Expression, purification and identification of the domain III of DENV II envelop protein in Escherichia coli / 南方医科大学学报

<p><b>OBJECTIVE</b>To express the domain III of DENV II envelop protein in Escherichia coli, obtain the purified recombinant protein and identify its immunoreactivity.</p><p><b>METHODS</b>Suckling mice were inoculated with live DENV II in the brain. The total RNA was extracted from the brain of the infected mice, and the envelope protein DNA fragment was amplified by RT-PCR and ligated into pMD 18-T to construct pMD 18-T-DV2-E. The domain III DNA fragment of the envelope protein was amplified by PCR with pMD 18-T-DV2-E as the template and cloned into pET-32a(+) to construct the expression plasmid pET-32a(+)-DV2-E-DIII. The recombinant plasmid was transformed into E.coli BL21(DE3) and induced by IPTG, and the expressed products were analyzed by SDS-PAGE and Western blotting.</p><p><b>RESULTS</b>After RT-PCR amplification, a specific DNA fragment of about 1.5 kb was obtained and ligated into pMD 18-T to construct pMD 18-T-DV2-E. With pMD 18-T-DV2-E as the template, the domain III DNA fragment about 320 bp in length was amplified and the expression plasmid pET-32a(+)-DV2-E-DIII was successfully constructed. After induction with IPTG, a specific soluble protein with a relative molecular mass of 29000 was obtained and the expression product accounted for 52.50 percent; of the total protein of the cell lysate. Western blotting demonstrated reactivity of the recombinant protein with His-Tag McAb and DENV (Type I-IV) McAb.</p><p><b>CONCLUSION</b>The recombinant plasmid can be highly expressed in E.coli BL21(DE3) in a soluble form and the recombinant protein can react with DENV (Type I-IV) McAb.</p>

Identification of necrophagous fly species from 12 different cities and regions in China using inter-simple sequence repeat melocular markers / 南方医科大学学报

<p><b>OBJECTIVE</b>To identify necrophagous fly species from different regions in China using inter-simple sequence repeat (ISSR) melocular markers and analyze their genetic difference and relationship.</p><p><b>METHODS</b>Five carrion fly species were collected from 12 cities and regions in China, including M.domestica, Lucilia sericata, Chrysomyia megacephala, Helicophagella melanura, and Boetthcherisca peregrina. Twenty-two ISSR primers were designed and synthesized, from which 8 were selected to identify the necrophagous fly species. Cluster analysis was conducted based on distance matrices using unweighted pair group method.</p><p><b>RESULTS</b>Totally 121 amplification samples were obtained using the 8 primers, and 679 clear and stable bands were visualized including 516 bands with polymorphisms. M.domestica, Lucilia sericata, Chrysomyia megacephala, Helicophagella melanura, and Boethcherisca peregrina from different regions in China produced their specific PCR band spectra. M. domestica from 10 different regions in China showed different inheritance patterns of the markers. Species-specific ISSR fragment was found among the necrophagous flys pecies. Cluster analysis among the most abundant carrion fly species demonstrated that M.domestica from 10 different regions could be divided into 4 groups at different levels. Most of the Chrysomyia megacephala and Lucilia sericata could be clustered in one tree.</p><p><b>CONCLUSION</b>This study represents the first identification of the common necrophagous fly species in China. ISSR-PCR-based identification of the species reveals the genetic diversity and genotypic difference among M.domestica from 10 cities and regions in China.</p>

SDS-PAGE analysis of the antibacterial activity of the hemolymph from housefly larva / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the inducible antibacterial activity of the hemolymph from housefly larva and analyze the antibacterial molecules.</p><p><b>METHODS</b>The hemolymph was collected from the third instar housefly larvae 48 h after pricking treatment. Nine standard bacterial strains were used for determination of the antibaterial activity of the collected hemolymph and its combination with ampicillin. The anti-yeast activity of the hemolymph and its mixture with fluconazol was also assayed. The antibacterial molecules in the hemolymph was analyzed by SDS-PAGE.</p><p><b>RESULTS</b>The growth of E.coli, Staphylococcus aureus, Staphylococcus albus, subserotypes of Shigeila flexneri, Bacillus proteus, Bacillus subtilis, Bacillus typhi, Bacillus paratyphosus, and Micrococcus lysodeikticus could be inhibited by the hemolymph collected from housefly larva, and the effect differed significantly between the groups (Plt;0.001). The hemolymph produced the strongest antibacterial activity against Micrococcus lysodeikticus, and the combination of the hemolymph with ampicillin most conspicuously inhibited the growth of Staphylococcus albus. The hemolyph and fluconazol exhibited obvious synergistic effect against yeast. SDS-PAGE identified some specific antibacterial molecules in the hemolymph.</p><p><b>CONCLUSION</b>The induced hemolymph from housefly larva possesses strong antibacterial and antifungal activities especially against Micrococcus lysodeikticus. The hemolymph in combination with ampicillin produces the strongest effect against Staphylococcus albus, and fluconazol can significantly enhance the anti-yeast activity of the hemolymph through a synergistic mechanism.</p>

Toxicity of photoactivated insecticide K-01 to the larvae of Aedes albopictus / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the toxicity of photoactivated insecticide K-01 to the larvae of Aedes albopictus and observe the histopathological changes in the larvae.</p><p><b>METHODS</b>The number of dead larvae was recorded after application of K-01 at different concentrations under different illumination conditions. The content variation of the midgut, malpighian tubules and fat bodies in the larvae was observed microscopically, and the genomic DNA of the larvae was extracted for electrophoresis to identify the target bands.</p><p><b>RESULTS</b>The maximum larvae-killing effect was achieved with 50 mg/ml K-01 applied under sunlight (100% killing 24 hours after application). Optical microscopic observation of the killed larvae revealed severe damage of the mid-intestinal cells that showed disintegration and elongation. Distinct vacuoles were observed in the fat body cells, in which red droplets were seen to assemble around the cell nuclei. The result of 0.8% agarose gel electrophoresis of the larvae genomic DNA presented typical ladder patterns, suggesting the presence of cell apoptosis.</p><p><b>CONCLUSION</b>K-01 is an effective photoactivated insecticide.</p>