藻蓝蛋白裂合酶催化藻红胆素(PEB)与CpcA和PecA共价偶联

 将由鱼腥藻Anabaenasp.PCC7120中cpcE/F构建的pCOLADuet-cpcE/F质粒和pACYCDuet-pebA质粒、pCDFDuet-hol-pebB质粒、pETDuet-pecA质粒、pETDuet-cpcA质粒转化入大肠杆菌,在IPTG的诱导下,实现了色素蛋白PEB-CpcA和PEB-PecA的成功表达.吸收光谱和荧光光谱研究表明,CpcE/F能够催化PEB共价连接到CpcA和PecA的Cys-84上形成色素蛋白PEB-PecA和PEB-CpcA,色素蛋白的最大吸收峰为555nm,最强荧光峰为570nm.Zn²⁺电泳分析结果表明,体内重组获得了目标色素蛋白,证明由cpcE/F所编码的蛋白裂合酶在体内重组中能够催化PEB与CpcA和PecA的共价偶联反应,生成非天然色素蛋白PEB-CpcA和PEB-PecA.     

藻蓝蛋白裂合酶基因的表达与酶活性研究

为了比较研究层理鞭枝藻藻蓝蛋白裂合酶PcE／F与藻红蓝蛋白裂合／异构酶PecE／F的结构与功能,将藻蓝蛋白裂合酶PcE／F氨基酸序列与其他蓝藻的相应序列进行比较,并进行大量表达,将表达的裂合酶PcE／F用于藻蓝胆素(PCB)与藻蓝蛋白α-亚基(α-PC)脱辅基蛋白(PcA)的体外重组,得到天然活性的α-PC,从而表明层理鞭枝藻中pcE／F所编码的蛋白质是α-PC生物合成的裂合酶．     

藻蓝蛋白裂合酶CpcS1与CpcT1的复合物初步研究

研究证实裂合酶CpcSl、CpcTl能分别催化藻蓝蛋白β亚基(简称CpcB)的两个色素结合位点(84位和155位)共价偶联藻蓝色素PCB(phycocyanobilin),但通过实验发现仅CpcSl和CpcTl共同催化CpcB无法生成天然构像的色素蛋白.为了挑选出辅助裂合酶,经过综合分析,筛选出6个与CpcSl、CpcTl同源的裂合酶.将编码裂合酶的基因按照本实验的需求,重新构建在其它载体上,在大肠杆菌BL21体内表达出蛋白后,分别与CpcSl、CpcTl混合·利用亲和层析分离,然后通过蛋白质电泳图谱初步分析裂合酶之间形成复合物的情况.能形成复合物的裂合酶,再与CpcSl、CpcTl及脱辅基蛋白共同转入大肠杆菌BL21体内进行体内重组研究.结果表明,CpcTl能分别与CpcSl、CpcT2、PecF形成复合物,但这些复合物均不能辅助催化CpcB生成天然产物.     

细菌光敏色素AphA的克隆表达及重组新方法

通过PCR技术，从蓝藻PCC7120中扩增出细菌光敏色素Apha的基因aphA，进而构建aphA的缺失突变体aphA(N-25/C-445)．二者分别转人表达载体pET30a中，在宿主菌中获得高效表达，获得的脱辅基蛋白与CpcA进行体外重组．在本体外重组体系中，以藻蓝蛋白α亚基(CpeA)，在光敏色素自身作用或者藻蓝蛋白裂合酶的帮助下从CpcA上捕获胆色素完成重组．藻蓝蛋白裂合酶CpcE／F能很大程度增强光敏色素脱辅基蛋白从CocA夺取PCB的能力．该结果对于研究细菌光敏色素的色素来源及生理生化机理都具有重要意义．     

螺旋藻藻蓝蛋白对小鼠免疫功能的影响

研究了螺旋藻藻蓝蛋白（Ｃ－ＰＣ）对正常和异常（经氢化可的松处理）的小鼠免疫功能的影响。实验结果表明,腹腔连续注射１５ｄＣ－ＰＣ,剂量为１００和２００ｍｇ／ｋｇ·ｄ－１能促进ＰＨＡ诱导的正常小鼠脾淋巴细胞的增殖,促进空斑形成细胞的溶血能力和血清中溶血素的含量,能显著对抗氢化可的松对机体免疫功能的损伤作用。     

集胞藻PCC6803基因slr2049定点突变及其体内重组功能研究

在Synechocystis sp.PCC 6803中找到与cpeS具有高度同源性的基因slr2049.采用PCR从Synechocystis sp.PCC 6803DNA中扩增出cpcB,slr2049,ho1,pcyA4个基因;定点突变试剂盒构建8个突变体slr2049(H21S)、slr2049(L23S)、slr2049(A24S)、slr2049(F25S)、slr2049(W72L)、slr2049(G84S)、slr2049(R107S)和slr2049(Y124S);将它们与表达载体pCDFDuet-1和pET-23a(+)相连构建pCDF-cpcB-slr2049野生型和pCDF-cpcB-slr2049突变体以及pET-ho1-pcyA,进行高效表达,用亲和层析柱纯化,产物用SDS-PAGE和光谱检测.研究表明:突变体slr2049(H21S)、slr2049(L23S)、slr2049(F25S)、slr2049(W72L)、slr2049(G84S)、slr2049(Y124S)与野生型slr2049催化获得的重组藻蓝蛋白β亚基具有与天然藻蓝蛋白β亚基相似的光谱特性,其他2个突变体slr2049(A24S)和slr2049(R107S)催化的产物则没有光谱特性.由此推测,第24位丙氨酸和第107位精氨酸可能是slr2049酶活性的必需氨基酸,其所处位置是slr2049的活性位点.     

层理鞭枝藻的藻蓝蛋白和藻红蓝蛋白α—亚基及其色素肽 …

用紫外可见吸收和圆二色光谱研究了层理鞭枝藻的藻蓝蛋白α－亚基色素肽和藻红蓝蛋白α－亚基色素肽的可逆光化学,这些研究表明藻蓝蛋白α－亚基色素钛和菏红蓝蛋白α－亚基色素肽分别相应于藻蓝蛋白和藻红蓝蛋白的辅基色素结构域,藻蓝蛋白α－亚基色素肽的光谱性质和可逆光化学与藻蓝蛋白α－亚基的相应性质很相似,藻红蓝蛋白α－亚基色素肽的光谱性质和可逆光化学与藻红蓝蛋白α－ 基的相应性质相似,不过藻红蓝蛋白α－亚基色     

海生红藻R-藻红蛋白和隐藻藻蓝蛋白光吸收系数测定

选用Bradford法和Lowry法,以牛血清蛋白和γ-球蛋白作为标准蛋白,分别对从海生红藻异管藻（Heterosiphonia japonica）和多管藻（Polysiphonia urceolata）以及蓝隐藻（Chroomonas placoidea）中分离纯化的异管藻R-藻红蛋白（HR-PE）、多管藻R-藻红蛋白（PR-PE）、蓝隐藻藻蓝蛋白（Cr-PC）进行了蛋白浓度及其特征吸收峰的光吸收系数测定.根据2种方法和2种蛋白针对3种藻胆蛋白的测定结果以及影响结果的主要因素进行了细致地分析和比较,Lowry法是进行藻胆蛋白,尤其是六聚体藻红蛋白浓度及光吸收系数测定的更适合、准确的检测方法;在进行同类或相同藻胆蛋白之间相对含量、比例的定量分析时,Bradford法是一种更便捷的测定方法.由于藻胆蛋白光吸收系数值与选用的蛋白定量检测方法及其所用的标准蛋白直接相关,在使用和比较藻胆蛋白光吸收系数值时必须明确检测方法和标准蛋白,否则藻胆蛋白光吸收系数值将不具有可比性.     

螺旋藻藻蓝蛋白对人血癌细胞K562生长的影响

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层理鞭枝藻藻红蓝蛋白裂合异构酶色氨酸环境的光谱探测

应用N-溴化琥珀酰亚胺(N-bromosuccinimide)研究了层理鞭枝藻藻红蓝蛋白裂合异构酶(PecE、PecF)色氨酸的化学修饰,用紫外吸收光谱、圆二色光谱和荧光光谱探测了色氨酸化学修饰过程中蛋白质结构微观环境,发现PecE和PecF的色氨酸都处在疏水区域或带负电荷区域,PecE的色氨酸残基更靠近分子表面.由于色氨酸是藻红蓝蛋白裂合异构酶的必需氨基酸,这些分析加深了解了色氨酸在藻红蓝蛋白裂合异构酶催化过程中的作用.     

Si-1基因在肿瘤中的缺失和突变状况初步研究

 通过对Si-1基因6,14和15号外显子区段的PCR扩增,分析了Si-1基因在116例恶性肿瘤组织、1例良性肿瘤和21例非肿瘤组织的缺失和突变.发现恶性肿瘤中有7例缺失,8例有内含子突变;良性肿瘤中没有缺失和突变;非肿瘤组织中没有缺失,1例有内含子突变.脑肿瘤的缺失率最高,达到57.1%,内含子突变率42.9%;肝癌的缺失率达15%,内含子突变率26.7%.上述结果表明,Si-1基因在脑肿瘤和肝癌组织中有较高的缺失和内含子突变,显示该基因有可能是这2种肿瘤的易感基因.     

Effect of internal deletion of Myosin II on growth and development ofDictyostelium discoideum

Four deletion mutantDictyostelium myosin II heavy chain genes, MyΔ824-941 (Δ1/ 3S2), MyΔ934-1454 (ΔS2), MyΔ934-1194 (ΔS2-1) and MyΔ1 157–1454 (ΔS2-2), were transformed by standard electfoporation into mhcA-cells (T-null), a mutantDictyostelium cell devoid of endogenous myosin II heavy chain gene. The growth, development and formation of fruiting bodies of cells expressing those mutant myosin II s under suspension culture were investigated by comparison with the wild type cell. The results indicate that internal deletion of myosin II affeds the growth and development ofDictyastelium. Furthermore, the longer the length of deletion, the more serious the defect in phenotype.     

瑞氏木霉木聚糖酶Ⅱ启动子功能区缺失分析的初探

木聚糖酶Ⅱ(Xylanase Ⅱ)是瑞氏木霉中两种主要的内切木聚糖酶之一．序列分析显示其1．1kh的启动子区含有多种丝状真菌转录因子结合位点，可受多种激活因子、抑制因子、增强子的转录调控．以编码β-葡糖苷酸酶(GUS)的E．coli uidA基因为报告基因，分别与木聚糖酶Ⅱ启动子及功能区(-312～-1080)缺失启动子融合，构建报告质粒pXIIPU与pXIIP△EXU，引入瑞氏木霉蛋白酶缺陷株Trichoderma reesei RutC30U4．先后对转化子进行发酵实验，测定GUS活性。结果显示功能区-312～-1080缺失后的启动子活性是原启动子活性的19％．结果表明在缺失的木聚糖酶Ⅱ启动子-312～-1080区仍存在调节转录活性的顺式作用元件，值得进一步研究．     

Frequency of the mtDNA 9-bp deletion in Chinese ethnic groups

The 9-bp deletion in the COIl/tRNALys intergenic region (region V) of human mitochondrial DNA was screened in 1521 Chinese from 16 ethnic groups and 9 Han geographic groups. The highest frequency was found in populations of Miao (32.4%) and Bouyei (30.8%) from Guizhou Province, whereas no deletion was found in Kazak population in Xinjiang. In the populations of Wa and Lahu from Yunnan Province, Uygur and Mongolian from Xinjiang,the deletion frequency was relatively low ( ≤ 4 % ), while in the remaining 18 groups, the frequency was moderate (6 % ～ 24% ). Except those Hans in Xinjiang, Guizhou and that reported in Taiwan, the deletion frequency in the Han geo graphic groups did not show a substantial difference. However, the deletion frequency in some ethnic groups from the same geographic region or with similar ethnohistory did not show similarity. A general decrease tendency in the deletion frequency was found from south to north and from coastal to inland. The frequency of the 9-bp deletion was approximate ly 17.20% in all Chinese we studied and reported elsewhere. Additionally, 4 individuals were found to carry the tripli cation of 9-bp segment in region V; one individual had X. II type of 9-bp deletion; and no other length polymorphisms were detected in this region in 27 randomly selected individuals with or without the deletion.     

Can the occurrence of rare insertion／deletion polymor—phisms in human mtDNA be verfied from phylogeny？

Due to its specific characteristics,such as ma-ternal inheritance and absence of recombination,each mtDNA belongs to certain monophyletic clade in the rooted mtDNA tree(haplogroup) according to the mutations it har-bors,Rare mutation(excluding parallel mutation) occurring at multiple times in different haplogroups could thus be a potential reading error according to the mtDNA phylogent.This experience has been widely used im double-checking the credibility of the rare mutations in human mtDNA sequences.However,no test has been performed so far for the feasibility of applying this strategy to the rare insertion/deletion(indel) events in mtDNA sequences.In this study,we attempted to relate the rare indels in mtDNAs to their haplogroup status in a total of 2352 individuals from 50 populations in China.Our results show that the insertion of A at position 16259 is restricted to a subclade of haplogroup Cand can be verified.The other indel polymorphisms,Which occur in the repeat of the deleted or inserted nucleotide(s),may not be distin-guished from phantom mutations from a phylogenetic point of view.Independently and multiply sequencing the frag-ment with the indel is the best and the most reliable way for confirmation.     

钒钛催化剂制备工艺参数对其表面结构与催化性能的影响

研究V2 O5/TiO2催化剂的制备工艺,如载钒量、煅烧时间、助催化剂和浸渍次数等,对催化剂表面结构及催化分解气相邻二氯苯的影响.采用漫反射紫外光谱(DRUVS)、X射线衍射(XRD)、BET等方法对催化剂进行表征.结果表明,随着载钒量的增大,催化剂表面活性物质含量增多,催化性能增强；延长煅烧时间会导致催化剂的团聚增加、比表面积减少,但有利于催化剂表面生成低聚态活性钒和V4+,从而提高其催化活性；添加助催化剂WO3和采用多次浸渍均能抑制活性钒的团聚,促进表面低聚态活性钒的分散；单聚和低聚钒酸盐是VOx/TiO2催化剂的活性物质,而高聚钒对催化分解气相邻二氯苯有抑制作用.