Prokaryotic Expression,Distribution And Function Of ATP-binding Cassette G5 Transporter Of Toxocara Canis

Toxocariasis is a zoonotic parasitic disease caused by the infestation of Toxocara canis?T.canis?in the small intestine of dogs.Many animals and humans can be infected by ingestion of infective eggs or infectious larvae of T.canis.The infective larvae of T.canis develop into adults in the intestine of definitive hosts,whereas the hepato-pulmonary migratory phase is followed by myotropic-neurotropic migration and undergo no further development in various tissues of paratenic hosts.Specifically,in humans,Toxocara canis infection manifests well-known clinical forms of visceral larva migrans?VLM?,ocular larva migrans?OLM?,neurological toxocariasis?NT?and covert toxocariasis?CT,in children?/common toxocariasis?CT,in adults?,which cause damage to various tissues and organs and seriously affect human health.ATP-binding cassette?ABC?transporters are highly conserved ubiquitous transmembrane proteins,which utilize the energy of ATP hydrolysis to translocate a wide variety of substrates across the membrane against a concentration gradient.ABC transporters can transport endogenous and heterologous biological macromolecules and small molecules across the cell membrane,including amino acids,sugars,peptides,cell metabolites,metal ions,and drugs,and play important roles in diverse biological processes such as osmotic stability,nutrient absorption,multidrug resistance,antigen processing,cell division,sporulation and reproduction.The function of ABC transporters has been extensively studied in various protozoa and helminths such as Plasmodium falciparum,Schistosoma japonicum and Caenorhabditis elegans.However,the function of ABC transporters of T.canis is still unknown.In study,the full-length of Tc-abcg-5 gene was cloned,the tissue distribution of TcABCG5 was investigated,and the biological function of Tc-abcg-5 gene was studied by RNA interference assays?RNAi?.The results were summarised as follows:1.Molecular cloning and sequence analysis of Tc-abcg-5 geneThe total RNA of T.canis was used as the template,the complete coding region?coding sequence,CDS?of Tc-abcg-5 was cloned and sequenced.The results showed that the CDS of Tc-abcg-5 gene was 1902 bp in length was obtained which encoded633 amino acids.The encoded protein was TcABCG5 with a theoretical molecular weight of 71.42 kDa and an isoelectric point of 8.07.Funtional domain analysis showed that the TcABCG5 protein contained an ABC transporter domain?aa 43-188?and six transmembrane regions?aa 350-372,aa 387-409,aa 422-453,aa 468-490,aa497-514,aa 600-622?.Hydrophobicity analysis showed that the TcABCG5 protein was a hydrophobic protein.Signal peptide predicted that TcABCG5 was a non-secreted protein.The alignment of the deduced Tc-abcg-5 amino-acid sequence with those predicted ABCG5 from C.elegans,Ascaris suum,Haemonchus contortus,Acanthocheilonema viteae,Anisakis simplex showed that Walker A and Walker B motifs for nucleotide binding of the ABCG sub-family were well conserved.Phylogenetic analysis indicated that TcABCG5 shared the highest levels of amino-acid sequence similarity with A.suum,and shared the lowest levels of amino-acid sequence similarity with mammals of Homo sapiens,Bos Taurus,Ovis aries,Cercocebus atys,Rattus norvegicus and Myotis brandtii.Biological function prediction showed that TcABCG5 had a wide range of substrate binding function and was involved in the transport of endogenous and heterologous biological macromolecules and small molecule compounds.2.Prokaryotic expression of TcABCG5 and polyclonal antibody preparationThe ABC transporter domain of Tc-abcg-5 gene was cloned and used for the construction of Tc-abcg-5/pET28a?+?expression plasmid,results of sequencing showed that 794 bp fragment had been inserted.After recombinant plasmids were transfected with Escherichia coli Transetta?DE3?,the recombinant plasmid expressed TcABCG5 in the form of inclusion bodies under the induction of IPTG,and the protein size was about 30 kDa.The optimized expression conditions were induced by 0.8 mM isopropylthiogalactoside?IPTG?and cultured at 30°C for 12 h.The target protein was purified by Ni²⁺-nitrilotriacetic-acid?Ni-NTA?resin,then used to produce rabbit anti-TcABCG5 polyclonal antibody.After testing,the prepared polyclonal antibody concentration was 0.86 mg/mL,the antibody titer was about 1:512000,and the purity was above 90%.Western Blot analysis showed that the TcABCG5 polyclonal antibody specifically binds to TcABCG5 protein,indicated a high specificity of the prepared rabbit anti-TcABCG5 polyclonal antibody.3.The specific transcription of Tc-abcg-5 and specific tissue distribution of TcABCG5Quantitative real-time PCR?qRT-PCR?was used to analyze the transcription of Tc-abcg-5 in T.canis females and males as well as in the tissues of females.High-level transcription of Tc-abcg-5 was detected in adult female T.canis.Transcriptional levels of Tc-abcg-5 among the reproductive tract,intestine and body wall of female worms were also examined,which showed higher level of transcription in the reproductive tract and relatively low transcription in the intestine and the body wall.Indirect fluorescence immunohistochemical analysis was used to determine the tissue distribution of ABCG5 in adult male and female T.canis.The results showed that TcABCG5 mainly distributed in the reproductive tissues and intestines of T.canis,especially in the ovary and uterus of females,and seminal vesicles of male,suggesting that TcABCG5 may be involved in the growth,development or reproduction of T.canis.4.RNA interference of Tc-abcg-5RNA interference?RNAi?technology was used to interfere with the transcription of the Tc-abcg-5 gene in T.canis adults.Gene knockdown analysis was performed using qRT-PCR to determine the transcriptional levels of Tc-abcg-5.Compared with siRNA-1024 group and siRNA-466 group,the transcriptional level of Tc-abcg-5 in siRNA-744 group was significantly reduced after 24 h and 48 h treatment,indicating that siRNA-744 had higher silencing efficiency.Subsequently,the delivery efficiency of siRNA-744 to eggs was compared by electroporation and immersion methods.The results showed that immersion method is more efficient.Interference with T.canis infective eggs was performed using siRNA-744 by soaking.Pathology study was used to analysis of mouse liver,brain and lung tissues on the seventh day after oral administration of interfered eggs,which showed that RNAi reduced the infectivity of T.canis infective eggs,thereby reducing the lung and liver lesions,indicating that TcABCG5 protein may participate in growth and development process of T.canis by affecting the development of eggs.