Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115

Human serum albumin (HSA) and human parathyroid hormone (1-34) [PTH (1-34)] fusion protein [HSA/PTH (1-34)] is a promising long-acting form of PTH (1-34) for osteoporosis treatment. Secretory expression of intact HSA/PTH (1-34) in Pichia pastoris GS115 was accompanied by two degradation fragments, with molecular weights around 66 kDa, in addition to the well-known ~45 kDa HSA-truncated fragment, resulting in a low yield of intact protein. In this study, two internal cleavage sites were identified in the PTH (1-34) portion of the fusion protein by Western Blot analysis. To minimize proteolytic cleavages, several protease genes including PEP4 (encoding proteinase A), PRB1 (proteinase B) and seven YPSs genes (yapsin family members) were knocked out respectively by disruption of the individual genes and the selective combinations. Reduced degradation was observed by single disruption of either PEP4 gene or YPS1 gene, and the lowest level of degradation was observed in a pep4△yps1△ double disruptant. After 72 h of induction, more than 80 % of the HSA/PTH (1-34) secreted by the pep4△yps1△ double disruptant remained intact, in comparison to only 30 % with the wild-type strain.     

Construction of prophylactic human papillomavirus type 16 L1 capsid protein vaccine delivered by live attenuated Shigella flexneri strain sh42

To express human papillomavirus （HPV） L 1 capsid protein in the recombinant strain of Shigella and study the potential of a live attenuated Shigella-based HPV prophylactic vaccine in preventing HPV infection, the icsA/virG fragment of Shigella-based prokaryotic expression plasmid pHS3199 was constructed. HPV type 16 L1 （HPV16L1） gene was inserted into plasmid pHS3199 to form the pHS3199-HPV16L1 construct, and pHS3199-HPV 16L1 was electroporated into a live attenuated Shigella strain sh42. Western blotting analysis showed that HPV 16L 1 could be expressed stably in the recombinant strain sh42-HPV 16L 1. Sereny test results were negative, which showed that the sh42-HPV16L1 lost virulence. However, the attenuated recombinant strain partially maintained the invasive property as indicated by the HeLa cell infection assay. Specific IgG, IgA antibody against HPV16L1 virus-like particles （VLPs） were detected in the sera, intestinal lavage and vaginal lavage from animals immunized by sh42-HPV 16L1. The number of antibodysecreting cells in the spleen and draining lymph nodes were increased significantly compared with the control group. Sera from immunized animals inhibited mufine hemagglutination induced by HPV 16L1 VLPs, which indicated that the candidate vaccine could stimulate an efficient immune response in guinea pig＇s mucosal sites. This may be an effective strategy for the development of an HPV prophylactic oral vaccine.     

豚草叶片和果实气体交换特性与11种土壤重金属相关性

对10个样地中Cu、Pb、Zn、Mn、Cr、Co、Ni、Cd、As、Sb和Hg11种土壤重金属含量及样地内豚草叶片和果实气体交换特性进行测定.结果表明,样地内豚草叶片的净光合速率在1·88~9·41μmol·m-2·s-1,而果实的净光合速率最高可达2·81μmol·m-2·s-1.叶片的呼吸速率、气孔导度、光合速率和水分利用效率的平均值分别为1·81μmol·m-2·s-1、75·7mmol·m-2·s-1、6·05μmol·m-2·s-1和4·72μmol·mmol-1,分别是果实的5·26、0·64、1·31和1·69倍,说明非同化器官幼嫩果实具有与叶片相当,甚至更强的呼吸、光合能力和水分利用效率;研究地点重金属Ni达到轻微污染水平,其它重金属含量都接近或者显著低于重金属污染的阈值.相关分析和多元回归分析显示,大部分土壤重金属(如Cu、Pb、Zn、Cd、As、Sb和Hg)含量的高低对豚草气体交换特性没有显著影响,仅部分重金属含量与豚草的叶片、果实气体交换特性密切相关,如Ni和Cr对豚草叶片、果实的气孔导度及水分利用效率显著相关;Cr与豚草叶片饱和光合速率显著相关;而As与豚草果实的气孔导度显著相关.表明大部分土壤重金属对叶片和球果的气体交换没有直接影响,而Ni、Cr和As可以在轻微污染甚至没有达到污染水平时影响豚草的气体交换特性.     

间断低氧对大鼠下丘脑超微结构及前增食欲素水平的影响

目的探讨睡眠中间断低氧对大鼠下丘脑前增食欲素及受体水平的影响以及下丘脑超微结构的变化。方法大鼠分成对照组、间断低氧组和持续低氧组,分别给予吸入空气,持续低氧和间断低氧气体,并在实验开始后1d、3d、1w和4w应用RT-PCR方法测定大鼠下丘脑前增食欲素及受体水平,分析其间的变化关系,电镜观察下丘脑的超微结构变化。结果与对照组和持续低氧组比较,间断低氧4w后大鼠下丘脑前增食欲素mRNA水平明显降低,受体水平升高,但在持续低氧和对照组之间无明显差异。在低氧后1d、3d、7d后大鼠下丘脑前增食欲素mRNA降低,受体水平升高,在4w后,持续低氧组则接近正常。急性持续低氧大鼠超微结构变化更严重,而慢性间断低氧变化更持久。结论慢性间断低氧可以引起下丘脑前增食欲素下降及受体水平升高,急性持续低氧也可引起上述变化,而慢性持续低氧未引起增食欲素改变;慢性间断低氧大鼠下丘脑超微结构表现为严重而持久的变化。     

羊草种群生物量生殖分配的初步研究

对羊草种群生物量生殖分配的研究表明,在生殖生长过程中,种群用于根茎和营养生长的生物量生殖分配占有绝对优势比例,而用于有性生殖的生物量生殖分配比例较小,在10%以下。在羊草种群构件生物量生殖分配中,根茎和营养枝的叶占有较大比例(20%～40%),而用于有性繁殖体种子生产的比例很小,在1.5%以下。对有性繁殖的小比例投资是导致羊草种群有性繁殖体种子产量低的重要原因.     

赶黄草总黄酮树脂精制工艺与检测方法

本研究以总黄酮吸附量和解析率为检测指标,结合动态洗脱考察结果,考察11种树脂对赶黄草总黄酮的富集精制能力。优化树脂类型,选定了对总黄酮进行富集纯化较好的树脂HPD450;采用单因素及正交实验,确定最佳工艺条件:上样量10 mL,以6 mL/min的流速8 BV水、6 BV 80%甲醇溶液洗脱,pH值为6~7。以此方法得到90%以上的赶黄草总黄酮,实验结果良好,总黄酮精制工艺成效显著。用HPLC法精确测定赶黄草中槲皮苷、PGHG和ThA三个指标性黄酮成分,最佳色谱条件为:C18色谱柱,检测波长280 nm,进样量5μL,流动相:乙腈-0.05%磷酸溶液(0~25 min:12%~45%乙腈;25~40 min:45%~70%乙腈)梯度洗脱;精确测定赶黄草全草中3种黄酮成分的含量。采用分光光度法检测,指导优化总黄酮精制工艺,并以HPLC法精确测定三种黄酮代表成分。比较两种方法测定结果相一致,互为补充;故在工业生产时,可以分光光度法指导生产,以HPLC法精确定量测定。     

荒漠沙蜥活动期间的体温节律

爬行动物的体温依赖于外界热环境的变化,本研究以荒漠沙蜥(Phrynocephalus przewalskii)为对象,收集其冬眠前活动期间的体温与地面和离地10 cm的空气温度,研究爬行动物体温节律性变化与环境温度的关系.研究结果表明,荒漠沙蜥在环境温度高于20℃后开始外出活动,其体温随着地面温度和空气温度的上升而逐步...     

色氨酸残基在内切葡聚糖酶分子中的作用

内切葡聚糖酶的化学修饰研究表明：色氨酸残基可能位于活性位点,与底物结合有关．荧光光谱测定指出该酶的荧光几乎都来自色氨酸残基,酶分子中色氨酸微环境对ｐＨ变化非常敏感,降低ｐＨ导致了酶分子构象发生了较大变化,配基结合使酶分子色氨酸微环境产生了改变,引发了与ｐＨ诱导不同的构象变化．