Expression and Purification of Human Keratinocyte Growth Factor 2 by Fusion with SUMO

Small ubiquitin-related modifier (SUMO) fusion system has been shown to be efficient for enhancing expression and preventing degradation of the target protein. We showed herein that SUMO fusion to human keratinocyte growth factor 2 (hKGF-2) gene was feasible and it significantly enhanced protein expression and its efficiency. The fusion DNA fragment composed of SUMO gene, which was fused to hexahistidine tag, and hKGF-2 gene was amplified by PCR and inserted into the expression vector pET28a to construct the recombinant plasmid, pET28a-SUMO-hKGF-2. The plasmid was then transformed into Escherichia coli Rosetta^TM2(DE3), and the recombinant fusion protein SUMO-hKGF-2 was expressed at 30°C for 6 h, with the induction of IPTG at the final concentration of 0.4 mM. The expression level of the fusion protein was up to 30% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography. After desalting by Sephadex G-25 size exclusion chromatography, the hexahistidine-SUMO-hKGF-2 was digested by SUMO proteases. The recombinant hKGF-2 was purified again with Ni-NTA column and the purity was about 95% with a total yield of 13.9 mg/l culture. The result of mitogenicity assay suggests that the recombinant hKGF-2 can significantly promote the proliferation of normal rat kidney epithelial (NRK-52E) cells. Xiaoping Wu, and Changjun Nie contributed equally to the work.     

Design of a novel class of biphenyl CETP inhibitors

A new class of CETP inhibitors was designed and prepared. These compounds are potent both in vitro and in vivo. The most active compound (12d) has shown an ability to raise HDL significantly in transgenic mouse PD model.     

营养保健型葛粉软糖的研制

利用优质葛粉和功能性甜味剂为主要原料,辅以适量的营养性凝胶剂,通过合理的制作工艺,制成了低热量、具有保健功能的葛粉软糖。     

花耳科二新种

本文报道了产于中国的花耳科Dacrymycetaceae二个新种,即莽山胶角菌Calocera mangshanensis Liu et Fan和云南花耳Dacrymyces yunnanensis Liu et Fan。     

县域生态资产核算研究——以云南省屏边县为例

生态资产(Eco-Assets)是生产与提供生态产品和服务的自然资源。以国家重点生态功能区县——屏边苗族自治县为例,研究了县域生态资产核算的指标和方法,以生态资产综合指数、负债表和损益表,开展屏边县生态资产实物量核算,以期能够切实反映出生态系统实物量的变化和生态补偿成效,结合生态补偿分析屏边县生态资产变化的原因和趋势,给生态补偿政策和离任干部审计提供技术支撑。结果表明:(1)屏边县生态资产核算可以分为森林、灌丛、草地、湿地自然生态系统和农田、城镇和荒漠人工生态系统实物量的核算。(2)2015年,屏边县生态资产指数为1.09,森林和灌丛占据主要位置,二者总和占当年生态资产的95.48%。15年来,屏边县生态资产综合指数降低了16.48%,其中森林和草地降低明显,分别减少了20.10%和6.67%,湿地指数增长了57.11%。(3)2000年以来,生态保护工程和城镇扩张是生态资产变化的主要原因,生态补偿投入持续增加为生态保护工程顺利建设实施提供了保障。本研究表明,现有的土地利用数据和生态环境监测数据基本可以支撑县域生态资产的核算,同时县域生态资产的变化能够体现出生态保护工程实行成效,并为实...     

阿帕替尼对人肝癌细胞增殖及凋亡的作用机制研究

目的:探讨阿帕替尼抑制肝癌细胞增殖促进凋亡的作用机制。方法:选取肝癌细胞系SNU739、HepG2,以CCK-8细胞增殖实验、平板克隆实验测定阿帕替尼对肝癌细胞增殖及克隆形成能力的影响;流式细胞术检测阿帕替尼对肝癌细胞凋亡的影响;蛋白免疫印迹法检测阿帕替尼影响肝癌细胞凋亡相关蛋白Bax、Bcl-2及Caspase3的表达情况。结果:与对照组相比,阿帕替尼可显著抑制肝癌细胞增殖(P0.05)。平板克隆实验提示与对照组相比,10μM和20μM阿帕替尼组肝癌细胞克隆数明显减少(P0.05)。流式细胞术结果提示10μM和20μM阿帕替尼处理组细胞凋亡率明显增加(P0.05)。蛋白免疫印迹法结果显示经阿帕替尼处理的肝癌细胞,促凋亡蛋白Bax及Caspase3的活性片段Cleaved-caspase3表达水平显著上调,抗凋亡蛋白Bcl-2显著下调(P0.01)。结论:阿帕替尼通过调节肝癌细胞凋亡相关蛋白从而抑制肝癌细胞增殖、促进其凋亡。     

Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ- ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27% ± 3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors development in the treated mice; and (iv) no local or systemic side-effects and toxicity associated with the gene transfer were found. It demonstrated the potential use of treating hemophilia B by recombinant adeno-associated viral vectors with mutant hFIXR338A gene, an alternative strategy for hemophilia B gene therapy to wild-type human factor IX.