Subjects: Biology >> Virology submitted time 2020-02-14
Abstract： Abstract: In 2019, the 2019 novel Coronavirus (2019-nCoV) has caused the pneumonia outbreak in Wuhan (a city of China). In our previous study, the analytical results showed that both 2019-nCoV and SARS coronavirus belongs to Betacoronavirus subgroup B (BB coronavirus), but have large differences. The most important finding was that the alternative translation of Nankai CDS could produce more than 17 putative proteins, which may be responsible for the host adaption. The genotyping of 13 viruses using the 17 putative proteins revealed the high mutation rate and diversity of betacoronavirus. The present study for the first time reported a very important mutation in the Spike (S) proteins of BB coronavirus. By this mutation, 2019-nCoV acquired a cleavage site for furin enzyme, which is not present in the S proteins of all other BB coronavirus (e.g. SARS coronavirus) except the Mouse Hepatitis coronavirus (MHV). This mutation may increase the efficiency of virus infection into cells, making 2019-nCoV has significantly stronger transmissibility than SARS coronavirus. Because of this mutation, the packing mechanism of the 2019-nCoV may be changed to being more similar to those of MHV, HIV, Ebola virus (EBoV) and some avian influenza viruses, other than those of all other BB coronavirus (e.g. SARS coronavirus) except the Mouse Hepatitis coronavirus (MHV). In addition, we unexpectedly found that some avian influenza viruses acquired a cleavage site for furin enzyme by mutation as 2019-nCoV. Further studies of this mutation will help to reveal the stronger transmissibility of 2019-nCoV and lay foundations for vaccine development and drug design of, but not limited to 2019-nCoV.
Peer Review Status:
Awaiting Review
Subjects: Biology >> Biophysics >> Oncology submitted time 2016-05-11
Wang, Anxin
Chen, Lisha
Li, Chunlin
Zhu, Yimin
Wang, Anxin
Chen, Lisha
Li, Chunlin
Wang, Anxin
Chen, Lisha
Li, Chunlin
Abstract： Accumulating evidence suggests that cancer stem cells (CSCs) are heterogeneous populations and their phenotypes are unstable. A number of intrinsic and extrinsic mechanisms contribute to CSC phenotypic variation. The existence of various CSC subpopulations which would lead to a rapid relapse after primary treatments might pose a problem for CSC targeted therapeutics. In order to develop more effective approaches to cancer therapeutics, more CSC-related surface markers or targeting molecules, as well as some novel targeting strategies should be explored. This review summarized the origin and performance of heterogeneity in CSCs and discussed their therapeutic implications. (c) 2014 Elsevier Ireland Ltd. All rights reserved.
Peer Review Status:Awaiting Review
Subjects: Biology >> Bioengineering submitted time 2019-04-21 Cooperative journals: 《中国生物工程杂志》
Abstract： 6-Hydroxynicotinic acid (6HNA) 3-monooxygenase (NicC) is the key enzyme for nicotinic degradation in Pseudomonas putida KT2440. NicC can catalyze the hydroxylation of pyridine ring to promote the ring cleavage reaction of pyridine ring. The expression level of NicC was enhanced by replace the rare codon in the N-terminal of NicC, and then the His-tagged NicC was purified to homogeneity. The optimal temperature reaction range of NicC is from 30℃ to 40℃, and the optimal reaction pH is 8.0. The Cd 2+ ion could significantly inhibit the activity of NicC. The apparent Km and Vmax values of the purified NicC for 6HNA were 51.8 μM and 14.1U/mg, respectively, and those for NADH were 15.0 μM and 10.79 U/mg, respectively. According to the HPLC and LC-MS analysis, NicC could catalyzes 6HNA to form 2,5-DHP and formic acid, and it could also transform 4-hydroxybenzoic acid to form hydroquinone. Isotope labeling experiments proved that the oxygen atom incorporated into 2,5-DHP is from dioxygen. The study will provide useful information for the microbial degradation of pyridinic compounds.
Subjects: Biology >> Bioengineering submitted time 2019-03-05 Cooperative journals: 《中国生物工程杂志》
Abstract： Objective: To construct a fusaruside-producing strain of Pichia pastoris, and to solve the source of the natural small molecule immunosuppressant fusaruside. METHODS: The genes related to the synthesis of fusaruside, the 3-position desaturase (Δ3(E)-SD) and 10-position desaturase (Δ10(E)-SD) genes, were amplified from Fusarium graminearum PH-1. ; And the co-expression vector of the two genes was constructed by the 2A peptide strategy, and transformed into Pichia GS115 for inducible expression of the two enzymes; after the induced Pichia cells were treated with methanol and dichloromethane, the efficient Liquid chromatography-mass spectrometry (HPLC-MS) was used to detect the product changes. Results: The 3-desaturase and 10-desaturase were successfully co-expressed in Pichia pastoris. SDS-PAGE showed that the molecular weight of the 3-desaturase was about 48 kDa, and the molecular weight of the 10-desaturase was about 65 kDa; HPLC- MS showed that recombinant yeast could produce fusaruside. Conclusion: Compared with the fusaruside native strain Fusarium, the yeast has a shorter fermentation time and higher yield, which lays a foundation for the further development and application of fusaruside.
Subjects: Biology >> Bioengineering submitted time 2019-03-05 Cooperative journals: 《中国生物工程杂志》
Abstract： Objective: A new material with ultra-large surface area named nano-forest is prepared by Micro-Electro-Mechanical System(MEMS) processing technology. Based on this material, a new microfluidic chip for point-of-care test with simple operation, rapid detection and high sensitivity is created. Methods: The fabrication of nano-forests in micro-channel on quartz substrate mainly includes: cleaning and drying of quartz substrate; spinning polyimide(PI) coating; re-spinning phenolic resin photoresist on PI coating; photolithography to expose the channel; treating the PI layer with oxygen plasma and argon plasma to synthesize nano-fiber forests structure; nano-fiber-quartz nanoforests are realized by using nano-fiber forests as nanomasks in anisotropic etching of quartz by using reactive ion etching (RIE); the micro-channel with nano-forests structure inside is achieved after removing upper nanofiber forests structure and phenolic resin photoresist coating.The height, width, density and specific surface area of nano-forest are studied and analyzed by scanning electron microscope(SEM). Optical properties are tested by ultraviolet-visible spectrophotometer. The driving force is characterized by the flow rate of PBS solution.The sensitization effect is evaluated by saturated fluorescence test through antibody and AbFluor 680 dye-labeled secondary antibody. The sample pad, bond pad, micro-channel with nano-forests structure, nitrocellulose membrane and absorbent material are assembled on PMMA substrate in sequence, which is the microfluidic chip. The chip based on the sandwich format with a polyclonal antibody and a AbFluor 680 dye-labeled secondary antibody is used to detect ricin toxin(RT). Results: The scanning electron microscope shows that the nanofiber forests structure is formed on quartz substrate after oxygen plasma and argon plasma bombardment. The single nanofiber is upright on the substrate with a diameter of about 50-100 nm, a height of 1.8 um and a density of about 20/μm2. The quartz nano-forests structure can be obtained after RIE with nano-fibre forests structure as mask and resist removal. The single structure is shaped like a cone. The diameter of the cone bottom is about 100-200 nm, the height is about 1.0 um, the density is about 10/μm2, and the surface area to bottom area is more than 5:1. Self-driven test provides information of the flow rate of PBS is to be about 5 mm/s in the micro-channel on the basis of nano-forests structure. The transmittance of the channel is 89.5%at 680 nm wavelength. It shows that the channel has good transmittance, which makes the loss of excitation light or emission light much less, and is conducive to the sensor capturing more signals. With same surface modification, the planar quartz structure has shortcomings of short lasting effect time and low saturation fluorescence intensity. To the contrary, nano-forests structure with ultra-large surface area has a good sensitization effect in the test. RT can be detected sensitively based on the significantly fluorescent intensity.The linear range of detection is from 10 pg/mL to 6250 pg/mL and the limit of detection (LOD) is lower than 10 pg/mL. Conclusion: The nano-forests structure with good optical properties reduces the requirements of sensor and also makes the choice of fluorescent dyes wider.The three-dimensional structure of the nano-forest has an ultra-large surface area, which increases the amount of antibody compared to the planar structure, and thus improves the sensitivity of detection greatly. Compared with the immunochromatographic test strip, the microfluidic chip has an advantage of high sensitivity, thus the quantitative analysis can be realized within a certain range. Most microfluidic chips require complex equipments to provide driving force, which will make them costly and bulky. Driven by the capillary force, the chip with nano-forests structure inside makes the detection simple and fast. Combined with the miniaturized detection terminal, the platform can be miniaturized, portable, and automated, achieving the goal of simple, fast and efficient analysis. These characteristics make the chip an ideal candidate for the development of rapid detection methods.
Subjects: Biology >> Bioengineering submitted time 2019-03-05 Cooperative journals: 《中国生物工程杂志》
Abstract： The third large-scale merger and reorganization of multinational seed companies have formed a new international seed pattern.，intellectual property protection is one of the biggest drivers of cross-border seed mergers and acquisitions. Studying the accumulation and reorganization of the core intellectual property rights formed by the merger of multinational seed companies, it is possible to further examine the changing trend of the new pattern of international seed industry competition. One of the trends, the United States, Japan and Europe are still the leaders and monopolists of global breeding research and development innovation activities, large-scale mergers and acquisitions, achieving the integration of technology and resources, and promoting the concentration of international seed industry, 42.75% of patent applications of the global seed industry is concentrated in the United States, Japan and the European Union, and holding 64.68% of the global DNA recombination technology patents. Trend 2, intellectual property has become a powerful weapon for multinational seed companies to maintain market competitive advantage, and multinational seed companies is the owner of core technology patents. Bayer/Monsanto, China Chemical/Syngenta and DuPont Pioneer/Dow's seed patent applications for global breeding is nearly 14%, and more than 81% of the global crop transformants are controlled by multinational seed companies. Research and analysis on the changing trend of the new pattern of international seed industry competition can provide valuable policy advice for China's seed industry mergers and acquisitions.
Subjects: Biology >> Bioengineering submitted time 2019-03-05 Cooperative journals: 《中国生物工程杂志》
Abstract： Objective To explore the role and mechanism of miR-17-5p in the autophagy pathway mediated by Mycobacterium tuberculosis by studying the regulatory mechanism of miR-17-5p on autophagy-related gene ATG7 and its effect on cell autophagy. Methods The target gene ATG7 of miR-17-5p was obtained by bioinformatics analysis. The wild-type(pMirGLO-ATG7-3'UTR-WT) and mutant vector of ATG7 were successfully constructed. The targeting relationship between miR-17-5p and ATG7 was verified by double luciferase reporting system and Western blot. THP-1-derived macrophages Infected by Mycobacterium tuberculosis (H37Ra) were divided into three groups: miR-17-5p mimics, miR-17-5p inhibitors, and miR-17-5p nc. The effect of H37Ra infection on the expression of miR-17-5p was detected by quantitative real-time PCR (qRT-PCR). The expression of LC3 protein and the number of autophagosomes were detected by Western blot and immunofluorescence. Results MTB infection can cause miR17-5p down-regulation, with the increase of infection plural decreased significantly. Bioinformatics predictions showed that miR-17-5p and ATG7 were targeted. Dual luciferase reporter assay and Western blot confirmed that miR-17-5p could bind to ATG7 and negatively regulate it. Western blot and immunofluorescence assay showed that the expression of LC3 II was down-regulated and the expression of autophagosomes was down-regulated in the miR-17-5p mimics group, but the reverse was found in the miR-17-5p inhibitor group. The expression of ATG7 and LC3 II protein in H37Ra infected group was higher than that in uninfected group. Conclusion MiR-17-5p directly targets ATG7 3'UTR to inhibit autophagy and plays a role in the anti-MTB effect of macrophages.
Subjects: Biology >> Bioengineering submitted time 2019-02-13 Cooperative journals: 《中国生物工程杂志》
Abstract：摘要：木聚糖（Xylan）在自然界中的含量极其丰富，在农作物和农林剩余物中大量存在。随着能源资源问题的日益凸显，对木聚糖的应用和研究越来越受到重视。木聚糖酶（Xylanase）是可以将木聚糖降解为低聚木糖和木糖的一类水解酶，近年来，为了实现木聚糖酶的高产、高酶活表达，科研工作者做了大量的研究工作，本文将就木聚糖酶异源表达（Heterologous expression）的研究进展进行综述。 关键词：木聚糖；木聚糖酶；异源表达
Subjects: Biology >> Bioengineering submitted time 2017-09-20
Abstract： Microbial Fuel Cells (MFCs for short) are bioreactors that use electricity-producing microorganisms as catalysts to directly convert chemical energy in organic matter (inorganic matter) into electrical energy. application prospects. The research group started and ran MFCs with sludge from Tianjin TEDA sewage treatment plant as inoculum. Multiple isolates were isolated from anode-enriched biofilms. Among them, isolates P2-A-1 and P2-A-5 showed high electrochemical activity, which were identified as Tolumonas osonensis and Kocuria rhizophila, respectively. This is the first report on the electricity production performance of this species. In addition to the optimization of electricity production conditions, the permeabilization of cells is an effective means to improve the output power of MFCs. Chemical treatment increases the adhesion efficiency of bacteria to electrodes, reduces the internal resistance of MFCs, increases cell permeability and Cell membrane fluidity increases the content of the key electron carrier CoQ10. By constructing membrane-encapsulated MFCs, cyclic voltammetry scanning of anolyte and GC-MS composition analysis, the extracellular electron transfer mechanism of the strain was clarified. Taking the model electrogenic microorganism Pseudomonas aeruginosa as the object, a series of endogenous and exogenous global transcription factors were introduced into Pseudomonas aeruginosa by global transcription mechanism engineering, and the idea of metabolic engineering was used to enhance the generation of intracellular electrons and the production of extracellular electrons. Electron transfer, to explore new strategies and methods for the construction of high-yielding electroactive engineered strains. Among them, the introduction of exogenous global transcription factor IrrE can significantly improve the electrical performance and environmental stress tolerance of Pseudomonas aeruginosa. Further analysis found that the introduction of this transcription factor had a significant impact on the expression levels of genes related to central metabolic pathways, biofilm-related genes, mediator synthesis-related genes, quorum sensing systems, and general stress response-related genes. This indicates that IrrE plays a global regulatory role in host organisms. The results of the project are of great significance for enriching the diversity of electricity-producing microorganisms and improving the output power of microbial fuel cells.
Subjects: Biology >> Bioengineering submitted time 2019-03-05 Cooperative journals: 《中国生物工程杂志》
Abstract：目的：建立OTUB1肝脏特异性基因敲除小鼠模型，初步分析其表型并研究OTUB1基因与肝脏代谢的关系。方法：利用Cre/Loxp系统构条件性基因敲除小鼠模型，即将OTUB1fl/fl转基因小鼠与Alb-Cre小鼠杂交,子代自交，得到OTUB1肝特异性基因敲除小鼠并进行鉴定。取同窝对照小鼠（control, NC）和肝特异型基因敲除（hepatic-specific OTUB1 knockout, HCKO）小鼠，通过PCR和免疫印迹（Western blot, WB），确证OTUB1肝脏特异性基因敲除小鼠模型是否成功构建。通过组织病理学方法，分析主要组织器官的形态以及是否存在自发的病变；通过血清生化指标检测肝脏脂代谢水平；通过血糖耐受实验（GTT）分析HCKO小鼠对血糖的控制。结果：基因组测序和WB检测结果显示HCKO小鼠肝脏中OTUB1被敲除，其他组织中OTUB1表达水平无变化，证明OTUB1肝脏特异性基因敲除小鼠模型构建成功。HCKO小鼠出生正常，各组织器官无异常，生化指标中总胆固醇水平明显降低，表明OTUB1影响肝脏脂代谢水平。糖耐受实验中HCKO小鼠血糖回落迅速，表明敲除OTUB1影响肝脏血糖调节稳态。结论：应用Cre/Loxp技术成功建立OTUB1肝脏特异性基因敲除小鼠模型,为研究OTUB1在肝脏的生理功能和调控机制提供了重要的动物模型。
Subjects: Biology >> Bioengineering submitted time 2019-02-13 Cooperative journals: 《中国生物工程杂志》
Abstract：摘要：以毕赤酵母为异源表达宿主合成人胰岛素前体，在实验室研究和工业生产中已有广泛应用。目前研究主要使用天然甲醇诱导型AOX1启动子，以甲醇为单一基础碳源进行胰岛素前体的诱导发酵生产。但在毕赤酵母高密度发酵生产过程中，甲醇代谢过程耗氧大、产热高，补料控制工艺复杂，限制了发酵生产的放大。本研究基于我们前期对启动子AOX1的转录调控设计研究，提出以人工设计的高效组成型转录调控器件CSAD_5驱动胰岛素前体基因表达，开发了葡萄糖为碳源的发酵生产工艺，以解决甲醇体系中的产热、耗氧以及工艺控制问题。在此基础上，通过增强筛选压力提高异源基因拷贝，获得了一株胰岛素前体高表达重组毕赤酵母，利用优化的培养工艺在5 L反应器水平发酵生产，胰岛素前体产量在108 h达到1.85 g/L，为目前报道以葡萄糖为碳源，生产人胰岛素前体的最高水平。该研究为胰岛素前体的工业生产以及毕赤酵母的应用提供了新的思路和方法。
Subjects: Biology >> Bioengineering submitted time 2019-01-17 Cooperative journals: 《中国生物工程杂志》
Abstract：外泌体是细胞内源性囊泡样生物纳米级膜结构，直径大小在40-100 nm之间，可由各种类型的细胞分泌释放。外泌体具有许多功能，如蛋白质、mRNA、miRNA和脂类的细胞间运输和传递，以及抗原递呈，还可能具有致癌的能力。肿瘤细胞所分泌释放的外泌体在肿瘤的发生、发展以及迁移等生理和病理过程中发挥重要的作用。目前从肿瘤外泌体中寻找特异性标志物已成为肿瘤研究者重点关注的方向，对肿瘤早期诊断、疗效评价和预后分析具有重要的意义。本文作者就近年来外泌体在肿瘤研究和诊断中研究进展进行综述。
Subjects: Biology >> Bioengineering submitted time 2019-02-13 Cooperative journals: 《中国生物工程杂志》
Abstract：摘要：高分子囊泡作为一种新型的纳米药物载体，具有生物可降解性、稳定性、生物相容性以及可修饰的多功能化等特点。改变聚合物种类和亲水-疏水嵌段的比例，可以制备具有不同形态和膜特性的高分子囊泡。经过修饰后的高分子囊泡，可赋予其更多的功能，从而实现药物的控释和药物靶向的能力。本文对高分子囊泡的结构、组成和制备方法以及在药物释放体系的应用等方面进行了较为详细的综述，目的是了解高分子囊泡最新研究进展以及未来科学家们亟需要解决的重要问题。
Subjects: Biology >> Bioengineering submitted time 2017-09-20
Abstract： Vitamin K2 (VK2) is a series of menaquinone compounds with isoprene side chains, which are represented by MK-n depending on the length of the side chains. Highly active VK2 is mainly synthesized by microorganisms and has the physiological function of preventing and treating diseases such as osteoporosis, hemorrhage, liver cirrhosis and Parkinson's disease. Flavobacterium is an important production strain and can synthesize a variety of VK2 homologs including MK4, MK5 and MK6. We found that by regulating the fermentation temperature, the type and yield of VK2 homologs synthesized by Flavobacterium can be controlled. In the range of 20~37℃, Flavobacterium sp. M1-14 grows best at 25℃, the biomass reaches 8.8 g/L, but the fermentation product is completely MK6, the yield is 13.9 mg/L, and the unit cell yield is 1.6 mg/L g. When the fermentation temperature is higher than 30°C, Flavobacterium can synthesize MK4, MK5 and MK6 at the same time. At 37°C, the yields of MK4 and MK5 are the highest, which are 1.6 mg/L and 1.7 mg/L, respectively, and the total amount of VK2 is 12.5 mg/L. At this time, the biomass was only 5.5g/L, and the unit cell yield was 2.3mg/g. In view of the difference in the optimum temperature for the growth of Flavobacterium cells and the synthesis of VK2 homologs, variable temperature fermentation was considered to increase biomass and VK2 production. After optimization of multiple factors, we developed a two-stage temperature change strategy with low temperature first and then high temperature, that is, fermentation at 25°C for 48 hours, and then fermented at 37°C for 96 hours, the VK2 yield reached 20.9 mg/L (among which MK4 was 2.1 mg). /L, MK5 is 2.3 mg/L, MK6 is 16.5 mg/L), the biomass is 8.8 g/L, and the unit cell yield is 2.4 mg/g. Then, on the 30L fermenter, we investigated the oxygen demand of fermentation at different temperatures by controlling the ventilation rate and rotation speed. It was found that at 25℃ and 37℃, the optimum kLa for VK2 synthesis by Flavobacterium fermentation was 360 h-1 and 60 h-1, respectively. A two-stage variable kLa control strategy was developed for the changes in the oxygen demand of bacteria during variable temperature fermentation. After optimization, the kLa was 360 h-1 in the first 24 hours of variable temperature fermentation, and the kLa was 60 h-1 in the next 120 hours. was 22.5 mg/L), which was 107% higher than the initial value, the biomass was 15.5 g/L, and the unit cell yield was 1.9 mg/g. The staged fermentation regulation strategy of changing temperature and kLa can change the type of homologues of Flavobacterium synthesizing VK2, significantly increase the production of VK2, and lay an optimized foundation for realizing the industrialization of VK2 bioproduction.
Subjects: Biology >> Bioengineering submitted time 2017-09-20
Abstract： Chromatography has been playing an important role in bioseparation. As one of the main components of chromatography, the structure and properties of chromatographic media have great effects on purification results. Among them, capacity is one of the most important properties to determine chromatographic media’s purification effects, and how to improve the capacity is one of the key points having been paid close attention to in bioseparation. This paper is based on high-capacity agarose-based chromatographic media, with hydrophobic and immobilized metal ion affinity chromatographic media being tailor-made, respectively. For hydrophobic chromatographic media, various lengths of spacer arm were introduced with a controllable hydrophobic property, and the media was used in CHO-rHBsAg purification. rHBsAg binding capacity was greatly improved by this media containing spacer-arms, and rHBsAg recovery was more than 90% and the purification factor was 65.8, both higher than the results from traditional chromatographic media. For immobilized metal ion affinity chromatographic media, dextran molecules were grafted with manipulation and its capacity of His-tagged recombinant proteins was enhanced by 1.5 times than that of traditional media. This novel tailor-made high-capacity chromatographic media is promising for large scale purification of recombinant proteins.
Subjects: Biology >> Bioengineering submitted time 2017-09-20
Abstract：琼脂糖微球是生化分离领域应用最广泛的分离介质基质，但尚存在微球粒径不均一、机械强度低以及原料的转化率低等问题，这导致生物大分子的分离纯化以及大规模层析生产效率低、工艺复杂化，成为了生化分离领域的发展瓶颈。常规搅拌等方法所制备的琼脂糖微球中琼脂糖含量大多小于6 wt%，当琼脂糖含量超过6 wt%时，水相粘度过大，传统的制备方法很难将高粘度的水相均匀地分散到油相中形成均一的乳液，导致生成的乳液粒径不均一。对于生化分离介质来说，小粒径介质能提供大比表面积，分辨率高；高琼脂糖含量不仅可以提高介质强度，还能丰富微球中的有效官能基团，为后续衍生应用提供基础。 本研究采用新型快速膜乳化技术，将琼脂糖加热溶解后，配制成所需浓度的琼脂糖溶液作为水相。将一定量的油溶性乳化剂溶于与水不相溶的有机相中并预热到一定温度下作油相。利用搅拌法将水相与油相混合制备得到W/O 型初乳液。利用快速膜乳化技术，通过调控压力，将初乳液迅速通过疏水修饰后SPG 膜得到粒径均一的W/O 型乳液，最后在缓慢搅拌的条件下冷却固化乳液胶凝，得到粒径均一的琼脂糖微球。 以6%琼脂糖浓度微球为例，在以石油醚及液体石蜡作为油相，与外水相体积比为1:6下，过膜压力0.3kgf/cm2的条件下，得到的微球平均粒径为30μm，Span值=0.62，CV 值=25%。对比市售Sepharose 6FF介质，粒径由90μm降到了30μm，而其CV 值提高了47%。而整个制球过程时间不超过10min，琼脂糖原料的成球转化率达到了100%。研究结果表明，当琼脂糖浓度分别达到8%、10%、12%、16%时，利用快速膜乳化法制备出的微球粒径可达28-32μm，Span值<0.9，CV 值<18%。 快速膜乳化制备的8%浓度琼脂糖微球经交联再通过烯丙基缩水甘油醚活化并偶联磺酸基制备成阳离子交换介质。上述介质用于纯化抗生素（万古霉素和螺旋霉素），粗品经柱上层析分离后，收集洗脱液，用HPLC分析纯品含量。研究结果表明，万古霉素纯度由88.7%提高至95.1%，螺旋霉素纯度提高至99.9%，纯度有了极大地提高。 快速膜乳化具有简单、高效等优点，在制备均一小粒径、高琼脂糖含量的分离介质方面具有突出优势。同时该方法也适用于其他载药纳微球的制备，有很好的应用前景。
Subjects: Biology >> Bioengineering submitted time 2017-09-20
Abstract：巴斯德毕赤酵母（Pichiapastoris）是近年来发展迅速的一种高效表达的宿主菌。毕赤酵母属甲醇营养型酵母，具有操作简便、易于高密度培养、生长迅速、对翻译后的蛋白进行加工、生产成本低等优点。毕赤酵母表达系统已有20多年的研究开发使用历史，有多种表达载体和改良的宿主菌可供选择，可进行胞内表达或分泌表达。利用基于同源重组的DNA转化系统将基因整合进基因组是目前最重要的基因编辑手段之一，已经被广泛应用于毕赤酵母中；但毕赤酵母面临着筛选标记不足的难题。 在Cre/loxP系统中，当两个loxP位点以顺时针相同的方向放在一段序列之间时，Cre酶能识别并切除该序列，留下一个loxP位点。当使用突变的loxP序列（例如lox71和lox66），当被Cre重组酶识别并进行作用后，会留下一个新的突变的lox72位点，并不会与下次引入的loxP位点进行作用，从而避免了残留的loxP位点与新引入的loxP位点被Cre重组酶识别的可能。 本文通过在毕赤酵母通用质粒pPICZA和pGAPZA的基础上，在博来霉素抗性基因表达盒后融合了诱导表达Cre的表达盒，并在这两个表达盒前后引入了lox71和lox66位点，分别构建了质粒pZACH和pGACH；进而进行筛选标记循坏利用。具体过程如下：1、质粒转化毕赤酵母，使用博来霉素进行抗性筛选得到阳性转化子；2、将阳性转化子接种于含有诱导剂甲醇的YPM培养基中，培养过夜；3、将培养过夜的含菌体的培养基划线于不含抗生素的YPD平板上，培养至酵母单菌落可见；4、将3中的酵母单菌落同时点种于不含抗生素的YPD平板和含博来霉素的YPDZ平板上，如能在YPD平板上生长而不能在YPDZ平板上生长，则说明该菌落的博来霉素抗性表达盒已丢失，再使用PCR鉴定进行验证。整个抗性素标记丢失需要约4-5天。 使用质粒pZACH分别在毕赤酵母中表达了来源于柠檬酸杆菌（Citrobacteramalonaticus）CGMCC 1696的植酸酶和来源于抗辐射不动杆菌（Acinetobacterradioresistens） CMC-1的脂肪酶，这些质粒的抗性素标记丢失效率均大于70%，而且植酸酶和脂肪酶的产量与菌体生长都和使用基础质粒pPICZA进行表达的结果相似。该质粒对外源蛋白表达以及菌体生长并无不良影响。 使用筛选标记循坏利用质粒pZACH和pGACH，可以克服毕赤酵母中筛选标记不足的缺点，便于更好地在毕赤酵母中进行基因改造，从而为毕赤酵母的进一步工业应用提供基础。
Subjects: Biology >> Bioengineering submitted time 2017-09-20
Abstract：毕赤酵母作为常用的蛋白表达系统，广泛应用于实验室规模的蛋白质制备、表征以及结构解析等方面，已有上千种蛋白在毕赤酵母系统中成功地得到表达。在工业酶制剂领域，也有许多酶制剂包括植酸酶、脂肪酶、甘露聚糖酶、木聚糖酶等利用毕赤酵母实现了产业化规模的生产[1]。 酵母细胞壁在保护细胞完整性方面起到非常重要的作用，能够维持细胞渗透压平衡，在形态发生过程中产生并维持细胞的形态，并且在环境压力中有保护细胞的作用[2]。细胞壁主要由多糖和蛋白组成，其中多糖为β-1,3-葡聚糖，β-1,6-葡聚糖和几丁质，蛋白主要由N-和O-修饰的蛋白组成，在电子显微镜下观察发现，酵母的细胞壁厚约 200nm，分为电子密度不同的两层结构，外层甘露糖蛋白层和内层葡聚糖骨架[3]。GPI修饰的蛋白是一种通过糖脂共价连接在蛋白C端，从而将蛋白定位在细胞表面的一类蛋白。GPI修饰的蛋白在所有的真核细胞中都存在，并且其基本结构在大部分生物中都是保守的。通过对毕赤酵母全基因组中所有编码的蛋白序列进行分析，结合GPI型细胞壁蛋白的结构特征，最终在毕赤酵母GS115中发现50个潜在的GPI型细胞壁蛋白[4]，对这些蛋白的生化分析及功能鉴定需要进一步的探索。 通过对毕赤酵母GS115潜在的50个GPI细胞壁蛋白逐一进行单基因敲除，构建了GPI型细胞壁蛋白缺陷菌株库，研究了各细胞壁缺陷菌株在不同浓度碳源条件下的生长情况及细胞形态变化，以及细胞表面亲疏水变化及对细胞壁干扰剂的耐受性情况。研究发现敲除细胞壁蛋白会引起细胞多方面的变化，如对不同碳源的利用差异，对不同细胞壁干扰剂的耐受差异等。挑取一株甲醇耐受的菌株进行转录组学的分析，发现敲除后细胞代谢路径发生了明显变化，如细胞壁各组分的合成路径，细胞膜甾醇合成路径相关基因明显上调，同时一些胁迫路径也被激活，以抵抗高浓度甲醇对细胞的损害。通过对细胞壁蛋白功能进行深入的研究，将为毕赤酵母作为宿主菌表达外源蛋白提供重要的参考价值。
Subjects: Biology >> Bioengineering submitted time 2017-09-20
Abstract： In this paper, the strain ZZ-46 which could assilimated glucose and xylose simultaneously producing lipid were screened from 10 wild yeast strains from the soil of Nanyang region of henan province. The cell biomass, lipid concentration, lipid content, lipid coefficient and lipid productivty of the strain reached 20.23g/L、9.89 g/L、48.91%、14.64g/100g and 0.083 g•L-1•h-1 respectively by fermenting 120h with 2: 1of glucose and xylose as carbon source. Simultaneous, Lipid-acid component was analyzed, there is almost no difference in lipid-acids component by using different mixed sugar ratios as carbon source and C16 and C18 series are main fatty acids. Oleic acid content is the highest in lipid-acid component, next are linoleic acid, palmitic acid and stearic acid in all samples and the sum account for over 90%. The lipid component of this strain is similar to plant oil and could be used as biodiesel source. The strain was primarily identified as Cutaneotrichosporon dermatis by the morphology feature and 26S rDNA D1/D2 domain sequence analysis, and Trichosporon dermatis homonym.
Subjects: Biology >> Bioengineering submitted time 2017-09-20
Abstract：熊胆粉作为保健食品和药品已经被使用了近千年。熊脱氧胆酸(Ursodeoxycholic acid, UDCA)是熊胆粉(需求量约为45吨/年)的主要活性成分，广泛用于脂肪性肝病、药物性肝炎、病毒性肝炎等胆汁淤积性肝病。7β-羟基甾醇脱氢酶(7β-HSDH, hydroxysteroid dehydrogenase)是双酶偶联法(图1)催化鹅脱氧胆酸(Chenodeoxycholic acid, CDCA)转化合成熊脱氧胆酸的关键瓶颈酶，其活性、稳定性以及辅酶亲和力均显著影响酶促生物转化路线的技术经济性。我们通过基因挖掘手段获得一个新酶7β-HSDHRt。然而, 迄今报道的所有7β-羟基甾醇脱氢酶大多具有活性低、稳定性差以及工作pH与7-HSDH不相容等共同缺陷, 从而导致目标产物UDCA的时空产率十分有限[1]。因此，采用酶工程技术大幅度提高相关酶的催化性能是决定生物技术能否拯救黑熊的关键所在。在此报告中我们提出了一种多目标共进化策略(MODE, multiobjective directed evolution)，用于改造7β-羟基甾醇脱氢酶的催化活性、热稳定性和最适pH值[2]。通过易错PCR、DNA shuffling和定点突变等方法，最终所得突变体V3−1的比活力比野生酶高 5.5倍，而半衰期延长3倍。此外，突变体的最适pH向弱碱性移动(pH 6.5pH 7.5)，从而更接近于前置酶7-HSDH所催化脱氢反应的酸碱度(pH 8.0)。使用上述共进化的突变酶V3−1进行级联转化反应时，UDCA 的时空产率达到 942 g L−1 d−1, 显著高于天然酶的 141 g L−1 d−1。由此可见，酶催化剂的蛋白质工程改造技术在提高酶的催化合成潜能，促进生物催化工艺在绿色化工制药领域的应用，以及在服务生态文明建设、推动环境保护和生态平衡等方面，均将发挥强大的威力。
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