.SDS-PAGE analysis showed that the soluble recombinant TcAQP1 c was expression in fusion at about 24 kDa in molecular weight.The expression conditions were optimized to gain more protein,under the inducement of 1.0 mM isopropylthiogalactoside(IPTG)and incubation of 6 h for 37 °C.The target protein of high purity was obtained using Ni-NTA chromatography with 50 mM imidazole solution,which was used to generate rabbit anti-TcAQP1 c polyclonal antiserum.The antiserum was characterized as concertration of 0.83 mg/mL and 0.86 mg/mL,titer >1: 64000.Western blot analysis indicated high specificity of the rabbit anti-TcAQP1 c polyclonal antiserum.3.Tissue differential expression of Tc-aqp-1 and tissue distribution of TcAQP1Quantitative real-time PCR was emplyed to reveal the profiles of Tc-aqp-1 in tissues of adult male and female worms.The results showed that Tc-aqp-1 was transcribed in multiple tissues of T.canis,with relatively high level in intestine of both male and female worms and low level in germline and body wall of both male and female worms.Fluorescence immunohistochemical analysis was carried out to detect the tissue distridution of AQP1 in T.cains.The results indicated that TcAQP1 distributed in the germinal and intestinal tissues of both adult male and female T.canis,especially in seminal vesicle of male T.canis and in ovary of female T.canis.All these indicated the functional roles of TcAQP1 in water and solute transport,also possible in reproductive process of T.canis,but the concrete mechanism remains to be further studied.4.RNA interference on Tc-aqp-1siRNA mediated RNA interference assays were performed to interfere the process of transcription-expression of Tc-aqp-1.Gene knockdown analysis was carried out to determine the transcriptional levels of Tc-aqp-1 using qRT-PCR.The results showed that the transcriptional level of Tc-aqp-1 in the siRNA-245 group was significantly decreased in the 3 groups.Subsequently,the T.canis in the siRNA-245 treatment group was dealed with 0.2 mg/mL albendazole.The result showed that the mortality of T.canis in siRNA-245 group was significantly lower than that in control group,indicating that TcAQP1 is involved in the transport of anti-parasite drugs and is a potential drug target. |
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