To express recombinant Ancylostoma caninum anticoagulant peptide-c2 (AcAPc2), a whole cDNA fragment encoding AcAPc2 was achieved by ligation- PCR and inserted into prokaryotic expression vector pTWIN1 for constructing the specific self-splicing prokaryotic expression vector, pTWIN1-AcAPc2; positive recombinants were transformed into E. coli ER2566 for expression research. The recombinant protein, AcAPc2-intein2-CBD, was soluble and expressed in E. coli ER2566 (about 30.1% fusion protein in total protein). AcAPc2-intein2-CBD was characterized to be 41 KD by SDS-PAGE and identified by Western-blot. The recombinant fusion protein was purified to a efficiently high degree by chitin affinity chromatography. After the process of specific self-splicing induced by beta-Mercaptoethanol, the target protein, AcAPc2, was obtained, characterized to be 21 KD by SDS-PAGE and migrated as a dimmer. Molecular weight of AcAPc2 conformed to native dimmer. Bio-information analysis indicated relationship between secondary construction of AcAPc2 and biologic function. These findings greatly facilitate the purification of AcAPc2 and are very important for the additional studies on its anti-coagulation mechanism and its clinical application as anti-coagulation medicine.
Animals ; Dogs ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genes, Helminth ; Genetic Vectors ; Helminth Proteins ; biosynthesis ; genetics ; Plasmids ; genetics ; Prokaryotic Cells ; metabolism ; RNA Splicing ; Recombinant Fusion Proteins ; chemistry ; pharmacology

To determine the molecular phylogenic location of Plagiorchis muris, 28S D1 ribosomal DNA (rDNA) and mitochondrial cytochrome C oxidase subunit I (mtCOI) were sequenced and compared with other trematodes in the family Plagiorchiidae. The 28S D1 tree of P. muris was found to be closely related to those of P. elegans and other Plagiorchis species. And, the mtCOI tree also showed that P. muris is in a separate clade with genus Glypthelmins. These results support a phylogenic relationship between members of the Plagiorchiidae, as suggested by morphologic features.
Animals ; Base Sequence ; DNA, Helminth/chemistry/genetics ; DNA, Mitochondrial/chemistry/genetics ; Electron Transport Complex IV/chemistry/*genetics ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction ; RNA, Ribosomal, 28S/chemistry/*genetics ; Sequence Alignment ; Support, Non-U.S. Gov't ; Trematoda/classification/*genetics

A complete cDNA sequence encoding a pore-forming subunit (Kir6.2) of ATP-senstive potassium channel in the adult worm, Clonorchis sinensis, termed CsKir6.2, was isolated from an adult cDNA library. The cDNA contained a single open-reading frame of 333 amino acids, which has a structural motif (a GFG-motif) of the putative pore-forming loop of the Kir6.2. Peculiarly, the CsKir6.2 shows a lack-sequence structure, which deleted 57 amino acids were deleted from its N-terminus. The predicted amino acid sequence revealed a highly conserved sequence as other known other Kir6.2 subunits. The mRNA was weekly expressed in the adult worm.
Adenosine Triphosphate/metabolism ; Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; Clonorchis sinensis/*genetics/metabolism ; Helminth Proteins/*genetics/metabolism ; Human ; Molecular Sequence Data ; Potassium Channels, Inwardly Rectifying/*genetics/metabolism ; RNA, Helminth/chemistry/genetics ; Sequence Alignment ; Support, Non-U.S. Gov't

The present study was performed to describe 2 human cases infected by the horsehair worm, Parachordodes sp., in Japan. Two gordiid worms were collected in the vomit and excreta of an 80-year-old woman in November 2009 in Kyoto city, and in the mouth of 1-year-old boy in December 2009 in Nara city, Japan, respectively. Both worms were males having bifurcated posterior ends and male gonads in cross sectional specimens. They were identified as Parachordodes sp. (Nematomorpha: Chordodidae) based on the characteristic morphologies of cross sections and areoles in the cuticle. DNA analysis on 18S rRNA partial sequence arrangements was also carried out and both worms were assumed to be close to the genus Paragordionus based on tree analysis, and far from Gordius sp. which has already been reported in humans in Japan. DNA sequencing of the Parachordodes worm does not appear on the database; therefore, more information on the gene sequences of the genus Parachordodes from humans, animals, or intermediates is required.
Aged, 80 and over ; Animals ; Cluster Analysis ; DNA, Helminth/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Female ; Helminthiasis/*diagnosis/*parasitology/pathology ; Helminths/anatomy & histology/classification/genetics/*isolation & purification ; Humans ; Infant ; Japan ; Male ; Microscopy ; Phylogeny ; RNA, Ribosomal, 18S/genetics ; Sequence Analysis, DNA

To gain information on retrotransposons in the genome of Paragonimus westermani, PCR was carried out with degenerate primers, specific to protease and reverse transcriptase (rt) genes of long-terminal-repeat (LTR) retrotransposons. The PCR products were cloned and sequenced, after which 12 different retrotransposon-related sequences were isolated from the trematode genome. These showed various degrees of identity to the polyprotein of divergent retrotransposon families. A phylogenetic analysis demonstrated that these sequences could be classified into three different families of LTR retrotransposons, namely, Xena, Bel, and Gypsy families. Of these, two mRNA transcripts were detected by reverse transcriptase-PCR, showing that these two elements preserved their mobile activities. The genomic distributions of these two sequences were found to be highly repetitive. These results suggest that there are diverse retrotransposons including the ancient Xena family in the genome of P. westermani, which may have been involved in the evolution of the host genome.
Amino Acid Sequence ; Animals ; Cloning, Molecular ; DNA, Helminth/analysis ; *Evolution, Molecular ; *Genome ; Molecular Sequence Data ; Paragonimus/*genetics ; Phylogeny ; RNA-Directed DNA Polymerase/chemistry/genetics ; Retroelements/*genetics ; Sequence Alignment ; Sequence Analysis, DNA ; Terminal Repeat Sequences/*genetics

We compared the DNA sequences of the genus Metagonimus: M. yokogawai, M. takahashii, and M. miyatai. We obtained 28S D1 ribosomal DNA (rDNA) and mitochondrial cytochrome c oxidase subunit I (mtCOI) fragments from the adult worms by PCR, that were cloned and sequenced. Phylogenetic relationships inferred from the nucleotide sequences of the 28S D1 rDNA and mtCOI gene. M. takahashii and M. yokogawai are placed in the same clade supported by DNA sequence and phylogenic tree analysis in 28S D1 rDNA and mtCOI gene region. The above findings tell us that M. takahashii is closer to M. yokogawai than to M. miyatai genetically. This phylogenetic data also support the nomination of M. miyatai as a separate species.
Animals ; Base Sequence ; Comparative Study ; DNA, Helminth/*chemistry/genetics ; DNA, Mitochondrial/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Electron Transport Complex IV/chemistry/*genetics ; Heterophyidae/classification/enzymology/*genetics ; Molecular Sequence Data ; Phylogeny ; RNA, Ribosomal, 28S/chemistry/*genetics ; Research Support, Non-U.S. Gov't ; Sequence Alignment ; Trematode Infections/*parasitology

We compared the DNA sequence difference of isolates of Clonorchis sinensis from one Korean (Kimhae) and two Chinese areas (Guangxi and Shenyang). The sequences of nuclear rDNA (18S, internal transcribed spacer 1 and 2: ITS1 and ITS2) and mitochondrial DNA (cytochrome c oxidase subunit 1: cox1) were compared. A very few intraspecific nucleotide substitution of the 18S, ITS1, ITS2 and cox1 was found among three isolates of C. sinensis and a few nucleotide insertion and deletion of ITS1 were detected. The 18S, ITS1, ITS2 and cox1 sequences were highly conserved among three isolates. These findings indicated that the Korean and two Chinese isolates are similar at the DNA sequence level.
Animals ; Base Sequence ; China ; Clonorchis sinensis/enzymology/*genetics ; Comparative Study ; DNA, Helminth/chemistry/*genetics ; DNA, Mitochondrial/chemistry/*genetics ; DNA, Ribosomal/chemistry/genetics ; DNA, Ribosomal Spacer/chemistry ; Electron Transport Complex IV/genetics ; Genetic Markers ; Korea ; Molecular Sequence Data ; RNA, Ribosomal, 18S/genetics ; Research Support, Non-U.S. Gov't ; Sequence Alignment ; Species Specificity ; *Variation (Genetics)

The first human case with trichinellosis was reported in 1964 in Tibet, China. However, up to the present, the etiological agent of trichinellosis has been unclear. The aim of this study was to identify a Tibet Trichinella isolate at a species level by PCR-based methods. Multiplex PCR revealed amplicon of the expected size (173 bp) for Trichinella spiralis in assays containing larval DNA from Tibet Trichinella isolate from a naturally infected pig. The Tibet Trichinella isolate was also identified by PCR amplification of the 5S ribosomal DNA intergenic spacer region (5S ISR) and mitochondrial large-subunit ribosomal RNA (mt-lsrDNA) gene sequences. The results showed that 2 DNA fragments (749 bp and 445 bp) of the Tibet Trichinella isolate were identical to that of the reference isolates of T. spiralis. The Tibet Trichinella isolate might be classifiable to T. spiralis. This is the first report on T. spiralis in southwestern China.
Animals ; DNA, Helminth/chemistry/genetics ; DNA, Mitochondrial/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; DNA, Ribosomal Spacer/genetics ; Genotype ; Humans ; Multiplex Polymerase Chain Reaction ; RNA, Ribosomal, 5S/genetics ; Sequence Analysis, DNA ; Swine ; Swine Diseases/*parasitology ; Tibet ; Trichinella spiralis/*classification/genetics/isolation & purification ; Trichinellosis/parasitology/*veterinary

The rumen parasite, Gastrothylax crumenifer (Platyhelminthes: Gastrothylacidae), is a highly pathogenic trematode parasite of goat (Capra hircus). It sucks blood that causes acute disease like anemia, and severe economic losses occur due to morbidity and mortality of the ruminant infected by these worms. The study of these rumen paramphistomes, their infection, and public health importance remains unclear in India especially in the western part of state Uttar Pradesh (U.P.), Meerut, India, where the goat meat consumption is very high. This paper provides the molecular characterization of G. crumenifer recovered from the rumen of Capra hircus from Meerut, U.P., India by the partial sequence of 28S rDNA. Nucleotide sequence similarity searching on BLAST of 28S rDNA from parasites showed the highest identity with those of G. crumenifer from the same host Capra hircus. This is the first report of molecular identification of G. crumenifer from this part of India.
Animals ; Cluster Analysis ; DNA, Helminth/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Goat Diseases/*parasitology ; Goats ; India ; Microscopy, Electron, Scanning ; Molecular Sequence Data ; Phylogeny ; Platyhelminths/*classification/genetics/*isolation & purification/ultrastructure ; RNA, Ribosomal, 28S/genetics ; Rumen/parasitology ; Sequence Analysis, DNA ; Trematode Infections/parasitology/*veterinary

Analysis of ancient DNA (aDNA) extracted from Ascaris is very important for understanding the phylogenetic lineage of the parasite species. When aDNAs obtained from a Joseon tomb (SN2-19-1) coprolite in which Ascaris eggs were identified were amplified with primers for cytochrome b (cyt b) and 18S small subunit ribosomal RNA (18S rRNA) gene, the outcome exhibited Ascaris specific amplicon bands. By cloning, sequencing, and analysis of the amplified DNA, we obtained information valuable for comprehending genetic lineage of Ascaris prevalent among pre-modern Joseon peoples.
Adult ; Animals ; Ascariasis/diagnosis/history/*parasitology ; Ascaris/classification/genetics/*isolation & purification ; Base Sequence ; Cytochromes b/genetics ; DNA Primers/genetics ; DNA, Helminth/*genetics ; DNA, Mitochondrial/*genetics/history ; Female ; Fossils/history/parasitology ; History, Ancient ; Humans ; Male ; Molecular Sequence Data ; Mummies/history/*parasitology ; Ovum/chemistry/classification ; Phylogeny ; RNA, Ribosomal, 18S/genetics