重组葡激酶基因在大肠杆菌内的高水平表达及生物学活性研究

将测序后的葡激酶重组质粒ＰＵＣ－ＳＡＫ经酶切后,组装于表达载体ｐＢＶ２２０,构建成ｐＢＶ－ＳＡＫ表达质粒,转化大肠杆菌。重组葡激酶表达水平达６０％～７０％,相对分子质量为 １５ ５００,主要以可溶状态存在于细胞中。生物活性测定证实,重组葡激酶具有很强的纤溶活性。     

重组葡激酶工程菌的构建方法

目的：利用N端缺失10个氨基酸的葡激酶（recombinant staphylokinase,rSaK）重组质粒,构建了表达可溶性rSaK126蛋白的工程菌,并研究不同条件下工程菌诱导表达目的蛋白含量的差异及纯化途径。 方法：采用细菌活化和培养方法诱导目的蛋白,并用SDS-PAGE测其含量,应用层析技术纯化蛋白。 结果：成功构建表达重组葡激酶的工程菌,表达的重组葡激酶蛋白约占菌体总蛋白的50％,经纯化后回收率为60%,纯度达99%以上。结论：成功构建高效表达重组葡激酶的工程菌,并获得了高含量、高纯度的目的蛋白。     

新型溶栓剂葡萄球菌激酶的研究进展

本综述从葡激酶的溶栓作用机制,包括与纤溶酶（原）等因子的结合与作用,葡激酶的高级结构,抗原性问题等方面概括了近年来有关葡激酶作为亲一代溶栓剂的研究成果,并指出进一步利用蛋白质工程,对葡激酶进行分子改造的设想。     

新型溶栓剂葡萄球菌激酶的研究进展

本综述从葡激酶的溶栓作用机制,包括与纤溶酶(原)等因子的结合与作用,葡激酶的高级结构,抗原性问题等方面概括了近年来有关葡激酶作为亲一代溶栓剂的研究成果,并指出进一步利用蛋白质工程,对葡激酶进行分子改造的设想。     

重组葡激酶的分离纯化

在构建出高表达,高活性的葡激酶工程菌株的基础上,对表达产物进行了分离纯化研究。经离子交换纯化后,纯度达８５％－９０％,回收率为５０％－６０％,经凝胶过滤后,纯度达９９％以上,回收率为６０％,经ＳＤＳ－ＰＡＧＥ测定,相对分子质量为１５．５＊１０＾３,比活为１．０＊１０＾５,Ｎ端氨基酸序列与献报道的相符。     

产生葡激酶的溶原性转换噬菌体的研究——Ⅰ.转换噬菌体的分离及宿主双溶原性的检出

从葡激酶阳性菌株66分离到两株噬菌体即α及β,α有转换葡激酶产生的能力,β则无。用去溶原方法由菌株66得到葡激酶阴性66SAK~-,由该菌株诱导得到噬菌体β。证明菌株66为带有两个前噬菌体的双溶原菌。在该菌株经过诱导后,得到的裂解液中,不能转换的噬菌体β多于转换噬菌体α。     

大肠杆菌表达的重组葡激酶-水蛭素融合蛋白的分离纯化及其二聚体分析

采用阴离子交换和凝胶过滤色谱法纯化大肠杆菌高密度培养所表达的重组葡激酶－水蛭素融合蛋白（rSFH）,经SDS-PAGE和RP-HPLC分析纯度达到98％以上,每升发酵液得率约0.7g。同时利用疏水色谱、MALDI-TOF对纯化过程中出现的rSFH同源二聚体的分析和表面疏水面积的计算及利用高效排阻色谱（HPSEC）分析NaCl、温度对rSFH的可逆二聚化行为的影响,认为疏水作用在rSFH的可逆二聚化行为中发挥着重要作用。     

葡萄球菌激酶作为新型溶栓剂的研究进展

近年来对溶栓剂的研究取得了很好的成果,主要集中在单链尿激酶型纤溶酶原激活剂(scuPA),组织型纤溶酶原激活剂(tPA),葡萄球菌激酶(SaK)等。葡萄球菌激酶(SaK)是一种激活溶纤维蛋白的制剂,它与纤溶酶原(Plg)形成1∶1的复合体,使后者转变为纤溶酶(Pli)后激活其他分子变为Pli。从葡激酶的溶栓作用机制,包括与纤溶酶(原)等因子的结合作用,葡激酶的高级结构,抗原性等问题以及近年来有关葡激酶作为新一代溶栓的研究进展进行了综述,并指出进一步利用蛋白质工程,对葡激酶进行分子改造的设想。     

葡激酶N端突变体的分子设计,高效表达与分离纯化

同源模建人微小纤溶酶原（ｍｉｃｒｏｐｌａｓｍｉｎｏｇｅｎ ,ｍｐｌｇ）的三维结构,建立葡激酶（ｓｔａｐｈｙｌｏｋｉｎａｓｅ,Ｓａｋ）,ｍｐｌｇ计算机分子对接的结构模型,模型与已有的实验结果基本相符。为研究葡激酶Ｎ端结构与功能的关系,进一步改造葡激酶分子,根据复合物结构模型设计了Ｎ端缺失１５个氨基酸的葡激酶突变体。     

具有抗血小板聚集功能的新型葡激酶突变体的表达、纯化及性质

[目的]为解决溶栓后再栓塞问题,构建N-端含RGD(Arg-Gly-Asp)序列的葡激酶双功能突变体.研究突变体的表达和纯化,并进行性质分析.[方法]将突变后的葡激酶突变体序列连入pBV220质粒,转化大肠杆菌BL21进行表达.阳离子交换、凝胶过滤和阴离子交换三步层析法纯化表达产物,采用溶圈法对纯化产物进行生物学活性测定,并测定纯化产物对血小板聚集的抑制效应.[结果]PAGE扫描结果显示,葡激酶突变体蛋白在大肠杆菌BL21中的表达量约占菌体蛋白总量的40%～50%;三步层析纯化后,HPLC测定其纯度可达95%.酪蛋白凝胶板溶圈法测得其比活性分别为10.8×104和11.0×104HU/mg,与野生型葡激酶活性相当;且具有明显的抗血小板聚集活性,血小板聚集仪测定其血小板聚集抑制率分别为10.72%和19.71%,明显高于野生型葡激酶血小板聚集抑制率.本实验利用pBV220载体高效表达了葡激酶突变体基因,得到了高纯度、高活性的突变体蛋白,为葡激酶生产产业化和临床应用奠定了良好的基础.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.     

Composition and diversity of bird communities in Swedish farmland–forest mosaic landscapes

CapsuleThe amount of forest (at local and landscape scales) and occurrence of residual habitats at the local scale are shown to be the major factors influencing bird community composition in farmland–forest landscapes in central Sweden.

Aims To investigate the importance of local habitat and landscape structure for breeding birds in farmland–forest landscapes in central Sweden.

Methods Breeding birds were censused at 292 points. A detailed habitat mapping was made within 300 m of the points. Within a 300–600 m radius only two major habitats (forests and arable fields) were identified.

Results Cluster analyses of bird communities identified three site types that also differed in habitat composition: (i) partially forested sites in forested landscapes; (ii) heterogeneous sites with residual habitats in mosaic landscapes; and (iii) field-dominated farmland sites in open landscapes. A total of 19 of 25 farmland bird species (restricted to farmland or using both farmland and forest) had the highest abundance in farmland sites with mosaics of forest and farmland, while only six farmland species had the highest abundance in field-dominated sites. The bird community changed from being dominated by farmland species to being dominated by forest species (common in forest landscapes without farmland) at small proportions (10–20%) of forest at the local scale. A major difference in habitat composition between heterogeneous and field-dominated sites was the occurrence of different residual habitats (e.g. shrubby areas and seminatural grasslands). These habitats seemed to influence bird community composition more than land-use, despite covering <10% of the area. Seminatural grasslands were important for bird community composition and species-richness, but grazing seemed to be less important. Among different land-use types, cereal crops were the least preferred fields. Set-asides with tall vegetation and short rotation coppices were positively associated with species-richness of farmland birds.

Conclusion In general, the composition of the landscape was important for bird community composition, although amount and distribution of forests, occurrence of residual habitats and land-use of fields at the local scale had the strongest influence on bird community composition. The possible implications of these patterns for managing farmland–forest landscapes are discussed.     

Chemical constituents from the fruits of Rhus typhina L. and their chemotaxonomic significance

This work describes the isolation and characterization of twenty-nine compounds from the fruits of Rhus typhina L., including eleven flavonoids (1–11), eleven phenols (12–22), two pentacyclic triterpenes (23–24), two organic acids (25–26), one lumichrome (27), one courmarin (28) and one pyrimidine (29) on the basis of their spectroscopic data. Compounds apigenin (1), daidzein (4), orobol (5), 3′, 5, 5′, 7-tetrahydroxyflavanone (6), naringenin (7), butein (8), (-)-catechin (9), quercetin-3-O-α-L-(3″-O-galloyl)-rhamnoside (11), 2-hydroxybenzoic acid (13), 4-hydroxybenzaldehyde (14), vanillin (15), methyl 3,4-dihydroxybenzoate (16), 3,5-dihydroxybenzamide (18), tyrosol (19), caffeic acid (20), 3-(2,4,6-trihydroxyphenyl)-1-(4-hydroxyphenyl)-propan-1-one (21), phlorizin (22), friedelin (23), oleanolic acid (24), 4,4-dimethyl-heptanedioic acid (25), anthranilic acid (26), lumichrome (27), scoparone (28) and uracil (29) have not been recorded before in this plant. This is the first report on the occurrence of compounds 4–7, 9, 11, 13–14, 16, 18–21, 25–29 from the genus Rhus. Moreover, the chemotaxonomic significance of these isolated compounds was also summarized.