柱串联法在凝胶过渡色谱中的应用一例

凝胶过滤色谱因其操作方法简单、重复性强等特点在大分子的分离化中被广泛使用,特别在基因工程蛋白质类药物的精细纯化中起着难以替代的作用。将Superdex75凝胶柱与SephacrylS-100凝胶柱串联在一起进行凝胶过滤,分离一种用普通凝胶过滤色谱难以分离的样品,成功地将分辨率（Rs）由0．71提高到1．70,得到了基线分离。     

蛇毒纤溶酶Fibrolase的凝胶过滤色谱复性研究

目的：研究蛇毒纤溶酶Fibrolase的最佳复性方法。方法：使用透析和凝胶色谱法对Fibrolase复性进行研究,比较2种复性方法的相对复性率及蛋白质回收率。结果：2种复性方法的复性率分别为20％、25％,蛋白质回收率分别为16％、5％。结论：凝胶过滤色谱复性优于透析法复性,凝胶过滤色谱复性使蛋白质的复性与纯化同步进行,简化了操作程序,提高了产品回收率。     

高效凝胶色谱测定蛋白质分子量

自从1959年凝胶过滤色谱出现以来,很快就提出这种方法可做为一种测定蛋白质分子量的手段。但经典的凝胶过滤色谱流速慢、柱效低、检测灵敏度和操作的自动化程度也不高。因此,长期以来凝胶过滤主要还是作为一种分离的手段,并不经常用来测定分子量。近年来高效凝胶过滤色谱法的出现,使这项技术有了进一步的发展。一些新型的高效液相色谱柱也表现出在一定的分子量范围内分配系数(Kd)与分子量对数呈近似线性的关系,因此利用高效液相色谱的高效、高速、高     

拟南芥蛋白质组研究中双向电泳技术条件的优化

双向电泳技术是蛋白质组学研究中的关键技术,是目前分辨率最高的工具之一.而提高双向电泳图蛋白质点的数目和分辨率,可以提高蛋白质组技术平台的信息完整性.通过对拟南芥双向电泳技术过程中的适当改进,如蛋白质的提取与溶解方法、上样量和聚丙烯酰胺凝胶浓度,加入硫脲,硫代硫酸钠等,对拟南芥双向电泳技术进行了优化,提高了双向电泳图谱的蛋白质点数目与分辨率.     

圆弧青霉碱性脂肪酶的分离纯化的特性

圆弧青霉突变株ＰＧ３７发酵液经离心、硫酸铵盐析、疏水层析、阴离子交换层析和凝胶过滤分离纯化得到了比活性为每毫克蛋白质５２００ｕ的碱性脂肪酶,纯化倍数１６．５,得率３３．２％,在聚丙烯酰胺凝胶电泳（ＰＡＧＥ）和ＳＤＳ－聚丙烯酰胺凝胶电脉（ＳＤＳ－ＰＡＧＥ）上均呈现单一 白质条带。ＳＤＳ－ＰＡＧＥ和凝胶过滤分别测得酶的分子量为２７．５ｋＤ和２９．ｋＤ,表明该酶以单体形式存在。Ｎ末端１０个氨基酸的序列测     

人用精制Vero细胞狂犬病疫苗纯化方法选择

人用精制Ｖｅｒｏ细胞狂犬病疫苗为一种安全、有效的新型疫苗,我们在研制该种疫苗时首先比较了密度梯度离心法和凝胶过滤柱层析法,并发现后者最佳。我们选择了以Ｓｅｐｈａｒｏｓｅ ４ＦＦ为介质的凝胶过滤柱层析的工艺,并对该方法的上样量、流速等指标进行了选择,确定了最佳方法,并在研制中使纯化疫苗的杂蛋白去除率达到９９．８％以上,为大规模生产提供了工艺方法。     

取代P物质类似物的合成及生物活性

使用固相法合成含有不同D-型氨基酸取代的八个P物质类似物。纯化采用凝胶过滤,硅胶柱层或反相高效液相色谱。化合物的SP样作用及拮抗SP样作用在离体豚鼠迴肠纵行肌上进行了试验。所有化合物都显示了不同程度的拮抗作用。其中化合物A-58[D-Arg~1,D-Phe~5,D-Trp~(7,9),Leu~(11)]SP拮抗活性最高。它拮抗SP样作用的剂量比率(EC_(50)之比)在10~(-6)M时为24,在3×10~(-6)M时为52.2。     

中国林蛙卵核糖核酸酶的分离纯化及其抗肿瘤作用

以中国林蛙卵为原料,采用丙酮分级沉淀、SP-Trisacryl阳离子交换色谱、Sephadex G-75凝胶过滤色谱、C8反相色谱等纯化方法,得到一种具有核糖核酸酶活性的蛋白质,采用SDS-PAGE电泳对该蛋白质进行了相对分子质量和纯度测定。结果表明：纯化的中国林蛙卵核糖核酸酶为相对分子质量13kDa的单一成分。该酶最适反应温度为65℃,最适反应pH为5.5~6.0,米氏常数为4.11μmol/L,最大反应速率为2.82 pmol/s。在体外细胞毒性实验中,对人三种肿瘤细胞HeLa、K562、MCF-7具有抑制作用,其IC50分别为0.6μmol/L、0.8μmol/L和4μmol/L,而对于正常人成纤维细胞在酶浓度达到8μmol/L时仍未见明显细胞毒性。这种从中国林蛙卵中分离纯化出的具有选择性细胞毒性的小分子量核糖核酸酶,为肿瘤的治疗提供了新的候选蛋白分子。     

人血清白蛋白纯化技术研究进展

本文综述了国内外关于血浆人血清白蛋白（pHSA）和基因重组人血清白蛋白（rHSA）分离纯化的进展和发展趋势。以冷乙醇沉淀为主的pHSA分离方法仍是目前多数工业采用的工艺,但近年来发展的离子交换色谱、凝胶过滤色谱、亲和色谱等多种色谱分离技术具有自动化程度高、生产周期短、更符合GMP等特点,正在逐步取代传统的冷乙醇沉淀法。当前用基因工程技术表达的人血清白蛋白对分离纯化提出了新的挑战。为获得高纯度、安全、稳定的产品,各种色谱分离技术得到更多的应用,其中扩张床离子交换色谱可以省去离心、过滤等传统固液分离操作。尽管rHSA的纯化技术已有一定的发展,但纯化过程仍需进一步优化以提高产品纯度及收率。     

大肠杆菌表达的单链抗体柱复性的研究

对包含体表达的抗乙肝病毒表面抗原(HBsAg)的单链抗体(ScFv)纯化复性进行了探索.尝试了利用金属螯合亲和层析和凝胶层析柱进行柱上在位复性的可行性. 对包含体表达的ScFv进行透析复性与柱复性,比较其相对复性率及蛋白质回收率,发现柱上复性效果优于传统的透析复性.抗HBsAg ScFv经凝胶色谱Sephacyl S-200柱复性的相对复性率为98%, 蛋白质回收率为81%.由于将纯化复性同步进行,简化了操作程序,提高了产品的回收率.     

房室海绵化石的微机研究:性状演化

利用微机对房室海绵进行了１）属的清理工作，２）标准化术语的建立与属的标准化术语描述，３）建数据库，４）性状的统计分析。并发现了房室海绵出水管类型、房室排列方式、房室充填物类型、孔和管、体型和房室形态方面的六大演化趋向。     

Super Hybrid Rice Breeding in China：Achievements and Prospects

Hybrid rice has contributed greatly to the self-sufficiency of food supply In China. To meet the future demand for rice production, a national program on super rice breeding was established In China In 1996. The corresponding targets, breeding strategies and most significant advances are reviewed In this paper. New plant type models have been modified to adjust to various rice growing regions. In recognition of the Importance of applying parents with Intermediate subspecies differentiation In Increasing F1 yield, medium type parental lines were selected from populations derived from Inter-subspecies crosses with the assistance of DNA markers for subspecies differentiation. Results also indicate that a substantial increase of blomass Is the basis for further enhancement of the grain yield potential, and amelioration of leaf characteristics Is helpful In Increasing the photosynthetic rate. Thirty-four super hybrid rice varieties have been released commercially, growing In a total area of 13.5 million hm2 and producing 6.7 thousand million kg more rice In 1998-2005. Although remarkable progress has been made In super hybrid rice breeding In China, selections on the root system and Integration of more blotechnologlcal tools remain a great challenge.     

Integrons found in different locations have identical 5' ends but variable 3' ends.

The positions of the outer boundaries of the 5'- and 3'-conserved segment sequences of integrons found at several different locations have been determined. The position of the 5' end of the 5'-conserved segment is the same for six independently located integrons, In1 (R46), In2 (Tn21), In3 (R388), In4 (Tn1696), In5 (pSCH884), and In0 (pVS1). However, the extent of the 3'-conserved segment differs in each integron. The sequences of In2 and In0 diverge first from the conserved sequence, and their divergence point corresponds to the 3'-conserved segment endpoint defined previously (H.W. Stokes and R.M. Hall, Mol. Microbiol. 3:1669-1683, 1989), which now represents the endpoint of a 359-base deletion in In0 and In2. The sequence identity in In3, In1, In4, and In5 extends beyond this point, but each sequence diverges from the conserved sequence at a different point within a short region. Insertions of IS6100 were identified adjacent to the end of the conserved region in In1 and 123 bases beyond the divergence point of In4. These 123 bases are identical to the sequence found at the mer end of the 11.2-kb insertion in Tn21 but are inverted. In5 and In0 are bounded by the same 25-base inverted repeat that bounds the 11.2-kb insert in Tn21, and this insert now corresponds to In2. However, while In0, In2, and In5 have features characteristic of transposable elements, differences in the structures of these three integrons and the absence of evidence of mobility currently preclude the identification of all of the sequences associated with a functional transposon of this type.     

Hydrogen cyanide synthesis and antifungal activity of the biocontrol strain Pseudomonas fluorescens In5 from Greenland is highly dependent on growth medium

Hydrogen cyanide (HCN) is a secondary metabolite produced by many antagonistic Pseudomonas species. In the present study, the gene cluster encoding HCN synthesis in a newly isolated Pseudomonas fluorescens strain, In5, from South Greenland was investigated. Sequence analysis showed that the Greenlandic hcn gene cluster comprises a novel hcn cluster. Transposon mutagenesis of strain In5 resulted in mutants In5-2E1 and In5-1H7 with no production of HCN, and mutant In5-6B9 with reduced HCN synthesis. In mutant In5-2E1, the transposon was inserted into the hcnC gene; in mutant In5-1H7, the Tn5 insertion was found in a region upstream of a putative malate:quinone oxidoreductase gene (mqo); and in mutant In5-6B9, the transposon disrupted a probable enoyl-CoA hydratase/isomerase gene. In vitro inhibition experiments with In5 (wild type) and In5-2E1 (mutant) showed that in nitrogen-rich Luria-Bertani medium, strain In5 but not the hcn mutant In5-2E1 produced HCN and inhibited the growth of hyphae of Rhizoctonia solani and Pythium aphanidermatum . In contrast, when cultivating the strains in the carbohydrate-rich potato dextrose medium, neither of the strains produced any HCN, and thus, they were unable to inhibit hyphal growth of fungi. These experiments strongly indicate that the synthesis of HCN is highly dependent on the growth medium used.     

An indium:calcium phosphate colloid that specifically targets fibrin

The ability of indium to target fibrin in vitro was evaluated. The radionuclide ^114mIndium (^114mIn) was prepared as a soluble and colloidal (In:In) form, as well as, a mixed indium:calcium phosphate (In:CaP) colloid. Soluble ^114mIn was prepared by maintaining acid pH (50 mM HCl). Colloidal ^114mIn (In:In) was prepared under slightly basic conditions (50 mM Tris-Cl, pH 7.6). The mixed In:CaP colloid was prepared by incubation of ^114mIn with calcium (10 mM) and phosphate (250 M) under slightly basic conditions (50 mM Tris-Cl, pH 7.6). To assess fibrin binding, the three ^114mIn preparations were mixed with diluted human plasma (source of fibrinogen). Fibrin polymerization was initiated by addition of calcium (5 mM) and thrombin (0.5 U/ml). Following incubation (15 min, 37 °C), the fibrin matrix was condensed, removed from the reaction mixture, and washed briefly. Fibrin uptake of ^114mIn (soluble, colloidal, or In:CaP) was determined by gamma counting. Results demonstrated that soluble ^114mIn exclusively bound a plasma protein electrophoretically and immunologically identified as transferrin. Although both colloidal ^114mIn and ^114mIn:CaP bound fibrin, the mixed ^114mIn:CaP colloid demonstrated substantially higher fibrin binding activity (about 2-fold). The target of indium binding was confirmed as fibrin due to the presence of characteristic cross-linked – dimers (100 kDa) and -monomers (58 kDa) by SDS-PAGE. ^114mIn colloid and the mixed ^114mIn:CaP colloid demonstrated no ability to bind fibrins precursor, fibrinogen. ^114mIn:CaP fibrin binding was associated with formation of CaP, as evidenced by its dependence on phosphate concentration. The biocompatibility of CaP including its ability to bind ^114mIn and specifically target fibrin may be of potential value for diagnostic imaging studies to identify regions of occult vascular stenosis (i.e., atherosclerotic plaques, deep vein thrombosis, pulmonary embolus).In memoriam.     

化学交联质谱技术的研究进展

化学交联质谱技术是解析蛋白质结构和研究蛋白质相互作用的重要工具。近5年以来,该技术在方法和应用上都取得了很大的进步。方法上,一方面可断裂交联剂与新型分离富集方法展现了较好的应用前景,另一方面更加高效的交联肽段搜索引擎和质量控制方法为交联质谱数据分析提供了有力的工具。应用上,一方面与冷冻电镜技术结合解析了大量蛋白质的结构,另一方面从研究蛋白质复合物的相互作用发展到研究全蛋白质组水平的相互作用网络。化学交联质谱技术在方法和应用上的蓬勃发展,体现了这一技术的重要作用。本文对化学交联质谱技术的各个环节进行了详细的综述,包括交联剂选择、交联反应、酶切、交联肽段富集、液质联用、交联肽段鉴定、质量控制和生物学应用,重点介绍了最近5年的研究进展。最后,讨论了化学交联质谱技术面临的挑战及未来的发展方向。     

番茄受精作用及其间隔期的研究

利用常规石蜡切片法研究了番茄受精作用的全过程,具体研究结果为:(1)授粉后2 h,花粉粒在柱头上萌发;约2~4 h,花粉管长入柱头,且末端膨大;约8 h后,生殖细胞进入分裂期;并于约两小时后,分裂为两个精细胞。(2)约14 h,花粉管进入子房腔;约18~24 h,花粉管进入胚囊,破坏一个助细胞,并在其珠孔端释放两个精子;随后被释放的精子移到卵细胞与次生核附近。(3)授粉后约30 h精核进入卵细胞;约34 h,精核与卵核融合,并在卵核内出现分散的雄性染色质,进而出现雄性核仁;44~50 h,雌、雄性核仁融合,形成合子;合子的休眠期为10 h左右。60 h之后,合子分裂形成二细胞原胚。(4)约26 h,另一个精子的精核与次生核核膜相贴伏,随后与之融合;约30~34 h,次生核内出现分散的雄性染色质,随之出现雄性核仁;约38~42 h,雌、雄性核仁融合,形成初生胚乳核。约44 h后,初生胚乳核进行有丝分裂,形成两个胚乳细胞。番茄胚乳发育属于细胞型。初生胚乳核无休眠期。(5)精子与次生核的融合比与卵核的融合快。(6)番茄的受精作用属于有丝分裂前配子融合类型。     

罗汉果双受精过程的细胞学观察

罗汉果（Ｓｉｒａｉｔｉａｇｒｏｓｖｅｎｏｒｉ（Ｓｗｉｎｇｌｅ）Ｃ．Ｊｅｍｅｙ）双受精过程属有丝分裂前配子融合类型,授粉后２４～４８ｈ,花粉管进入胚囊,穿过一个助细胞,放出两个精子。雌雄核融合和雄核与次生核融合同时发生在授粉后６２～７２,雄核与次生核融合速度快于配子融合,７２ｈ后即可见到初生胚乳核分裂。合子中的雌雄核仁在授粉后第５～６ｄ融合,授粉后８～９ｄ合成分裂形成二细胞胚。在双受精过程中,多次观察到有多条花粉管进入胚囊和多精入极核现象。原胚期有附加花粉管从珠孔进入。     

The association between inversion In(3R)Payne and clinally varying traits in Drosophila melanogaster

In Drosophila melanogaster, inversion In(3R)Payne increases in frequency towards low latitudes and has been putatively associated with variation in size and thermal resistance, traits that also vary clinally. To assess the association between size and inversion, we obtained isofemale lines of inverted and standard karyotype of In(3R)Payne from the ends of the Australian D. melanogaster east coast cline. In the northern population, there was a significant association between In(3R)Payne and body size, with standard lines from this population being relatively larger than inverted lines. In contrast, the inversion had no influence on development time or cold resistance. We strengthened our findings further in a separate study with flies from populations from the middle of the cline as well as from the cline ends. These flies were scored for wing size and the presence of In(3R)Payne using a molecular marker. In females, the inversion accounted for around 30% of the size difference between cline ends, while in males the equivalent figure was 60%. Adaptive shifts in size but not in the other traits are therefore likely to have involved genes closely associated with In(3R)Payne. Because the size difference between karyotypes was similar in different populations, there was no evidence for coadaptation within populations.     

不同生境内濒危植物长喙毛茛泽泻种群数量动态比较

湖南茶陵自然种群动态在一年内表现为春季数量下降，以后振荡变化。在以莼菜为主的样方中，种群在夏季以后一直呈增加趋势，至冬季末数量下降；在杂草拥挤的样方内，种群基本呈下降趋势。湖北武汉，网室中迁地保护的种群生长在理想条件下，单一种群呈逻辑斯缔（Ｌｏｇｉｓｔｉｃ）增长，方程为Ｎ（株／ｍ２）＝８４０１＋ｅ４．４７７－０．２６４ｔ。江西东乡回归原产地的种群在回归当年经过精心照顾，种群呈增加趋势，次年随着杂草茂盛生长，种群数量迅速下降，种群具消失的危险。长喙毛茛泽泻种群数量的变动很大程度上受水位变化和杂草干扰的影响。实验结果表明，长喙毛茛泽泻种群能成功地实施迁地保护。