Cloning, sequencing, expression, and purification of aspartic proteases isolated from two human Demodex species.

Aspartic proteases (ASPs) are important hydrolases for parasitic invasion of host tissues or cells. This was the first study on Demodex ASP. First, the complete coding sequence (CDS) was amplified, cloned and sequenced. Then, the protein physical and chemical properties was analysed. Finally, the recombinant plasmid, expression and purification system was established. Results showed that the lengths of CDS of Demodex folliculorum and D. brevis were 1161 and 1173 bp, respectively. The molecular weight of the protein was approximately 40 KDa. It contained an aspartic acid residue, a substrate-binding site and signal peptide, yet lacked a transmembrane domain and was located in the membrane or extracellular matrix. The phylogenetic and conserved motif analyses showed that D. folliculorum and D. brevis clustered separately and then formed a single branch, which finally clustered with other Acariformes species. The prokaryotic expression systems for recombinant ASP with His-tag (rASP-His) and GST-tag (rASP-GST) were constructed. The inclusion bodies of rASP-His were renaturated by gradient urea and purified using NI beads, while those of rASP-GST were renaturated by sarkosyl and Triton X-100 and purified using GST beads. Conclusively, the prokaryotic expression and purification system of Demodex rASP was successfully established for further pathogenic mechanism research.

Classification and identification of mosquitoes in China based on rDNA 28S D5 region.

Accurate classification and identification of mosquitoes are essential for the prevention and control of mosquito-borne diseases. In this study, adult mosquitoes were collected from 15 cities across 14 provinces in China. They were identified morphologically with the dominant species determined. Furthermore, representative samples were identified at the molecular level based on rDNA 28S D5. In total, 880 adult mosquitoes were collected belonging to Culex (266), Aedes (473), Armigeres (13), and Anopheles (5). Aedes albopictus and "C. pipiens subgroup" were the dominant species. A total of 140 sequences of 28S D5 region (68 for "C. pipiens subgroup", 51 for Ae. albopictus, 18 for Ar. subalbatus, and three for An. sinensis) ranging from 148 to 161 bp were obtained, with 100 % success of amplification and sequencing. Molecular identification were consistent with morphological classification. Sequence analysis showed that "C. pipiens subgroup" was identified into three clades: the traditional C. pipiens subgroup (Clade I), the newly discovered C. cf. perexiguus (Clade II), and C. new sp. (Clade III). Clade I contained the most abundant haplotypes (16) widely distributed without geographical differences. Clade II included six haplotypes that were aggregately distributed south of the Yangtze River. Only three sequences in Clade III showed two haplotypes with no geographical differences. Further morphological comparisons demonstrated differences in body color, beaks, and abdomens among the three clades. In conclusion, the rDNA 28S D5 region could effectively distinguish Culex, Aedes, Armigeres, and Anopheles species at the lower category level, demonstrating its potential as a mini-DNA barcode for mosquito identification.

Secondary structure construction and molecular identification of rRNA 18S V4 region E23-5-E23-6 of parasitic lice of Hominidae.

The parasitic lice of Hominidae are a class of blood-sucking insects, having a large fragment expansion region in ribosome 18S V4 region. In this study, the value of the E23-5-E23-6 stem-loop structure in the insertion region for molecular identification of lice were explored through motif analysis and secondary structure construction. Five pubic lice samples from China were morphologically identified, and primers for the rRNA 18S V4 region were designed for molecular identification. The V4 sequence of the parasitic lice of Hominidae was retrieved from GenBank for sequence analysis. The five samples were identified as pubic lice based on V4 region, which were of the same specie but geographically different from Australian strains in Genbank, with the identity of 99.06-99.46%. Compared with the human lice, both the chimpanzee lice and pubic lice had large indel fragments in the V4 region. Comparison results showed that Muscle and MAFFT had better alignment and phylogeny results than Clustal. The large expansion region, comprising E23-5 and E23-6, was located between E23-4 and E23-7. The V4 secondary structure showed that the stem-loop structures of the lice parasitizing on pubic area, human, and chimpanzee were different in the E23-5 and E23-6, which could effectively distinguish the three parasitic lice and divide the human lice into five genotypes. This is suitable not only for the identification of three lice species in higher taxonomic ranks but also for genotype identification of human lice in lower taxonomic ranks. The difference between the stem-loop structure is more intuitive than that between the primary sequences.

Morphological and Molecular Identification of Oestrus ovis (Diptera: Oestridae) Larvae Collected from a Chinese Patient with Conjunctival Myiasis.

PURPOSE: Human conjunctival myiasis, which is often misdiagnosed or missed clinically, is commonly caused by Oestrus ovis larvae. Here, pathogenic identification was performed for two maggots collected from a patient from China, to provide a clinical scientific basis for diagnosis and treatment. METHODS: Morphological identification was performed using a microscope. Oestrus mtDNA cox1 and rDNA 28S were selected as target genes for duplex PCR amplification, followed by cloning, sequencing, and identification. RESULTS: Morphological examination showed that the maggots were approximately 1.0-1.5 mm long, long-oval-shaped, segmented, and covered with small spines, with a pair of hooks in the scolex and claw-like spines at the telson. Therefore, they were identified as the first-instar larvae of O. ovis. Duplex PCR detected products of approximately 400 and 200 bp, consistent with the size of designed cox1 and 28S D7a gene fragments, respectively. Sequences of cox1 and 28S D7a from the samples in question had 99.5-100.0% and 96.2-100.0% similarity (respectively) to GenBank sequences of O. ovis specimens known to parasitize sheep, goats, and humans. However, some 28S D7a sequences exhibited 89.7-90.6% similarity to GenBank sequences of Oestrus sp. known to parasitize Capra pyrenaica (Artiodactyla: Bovidae) (Iberian ibex). Therefore, we considered that the larvae infecting the patient originated from sheep or goats, not Iberian ibex. The phylogenetic trees supported this conclusion. CONCLUSION: This study implemented the first duplex PCR molecular identification of O. ovis larvae parasitizing human eyes in China as a complementary approach to morphological identification. Our results indicate that molecular tools can be utilized to aid in the diagnosis of opthalmomyiasis.

Establishment of purification method for prokaryotic expression of Serpin gene for Dermatophagoides farinae.

This study aimed to develop an effective method for the expression and purification of the Dermatophagoides farinae serpin protein and to establish an experimental foundation for elucidating its role in the temperature stress response. The total RNA of D. farinae was extracted, and specific primers were designed for serpin amplification. Serpin was joined with pET32a vector and transformed into BL21 (DE3) cells. Expression of recombinant proteins was induced. Proteins were extracted by enzymatic lysis or enzymatic lysis combined with ultrasonication. Recombinant proteins were purified by Ni-NTA method. SDS-PAGE was conducted to evaluate protein expression, extraction, and purification efficiency. Agarose gel electrophoresis and sequencing analysis showed that the amplified serpin open reading frame was 1284 bp, encoding a hydrophilic and stable protein with a relative molecular weight of 48.30 kD. SDS-PAGE demonstrated that there was a specific band at 55-70 kD, which was consistent with the predicted size of the recombinant pET32a-Serpin protein. Enzymatic lysis combined with 30% ultrasonic power promoted the release of soluble protein more effectively than enzymatic lysis alone. 16 °C for 4 h was optimal for inducing expression. The optimal imidazole concentrations for washing non-His-tagged protein and eluting His-tagged protein were determined to be 20 mM and 200 mM, respectively. In this study, A prokaryotic expression and purification system for the D. farinae serpin protein was successfully established, providing a technical reference for functional gene research in mites at the protein level.

Bacterial and diazotrophic diversities of endophytes in Dendrobium catenatum determined through barcoded pyrosequencing.

As an epiphyte orchid, Dendrobium catenatum relies on microorganisms for requisite nutrients. Metagenome pyrosequencing based on 16S rRNA and nifH genes was used to characterize the bacterial and diazotrophic communities associated with D. catenatum collected from 5 districts in China. Based on Meta-16S rRNA sequencing, 22 bacterial phyla and 699 genera were identified, distributed as 125 genera from 8 phyla and 319 genera from 10 phyla shared by all the planting bases and all the tissues, respectively. The predominant Proteobacteria varied from 71.81% (GZ) to 96.08% (YN), and Delftia (10.39-38.42%), Burkholderia (2.71-15.98%), Escherichia/Shigella (4.90-25.12%), Pseudomonas (2.68-30.72%) and Sphingomonas (1.83-2.05%) dominated in four planting bases. Pseudomonas (17.94-22.06%), Escherichia/Shigella (6.59-11.59%), Delftia (9.65-22.14%) and Burkholderia (3.12-11.05%) dominated in all the tissues. According to Meta-nifH sequencing, 4 phyla and 45 genera were identified, while 17 genera and 24 genera from 4 phyla were shared by all the planting bases and all the tissues, respectively. Burkholderia and Bradyrhizobium were the most popular in the planting bases, followed by Methylovirgula and Mesorhizobium. Mesorhizobium was the most popular in different tissues, followed by Beijerinckia, Xanthobacter, and Burkholderia. Among the genera, 39 were completely overlapped with the results based on the 16S rRNA gene. In conclusion, abundant bacteria and diazotrophs were identified in common in different tissues of D. catenatum from five planting bases, which might play a great role in the supply of nutrients such as nitrogen. The exact abundance of phylum and genus on the different tissues from different planting bases need deeper sequencing with more samples.