| Taenia multiceps is a widespread parasite in many areas of the world. Adult T. multiceps inhabit in the small intestine of dogs, wolves, foxes and other canids. The larval stage, known as coenurus, mainly parasitizes the brain or spinal cord of cloven-hoofed animals such as buffalo, cattle, goats, sheep, and yak, as well as wild species, causing coenuriasis. Coenurosis occurs almost all over the world, causing enormous economic losses to the livestock industry and threatening human health.Here, synonymous codon usage bias was studied. Besides, cloning, expression and recombinant protein analysis of four functional genes from Taenia multiceps including GP50, P2, dUTPase and Cu/Zn-SOD were performed as well. The main results of researches are summarized as following:1. Analysis of codon usage pattern in Taenia multiceps based on a transcriptome datasetSynonymous codon usage of Taenia multiceps was examined based on 8620 reconstructed annotations of transcriptomic sequences, revealing there is only a weak codon bias, and the mean values of GC and GC3 contents for the reconstructed genes were 49.29% and 51.43%, respectively. We inferred that the nucleotide composition of genes, mutational pressure, natural selection, gene expression level, the amino acids with grand average of hydropathicity (GRAVY) and aromaticity (Aromo) and the effective selection of amino-acid also influence the codon usage in Taenia multiceps. However, natural selection might play a major role in shaping codon usage variation. The codon usage of ribosome genes was mainly affected by mutations, while the essential genes were mainly by selections. Twenty-two codons were determined as "optimal codons", it is speculated that the genes of Taenia multiceps was affected by positive selections during evolution. The optimal codons overall present GC-rich (GC:AU=43:23), and the optimal codons (except CGU) ended with G or C. In addition, the study also found that there are different degrees of variation in codon usage between Taenia multiceps, Escherichia coli, yeast and Homo sapiens, little difference was found between Taenia multiceps and Taenia pisiformis. Knowledge of the codon usage pattern in Taenia multiceps can provide valuable information for the discovery of new genes, molecular genetic engineering and evolutionary studies.2. Expression, tissue localization of GP50 gene and construction of indirect ELISA diagnostic method for coenurosis cerebralis in goatsIn the present study, a full-length cDNA that encodes GP50 was selected from the transcriptome of T. multiceps (TmGP50). Cloned and expressed in prokaryocyte and then analyzed by immunofluorescence localization. The GP50 contains an 897 bp open reading frame, in which signal sequence resides in 1?48 sites and mature polypeptide consists of 282 amino acid residues, and it was predicted to have a molecular weight of 31.02 kDa, pI=7.58. Immunofluorescence staining showed that TmGP50 were highly localized to the microthrix and parenchymatous zone of both the adult parasite and the coenurus; besides, it was widely distributed in cystic wall of coenurus. Building on good immunogenic properties, rTmGP50-based ELISA exhibited a sensitivity of 95.0%(19/20) and a specificity of 92.6%(25/27) in detecting anti-GP50 antibodies in the sera of naturally infected goats and sheep, and there were cross-reactivity with serum from Cysticercus tenuicollis-positive goat and Echinococcus granulosus-positive sheep. In goats experimentally infected with T. multiceps, anti-TmGP50 antibody was detectable in the control group from 2 to 17 weeks p.i. In the drug-treated group, the anti-TmGP50 antibody dropped below the cut-off value about 3 weeks after treatment with praziquantel and remained below this critical value until the end of the experiment. This study suggests that TmGP50 has the potential to be selected as diagnostic antigen of coenurosis cerebralis.3. Expression, tissue localization and serodiagnostic potential of Taenia multiceps acidic ribosomal protein P2We identified a full-length cDNA that encodes acidic ribosomal protein P2 from the transcriptome of T. multiceps (TmP2). The open reading frame (366 bp) of the target gene encodes a 12.62 kDa protein, which showed high homology to that from Taenia solium (93% identity) and lacked a signal peptide. Immunofluorescence staining showed that TmP2 was highly localized to the parenchymatous zone of both the adult parasite and the coenurus; besides, it was widely distributed in cystic wall of coenurus. The established indirect ELISA based on rTmP2 (recombinant TmP2) exhibited a sensitivity of 95.0%(19/20) and a specificity of 96.3%(26/27) in detecting anti-P2 antibodies in the sera of naturally infected goats and sheep, and there is cross reaction with positive sera of cysticercus tenicollis from goats. In goats experimentally infected with T. multiceps, anti-TmP2 antibody was detectable in the control group from 3 to 10 weeks and 15 to 17 weeks p.i. In the drug-treated group, the anti-TmP2 antibody dropped below the cut-off value about 2 weeks after treatment with praziquantel and remained below this critical value until the end of the experiment. This study suggests that TmP2 has the potential to be selected as diagnostic antigen of coenurosis cerebralis.4. Cloning, expression and enzymatic activity analysis of Taenia multiceps dUTPaseWe identified a full-length cDNA that encodes dUTPase from the transcriptome of T. multiceps (TmdUTPase). The open reading frame (447 bp) of the target gene encodes a 16.39 kDa protein. The protein sequence of TmdUTPase was found to be highly homologous to those from T. solium (100% identity) and Taenia pisiformis (96%). A specific band was observed when using goat anti-T. multiceps serum by Western blot analysis, and the protein concentration of dUTPase was 0.907mg/mL. Immunofluorescence staining showed that TmdUTPase was highly localized to the parenchymatous zone of the adult parasite while distributed sporadically in the microthrix; besides, it was widely distributed in cephalomeres and cystic wall of coenurus. The analysis of the enzymatic activity indicated that the recombinant TmdUTPase could hydrolysis dUTP and the activity was 986.29U.mg-1. Furthermore, the study also found that the activity of enzyme can be inhibited by EDTA and enhanced by Mg2+.5. Cloning, expression and enzymatic activity analysis of Taenia multiceps copper-zinc superoxide dismutaseWe identified a full-length cDNA that encodes Cu/Zn-SOD from the transcriptome of T. multiceps(TmCu/Zn-SOD). The open reading frame (459 bp) of the target gene encodes a 16.72 kDa protein. The protein sequence of TmCu/Zn-SOD was found to be highly homologous to those from T. solium (98% identity) and Taenia crassiceps (92%). A specific band was observed when using goat anti-T. multiceps serum by Western blot analysis. The protein concentration of TmCu/Zn-SOD was 1.587mg/mL, and its activity was up to (3138±25.67 IU/mg prot) measured by hydroxylamine method. Furthermore, the study also found that the activity of enzyme can be inhibited by H2O2, SDS and EDTA, while C2H5OH-CHCl3 and urea had no effect. |
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