Enterovirus （EV71） can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 （VP1）, plays a critical role in the pathogenicity of EVT1. High level...Enterovirus （EV71） can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 （VP1）, plays a critical role in the pathogenicity of EVT1. High level expression and secretion ofVP 1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP 1 DNA vaccines, including wt-VP 1, tPA-VP 1, VP l-d, VP 1-hFc and VP 1 - mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different （P〈0.05）. In this study, we further investigated the protein lev- els of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc （hFc） to VP1. VP 1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VPI-hFc protein for additional studies.展开更多
A method for the determination of human immunoglobulin G（IgG） based on a colloidal gold label by fluorospectrophotometry was developed. The sandwich immunoreaction among goat-anti-human IgG, human IgG and goat-anti-...A method for the determination of human immunoglobulin G（IgG） based on a colloidal gold label by fluorospectrophotometry was developed. The sandwich immunoreaction among goat-anti-human IgG, human IgG and goat-anti-human IgG labeled with colloidal gold nanoparticles was applied in this experiment. First, a sandwich im- munocomplex was formed on the surface of 96 well clear polystyrene high bind stripwellTM microplate. After the formation of the sandwich immunocomplex, a solution was added to dissociate the immunocomplex at room temperature. Then the solution of each well was transferred into the corresponding test tube. Thirdly, the rhodamine B solution was injected into each test tube. The rhodamine B chloraurate was extracted into ether that was measured with a spectrofluorometer. The experimental results indicate that the fluorescence intensity increased with the increase of human IgG concentration. The fluorescence intensity of rhodamine B chloraurate at 570 nm was proportional to the logarithm of human IgG concentration in a range from 10 to 5 × 10^5 ng/mL. It was shown that the determination of human IgG was easily made with the proposed fluorospectrophotometry.展开更多
A simple, and homogeneous detection system for human IgG based on the optical properties of aggregated gold nanoparticles probes were investigated. When gold nanoparticles with about 13 nm in diameter were modified by...A simple, and homogeneous detection system for human IgG based on the optical properties of aggregated gold nanoparticles probes were investigated. When gold nanoparticles with about 13 nm in diameter were modified by goat anti-human IgG, the addition of human IgG could change the absorption of colloidal gold solution, and the absorption intensity at 740 nm depended on the amount of human IgG. The aggregation of gold nanoparticles was also validated using transmission electron microscopy （TEM）. A series of experiments were carried out to study the effects of pH value, the reaction temperature, and non-specific adsorption on the assay. A dynamic range of 10-500 μg/3 mL human IgG was observed. The new bioassay could be used for the rapid and homogeneous detection of antibodies in bioanalytical chemistry.展开更多
基金supported by the National Natural Science Foundation of China(Grant No.81000725 and 31470889)the Priority Academic Program of Basic Medical Science of Nanjing Medical University(Grant No.JX10131801060)
文摘Enterovirus （EV71） can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 （VP1）, plays a critical role in the pathogenicity of EVT1. High level expression and secretion ofVP 1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP 1 DNA vaccines, including wt-VP 1, tPA-VP 1, VP l-d, VP 1-hFc and VP 1 - mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different （P〈0.05）. In this study, we further investigated the protein lev- els of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc （hFc） to VP1. VP 1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VPI-hFc protein for additional studies.
文摘A method for the determination of human immunoglobulin G（IgG） based on a colloidal gold label by fluorospectrophotometry was developed. The sandwich immunoreaction among goat-anti-human IgG, human IgG and goat-anti-human IgG labeled with colloidal gold nanoparticles was applied in this experiment. First, a sandwich im- munocomplex was formed on the surface of 96 well clear polystyrene high bind stripwellTM microplate. After the formation of the sandwich immunocomplex, a solution was added to dissociate the immunocomplex at room temperature. Then the solution of each well was transferred into the corresponding test tube. Thirdly, the rhodamine B solution was injected into each test tube. The rhodamine B chloraurate was extracted into ether that was measured with a spectrofluorometer. The experimental results indicate that the fluorescence intensity increased with the increase of human IgG concentration. The fluorescence intensity of rhodamine B chloraurate at 570 nm was proportional to the logarithm of human IgG concentration in a range from 10 to 5 × 10^5 ng/mL. It was shown that the determination of human IgG was easily made with the proposed fluorospectrophotometry.
基金Funded by the Major Project of Wuhan (No. 200016009107)the Scientific Research Fund from Huazhong Agricultural University (No.52204-08070)
文摘A simple, and homogeneous detection system for human IgG based on the optical properties of aggregated gold nanoparticles probes were investigated. When gold nanoparticles with about 13 nm in diameter were modified by goat anti-human IgG, the addition of human IgG could change the absorption of colloidal gold solution, and the absorption intensity at 740 nm depended on the amount of human IgG. The aggregation of gold nanoparticles was also validated using transmission electron microscopy （TEM）. A series of experiments were carried out to study the effects of pH value, the reaction temperature, and non-specific adsorption on the assay. A dynamic range of 10-500 μg/3 mL human IgG was observed. The new bioassay could be used for the rapid and homogeneous detection of antibodies in bioanalytical chemistry.
