戊型肝炎病毒样颗粒在重组汉逊酵母中的发酵表达、纯化及其免疫原性分析

为了研制戊型肝炎新型基因工程疫苗,利用汉逊酵母表达系统表达重组戊型肝炎病毒样颗粒,成功构建了重组戊型肝炎疫苗工程菌株HP/HEV2.3,对该菌株的发酵条件和纯化工艺进行了研究。先将工作种子批进行发酵培养,收集发酵后的细胞培养物;对其先后进行细胞破碎、澄清和超滤、硅胶吸附和解吸附、超滤浓缩换液、色谱纯化及除菌过滤,制得重组汉逊酵母戊型肝炎病毒样颗粒,纯化收率为33%,纯度达99%;电镜观察显示该重组汉逊酵母戊型肝炎病毒样颗粒与天然戊型肝炎病毒颗粒理论大小一致,为32 nm;基因序列与理论一致;SDS-PAGE分析结果表明其表达的外源蛋白质分子量与预期的目的蛋白质分子量大小一致,均为56 k Da,表达量占细胞总蛋白的26%,表达水平为1.0 g/L发酵液;Western blotting、ELISA活性检测及小鼠免疫接种效力试验ED_(50)结果表明,此重组汉逊酵母戊型肝炎病毒样颗粒具有良好的抗原性和免疫原性,可用于制造戊型肝炎新型基因工程疫苗。     

重组乙型肝炎疫苗免疫小鼠后早期免疫相关因子的反应

目的分析乙肝疫苗免疫后早期小鼠体内细胞因子、趋化因子、转录调节因子等多种免疫相关因子在mRNA及蛋白水平的反应,寻求早期评价乙肝疫苗免疫效果的指标。方法采用皮下免疫方式,每只BALB/c小鼠注射含2μg HBs Ag的汉逊酵母重组乙肝疫苗,免疫后3 h、24 h、48 h、96 h、168 h收集处理小鼠脾细胞和血清,使用Luminex方法测定多种免疫相关因子的mRNA表达和血清中蛋白类因子的分泌水平。结果脾细胞中IFN-α1、IFN-β1、IFN-γ、IL-2、IL-5、IL-6、IL-12p40、CCR1、CCR5、CCL3、CCL4 mRNA在免疫后3 h检测无表达,之后逐渐升高,在24 h达到表达高峰。CXCL10、IRF7 mRNA在免疫后3 h即出现表达,至24 h时达到表达高峰,分别为对照组的6.09倍和9.01倍。血清中CXCL10免疫后3 h即可检测,在24 h达到表达高峰。IFN-γ在96 h开始分泌,168 h时分泌水平最高。IL-12p70的分泌趋势与IFN-γ近似,在96 h之前的3个时间点分泌水平较低,168 h时达到分泌高峰。结论汉逊酵母重组乙肝疫苗免疫后3 h到168 h可检测到多种免疫相关因子表达,为早期评价乙肝疫苗免疫效果提供了指标。     

粟酒裂殖酵母N-糖酰胺酶在大肠杆菌中的表达、纯化及活性分析

根据GenBank中公布的粟酒裂殖酵母(Schizosaccharomyces pombe)N-糖酰胺酶(Png1p)cDNA序列, 设计并合成一对特异性引物, 利用RT-PCR技术从粟酒裂殖酵母中克隆出糖酰胺酶cDNA。将得到的基因克隆到表达载体pET-15b中。重组质粒转入大肠杆菌BL21(DE3)中, 经诱导表达和纯化提取后, 进行酶活测定。实验结果表明, 该酶的分子量约为39 kD, 纯化后的重组N-糖酰胺酶可以对变性处理的糖蛋白进行糖链的切除, 且这种作用需要还原剂DTT的辅助作用; N-糖酰胺酶只对错误折叠的糖蛋白有作用, 对天然的糖蛋白没有作用。等量粟酒裂殖酵母Png1p在不同温度、pH、DTT浓度和底物变性温度下对等量核糖核酸酶B(RNase B)的脱糖基化检测发现, 重组酶的最适反应温度30°C, 最适反应pH为7.0, 需要的最适DTT浓度为10 mmol/L, 底物在100°C处理10 min时酶的脱糖基化率最高。     

粟酒裂殖酵母N-糖酰胺酶在大肠杆菌中的表达、纯化及活性分析

根据GenBank中公布的粟酒裂殖酵母(Schizosaccharomyces pombe)N-糖酰胺酶(Png1p)cDNA序列, 设计并合成一对特异性引物, 利用RT-PCR技术从粟酒裂殖酵母中克隆出糖酰胺酶cDNA。将得到的基因克隆到表达载体pET-15b中。重组质粒转入大肠杆菌BL21(DE3)中, 经诱导表达和纯化提取后, 进行酶活测定。实验结果表明, 该酶的分子量约为39 kD, 纯化后的重组N-糖酰胺酶可以对变性处理的糖蛋白进行糖链的切除, 且这种作用需要还原剂DTT的辅助作用; N-糖酰胺酶只对错误折叠的糖蛋白有作用, 对天然的糖蛋白没有作用。等量粟酒裂殖酵母Png1p在不同温度、pH、DTT浓度和底物变性温度下对等量核糖核酸酶B(RNase B)的脱糖基化检测发现, 重组酶的最适反应温度30°C, 最适反应pH为7.0, 需要的最适DTT浓度为10 mmol/L, 底物在100°C处理10 min时酶的脱糖基化率最高。     

大肠杆菌表达的人IL-6复性条件研究

采用稀释法复性,筛选重组人白细胞介素-6(rhIL-6)包涵体蛋白的复性条件。结果显示,复性液的浓度、pH值、复性时间和复性蛋白浓度,对重组人白细胞介素-6复性效果有很大影响。在复性液为2mol/L尿素、pH8.5、复性时间为24h和复性蛋白浓度50μg/ml条件下,重组人白细胞介素-6包涵体蛋白的复性效果最佳。     

人源性抗HBsAg抗体Fab段在酵母中的表达

通过分步整合的方式,将人源性抗乙肝表面抗原（HBsAg）抗体Fab的轻、重链基因分步整合到巴斯德毕赤（Pichia pastoris）酵母GS115菌株的染色体上,经甲醇诱导,成功地分泌表达出抗HBsAg抗体的Fab片段,表达量达50～80mg/L。ELISA结果显示重组酵母分泌表达出的Fab具有较强的结合HBsAg的能力。通过抗Fab的抗体柱亲和层析,纯化出了纯度较高的Fab产品。     

应用CTAB法对砂梨品种DNA提取效果的研究

为了获得质量较好的DNA,我们采用CTAB法对11个砂梨品种的叶片DNA提取情况进行了研究,发现参试样品中一些样品的蛋白质含量较多,而另外一些样品的多糖、酚类物质含量较高。对于蛋。白质含量较多的这类材料,适当增加24：1的处理次数能够使蛋白质沉淀下来;对于多糖、酚类物质含量较高的材料,用5mol／L的NaCl和3mol／L的NaAc等处理能够有效的降低多糖含量。另外,对研磨样品的加入量和PVP的加入时间进行了对比,最后用双酶切、电脉检测等不同的分析方法对所得到的DNA提取物的浓度和纯度进行检测,发现加入样品量在0．3g左右、在研磨后的粗提液中加入PVP,所得到的DNA质量较好.     

毕赤酵母工程菌表达乙肝表面抗原结合蛋白(SBP)的发酵纯化工艺研究

乙肝表面抗原结合蛋白(HBsAg binding protein,SBP)是以HBsAg为探针,通过人肝cDNA噬菌体表达库筛选的一种人源蛋白。SBP可特异性结合乙肝表面抗原(Hepatitis B virus surface antigen, HBsAg),增强乙肝疫苗的免疫效果,是一种潜在快速高效的免疫佐剂。成功构建了分泌型高表达SBP的毕赤酵母工程菌。对该菌株进行了放大规模发酵表达,并对纯化工艺进行了研究。在发酵实时检测过程中,自诱导剂甲醇加入开始,SBP蛋白的分泌表达量随时间推移而逐渐增加,发现于38h达到最佳水平且此时杂蛋白含量最少,是放罐收集菌液的最佳时间。18L发酵液在低温离心除菌体和沉淀物后可得到15L的上清液,再利用截留量为5kDa的超滤膜包将上清液浓缩至2L,将浓缩后的上清液依次通过S200分子筛柱和TDEAE阴离子交换柱进行分离纯化,可得到300ml浓度为1.125mg/ml、纯度达98%的目标蛋白液,发酵液得率为22.5mg/L;最后将蛋白液定量分装冻干低温保存。所获得的放大规模SBP发酵诱导表达条件和SBP蛋白的分离纯化工艺,为SBP蛋白大规模生产奠定了坚实的基础。     

小鼠肠道菌群HPLC分析中色谱分离条件的优化

研究了在小鼠肠道菌群的高效离子交换色谱（HPLC）分析中,不同色谱分离条件对分离效果的影响,确立了最佳色谱条件：样品上样于Toyopearl TSKgel SuperQ-650c强阴离子交换树脂柱,以0.02mol/L哌嗪-HCl缓冲液（pH8.0）平衡,洗脱盐浓度为1.0mol/L NaCl,洗脱梯度为0.1-0.5mol/L NaCl线性梯度洗脱80min,再经0.5-0.75mol/L NaCl线性梯度洗脱25min,流速1mL/min,进样量为1mL。小鼠肠道菌群HPLC分析方法的建立,为深入研究小鼠肠道菌群的组成和动态变化奠定了基础。     

人呼吸道合胞病毒截短F1重组蛋白包涵体的制备、纯化和复性

目的将前期在大肠埃希杆菌中获得表达的A型人呼吸道合胞病毒兰州分离株截短的F1重组蛋白进行纯化和复性,为后期动物免疫制备抗原。方法 37℃诱导重组菌体p ET-42b-F1J/Rossata,诱导完毕后离心收集菌体,高压破碎菌体并收集包涵体后用不同浓度的Triton X-100(细胞裂解液)洗涤包涵体3次。洗涤的包涵体用8 mol/L尿素进行溶解并用镍离子亲和层析方法进行初步纯化,用阳离子交换层析方法对初步纯化蛋白进行最终的纯化。亲和层析纯化蛋白用3种不同的复性液进行了稀释复性。结果 37℃诱导5 000 m L重组菌p ET-42b-F1J/Rossata共收获37 g湿菌体,经过不同浓度Triton X-100洗涤包涵体后纯度可达75%。包涵体用8 mol/L尿素溶解后经镍离子亲和层析纯化纯度约为40%,再用阳离子交换层析介质SP HP进一步纯化样品后纯度可达90%。纯化蛋白以3种不同的复性液都能得到复性,其中复性液3的复性效果相对较好。结论实验中探索了人呼吸道合胞病毒截短F1重组蛋白包涵体的纯化方法及步骤,为后期的蛋白制备及动物免疫奠定了基础。     

Effect of transglutaminase and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide on the solubility of fish gelatin–chitosan films

The possibility of decreasing the water solubility of the films made from fish gelatin and chitosan by modification with TGase was investigated. The effectiveness of enzymatic treatment was also compared with chemical crosslinking using EDC. The treatment of the components with TGase in concentration of 0.2 mg/ml of the film-forming solution limited the solubility of the films at 25 °C from 65% to 28% at pH 6 and from 96% to 37% at pH 3. After 15 min of heating at 100 °C, the modified films were soluble in 23% at pH 6 and in 41% at pH 3. Further decrease of the solubility of the fish gelatin–chitosan films was achieved when enzymatic modification was conducted in the presence of 5–10 mM DTT; the solubility was about twice lower than that without DTT at both studied temperatures and pH values. Generally, the composite films modified with EDC in concentration of 30 mM were distinctly less soluble than films made from the components modified with TGase in the presence of DTT.     

Lipoprotein separation and low density lipoprotein molecular weight determination using high performance gel-filtration chromatography

Plasma lipoproteins were isolated at d less than 1.225 g/ml from nonhuman primates of three species, cynomolgus, rhesus, and African green (vervet) monkeys. Individual lipoprotein classes were separated by high performance gelfiltration chromatography and low density lipoprotein (LDL) molecular weight was determined. A comparison was made using column configurations including TSK 3000 SW, 4000 SW, and 5000 PW columns. Due to its relative simplicity, stability, and economy, a single 5000 PW column was selected for most of the work. The recovery of lipoprotein cholesterol from the column averaged 91 +/- 2.5%. A comparison of the immunologic, chemical, and electrophoretic properties of high density lipoproteins (HDL) and LDL isolated by this technique with those of HDL and LDL isolated by conventional agarose column chromatography indicated that lipoproteins isolated by high performance gel-filtration chromatography were intact and reasonably free of cross contamination. A standard preparation of 125I-labeled LDL was added to the d less than 1.225 g/ml lipoprotein fraction just prior to separation and a relative size index, r1, was determined. When r1 values for a large number of samples were compared with the log of the LDL molecular weight (determined by agarose column chromatography) a linear relationship was found with a correlation coefficient, r = 0.85. The regression equation for this relationship could be used to calculate LDL molecular weights from the r1 value. These values agreed with LDL molecular weight determined by flotation equilibrium analysis in the analytical ultracentrifuge. We conclude that high performance gel-filtration chromatography using the TSK 5000 PW column provides an analytical and preparative technique for simultaneous separation of individual lipoproteins and determination of LDL molecular weight.     

Adaptation of the tetrazolium method for testing the seed viability, and scanning electron microscopy study of some Western European orchids

A modified tetrazolium method was formulated for use with seeds of Western European orchids. The sequence of treatments which gave the highest percentage of coloured (i.e. viable) embryos was: (1) pretreatment in a solution of 5% (w/v) Ca(OCl)₂+ 1% (v/v) Tween-80, (2) soaking for 1 day in sterile water, (3) the classical tetrazolium test. The optimal duration of the pretreatment in Ca(OCl)₂+ Tween-80 depends upon the species, and to investigate the effect a scanning electron microscopy study was performed on the testa of 3 species. For a given species, the optimal pretreatment period was not affected by the year of harvest or the source of the seed lots.     

Reduction of DL-selenocystine and isolation of L-seleoncysteine

Cystine, selenocytsine, and several analogs were reduced by dithiothreitol (DTT), beta-mercaptoethanol (ME) and sodium borohydride (NaBH4). DTT was the most effective; DTT to cystine ratios from 10 to 80 were equally effective. With selenocysteine, however, absorption was considerably reduced at all ratios. Selenocysteine was identified as the reduction product by reaction with Gaitonde's reagent, comparison of absorption spectra, paper chromatograhy, utilization by cysteinyl-tRNA synthetase fro Paracoccus denitrificans and Vigna radiata, changes in solubility after DTT treatment, and comparison of infrared spectra. During the ATP-PPi exchange assay, DTT and ME convert cysteine and selenocysteine derivatives to cysteine and selenocysteine which serve as substrates for cysteinyl-tRNA synthetase.     

Concerns in the development of an assay for determination of a highly conjugated adsorption-prone compound in human urine

Concerns in pre-analytical handling of urine samples are discussed using a new KDR kinase inhibitor, 3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one (compound A), as an example of a case where high light sensitivity and low analyte recovery (high affinity for container surface) were found. The absence of these problems in plasma samples may be a result of the plasma protein content. Low recovery of the analyte from urine can be remedied by either changing the container or by using additives, such as bovine serum albumin (BSA) or non-ionic surfactant Tween-20. In the case of compound A, changing containers (polypropylene versus glass vial) or addition of BSA did bring analyte recovery up to 80%. However, the addition of 0.2% Tween-20 into urine quality controls (QCs) gave more than 95% analyte recovery, indicating effective reduction of analyte loss to the surface of containers. The urine assay using mixed-mode SPE and LC-MS/MS was not affected significantly by introducing Tween-20 into the samples. The mean SPE extraction recovery was 68.4% and matrix suppression of ionization on MS was less than 8% at all analyte concentrations. The linear range of the calibration curve was 0.5-400 ng/mL on PE Sciex API 3000 LC-MS/MS system. The assay intraday accuracy and precision were 92.1-104.8% and <4.2% (%CV), respectively. Urine QC samples, containing 0.2% Tween-20, gave excellent recovery after three cycles of freeze and thaw. Since analyte loss to its urine container surface is not unique to compound A (M. Schwartz, W. Kline, B. Matuszewski, Anal. Chim. Acta 352 (1997) 299-307; A.L. Fisher, E. DePuy, T. Shih, R. Stearns, Y. Lee, K. Gottesdiener, S. Flattery, M. De Smet, B. Keymeulen, D.G. Musson, J. Pharm. Biomed. Anal. 26 (2001) 739-752), we suggest an evaluation of the potential problem in the early stages of urine assay development to ensure reliable quantitation of analytes. The addition of Tween-20 can serve as a useful analytical tool to other analytes with similar situations.     

Heat stable lipase activity of thermotolerant bacteria from hot springs at Orissa, India

Thermotolerant bacteria (35 in toto) isolated from three hot springs (Atri, Taptapani and Deuljhari, Orissa), were screened for lipase activities. Of these, nine strains of Bacillus spp. and three strains of Pseudomonas spp. showed heat stable lipase activity at 60 degrees C. The hydrolytic activity of these bacteria was tested using Tween-20 and Tween-80 as substrates at different temperatures using plate assay and titration techniques. The hydrolytic activity at different pH values and salt concentrations was investigated.     

基于吐温-80下2株功能菌协同降解多环芳烃的特性研究

【目的】研究恶臭假单胞菌B6-2和克雷伯氏菌CW-D3T构建的混合功能菌对多环芳烃的协同修复效能,并探究非离子表面活性剂吐温-80对混菌降解多环芳烃的影响,以期为芳烃化合物的生物修复提供技术参考和理论依据。【方法】通过生长曲线及平板菌落计数法反映混菌生长情况及比例,从而评估混菌降解体系的可行性;通过高效液相色谱法探究各体系以及不同吐温-80浓度下混培体系对多环芳烃的降解效能;最后通过烷烃吸附法测定细胞表面疏水性,以探究吐温-80对混合功能菌降解多环芳烃的影响机制。【结果】等比例混合的2株菌共培养生长状态优于纯培体系,对混合多环芳烃(菲、荧蒽、芘)的降解率分别为33.4%、30.1%、28.6%(7 d),相较于菌CW-D3T,分别提高了1.31倍、1.46倍、1.42倍。混培体系中加入500 mg/L的吐温-80对菲、荧蒽、芘的降解率分别为47.7%、43.2%、38.8%(7 d),相较于对照组各提高了1.55倍、1.38倍、1.31倍,而更高浓度的吐温-80无明显促进作用或轻微抑制。添加吐温-80使菌CW-D3T和混菌的表面疏水性提高,而菌B6-2表面疏水性降低。结合细菌生长量分析...     

双组份转运系统BrnFE的过表达和表面活性剂的添加对谷氨酸棒杆菌发酵生产L-异亮氨酸的影响

为了提高L-异亮氨酸生产菌株Corynebacterium glutamicum LD320的产酸水平,通过改善其分泌系统,在C. glutamicum LD320中分别过表达突变型和野生型的双组份转运系统BrnFE操纵子,构建了重组菌LD320/pXMJ19-brnFE和LD320/pXMJ19-brnFE1。通过对两株重组菌的L-异亮氨酸生产分析比较,发现突变型比野生型能更有效地提高 L-异亮氨酸产量。同时对 LD320/pXMJ19-brnFE1进行表面活性剂添加实验,发现Tween-80为最佳选择,其最佳添加量为0.5 g/L,最佳添加时间为对数期的16 h。最后通过7 L发酵罐放大实验,LD320/pXMJ19-brnFE1的L-异亮氨酸产量由18.53 g/L提高到25.45 g/L,比对照组提高了37%。     

The disulfide linkage and the free sulfhydryl accessibility of acyl-coenzyme A:cholesterol acyltransferase 1 as studied by using mPEG5000-maleimide

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is a membrane protein located in the endoplasmic reticulum (ER). It plays important roles in cellular cholesterol homeostasis. Human ACAT1 (hACAT1) contains nine cysteines (C). To quantify and map its disulfide linkage, we performed thiol-specific modifications by mPEG(5000)-maleimide (PEG-mal) and iodoacetamide (IA) under denatured condition, using extracts that contain wild-type or various single C to A mutant hACAT1s. With the wild-type enzyme, seven Cs could be modified before dithiothreitol (DTT) treatment; nine Cs could be modified after DTT treatment. With the C528A or the C546A enzyme, all eight Cs could be modified before or after DTT treatment. With all other remaining single C to A mutant enzymes, six Cs could be modified before DTT treatment, and eight Cs could be modified after DTT treatment. We next performed Lys-C protease digestion on hACAT1 with a hemagglutinin (HA) tag at the C-terminus. The digests were treated with or without DTT and analyzed by SDS-PAGE and Western blotting. The two predicted C-terminal fragments (K496-K531 and N532-F550-HA tag) were trapped as a single peptide band, but only when the digests were treated without DTT. Thus, C528 and C546 near the enzyme's C-terminus form a disulfide. PEG-mal is impermeable to ER membranes. We used PEG-mal to map the localizations of the seven free sulfhydryls and the disulfide bond of hACAT1 present in microsomal vesicles. The results show that C92 is located on the cytoplasmic side of the ER membrane and the disulfide is located in the ER lumen, while all other free Cs are located within the hydrophobic region(s) of the enzyme.     

Study of humoral hemolytic factors in hemorrhage using chromatographic methods]

The kidney-dependent increase of the haemolytic activity of blood serum after acute decompensated blood loss was demonstrated in the experiments on rats. The preparative ion-exchange chromatography on DEAE-Toyoperal was used to separate the haemolytic active component of the posthaemorrhagic blood serum with the max value of the specific activity of 6.86 A/micrograms protein. The analysis of the separated component by size-exclusion chromatography on TSK-G3000-SW column indicated a molecular mass of 80-100 kDa. In injection in the circulatory system in in vivo experiments the dose-dependent effect of the action of separated component was demonstrated.