优化培养基增加层理鞭枝藻藻胆蛋白产量

藻胆蛋白(phycobiliprotein)是蓝藻和红藻藻胆体的组成部分,是光合作用集光复合体的组成部分,一般由α和β亚基构成,每个亚基含1～4个辅基色素,从而使藻胆蛋白具有特定的光谱吸收性质。根据这些吸收光谱性质,可以将藻胆蛋白分为:别藻蓝蛋白(APC)、藻蓝蛋白(PC)和藻红蛋白(PE)等,在某些缺乏PE而有异形胞的蓝藻中存在充当PE天线捕光功能的藻红蓝蛋白(PEC)〔1〕。藻胆蛋白可用于天然食用色素、化妆品色素和制药行业,还可作为免疫检测、荧光显微技术和流式细胞荧光测定法技术方面的荧光探针。特别是本工作研究的层理鞭枝藻(简称M.laminosu…     

蔷薇藻藻蓝蛋白的稳定性研究

蔷薇藻Rhodella reticulata是属于红藻门的一种海洋单细胞微藻,其生长过程中产生藻胆蛋白、胞外多糖等生物活性物质。蔷薇藻经过破碎,通过硫酸铵沉淀和DEAE Sepharose FF柱层析后,可以分离出较纯的藻蓝蛋白。本文从pH、温度、光照、食品添加剂、金属离子等方面对蔷薇藻藻蓝蛋白稳定性作了较全面的研究：藻蓝蛋白在pH为7时最稳定;低温有利于保持藻蓝蛋白的活性;相对于光照,在避光条件下,藻蓝蛋白有较高的稳定性;适当浓度的蔗糖和葡萄糖都有利于藻蓝蛋白的保存;金属离子影响藻蓝蛋白的稳定性。     

一步柱层析纯化螺旋藻藻蓝蛋白

采用硫酸铵盐析结合疏水层析技术分离纯化螺旋藻中的藻蓝蛋白.试验结果表明,在磷酸盐缓冲体系下藻蓝蛋白粗提液经1.25 mol/L硫酸铵盐析处理后离心脱气,只需采用一步Macro-Prep Methyl 疏水层析,藻蓝蛋白的纯度（A620/A280）可提高到4.017,回收率为19.38%.特征吸收峰和荧光光谱证实纯化后的产物符合藻蓝蛋白的性质,Native-PAGE电泳只出现单一染色带,表明纯化得到的藻蓝蛋白是均一的;SDS-PAGE电泳出现分子量为15.4 kDa、17.3 kDa的2条染色带,分别为藻蓝蛋白的α亚基与β亚基.     

条斑紫菜藻胆蛋白提纯方法优化探索

采用溶胀法与组织捣碎法相结合的紫菜叶状体细胞破碎方法.研究了不同物液比、缓冲液浸泡时间和硫酸氨盐析次数对藻胆蛋白纯度和产率的影响,对比了各种羟基磷灰石的层析效果,并对所得藻红蛋白和藻蓝蛋白做了吸收光谱、荧光发射光谱和SDS-PAGE鉴定.结果表明,在物液比为1:5,浸泡时间为36h时,紫菜综合破碎效果最佳;经过4次硫酸氨盐析后,藻红蛋白和藻蓝蛋白的纯度最高,分别达到1.71和0.98,采用Siegelman的方法制备的羟基磷灰石一次层析所得藻红蛋白和藻蓝蛋白的纯度最高,分别达到4.73和4.42,产率分别为0.144%和0.042%,且光谱和电泳鉴定结果均达到商品化要求.     

螺旋藻别藻蓝蛋白的纯化、理化特性与结晶

硫酸铵分级沉淀结合多种层析技术,从螺旋藻（SP—Dz）中纯化到电泳纯别藻蓝蛋白（Allophycocyanin,APC）,纯度（A650／A280）达4．83。APC在30min内的荧光扫描曲线为直线;30min连续光照其相对荧光强度仍为原来的98％以上,其抗荧光淬灭能力强于同样条件下的藻蓝蛋白（PC）及荧光素TRITC。用悬滴气相扩散法培养获得了APC晶体。     

具尾蓝隐藻(Chroomonas caudata Geitler)研究──Ⅱ.藻蓝蛋白(Phycocyanin)的初步分析

初步分析了具尾蓝隐藻（ChroomonascaudataGeitler）的藻蓝蛋白,其吸收光谱为一双峰曲线,两个吸收峰分别为590nm和640nm。按A．N．Glazer等关于隐藻藻蓝蛋白分型的意见,具尾蓝隐藻的藻蓝蛋白属于Ⅱ型PC－645。     

钝顶螺旋藻藻胆蛋白的分离,纯化及其理化特性

钝顶螺旋藻(Spirulina Platensis var.nanjingensis)一变异株的水溶性色素精提物,经固体硫酸铵沉淀,羟基磷灰石(HA)和Sephadex G-100柱层析后可分离、纯化出藻蓝蛋白(C-PC)和别藻蛋白(APC)。它们的纯度可分别达到AS 620/A_(277)=4.71;A_(650)/A_(270)=5.62。纯化后的C—PC和APC在聚丙烯酰胺凝胶电泳(PAGE)中仅见一条色带,其最大吸收峰分别在620nm和050nm。经12％的十二烷基硫酸钠—聚丙烯酰胺凝胶电泳(SDS—PAGE),以及高效液相色谱(HPLC)分离,C—PC和APC均可分为α和β两个亚单位。两者的亚单位分子量分别为:C—PC—α,15000;C—PC—β,14500;APC—α,15000;APC—β,13500。依此推算,该藻的C—PC和APC的最小分子量应为29.5kD和28.5kD。经等电电泳法测定,其C—PC和APC的等电点分别在4.8和4.9。氨基酸组成和含量分析结果表明,除色氨酸(Try)未测外,c—PC含有14种氨基酸,APC含有15种氨基酸,两者都缺乏组氨酸(His)和脯氨酸(Pro),C—PC还缺少蛋氨酸(Met)。     

硫酸铵三步盐析对藻胆蛋白纯化的影响

主要研究了多次硫酸铵盐析对条斑紫菜藻胆蛋白提取纯化效果。对分离提取的对条斑紫菜藻胆蛋白溶液进行了3次硫酸氨溶液盐析,实验结果表明:55%饱和度可以将绝大部分藻胆蛋白盐析;采用不同组合(15%、20%、25%、30%、35%、40%、45%7个饱和度分别与50%、55%、60%3个饱和度两两组合)二步硫酸铵盐析沉淀藻胆蛋白,使R-藻红蛋白和C-藻蓝蛋白的盐析后纯度(A564/A280)分别达到了1.0和0.45以上,得率分别为1.4%和0.95%;第3次硫酸铵盐析使R-藻红蛋白、C-藻蓝蛋白的纯度分别达到了1.4和0.4以上,最终产率分别为1.3%和0.8%,而变藻蓝蛋白产率有所下降(从0.65%到0.49%),但纯度变化不大。实验证明了采用多次盐析方法可以很大程度提高藻胆蛋白纯度。     

具尾蓝隐藻(Chroomonas caudata Geitler)研究──Ⅱ.藻蓝蛋白(Phycocyanin)的初步分析

初步分析了具尾蓝隐藻(Chroomonas caudata Geitler)的藻蓝蛋白,其吸收光谱为一双峰曲线,两个吸收峰分别为590nm和640nm。按A.N.Glazer等关于隐藻藻蓝蛋白分型的意见,具尾蓝隐藻的藻蓝蛋白属于Ⅱ型PC－645。     

藻蓝蛋白和藻红蓝蛋白β亚基半胱氨酸的定点突变及体外重组研究

为研究藻蓝蛋白 (PC)和藻红蓝蛋白 (PEC) β亚基 (分别简称为 β PC、β PEC)生物合成、结构与功能的关系 ,用MegaprimerPCR定点突变技术设计 β PC、β PEC中与藻蓝胆素连接的第二个半胱氨酸的定点突变蛋白质 β PC(C15 5I)和 β PEC(C15 5I)。将相应的基因片段亚克隆于表达载体pET 30a,并转化大肠杆菌BL2l(DE3)。经IPTG诱导后 ,β PC(C15 5I)和 β PEC(C15 5I)在大肠杆菌中均得到了高效表达。β PC(C15 5I)和 β PEC(C15 5I)与藻蓝胆素的重组结果表明两个突变体的结构基本没发生改变 ,有利于对 β PC和 β PEC的生物合成进行进一步研究。     

7B2 facilitates the maturation of proPC2 in neuroendocrine cells and is required for the expression of enzymatic activity

The prohormone convertase PC2, which is thought to mediate the proteolytic conversion of many peptide hormones, has recently been shown to interact with the neuroendocrine-specific polypeptide 7B2 in Xenopus intermediate lobe (Braks, J. A. M., and G. J. M. Martens. Cell. 78:263. 1994). In the present work we have stably transfected neuroendocrine cell lines with rat 7B2 constructs and found that overexpression of 27 kD 7B2 greatly facilitates the kinetics of maturation of proPC2, both in AtT-20/PC2 cells and in Rin5f cells. The half-life of conversion of proPC2 was reduced from 2.7 to 1.7 h in AtT- 20/PC2 cells stably transfected with 27 kD 7B2 cDNA. The previously proposed "chaperone" domain was not sufficient for this facilitation event; however, a construct corresponding to the 21-kD 7B2 protein (which represents the naturally occurring maturation product) functioned well. A 7B2 construct in which maturation of 27 kD 7B2 to its 21-kD form was blocked was unable to facilitate maturation of proPC2. To correlate effects on PC2 maturation with the actual generation of PC2 enzymatic activity, a similar transfection of 21 kD 7B2 was performed using CHO cells previously amplified for the expression of proPC2. Enzymatic activity cleaving the fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-AMC was highly correlated with the expression of immunoreactive 21 kD 7B2 in the conditioned medium; medium obtained from the parent cell line was completely inactive. Enzymatic activity was identified as PC2 on the basis of inhibition by the carboxy-terminal peptide of 7B2, which has previously been shown to represent a potent and specific PC2 inhibitor. Taken together, our in vivo results indicate that the interesting secretory protein 7B2 is a bifunctional molecule with an amino-terminal domain involved in proPC2 transport as well as activation.     

Circulating parasite antigen(s) in lymphatic filariasis: use of monoclonal antibodies to phosphocholine for immunodiagnosis

Hybridoma cell lines producing monoclonal antibodies (MAb) against a 200 kD antigen found circulating in the sera of microfilaremic patients infected with Wuchereria bancrofti were obtained by immunizing mice with a partially purified antigen preparation. A sensitive MAb (CA101)-based ELISA for measuring circulating parasite antigen was capable of detecting antigen in the sera of 93% of patients with microfilaremia, 46% of those with lymphatic obstruction, and 56% of patients with tropical pulmonary eosinophilia syndrome. Circulating antigen was absent from sera of normal controls, and "false positives" were recorded in only two of 17 patients with nonfilarial helminth infections. By ELISA and immunoblot analysis, it was shown that three of the monoclonals raised to this 200 kD antigen were directed to epitopes of phosphocholine (PC). Two MAb (CA86, CA101) were identified as having the T15 idiotype previously associated with antibodies to the PC of pneumococcal teichoic acid; one was untypeable. All three of these anti-PC MAb reacted with adult, microfilaria, and larval antigen preparations, and by immunoblotting showed multiple banding patterns that indicated the presence of PC determinants on many different antigenic molecules. On the other hand, target antigens of CA101 which were found in the circulation of infected patients were limited to three species with apparent m.w. of 200, 160, and 78 kD. The 200 kD antigen was seen more frequently than the other two antigens. Other T15 anti-PC MAb derived from mice not immunized with filarial antigen showed similar patterns of reactivity with circulating filarial antigen.     

Astrotactin: a novel neuronal cell surface antigen that mediates neuron- astroglial interactions in cerebellar microcultures

A microculture system for mouse cerebellar cells has been used to identify an immune activity, raised in rabbits against postnatal cerebellar cells, that blocks neuron-glial interactions in vitro. In the presence of blocking antibodies, stable neuron-glial contacts did not form and neuronal induction of glial process outgrowth did not occur. Subsequently, neurons were randomly arranged in the cultures rather than organized along the arms of astroglia. We have named the immune activity that blocks neuron-astroglial interactions anti-astrotactin. Partial purification of the anti-astrotactin blocking antibodies was obtained by cellular absorption with PC12 cells, a clonal cell line which expresses both the N-CAM and NILE (Ng-CAM, L1) glycoproteins. Subsequent absorption with purified cerebellar granule cells, but not with astroglial cells, removed the blocking activity, suggesting that the antigen(s) bound by blocking antibodies are neuronal. Immunoprecipitation of [35S]methionine- or [3H]fucose-radiolabeled Triton extracts of early postnatal cerebellar cells showed that the unabsorbed antiserum recognized a large number of proteins. Among these were bands with apparent molecular masses of N-CAM (180 and 140 kD) and NILE (230 kD). After absorption of the immune serum with PC12 cells, the number of bands recognized by the antiserum was reduced to a prominent band at 100 kD and a diffuse smear of material between 80 and 90 kD. The prominent band at 100 kD was removed by subsequent absorption of the immune serum with granule cells, a step which removed the blocking activity in the cerebellar microculture assay. Further evidence suggests that the astrotactin activity is missing or defective on granule cells from the neurological mutant mouse weaver, an animal that suffers a failure of glial-guided neuronal migration. When anti-astrotactin Fab fragments were pre-absorbed with weaver cerebellar neurons and then tested in the functional assay of neuron-glial interactions, the immune blocking activity was not removed. In contrast, wild-type cerebellar neurons removed the anti-astrotactin blocking activity under the same conditions. Subsequently, when [3H]fucose-radiolabeled Triton extracts of weaver and normal cells were immunoprecipitated with whole or PC12-absorbed anti-astrotactin antiserum, the intensity of the band at 100 kD was reduced by 95% in weaver cells.     

大鼠脊神经节感觉神经特异蛋白35kD(SSP-35)的纯化及鉴定(英文)

 以大鼠脊髓背根神经节及背根纤维组织为材料 ,通过离子交换层析 ,FPLC凝胶过滤等技术分离纯化了脊感觉神经特异蛋白 35kD(SSP 35) .经SDS PAGE分析 ,该蛋白特异地存在于脊感觉神经而不存在于脊运动神经 .非还原电泳结果表明 ,该蛋白分子内含有巯基 ,有二聚体存在 .根据HPLC测定 ,蛋白纯度达 90 %以上 .将此蛋白给予培养的PC 1 2细胞 ,观察到它具有神经营养作用 .     

Analysis of individual CNS protein synthesis

A procedure for labeling rat CNS proteins in vivo which is useful for behavioral and pharmacological studies has been developed. Intraventricular administration of³⁵S-methionine through bilateral indwelling cannulae provided reproducible and highly specific radiolabeling of proteins from frontal cortex (FC), parietal cortex (PC), occipital cortex (OC), striatum (ST), septal nuclei (SN), amygdala (AM), hippocampus (HIP), thalamus (TH), brain stem (BS) and cerebellum (CB). Relative rates of synthesis of over 200 individual proteins were subsequently analyzed by 2DGE. Regional analysis demonstrated increased labeling of a protein of MW 28 kD and pI 6.4 in the hippocampus that was barely detectable in striatum of control rats. In heat-shocked animals, there was increased relative synthesis of the 74 kD Heat Shock Protein in both the septal nuclei and hippocampus.     

Effect of angiotensin II on protein phosphorylation in PC12 cell line

Two subtypes of angiotensin II receptors have been characterised so far: AT1 and AT2. In PC12W pheochromocytoma cells, only AT2 receptors have been found (acting probably through G1 proteins or via G protein-independent mechanism). Here, dynamic changes in phosphorylation pattern in PC12W cells upon induction of angiotensin II and under influence of redox agents were investigated. PC12W pheochromocytoma cell line was preincubated with angiotensin II, then incubated with redox agents. After lysis the cells were subjected to Western-Blotting technique with antiphosphotyrosine and anti-ERK2 antibodies, as well as phosphotyrosine phosphatases and kinases activity was measured. Angiotensin II through its AT2 receptor induced dephosphorylation of tyrosines of the proteins in the range of 60 to 150 kD in PC12W cells. The obtained phosphorylation pattern suggests that AT2 receptors may act comparably to leukocyte CD45 receptor pathway. Treatment of PC12W cells with H2O2 resulted in significant decrease in phosphotyrosine phosphatases activity. It could be assumed that signal transduction based on protein phosphorylation might be controlled by cellular redox mechanisms.     

Nischarin as a functional imidazoline (I1) receptor

Gene matching shows that Nischarin is a mouse homologue of human imidazoline receptor antisera-selective (IRAS) protein, a viable candidate of the imidazoline (I1) receptor. Nischarin and IRAS share the functions of enhancing cell survival, growth and migration. Bioinformatics modeling indicates that the IRAS and Nischarin may be transmembrane proteins and the convergence information raises the interesting possibility that Nischarin might serve as the I1-receptor. To test this hypothesis, we developed antibodies against the Nischarin protein, and conducted signal transduction (functional) studies with the I1-receptor agonist rilmenidine in the presence and absence of Nischarin antisense oligodeoxynucleotides (ODNs). NIH3T3 cells transfected with the Nischarin cDNA and incubated with the newly synthesized antibody expressed a 190 kD band. The antibody identified endogenous Nischarin in differentiated PC12 cells around 210 kD, which is consistent with reported findings in other cells of neuronal origin. The immunoflourescence findings showed the targeted protein to be associated with the cell membrane in PC12 cells. Nischarin ODNs abolished the expression of Nischarin in PC12 cells. Equally important, the Nischarin ODNs eliminated the production of MAPK(p42/44), a recognized signal transduction product generated by I1-receptor activation in differentiated PC12 cells. Together, the present findings suggest that Nischarin may serve as the functional I1-receptor or at least share a common signaling pathway in the differentiated PC12 cells.     

猪Ⅱ型圆环病毒ORF1基因克隆及在E.coli中的表达

猪圆环病毒(porcinecircovirus ,PCV)属圆环病毒科(Circoviridae) ,为单股负链DNA病毒,以滚环方式进行复制,是目前已知的最小的动物病毒之一.PCV有2种基因型即PCV 1和PCV 2 ,两者细胞培养均不引起病变.前者广泛存在于猪源肾细胞中,但并不引起感染猪发病,其基因组为1 75 9bp ;后者首先由Allan等[1 ] 从患断奶猪多系统衰弱综合症(postweaningmultisystemicwastingsyndrome ,PMWS)的猪群中分离到,被证明为PMWS的重要病原.PCV 2主要侵害感染猪的免疫系统[2 ] ,从而诱发猪体的免疫抑制.PCV 2常和呼吸与繁殖障碍综合征病毒(PRRSV)…     

Effect of coexisting cells on the NGF-inducing neurite growth from PC12h-R

Possible roles of coexisting cells in inducing neurite growth from a nerve cell were studied. Nerve growth factor (NGF)-inducing neurite growth from PC12h-R (a cell line derived from cultured nerve cells) was investigated at various cell densities. At the cell density 10²10⁴ cells/ml neurites appeared even without NGF. In contrast, no neurite appeared without NGF in single cell culture. The neurite growth observed in plural cell culture without NGF was only partially inhibited by antibody to NGF receptor (Ab-NGFR). However, the effect of the used medium alone was mostly inhibited by Ab-NGFR. These results suggest that the neurite inducing potency of coexisting cells is via different sites than the NGF receptor.Abbreviations Ab-IgG-FITC anti-mouse-IgG labeled with fluorescein isothiocyanate - Ab-NF monoclonal antibody to neurofilament 160 kD - Ab-NGFR monoclonal antibody to NGF receptor - BDNF brain-derived neurotrophic factor - D-medium medium for differentiation culture - DMEM Dulbecco's modified Eagle's medium - M-medium medium for multiplication culture - NGF nerve growth factor - NGFR NGF receptor - NT-3 neurotrophin-3 - PC12 pheochromocytoma cell line - PC12h-R subclone of PC12 - Sup-D supernatant of D-medium