GM—CSF和G—CSF的协同抗肿瘤作用

GM－CSF抗肿瘤方面的主要作用是与免疫分子联合使用治疗肿瘤;GM－CSF基因转入肿瘤细胞和树突状细胞中分别作肿瘤疫苗和细胞疫苗发挥抗肿瘤作用;GM－CSF和G－CSF用于缓解放疗和化疗后骨髓抑制。     

枸橼酸酐对原核表达重组GM—CSF的修饰作用

根据枸橼酸酐对蛋白质中的游离氨基进行化学修饰后可使蛋白质溶解度提高的原理,以大肠杆菌表达的人重组ＧＭ－ＣＳＦ为模型,研究了枸橼酸酐修饰对含凝血酶识别位点的融合蛋白的作用,发现用微量的枸橼酸酐修饰的重组ＧＭ－ＣＳＦ变性,复性更容易,溶解度明显提高,并对凝血酶的消化更为敏感,使凝血酶用量降低１００倍,ＧＭ－ＣＳＦ活性测定结果证明枸橼酸酐修饰不影响其生物学活性,这些结果为枸橼酸酐修饰法在大肠杆菌表达重线     

优化mRNA翻译起始区提高人粒—巨噬细胞集落刺激因子在E.coli…

人粒－巨噬细胞集落刺激因子（ｈＧＭ－ＣＳＦ）是一种重要的造血生长因子。利用基因重组技术构建两个ｈＧＭ－ＣＳＦ的Ｅ．ｃｏｌｉ表达菌株,一个为在不改变氨基酸顺序的前提下,对ｍＲＮＡ翻译起始区核苷酸顺序进行优化突变（ｈＧＭ－ＣＳＦ（Ｍ））,另一个为未突变的对照（ｈＧＭ－ＣＳＦ（Ｎ））。经酶切电泳、ＤＮＡ测序、ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ ｂｌｏｔ等未突变的对照（ｈＧＭ－ＣＳＦ（Ｎ））。经酶切电泳、Ｄ     

人巨噬细胞集落刺激因子（hM—CSF）基因的合成及表达

按照ｈＭ－ＣＳＦ基因的序列,适当采用大肠杆菌的优选密码子,人工合成了Ｍ－ＣＳＦ的基因。在合成中我们采用了分段克隆和顺次克隆的方法,在次级克隆中我们成功地使用多片段分组连接和多片段一次克隆战略,还尝试了多种单链战术,在表达载体的构建中,充分利用人工合成基因的灵活性,通过对Ｎ端６个氨基酸编码的变换及ＳＤ序列－ＡＴＧ间距离的改变,获得了大肠杆菌中高效表达的重组体,表达的蛋白量的变换及ＳＤ序列－ＡＴＧ间距     

人GM—CSF cDNA的克隆和在大肠杆菌中的表达

从诱导的人胚肺细胞ＨＦＬ株中提取总ＲＮＡ．经ＲＴ－ＰＣＲ反应获取了人ＧＭ－ＣＳＦｃＤＮＡ,ＤＮＡ序列测定表明其顺序与文献报道完全一致。为了获得高效表达,应用ＰＣＲ改造了人ＧＭ－ＣＳＦ的ｃＤＮＡ５’端核苷酸序列,并将改造的人ＧＭ－ＣＳＦ基因插入含Ｔ７启动子的质粒ｐＥＴ－１１ｄ构建成表达质粒ｐＥＴＣ－５,将此质粒转化大肠杆菌株ＢＬ２１（ＤＥ３）得到表达菌株ＢＬＥＣ４。表达菌株用０．５ｍｏｌ／ＬＩＰＴＧ诱导２小时后,产生大量重组蛋白并形成包涵体。ＳＤＳ—ＰＡＧＥ电泳图谱扫描结果表明,ｒｈＧＭ－ＣＳＦ产量占菌体总蛋白量的１６％。ＥＬＩＳＡ和ＴＦ－１细胞培养测定表明,初步纯化和复性的ｒｈＧＭ－ＣＳＦ具有天然的ｈＧＭ－ＣＳＦ生物活性。     

人粒细胞集落刺激因子（hG—CSF）cDNA3‘—UTR对其表达的影响

利用人粒细胞集落刺激因子（ｈＧ－ＣＳＦ（）ｃＤＮＡ３＇端非翻译区（３＇ＵＴＲ）中存在的ＤｒａＩ酶切位点,通过部分酶切与完全酶切,删除３＇－ＵＴＲ不同长度构建了四种ｈＧ－ＣＳＦ ｃＤＮＡ瞬时重组表达质粒,转染ＣＯＳ－７细胞手,生物活性测定结果提示,ｈＧ－ＣＳＦｃＤＮＡ３＇－ＵＴＲ对其表达起负调控作用,其关键性序列位于紧接终止密码子ＴＧＡ下游的６５ｂｐ范围内,３＇－ＵＴＲ对ｈＧ－ＣＳＦ ｃＤＮＡ表达的     

重组人GM—CSF基因在昆虫细胞中的表达

利用苜蓿夜蛾核型多角体病毒（ＡｃＮＰＶ）带β－Ｇａｌａｃｔｏｓｉｄａｓｅ基因标记的非融合蛋白基因转移载体ｐＢｌｕｅＢａｃ将人粒细胞巨噬细胞集落刺激因子（ｈＧＭ－ＣＳＦ）基因成功地插入病毒ＡｃＮＰＶ的基因组中．ｈＧＭ－ＣＳＦ基因在感染重组病毒的草地夜蛾（Ｓｐｏｄｏｐｔｅｒａｆｒｕｇｉｐｅｒｄａ）培养细胞Ｓｆ９中得到表达,感染后的Ｓｆ９细胞培养液能刺激人骨髓细胞在体外形成典型的集落,表达水平可达２．７×１０５５ＣＦＵ／ｍｌ。以ｈＧＭ－ＣＳＦ单抗所作的ＷｅｓｔｅｒｎＢｌｏｔｔｉｎｇ表明,表达的ｈＧＭ－ＣＳＦ对是３种糖基化程度不同的产物,分子量分别约为１５ｋｄ,１８ｋｄ和２０ｋｄ。     

大肠杆菌表达的重组人粒细胞-巨噬细胞集落刺激因子的纯化

对重组人粒细胞－巨噬细胞集落刺激因子（ｒｈＧＭ－ＣＳＦ）高效表达克隆ｐＺＷ．ＧＭ的表达产物进行了纯化,并对纯化的ＧＭ－ＣＳＦ进行了Ｎ端氨基酸序列分析。人ＧＭ－ＣＳＦ基因表达产物在大肠杆菌中以不溶性包涵体形式存在,经过超声破菌、包涵体抽提、凝胶过滤层析、复性、离子交换一系列化步骤,终产物纯度达９９％,按蛋白总量计算回收率达１０％,比活性达１×１０＾７ｕ／ｍｇ蛋白质。通过测定纯化人ＧＭ－ＣＳＦ的Ｎ端１     

人重组GM—CSF／HSA—D3嵌合蛋白（GM—HSA）在大肠杆菌中的表达及其产…

将人粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）和人血清白蛋白第三功能区（ＨＳＡ－Ｄ３）的基因串联后,在Ｅ．ｃｏｌｉ中获高效表达,表达量占菌体蛋白的３２．６％。利用ＴＦ－１体外细胞活性测定表明,ＧＭ－ＨＳＡ的活性单位为１．０４×１０＾６Ｕ／ｍｇ,虽然其比活性低于ＧＭ－ＣＳＦ,但比后者具有更高的体外热稳定性和储藏稳定性。     

rhGM—CSF／LIF融合蛋白基因的克隆及表达

利用基因重组技术,人工构建了一个编码五肽Ｇ－Ｓ－Ｇ－Ｇ－Ｓ的基因接头,将ＧＭ－ＣＳＦ和ＬＩＦ的ｃＤＮＡ相连而构成融合基因,将融合基因载入原核表达载体ｐＢＶ２２０后转化大肠杆菌,经热诱导后进行Ｗｅｓｔｅｒｎ印迹反应鉴定证实获得ｒｈＧＭ－ＣＳＦ／ＬＩＦ融合蛋白（简称ｒｈｇＭ－ＬＩＦ）活性测定表明重组的融合蛋白具有两因子双重活性。     

甘蔗品质指标的通径分析和因子分析

应用通径分析和因子分析方法,对36个甘蔗品种（系）的11个品质指标数据进行分析。简单相关分析结果表明,甘蔗蔗糖分除了与蔗渣蒸煮液失水量没有显著相关外,与其他9个品质指标均有显著或极显著相关性。甘蔗纤维分与甘蔗出汁率、蔗汁折光锤度、蔗汁旋光读数、蔗汁直接转光度、蔗汁间接转光度和蔗渣含水量有极显著相关性,而与蔗渣蒸煮液失水量、蒸煮液的折光锤度和蒸煮液的旋光读数的相关性不显著。通径分析结果表明,甘蔗蔗糖分主要受甘蔗出汁率、蔗汁直接转光度、蔗汁间接转光度、蔗渣蒸煮液失水量和蒸煮液的旋光读数等5个指标影响较大,而甘蔗纤维分主要受甘蔗出汁率、蔗渣含水量、蔗渣蒸煮液失水量和蒸煮液的折光锤度等4个指标影响较大。因子分析结果表明,9个甘蔗品质指标可由4个主因子所代表。其特征值的累计贡献率迭94．30％。前3个主因子分别为蔗汁糖分指标因子、蔗渣糖分指标因子和蔗渣水分因子。其特征值的累计贡献率达83．06％,第4个主因子只有蔗渣蒸煮液失水量载荷值较大。     

甘蔗蔗汁品质性状的回归分析及模型

本文对47份不同甘蔗基因型的蔗汁品质性状进行简单回归和逐步回归分析.结果表明,蔗汁蔗糖分(Suc)与锤度(BX)、温度校正后锤度(BX′)、蔗汁旋光读数(0Z)以及转光度(pol)之间均呈线性回归,简单回归模型均达极显著水平(P＜0.01),决定系数(R2)分别为0.9393、0.9199、0.9861及0.9839.通过逐步回归分析和残差分析建立多重线性回归方程为Suc=0.05706 0.21488BX 0.181030Z,方差分析表明多重线性回归模型达极显著水平(P＜0.01),决定系数为0.9931.t测验表明,蔗汁蔗糖分的模型预测值与实测值间的差异不显著,相对误差平均为0.49%.     

Extracellular Calcium Has Distinct Effects on Fast and Slow Components of the Depolarization-Induced Secretory Response from Chromaffin Cells

Abstract: An increase in extracellular Ca²⁺ concentration from 0.25 to 10 m M enhanced secretion of norepinephrine and epinephrine induced by a high extracellular K⁺ concentration (75 m M ). The increment in extracellular Ca²⁺ concentration also increased the observed peak inward Ca²⁺ current in response to long (10-s) depolarizing pulses from a holding potential of −55 mV to +5 mV, from about −26 to −400 pA. However, the total amount of Ca²⁺ influx into the cell only increased when the extracellular Ca²⁺ concentration was raised from 0.25 to 1 m M and then remained constant up to 10 m M extracellular Ca²⁺. ATP is cosecreted with catecholamines following a depolarizing stimulus. Kinetic studies indicated that ATP secretion had two components with time constants, in the presence of 2.5 m M extracellular Ca²⁺, of ∼4 and 41 s, being the fast component of secretion produced by the exocytosis of ∼220 chromaffin granules. The results suggest that, for a given depolarizing stimulus, the size and rate of release for the fast and slow components of secretion are dependent on extracellular Ca²⁺ concentration.     

小麦农艺性状与品质特性的多元分析与评价

估算96个小麦品种(系)的11个农艺性状和10个品质特性参数的主成分,并以主成分和欧氏距离为基础,分别作二维排序分析和聚类分析。农艺性状的前4个主成分反映了85.3450%的原始数据信息量;品质特性的前4个主成分代表了89.1483%的原始数据信息量。以96个材料的主成分得分绘制二维排序图,27个小麦品种(系)表现为矮秆、子粒和旗叶较大,丰产性较好、综合农艺性状优良;32个小麦品种(系)表现为铁、锌含量较高,加工品质较好、综合品质特性优良。在系统聚类图中,农艺性状和品质特性分别被聚成5类。综合农艺性状较好的材料主要集中在第Ⅲ类和第Ⅳ类;综合品质特性较好的材料主要集中在第Ⅰ类和第Ⅱ类。综合分析发现,同时兼顾丰产性较好且子粒铁、锌含量较高,品质特性较好的小麦品种(系)有:泰山9818、西农822、轮选719、杨-31、西安837和中育9383。将聚类分析和二维排序分析结合起来,能较好的对小麦的性状组成做出综合评价,鉴定和评价出优质、高产、综合性状优良的小麦品种(系),为小麦遗传育种提供优良的种质资源,为合理选配亲本提供参考。     

Studies on the Development of Glandular Hairs in Nymphoides peltatum and the Ultrastructure During Their Secretory P

Each glandular hair of Nyrnphoides peltaturn (Gmel.) O. Kuntz consisted of only one row of cylindar cells with secretory function. The hairs originated from the protoderm cells on the adaxial surface of the second leaf primordium from the shoot apex. Cells of the glandular hairs prossessed dense cytoplast during the secretory period, but the vacuoles were very small. There were not only abundant mitochondria, Golgi bodies and endoplasmic reticulum in the glandular hair cells, but also many plasmodesmata. The authors' research indicated that the mucilage was carried to the edge of the cells by the membranous multilamellar bodies and the vesicles from both Golgi bodies and endoplasmic reticulum. The mucilage was secreted extracellularly by either exocytosis or ecrine secretion. The side walls of the glandular hairs swelled because of mucilage mass accumulation in the walls. The mucilage, being tested to be composed of polysaccharides and a trace of protein, played an important role in protecting the development of the vegetative buds of N. peltatum.     

AIAP: A Quality Control and Integrative Analysis Package to Improve ATAC-seq Data Analysis

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is a technique widely used to investigate genome-wide chromatin accessibility. The recently published Omni-ATAC-seq protocol substantially improves the signal/noise ratio and reduces the input cell number. High-quality data are critical to ensure accurate analysis. Several tools have been developed for assessing sequencing quality and insertion size distribution for ATAC-seq data; however, key quality control (QC) metrics have not yet been established to accurately determine the quality of ATAC-seq data. Here, we optimized the analysis strategy for ATAC-seq and defined a series of QC metrics for ATAC-seq data, including reads under peak ratio (RUPr), background (BG), promoter enrichment (ProEn), subsampling enrichment (SubEn), and other measurements. We incorporated these QC tests into our recently developed ATAC-seq Integrative Analysis Package (AIAP) to provide a complete ATAC-seq analysis system, including quality assurance, improved peak calling, and downstream differential analysis. We demonstrated a significant improvement of sensitivity (20%–60%) in both peak calling and differential analysis by processing paired-end ATAC-seq datasets using AIAP. AIAP is compiled into Docker/Singularity, and it can be executed by one command line to generate a comprehensive QC report. We used ENCODE ATAC-seq data to benchmark and generate QC recommendations, and developed qATACViewer for the user-friendly interaction with the QC report. The software, source code, and documentation of AIAP are freely available at https://github.com/Zhang-lab/ATAC-seq_QC_analysis.     

四川省的小麦地方品种品质分析

以67份四川省的小麦地方品种为试验材料,测定其19个品质指标并进行品质评价,旨在为小麦品质改良提供信息。结果表明,蛋白质和赖氨酸含量偏低,湿面筋含量、沉降值和面筋指数较高。粉质仪参数——形成时间、稳定时间和评价值偏低,面团流变学特性差。但也发现一些优质或专用小麦材料,如蛋白质含量〉14％的1份,湿面筋含量〉40％的10份,沉降值〉40ml的29份,中筋和弱筋小麦分别为7份和5份,德阳天绿场小麦、南部棒槌麦、酉阳光头、安岳红小麦和蓬溪红花光头麦5份材料具有相对较好的综合品质。相关分析表明,蛋白质含量、干湿面筋含量、沉降值、形成时间、稳定时间和评价值两两问简单相关（极）显著,蛋白质含量与湿面筋含量间和沉降值与稳定时间间偏相关显著。蛋白质含量与总淀粉含量间简单相关为极显著负相关。偏相关不显著。总淀粉含量与支链淀粉含量问简单相关和偏相关均极显著,总淀粉含量与直链淀粉含量间简单相关不显著,偏相关极显著,直链淀粉含量与支链淀粉含量间简单相关和偏相关均为极显著负相关。因此,在小麦育种早代进行蛋白质含量、沉降值和直链淀粉含量的选择为好。     

Secretory and GM1 receptor binding role of N-terminal region of LTB in Vibrio cholerae

Heat labile enterotoxin from enterotoxigenic Escherichia coli is similar to cholera toxin (CT) and is a leading cause of diarrhea in developing countries. It consists of an enzymatically active A subunit (LTA) and a carrier pentameric B subunit (LTB). In the current study, we evaluated the importance of the N-terminal region of LTB by mutation analysis. Deletion of the glutamine (ΔQ3) residue and a substitution mutation E7G in the α1 helix region led to defects in LTB protein secretion. Deletion of the proline residue (ΔP2) caused a decrease in α helicity. The ΔP2 mutant affected GM₁ ganglioside receptor binding activity without affecting LTB pentamer formation. Upon refolding/reassembly, the ΔP2 mutant showed defective biological activity. The single substitution mutation (E7D) strengthened the helix, imparting structural stability and thereby improved the GM₁ ganglioside receptor binding activity. Our results demonstrate the important role of N-terminal α1 helix in maintaining the structural stability and the integrity of GM₁ ganglioside receptor binding activity.     

Expression, purification and characterization of a photoprotein, clytin, from Clytia gregarium

A novel histidine-tagged secretion vector in Escherichia coli was constructed and large amounts of highly pure clytin, a calcium-binding photoprotein, was prepared. The histidine-tagged apoclytin expressed into the periplasmic space in E. coli was purified by nickel chelate affinity chromatography. Recombinant clytin was regenerated from apoclytin by incubation with coelenterazine in the presence of dithiothreitol and then purified by anion-exchange chromatography and hydrophobic chromatography. The yield of recombinant clytin was 20mg from 2L of cultured cells with purity greater than 95%. Luminescence properties of recombinant clytin were identical to that of native clytin (phialidin). The Ca(2+) sensitivity of recombinant clytin is lower than that of aequorin and clytin is suited for measuring higher concentration of Ca(2+). Semi-synthetic clytins were also prepared with coelenterazine analogues, and the initial intensity, luminescence capacity and half decay time were characterized.