The experiments were carried out in paddy field of CNRRI in 1985-1989. Seedlings of the indica variety Erjiufeng were raised in a seedbed and transplanted to pots with area of 0.1 m~2 at seedling age of 30 d. Treatmen...The experiments were carried out in paddy field of CNRRI in 1985-1989. Seedlings of the indica variety Erjiufeng were raised in a seedbed and transplanted to pots with area of 0.1 m~2 at seedling age of 30 d. Treatments with (CRMS) were conducted at different stages: pistil and stigma primordium formation展开更多
According to the standard of GB/T1500.3-2008 and the guidelines of ISO/REMCO, the preparation of National Certified Reference Materials （CRMs） of active compounds was composed of the following two aspects： preparat...According to the standard of GB/T1500.3-2008 and the guidelines of ISO/REMCO, the preparation of National Certified Reference Materials （CRMs） of active compounds was composed of the following two aspects： preparation and certification. This paper presents an example of the preparation of biochanin A to illustrate the preparation of National CRMs of active compounds from natural products. Firstly, the target compound was extracted and purified from crude materials. Then, its chemical structure was identified by UV spectrum, IR spectrum, ESI-MS, and NMR. The purity of the compound was determined by HPLC with the area normalization method. The certified value assessed by comparing the purity values from six independent qualified laboratories and the value of uncertainty were given. Thermal analysis, residual solvent, homogeneity testing and stability testing was tested in the process.展开更多
白喉毒素突变体蛋白(cross-reacting material 197, CRM197)是一种常用的多糖结合疫苗载体,其在大肠杆菌体系中往往以包涵体形式表达。为了改善通过包涵体复性来制备CRM197而导致的收率低、操作烦琐等不足,研究设计了一种基于SUMO标签...白喉毒素突变体蛋白(cross-reacting material 197, CRM197)是一种常用的多糖结合疫苗载体,其在大肠杆菌体系中往往以包涵体形式表达。为了改善通过包涵体复性来制备CRM197而导致的收率低、操作烦琐等不足,研究设计了一种基于SUMO标签串联的策略,改善重组CRM197的可溶性,同时使用Origami B (DE3)大肠杆菌宿主,成功使重组CRM197蛋白上清液可溶比例提高至95%以上。同时简化了CRM197蛋白的获取途径,经两步Ni亲和层析和一步酶切除去融合标签等手段建立了从破菌上清液中纯化CRM197的方法,获得纯度高于98%的CRM197且收率大于80%。通过圆二色光谱及荧光发射光谱分析等证实纯化所得CRM197空间结构折叠正确。抗原性ELISA实验结果表明所制得的CRM197蛋白与CRM197标准品蛋白抗原性相当。研究结果表明串联双SUMO标签对重组CRM197在大肠杆菌中高效、可溶表达具有促进作用,并建立了快速纯化体系,获得了具有较好免疫原性的CRM197蛋白,为改善重组蛋白在原核表达体系的可溶表达设计提供了新思路。展开更多
CRM197(cross-reacting material 197)是白喉毒素的天然突变体,它的酶活性丧失但受体结合能力及免疫原性却完全保留,这一特性使其成为安全且高效的疫苗载体,被广泛应用于偶联或结合疫苗的开发。本研究利用基因工程技术分别制备白喉毒素...CRM197(cross-reacting material 197)是白喉毒素的天然突变体,它的酶活性丧失但受体结合能力及免疫原性却完全保留,这一特性使其成为安全且高效的疫苗载体,被广泛应用于偶联或结合疫苗的开发。本研究利用基因工程技术分别制备白喉毒素突变体CRM197及它与E型肉毒毒素受体结合区(EHc)的2种融合蛋白质分子,对获得的重组蛋白质分子进行SDS-PAGE和免疫印迹鉴定,继而免疫BALB/c小鼠,评价它们作为融合蛋白质亚单位疫苗的免疫效果。结果表明,制备了无毒性的白喉毒素突变体CRM197和CRM197-EHc与EHc-CRM197两种融合蛋白质分子,这两种融合蛋白质分子作为亚单位疫苗诱导免疫小鼠产生了强的抗CRM197和EHc抗原的体液免疫反应;与CRM197与EHc联合组相比,CRM197-EHc与EHc-CRM197融合蛋白质疫苗组能够诱导更高的抗CRM197抗体效应(P<0.01),EHc-CRM197融合蛋白质低剂量的疫苗组也能够诱导更高的抗EHc抗体效应(P<0.05)。因此,EHc在融合蛋白质疫苗中发挥了载体分子效应,增强了CRM197的免疫原性;CRM197在EHc-CRM197融合蛋白质分子疫苗中作为载体分子增强了EHc的免疫原性,作为融合亚单位疫苗诱导产生了最高的抗CRM197和EHc抗原的体液免疫反应,是个高效的二价疫苗分子。展开更多
文摘The experiments were carried out in paddy field of CNRRI in 1985-1989. Seedlings of the indica variety Erjiufeng were raised in a seedbed and transplanted to pots with area of 0.1 m~2 at seedling age of 30 d. Treatments with (CRMS) were conducted at different stages: pistil and stigma primordium formation
基金The Natural Science Foundation of China (20872083)the Key Science and Technology Program of Shandong Province and the State Key Laboratory of Crop Biology (2008kf05)
文摘According to the standard of GB/T1500.3-2008 and the guidelines of ISO/REMCO, the preparation of National Certified Reference Materials （CRMs） of active compounds was composed of the following two aspects： preparation and certification. This paper presents an example of the preparation of biochanin A to illustrate the preparation of National CRMs of active compounds from natural products. Firstly, the target compound was extracted and purified from crude materials. Then, its chemical structure was identified by UV spectrum, IR spectrum, ESI-MS, and NMR. The purity of the compound was determined by HPLC with the area normalization method. The certified value assessed by comparing the purity values from six independent qualified laboratories and the value of uncertainty were given. Thermal analysis, residual solvent, homogeneity testing and stability testing was tested in the process.
文摘白喉毒素突变体蛋白(cross-reacting material 197, CRM197)是一种常用的多糖结合疫苗载体,其在大肠杆菌体系中往往以包涵体形式表达。为了改善通过包涵体复性来制备CRM197而导致的收率低、操作烦琐等不足,研究设计了一种基于SUMO标签串联的策略,改善重组CRM197的可溶性,同时使用Origami B (DE3)大肠杆菌宿主,成功使重组CRM197蛋白上清液可溶比例提高至95%以上。同时简化了CRM197蛋白的获取途径,经两步Ni亲和层析和一步酶切除去融合标签等手段建立了从破菌上清液中纯化CRM197的方法,获得纯度高于98%的CRM197且收率大于80%。通过圆二色光谱及荧光发射光谱分析等证实纯化所得CRM197空间结构折叠正确。抗原性ELISA实验结果表明所制得的CRM197蛋白与CRM197标准品蛋白抗原性相当。研究结果表明串联双SUMO标签对重组CRM197在大肠杆菌中高效、可溶表达具有促进作用,并建立了快速纯化体系,获得了具有较好免疫原性的CRM197蛋白,为改善重组蛋白在原核表达体系的可溶表达设计提供了新思路。
文摘CRM197(cross-reacting material 197)是白喉毒素的天然突变体,它的酶活性丧失但受体结合能力及免疫原性却完全保留,这一特性使其成为安全且高效的疫苗载体,被广泛应用于偶联或结合疫苗的开发。本研究利用基因工程技术分别制备白喉毒素突变体CRM197及它与E型肉毒毒素受体结合区(EHc)的2种融合蛋白质分子,对获得的重组蛋白质分子进行SDS-PAGE和免疫印迹鉴定,继而免疫BALB/c小鼠,评价它们作为融合蛋白质亚单位疫苗的免疫效果。结果表明,制备了无毒性的白喉毒素突变体CRM197和CRM197-EHc与EHc-CRM197两种融合蛋白质分子,这两种融合蛋白质分子作为亚单位疫苗诱导免疫小鼠产生了强的抗CRM197和EHc抗原的体液免疫反应;与CRM197与EHc联合组相比,CRM197-EHc与EHc-CRM197融合蛋白质疫苗组能够诱导更高的抗CRM197抗体效应(P<0.01),EHc-CRM197融合蛋白质低剂量的疫苗组也能够诱导更高的抗EHc抗体效应(P<0.05)。因此,EHc在融合蛋白质疫苗中发挥了载体分子效应,增强了CRM197的免疫原性;CRM197在EHc-CRM197融合蛋白质分子疫苗中作为载体分子增强了EHc的免疫原性,作为融合亚单位疫苗诱导产生了最高的抗CRM197和EHc抗原的体液免疫反应,是个高效的二价疫苗分子。
