纳豆激酶基因的克隆及其在毕赤酵母中的表达

纳豆激酶纳是从日本传统食品纳豆中发现的一类具有溶栓效果的蛋白酶,由于其具有安全,高效,作用时间长,易吸收,廉价等优点,现在正成为一个开发治疗血栓类疾病药物的研究热点。从本实验室保存的一株高溶栓的纳豆杆菌N07出发,提取总基因组DNA,利用PCR手段扩增获得了纳豆激酶长为825bp的成熟肽基因片段。构建重组表达质粒pPICZaA-NK,经EcoR I、Xba I双酶切、PCR、测序验证得出重组表达质粒上的外源基因即为825bp的目的片段;将重组质粒pPICZaA-NK用内切酶Sac I线性化后电击导入毕赤酵母X33,通过含Zeocin的YPDS平板筛选获得重组酵母。重组酵母在BMMY培养基中发酵培养,用1%甲醇诱导目的蛋白表达。用纤维蛋白平板法检测发现发酵上清具有纤溶活性,经硫酸铵盐析、透析、Sephadex-G50过柱等步骤分离得到纳豆激酶蛋白,进行SDS-PAGE鉴定表明,表达的纳豆激酶蛋白分子量为27KD。以尿激酶为标准,实验所得纳豆激酶发酵上清液溶栓活性约为195U/mL。成功的将纳豆激酶成熟肽基因在毕赤酵母X33中表达,为纳豆激酶基因工程进一步研究奠定基础。     

生黑醋酸杆菌的太空诱变选育

目的:通过太空育种的方法选育获得高活性的生黑醋酸杆菌,实现维生素C的高效发酵.方法:采用太空育种的方法,对VC一步发酵菌生黑醋酸杆菌进行神州七号搭载诱变.结果:经过多轮试管初筛、摇瓶复筛,获得1株高效菌,编号为G454.23%醇浓摇瓶发酵,发酵周期缩短约2h;在适度提高通风下,可完成高醇浓度(33%)发酵,发酵周期约24h.该菌经发酵条件优化,连续5批常规生产.结果:新菌株G454的发酵稳定性好,周期均在20h以内,平均周期18.4h;与对照相比,发酵周期缩短2h,山梨糖产率12.34mg/ml/h,较对照提高7.68%.结论:筛选所得菌株454可缩短发酵周期10%.,提高山梨糖产率7.68%.     

阿维菌素对棉铃虫的抗性选育及其对解毒酶活性的影响

为了了解棉铃虫对阿维菌素的抗药性机理,用阿维菌素对棉铃虫(Heliothis armigera)进行了抗性选育,并对选育的27代棉铃虫解毒酶的活性进行了检测.选育从F0至F21代,抗性缓慢波动上升,达到选育前的121.19倍;F0至F27代抗性迅速增长,达到选育前的821.73倍,抗性发展趋势呈现S型曲线.随着选育代数的增加,对乙酰胆碱酯酶(AChE)没有明显的影响;羧酸酯酶(CarE)活性,F27是F0的2.52倍(α-NA)和2.83倍(β- NA),从20开始,活性在较高水平上波动;谷胱甘肽转移酶(GST)活性,F27是F0的2.12倍,且从F16开始,活性在较高水平上波动.选育的抗性品系,增效醚对阿维菌素增效6.77倍.上述结果说明棉铃虫对abamectin的抗性机制也涉及MFO、GSTs、CarE等多个酶系统,但与乙酰胆碱酯酶(AChE)无关.     

Biosynthesis of a novel recombinant peptide derived from hPTH(1-34)

A recombinant human parathyroid hormone fragment, Pro-Pro-[Arg(11)]hPTH(1-34)-Pro-Pro (MW=4550 Da), was developed by substituting Arg for Leu at position 11 and adding Pro-Pro at the carboxyl terminus. Following a single injection (0.5-13.5 μg/bird) of the rhPTH fragment, the serum calcium level in chickens increased 12-42% (P<0.05) after 1h as determined by the Parson's Chicken Assay. The functional activity of Pro-Pro-[Arg(11)]hPTH(1-34)-Pro-Pro may be due to removal of the N-terminus Pro-Pro- by X-prolyl dipeptidyl peptidase IV (DPPIV) in vivo, increasing its activity compared to Pro-Pro-hPTH(1-34). This artificial rhPTH fragment could be used to increase calcium mobilization and potentially improve bone health.     

Differential responses in two varieties of winter wheat to elevated ozone concentration under fully open‐air field conditions

Two modern cultivars [Yangmai16 (Y16) and Yangfumai 2 (Y2)] of winter wheat (Triticum aestivum L.) with almost identical phenology were investigated to determine the impacts of elevated ozone concentration (E‐O₃) on physiological characters related to photosynthesis under fully open‐air field conditions in China. The plants were exposed from the initiation of tillering to final harvest, with E‐O₃ of 127% of the ambient ozone concentration (A‐O₃). Measurements of pigments, gas exchange rates, chlorophyll a fluorescence and lipid oxidation were made in three replicated plots throughout flag leaf development. In cultivar Y2, E‐O₃ significantly accelerated leaf senescence, as indicated by increased lipid oxidation as well as faster declines in pigment amounts and photosynthetic rates. The lower photosynthetic rates were mainly due to nonstomatal factors, e.g. lower maximum carboxylation capacity, electron transport rates and light energy distribution. In cultivar Y16, by contrast, the effects of E‐O₃ were observed only at the very last stage of flag leaf ageing. Since the two cultivars had almost identical phenology and very similar leaf stomatal conductance before senescence, the greater impacts of E‐O₃ on cultivars Y2 than Y16 cannot be explained by differential ozone uptake. Our findings will be useful for scientists to select O₃‐tolerant wheat cultivars against the rising surface [O₃] in East and South Asia.     

蛋白质表面氨基酸特性研究进展

分布在蛋白质分子表面的暴露于溶剂的氨基酸所具有的一些特性对蛋白质的折叠和聚合过程、蛋白质一蛋白质相互作用以及蛋白质的功能具有重要影响。本文分析了蛋白质表面氨基酸在疏水性、保守性、静电势及结构方面的一些特性,阐述了近年来国际上利用这些特性对蛋白质一蛋白质相互作用界面进行预测的方法,最后介绍了几款预测蛋白质表面氨基酸的软件。     

后磨牙模拟根管模型制备与研究

目的：制备后磨牙模拟根管模型。方法：依照预实验中测试的牙本质和树脂根管模型材料的布氏硬度值（牙本质HB72,树脂块HB54）选取纯铜质铜管为材料（纯铜HB58）同时利用预实验中确认的后磨牙模拟根管模型弯曲角度的范围（70°-60°）并参照树脂根管模型相关数据,设计不同弯曲角度（70°,64°,60°）的纯铜质根管模型,将20套镍钛根管锉随机分为4组,其中3组根管锉分别预备相应角度的10个纯铜质后磨牙根管模型,另一组在临床预备10个后磨牙根管作为对照组。所有镍钛根管锉均在疲劳寿命测试装置中测试疲劳寿命。结果：选用根管长度为16mm±1mm,弯曲半径为5mm,内径为0.70mm的纯铜质铜管制作后磨牙根管模型。其中在弯曲角度为70°的后磨牙根管模型中有33%（2/6）近似临床;在弯曲角度为64°的后磨牙根管模型中有67%（4/6）近似临床;在弯曲角度为60°的后磨牙根管模型中有33%（2/6）近似临床。结论：弯曲角度为64°的纯铜制后磨牙模拟根管模型更接近于临床实际,可为体外研究镍钛根管锉各项指标提供一个较为理想的模型。     

基于钙黄绿素- 铜(Ⅱ)荧光体系测定乙酰半胱氨酸

目的:基于钙黄绿素-铜(Ⅱ)荧光体系测定乙酰半胱氨酸。方法:在pH=8.0的Na2HPO.412H2O-KH2PO4缓冲液中,以492 nm为激发波长,520 nm为发射波长测定乙酰半胱氨酸溶液的荧光强度。结果:在pH=8.0的Na2HPO.412H2O-KH2PO4缓冲液中,二价铜离子与钙黄绿素配位引起荧光猝灭。由于乙酰半胱氨酸中巯基上的硫离子与Cu2+的亲和力很强,可从钙黄绿素-铜(Ⅱ)的络合物中夺取铜离子而使钙黄绿素游离出来,从而使体系的荧光得以恢复,并且荧光恢复的程度与加入乙酰半胱氨酸的量在一定范围内成线性。结论:建立了一种测定乙酰半胱氨酸的荧光分析新方法,该方法的线性范围为6.0 10-6～1.4 10-5 mol/L,检出限为4.010-6 mol/L。     

高效液相色谱法测定大豆乳清提取物中大豆异黄酮的含量

建立了大豆乳清提取物中大豆异黄酮含量的高效液相色谱测定方法。采用Nova-Pak C₁₈（3.9×150 mm,4 μm）色谱柱;以甲醇：0.4%磷酸=30：70（v/v）为流动相分析染料木苷和黄豆苷;流速为0.7 mL·min^-1;柱温为30℃;检测波长为260 nm。试验结果表明,大豆乳清提取物中的大豆异黄酮含量为72.5%,其组成以染料木苷和黄豆苷为主,二者比例接近1∶1,苷元型大豆异黄酮未检出。染料木苷和黄豆苷的平均回收率分别为98.1%和98.4%,相对标准偏差（RSD）分别为0.7%（n=5）和0.8%(n=5)。该方法快速、准确、重复性好。