Prokaryotic expression, polyclonal antibody preparation, spatio-temporal expression profile and functional analysis of c-Myc of Helicoverpa armigera (Lepidoptera: Noctuidae) / 生物工程学报

c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/β-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni2+-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells. The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.

Advances in receptor-mediated resistance mechanisms of Lepidopteran insects to Bacillus thuringiensis toxin / 生物工程学报

Bacillus thuringiensis is widely used as an insecticide which is safe and environmentally friendly to humans and animals. One of the important insecticidal mechanisms is the binding of Bt toxins to specific toxin receptors in insect midgut and forming a toxin perforation which eventually leads to insect death. The resistance of target pests to Bt toxins is an important factor hampering the long-term effective cultivation of Bt crops and the continuous use of Bt toxins. This review summarizes the mechanism of insect resistance to Bt toxins from the perspective of important Bt toxin receptors in midgut cells of Lepidopteran insects, which may facilitate the in-depth study of Bt resistance mechanism and pest control.

Development and application of a motor rehabilitation system based on Kinect somatosensory interaction technology / 中华物理医学与康复杂志

Objective To develop a motor rehabilitation system based on Kinect somatosensory interaction technology to be used in families.Methods The Kinect skeleton real-time tracking technique was applied to develop three motor rehabilitation protocols to instruct patients in how to perform rehabilitation training and to evaluate their performance.Results Five subjects participated in the experiment.They achieved average scores of 79.15 ±4.89 and 98.89±0.67 for 3D movement and arm lifting respectively.In the pose recognition experiment,their average recognition rate was 90.37 ± 5.21％.Conclusion The proposed rehabilitation system can instruct patients in performing training exercises and evaluate their performance at home.

Genome cloning of human bocavirus (HBoV1) and analysis of viral promoter activity / 生物工程学报

Human bocavirus (HBoV) is a recently discovered parvovirus, which is suspected to be an etiologic agent of respiratory disease and gastrointestinal disease in human. In the present study, we screened 941 nasopharyngeal aspirates collected from hospitalized children with lower respiratory tract infections from October 9, 2007 to March 20, 2009 in the Children's Hospital of Hubei Province. Our results showed that 33 of 941 samples (3.51%) were detected positive for HBoV. To obtain a full-length HBoV clone, three segments which covered the nearly full-length genome were amplified by PCR from HBoV positive samples separately and cloned into pBluescript SK II vector, and the resulting plasmid was designated as pWHL-1 (GenBank Acession No. GU139423). We constructed the both EGFP and luciferase reporter gene vectors under the control of the HBoV unique promoter, respectively. Our data demonstrated that the HBoV promoter exhibited very high activity in all mammalian cells tested by fluorescent microscopy observation of the EGFP and luciferase activity assay and its strength was 4-5 fold higher compared to that of the CMV promoter. This work provided an excellent tool for further study of the mechanism of transcription and expression of the viral genome.