人RHD基因的克隆和鉴定

目的克隆人RHD基因,并对其进行鉴定。方法以RhD阳性志愿者骨髓为材料,用TRIzol试剂提取总RNA;设计、合成人RHD基因扩增引物,RT-PCR方法扩增RHD基因片段;T／A克隆后将其亚克隆人pET28a（＋）载体中,经酶切、PCR和测序对重组质粒进行鉴定。结果骨髓总RNA被成功提取;RT-PCR成功扩增出RHD基因片段,其大小与预期约509bp基本一致;T／A克隆后再将其亚克隆,通过酶切和PCR证明RHD基因成功亚克隆入pET28a（＋）载体中;基因测序结果比对显示,与已公布的RHD基因（GenBank登录号为NM016124）序列基本一致,同源性为98％。结论成功克隆了RHD基因,这将为进一步研究奠定基础。     

铃兰的胚胎学研究

铃兰（Ｃｏｎｖａｌｌａｒｉａ ｍａｊａｌｉｓＬ．）的花药四室,药壁发育为单子叶型。腺质绒毡层发育后期出现双核至多核。小孢子四分体主要呈左右对称型,偶见四面体型。胞质分裂为连续型。二细胞型花粉。子房三室,中轴胎座。胚珠倒生,双珠被,厚珠心。珠孔由内珠被形成。胚囊发育为葱型和英地百合型。受精以后,在子房壁、珠柄和外珠被细胞中含有草酸钙针晶。胚发育类似于石竹型,但基细胞发生纵裂形成两个子细胞。核型胚乳;合点端具巨形胚乳核     

Cadaveric study of the arterial anatomy of the upper lip

Arterial distribution of the upper lip was investigated in this study. The location, course, length, and diameter of the superior labial artery and its alar and septal branches were determined on 14 preserved cadaver heads. Another cadaver head was used to show the arterial tree by the colored silicone injection technique. The superior labial artery was the main artery of the upper lip and always originated from the facial artery. The superior labial artery was 45.4 mm in length, with a range from 29 to 85 mm. The mean distance of the origin of the superior labial artery from the labial commissura was 12.1 mm. The superior labial artery was 1.3 mm in external diameter at its origin. The mean distance of origin of the superior labial artery from the lower border of the mandible was 46.4 mm. The alar division of the superior labial artery was mostly found as a single branch (82 percent). Its mean length was 14.8 mm and the mean diameter at the origin was 0.5 mm. The distance between the origins of the superior labial artery and the septal branch was 33.3 mm. The septal branch was single in most of the cases (90 percent). The mean length of the septal branch was 18.0 mm and the diameter at its origin was 0.9 mm. After all dissections, it was concluded that the arterial distribution of the upper lip was not constant. The superior labial artery can occur in different locations unilaterally and bilaterally, with the branches showing variability.     

内蒙古地区刺叶柄棘豆(Oxytropis aciphylla Ledeb.)种群遗传多样性分析

用ISSR分子标记对内蒙古地区刺叶柄棘豆(Oxytorpis aciphylla Ledeb.) 5个地理种群进行了种群遗传多样性分析。结果表明：刺叶柄棘豆种群具有较高的遗传多样性,11个ISSR引物扩增出215条带,总的多态位点百分率为98.14%,Shannon多样性指数I=0.2108,Nei基因多样性指数 H=0.341 6,种内总基因多样性(H_t) 为0.2108,种群内基因多样性(H_s)为0.160 4, 大部分遗传变异(76.11%)的遗传变异存在于种群内, 23.89%的遗传变异存在于种群间。遗传分化系数(Gst)为0.238 9,基因流(Nm)为1.592 9。5个居群间已有遗传分化趋势,遗传漂变不会引起遗传分化。UPGMA遗传距离聚类结果表明, 5个地理种群中,植被类型为荒漠草原的4个种群之间遗传距离较近,与1个荒漠种群距离较远。     

穴居狼蛛毒中一个抗菌活性多肽的鉴定和纯化

从我国新疆地区产穴居狼蛛的毒液中分离纯化了一种多肽——狼蛛抗菌肽(Lycosin)。穴居狼蛛的粗毒在酸性聚丙烯酰胺凝胶电泳中可分出11条蛋白带,用0.6％的大肠杆菌琼脂胶覆盖在凝胶上进行鉴定表明,电泳迁移率最快的区带有抗菌活性。经鉴定该多肽分子系由43至45个氨基酸残基组成,N-末端为丙氨酸,分子中的碱性和疏水性氨基酸分别占总氨基酸残基数的1/4和1/3。     

逆相蒸发法制备茶多酚脂质体及质量评价

采用逆相蒸发法制备茶多酚脂质体并进行质量评价。通过二次回归旋转组合设计优化茶多酚脂质体制备工艺及配方,对其形态、结构、粒径分布等性质进行考察。研究结果表明,最佳配方为m（大豆卵磷脂）：m（胆固醇）=3：1、茶多酚质量浓度为7mg／mL、V（有机相）：V（水相）=4：1、磷酸盐缓冲液浓度15mmoL／L,此条件下包封率为50．37％;所制备的茶多酚脂质体形态呈圆球形或椭球形,为大单室脂质体,有效粒径为165．3nm,Zeta电位为-69．3mV。逆相蒸发法制备茶多酚脂质体方法简单可行,所制备的脂质体具有一定缓释性。     

Flower-inducing and -inhibiting activities of Phloem Exudate from Cotyledons of Pharbitis Seedlings

The flower-inducing and -inhibiting activities of phloem exudate (PE) prepared from cotyledons of Pharbitis seedlings were examined, using apex cultures in vitro from Pharbitis as a bioassay system.The PE was prepared from photoperiodically-induced cotyledons (SD-PE). The SD-PE was subjected to the following fractionations: When the SD-PE was extracted with CHCl₃ and then ethyl acetate, the inducing activity was located in the final aqueous fraction. The activity was localized in the diffusate when the aqueous fraction was dialyzed (molecular weight cut off was 10,000). The diffusate was fractionated by ion exchange chromatography, and flower-inducing activity was found in the fraction adsorbed onto anion exchange resin. When the fraction was applied to a Sep-Pak C18 cartridge, the activity eluted with 25% MeOH. As a result of the above fractionation, activity was increased about 30-fold.The nature of the flower-inhibiting activity of the PE taken from cotyledons exposed to continuous-light conditions was examined (CL-PE). The inhibiting activity was decreased as the cotyledons were exposed to longer dark periods; it appeared to be heat-stable. The CL-PE also inhibited flowering in Lemna. The CL-PE was subjected to the following fractionations: When the CL-PE was extracted with CHCl₃ and ethyl acetate, activity was located in the final aqueous fraction. Activity was localized in the diffusate when the aqueous fraction was dialyzed (molecular weight cut off was 10,000). When the diffusate was fractionated by ion exchange chromatography, the activity was found in the flow-through fraction. When the fraction was applied to a hydroxyapatite cartridge, the activity eluted with 25 mM sodium phosphate buffer. When the fraction was re-dialyzed (molecular weight cut off was 1,000), the diffusate contained the activity. As a result of the above fractionation, activity was increased about 10-fold.     

小玉竹的胚胎学研究

小玉竹Polygonatum humile Fisch．ex Maxim．的花药四室．绒毡层腺质型,发育后期出现双核至多核。小孢子四分体左右对称型,偶见四面体型,胞质分裂连续型。成熟花粉具2细胞。子房3室,中轴胎座。胚珠倒生,双珠被,厚珠心,珠孔由内珠被形成。胚囊发育为葱型。受精后,子房壁和外珠被细胞中含有草酸钙针晶。胚发育类似于紫菀型,基细胞有时纵裂形成两个子细胞。胚乳核型。根据实验结果,并结合前人的资料,本文提出了黄精属的胚胎学特征,并在此基础上对黄精族的概念以及属间系统关系进行了探讨。     

枯草芽胞杆菌微生态制剂的研制

采用液体发酵工艺,确定枯草芽胞杆菌的最适发酵条件为:发酵温度30℃,初始pH值7.2,并以1%海藻酸钠和3%明胶组成的混合胶体溶液为囊壁材料,以4%氯化钙作固化剂将枯草芽胞杆菌制成微胶囊剂,稳定性试验结果显示经微胶囊包埋的枯草芽胞杆菌制剂,室温下保存1个月,活菌存活率为98.8%,保存3个月,活菌存活率为50.6%,保存6个月,活菌存活率为15.7%,均高于未经微胶囊化的样品;在4℃冷藏下保存3个月,未经微胶囊化的样品活菌存活率仅为经微胶囊包埋制剂的66.2%。该微胶囊制剂提高了活菌存活率,延长了活菌常温保存期。     

Purification and properties of thiosulfate dehydrogenase from Acidithiobacillus thiooxidans JCM7814

A key enzyme of the thiosulfate oxidation pathway in Acidithiobacillus thiooxidans JCM7814 was investigated. As a result of assaying the enzymatic activities of thiosulfate dehydrogenase, rhodanese, and thiosulfate reductase at 5.5 of intracellular pH, the activity of thiosulfate dehydrogenase was measured as the key enzyme. The thiosulfate dehydrogenase of A. thiooxidans JCM7814 was purified using three chromatographies. The purified sample was electrophoretically homogeneous. The molecular mass of the enzyme was 27.9 kDa and it was a monomer. This enzyme had cytochrome c. The optimum pH and temperature of this enzyme were 3.5 and 35 degrees C. The enzyme was stable in the pH range from 5 to 7, and it was stable up to 45 degrees C. The isoelectric point of the enzyme was 8.9. This enzyme reacted with thiosulfate as a substrate. The Km was 0.81 mM.     

乌鸡酶解技术研究

采用猪胰脏作为胰酶的主要来源,对乌鸡的酶解工艺进行了研究。实验结果表明：酶反应的最适温度为４５℃,胰酶混合物的合适用量为乌鸡鲜肉重量的５％,反应１８小时达反应平衡,反应浓度以１ｋｇ鲜乌鸡２０００ｍｌ水为宜,反应过程中维持ｐＨ７．３即可,酶解产物经测定：氨基酸及可溶性短肽总含量高达１６．６１％,其中必须氨基酸含量占氨基酸总含量的５３％。     

内切葡聚糖酶基因在巨大芽孢杆菌中的表达及其酶学性质研究

通过PCR方法将已克隆的内切葡聚糖酶基因(GenBank No. DQ782954)信号肽编码序列去除, 然后与表达载体pHIS1525连接后转化大肠杆菌DH5a, 筛选出阳性转化子DH5 a -pHIS1525-G7并提取质粒进一步转化巨大芽孢杆菌WH320原生质体, 获得基因工程菌WH320-pHIS1525-G7。刚果红染色和SDS-PAGE分析表明该基因在巨大芽孢杆菌中得到了有效表达。基因工程菌经优化培养后, 胞外上清液中的酶活力可达889 U, 是出发菌株(即枯草芽孢杆菌C-36)的11.22倍。酶学性质研究表明: 该酶的最适反应温度与pH值分别为65℃与pH 6.0, 在pH 4.5~10.0范围内50℃保温30 min可保持在最高酶活的80%以上。     

毕赤酵母发酵生产T4溶菌酶的中试研究

研究了T4溶菌酶的中试发酵放大生产。在200L发酵罐中毕赤酵母诱导表达T4溶菌酶蛋白,发酵液蛋白表达量达到1.69g/L,酶活达到192U/mg;制得冻干酶粉211.1g。经测定粗酶粉中T4溶菌酶的含量达到68.9%,酶活为182U/mg,总收率达到66%,成功建立了一条T4溶菌酶的中试发酵生产工艺线路。     

γ-Fe_2O_3-凹土超顺磁性纳米复合材料固载猪胰脂肪酶

以凹凸棒石黏土为原料,制备γ-Fe2O3-凹土超顺磁性纳米复合材料(γ-Fe2O3-ATP)作为猪胰脂肪酶(PPL)固定化的载体,利用透射电子显微镜(TEM)、N2吸附脱附等温图(BET)、振动试样磁强计(VSM)等对材料进行表征,同时对固定化条件和固定化酶的相关性质进行了研究。结果表明:制备的γ-Fe2O3-ATP是介孔材料,比表面积为102.63 m2/g,平均孔径为10.862 nm,饱和磁化强度为8.915 emu/g,其作为载体能实现固定化酶与反应介质简单、快速分离回收和重复利用。在固定化时间为4 h及pH 6.0时制备的固定化酶效果最佳;经过6 h高温保存后固定化酶可保留初始酶活的52%,而游离酶仅保留初始酶活的19%,同时固定化酶在重复使用5次后酶活仍保留初始酶活的60%。     

LIM结构域蛋白FHL1C抑制Notch信号途径的转录激活研究

目的：克隆人表达FHL1C基因以及建立真核表达载体和慢病毒载体。方法：从人的骨骼肌细胞来源的cDNA中扩增并克隆人FHL1C基因的编码区,连接至pMD-18T载体酶切鉴定后测序。序列测定确认后,双酶切pMD18T-FHL1C回收片段,插入真核表达载体,构建真核表达载体pCMV-Myc-FHL1C酶切鉴定正确后,转染Hela细胞及Cos7细胞,用Western Blot检测其在转染细胞中的表达,用双荧光素报告基因系统检测其对Notch信号作用。构建FHL1C-IRES-GFP表达单元,建立慢病毒表达载体plen-ti6/V5-FHL1C-IRES-GFP,包装慢病毒后感染培养的细胞,用免疫荧光显微镜、Western Blot检测分析其在被感染的细胞中的表达。结果：通过PCR方法成功扩增了人FHL1C基因的编码区。通过转染Hela及Cos7细胞,使用Western Blot检测其蛋白水平表达,双荧光报告基因系统分析均能够下调激活的Notch信号。成功构建了FHL1C慢病毒表达载体pLenti6/V5-FHL1C-IRES-GFP,包装慢病毒,把获取的慢病毒感染细胞后通过荧光显微镜证实被感染的细胞绿色荧光蛋白正确表达,Western Blot检测证实其表达。结论：成功建立起FHL1C真核表达载体及慢病毒表达系统,为研究急性T淋巴细胞白血病与Notch信号转导通路之间的关系奠定了基础。     

β-Galactosidase from a cold-adapted bacterium: purification, characterization and application for lactose hydrolysis

The enzyme beta-galactosidase was purified from a cold-adapted organism isolated from Antarctica. The organism was identified as a psychotrophic Pseudoalteromonas sp. The enzyme was purified with high yields by a rapid purification scheme involving extraction in an aqueous two-phase system followed by hydrophobic interaction chromatography and ultrafiltration. The beta-galactosidase was optimally active at pH 9 and at 26 degrees C when assayed with o-nitrophenyl-beta-D-galactopyranoside as substrate for 2 min. The enzyme activity was highly sensitive to temperature above 30 degrees C and was undetectable at 40 degrees C. The cations Na+, K+, Mg2+ and Mn2+ activated the enzyme while Ca2+, Hg2+, Cu2+ and Zn2+ inhibited activity. The shelf life of the pure enzyme at 4 degrees C was significantly enhanced in the presence of 0.1% (w/v) polyethyleneimine. The pure beta-galactosidase was also evaluated for lactose hydrolysis. More than 50% lactose hydrolysis was achieved in 8 h in buffer at an enzyme concentration of 1 U/ml, and was increased to 70% in the presence of 0.1% (w/v) polyethyleneimine. The extent of lactose hydrolysis was 40-50% in milk. The enzyme could be immobilized to Sepharose via different chemistries with 60-70% retention of activity. The immobilized enzyme was more stable and its ability to hydrolyze lactose was similar to that of the soluble enzyme.     

Isolation and study of active ATP-dependent phosphofructokinase from apple fruits Pyrus domestica Borkh

The activity of ATP-dependent phosphofructokinase (PFK) from subepidermal tissue of apple fruits was studied. The enzyme extracted under optimal conditions was stable for 14 h at room temperature. The enzyme was partially purified by ammonium sulfate fractionation and dialysis. PFK from apple fruits was found to be ATP-, UTP-, and CTP-specific. It was inhibited by PEP, Gly-2-P, Gly-1,3-DP, and ADP. The effect of the listed inhibitors was diminished by the presence of phosphate. The activity of PFK was stimulated by magnesium cations. The activity of the enzyme in fruits of an Antonovka cultivar was higher than in the Simirenko rennet cultivar by a factor of 1.3.     

Isolation and characteristics of carboxypeptidase B from the pyloric ceca of the starfish Asterias amurensis

Carboxypeptidase B was purified from the pyloric ceca of the starfish Asterias amurensis. The final enzyme preparation was nearly homogeneous in polyacrylamide gel electrophoresis and its molecular weight was estimated as approximately 34,000. The optimum pH and temperature of the enzyme for hydrolysis of benzoyl-glycyl-L-arginine were at approximately pH 7.5 and 55 degrees C, respectively. The enzyme was unstable at above 50 degrees C and at below pH 5.0. The enzyme was activated by Co(2+), but was inhibited by EDTA and Hg(2+). The N-terminal amino acid sequence of A. amurensis carboxypeptidase B was ASFDYNVYHSYQEIMNWITN.     

脂肪储存小滴蛋白5重组腺病毒的制备

目的：利用AdEasy腺病毒表达系统构建含有小鼠脂肪储存小滴蛋白5（LSDP5）基因的重组腺病毒。方法：从小鼠肝脏cDNA克隆出LSDP5基因全长,克隆至pMD18-T载体中,酶切测序。回收酶切产物,连接到腺病毒穿梭载体pShuttle-CMV,构建pShuttle-CMV-LSDP5重组质粒,经PmeI酶切线性化后转化至含有腺病毒骨架质粒pAdEasy-1的BJ5183中。筛选阳性克隆,提取重组质粒,PacI酶切线性化并转染AD293细胞进行包装,提取病毒DNA,鉴定重组病毒并检测病毒滴度。结果：LSDP5基因克隆经测序证实与Genebank公布一致,双酶切重组pMD18-T载体得到1400 bp左右的片段。重组穿梭载体经Kpn I和Sal I双酶切后得到预期片段。PacI酶切得到30 Kb大片段和4.5 Kb小片段。转染AD293细胞后收集病毒,经PCR鉴定,获得理想的目的片段。取病毒上清反复感染AD293细胞以扩增病毒,最后所得病毒滴度为2.5×109pfu/ml。结论：成功构建了携带脂肪储存小滴蛋白5基因的重组腺病毒载体,为进一步研究LSDP5基因功能奠定基础。     

β-葡萄糖苷酶在酿酒酵母表面的表达

应用表面表达技术对来自Trichodermareesei的β-葡萄糖苷酶在酿酒酵母表面的表达及后期性质进行了研究。实验结果表明酵母表面表达酶有活性,该酶的最佳诱导时间为24h,最适温度是70℃,而酶活的最适pH是5．5。使异源表面表达了Bgl1的酵母在以纤维二糖为唯一碳源的培养基中生长,发酵结果表明纤维二糖被明显利用了,但在培养186h后,发酵液中仍残留一定量的纤维二糖。这种技术对纤维素发酵系统中纤维二糖酶活性低的现状有所帮助。