Characterization of the Biological Activities of Recombinant Fusion Protein Green Fluorescent Protein/Human Zona Pellucida Protein 3 (GFP/HZP3)

Despite numerous reports indicating the successful production of bioactive recombinant ZP3, no report has shown the rhZP3 having direct binding activity with human sperm. Recombinant ZP3 generated from our previous study displayed binding activity with human sperm through indirect evidence from hemizona assay (HZA). This present study focused on the production of recombinant ZP3 with direct binding activity with human sperm. Through the application of a pEGFP expression vector, fusion protein GFP/ZP3 was successfully generated and expressed. The expression of GFP/ZP3 was evidenced by RT-PCR and western blot. The fusion protein was partially purified by Ni-NTA affinity column from cell culture medium of stably transfected cells generated from a single cell clone. Immuno-blotting analysis of the fusion protein indicated that the GFP/ZP3 was about 90–92 kD in molecular mass which is close to the sum of the size of GFP and ZP3. Flow cytometry was conducted to evaluate the direct binding activity of fusion protein with human sperm. The GFP/ZP3 showed dose-dependence in the binding assay. GFP tag removal resulted in almost no detectable binding as assayed by flow cytometry, thereby indicating that ZP3 binding was specific. Furthermore, immunofluorescence demonstrated that the fusion protein interacted with human sperm on the acrosome region. In the acrosome reaction assay, fusion protein was proved to be able to induce the sperm acrosome reaction (19.2% ± 3.4% as compared to basal line 10.8% ± 2.3%) at the concentration of 1ug/ml. Therefore, the fusion protein displayed full spectrum biological activity of ZP3, and, most importantly unlike prior studies, this binding activity was elucidated in a direct way

Optimizing Urban Distribution Routes for Perishable Foods Considering Carbon Emission Reduction

The increasing demand for urban distribution increases the number of transportation vehicles which intensifies the congestion of urban traffic and leads to a lot of carbon emissions. This paper focuses on carbon emission reduction in urban distribution, taking perishable foods as the object. It carries out optimization analysis of urban distribution routes to explore the impact of low carbon policy on urban distribution routes planning. On the base of analysis of the cost components and corresponding constraints of urban distribution, two optimization models of urban distribution route with and without carbon emissions cost are constructed, and fuel quantity related to cost and carbon emissions in the model is calculated based on traffic speed, vehicle fuel quantity and passable time period of distribution. Then an improved algorithm which combines genetic algorithm and tabu search algorithm is designed to solve models. Moreover, an analysis of the influence of carbon tax price is also carried out. It is concluded that in the process of urban distribution based on the actual network information, the path optimization considering the low carbon factor can effectively reduce the distribution process of CO2, and reduce the total cost of the enterprise and society, thus achieving greater social benefits at a lower cost. In addition, the government can encourage low-carbon distribution by rationally adjusting the price of carbon tax to achieve a higher social benefit