人钙调素在大肠杆菌中的表达、纯化及其活性研究

利用基因重组技术,将经PCR扩增获得的人钙调素基因(hCaMcDNA)插入质粒pBV220,构建重组表达载体hCaM/pBV220,用酶切、DNA测序、PCR扩增鉴定阳性克隆.阳性重组子在大肠杆菌DH5α中经温度诱导可高效表达CaM蛋白,经15%SDS-PAGE分析,可观察到一与CaM分子量相符(约17kD)的诱导表达条带,其表达量占菌体蛋白总量20%,并主要以可溶性形式表达.Westernblot结果证实,17kD的表达条带可与标准鼠抗人CaM单克隆抗体起特异反应.用Pheny1-SepharoseCL-4B疏水亲和层析法纯化重组菌超声上清表达产物,每1L菌液可获CaM纯品3～4mg.重组人CaM(rhCaM)与牛脑CaM的氨基酸组成基本一致.生物活性测定结果提示,rhCaM具有激活NAD激酶的活性,其激活程度与标准人脑CaM几乎一致.     

豌豆NAD激酶的提纯及活力测定

NAD激酶(ATP:NAD 2′-磷酸转移酶EC 2.7.1.23简称NADK)催化NAD磷酸化生成NADP,其活性随着酶的纯化而降低,其激活依赖于Ca~(2 )激活的钙调素(Calmod-ulin简称CaM),两者之间成剂量关系,因此,可用该酶定量CaM。活性CaM一般采用酶法[如环腺苷酸磷酸二酯酶(PDE)和红血球Ca~(2 )-ATP酶等]定量,但采用这类酶时,酶活性易受磷酯与脂     

小麦黄化胚芽鞘的细胞壁钙调素和钙调素结合蛋白

小麦黄化胚芽鞘经苯基琼脂糖亲和层析提取和纯化,其细胞壁CaM在有钙和缺钙时SDS电泳呈现不同的迁移率;依赖Ca~(2 )与苯基疏水结合;在紫外吸收光谱上具有五个特征峰;对PDE的激活剂量反应曲线和从非活性状态向活性状态转变时所需的Ca~(2 )浓度均和胞内CaM相同,说明细胞壁CaM和胞内CaM具有相同的基本理化特性。采用CaM琼脂糖亲和层析,发现在小麦细胞壁中存在CaM结合蛋白,其中以分子量为40.7 kD的 CaM结合多肽为主。细胞壁CaM结合蛋白不具有过氧化物酶、ATP酶或酸性磷酸酯酶的活性。     

重组人钙调素的制备及其对细胞增殖作用影响的研究

用PCR方法获得人钙调素基因Ⅲ(hCaMⅢcDNA),将其插入表达载体pBV220,构建重组表达质粒hCaMⅢ/pBV220,阳性重组子在大肠杆菌DH5α中经温度诱导可高效表达CaM蛋白,经15%SDS-PAGE分析,可观察到一分子量与理论值相符(约17kD)的诱导表达条带.进一步分析表达产物的性质表明,CaM主要以可溶性形式表达.Westernblot结果证实,17kD的表达条带可与标准鼠抗CaMMcAb起特异反应.用Phenyl-SepharoseCL-4B疏水亲和层析法纯化重组菌超声上清表达产物,可获得纯度达95%以上的CaM,每1L菌液可获CaM纯品3～4mg.生物活性测定结果提示,rhCaM具有与标准人脑CaM(Sigma)同样的激活NAD激酶的活性.将K562细胞及SP2/0细胞分别接种于24孔或96孔培养板,加入不同浓度rhCaM、CaM拮抗剂三氟拉嗪(TFP),培养48h后,用MTI比色法检测细胞增殖状况.rhCaM在一定浓度范围内与细胞增殖率成显著正相关;CaM拮抗剂TFP可抑制细胞增殖.将rhCaM加入已受TFP抑制的细胞,可恢复正常的细胞增殖功能.     

检测钙调素的NAD激酶法

drClinmillWUShu－Pins,ZHANGXue－on,WANGJian－Ping,WANGYou－Al（HdriN～］ned．8彻加部办呼050016）磷酸二酯酶（PDE）定量测定活性钙调素（CaM）是国内外普遍应用的酶学方法［1］,而用NAD激酶（NADK）法检测活性CaM,虽早已提出[3],但应用并不普遍,国内尤为如此。多年来,我们研究了提取植物NADK及测定活性的方法[2],并用此法于红萝卜,玉米、豌豆、水稻、白茫、衣藻、四膜虫,人血细胞等多种生物的活性CaM的定性和定量检测,证实它与PDE法相比,有提取较为简易,灵敏度高,检测程序简便等多种优点;此外,…     

昆虫抗杆状病毒活性物质的诱导及鉴定

用BmNPV感染家蚕3龄幼虫,取其血淋巴经(NH4)2SO4分级沉淀,得到病毒抑制因子(viral iinhibitory factor,VIF)粗制样品Ⅰ和Ⅱ。通过TCID 50测定检测其抗病毒活性,结果VIF样品Ⅰ和样品Ⅱ均表现出抗病毒活性。将样品Ⅰ、Ⅱ经DEAE—SepharoseFF柱初步纯化,测得主要活性峰分别为峰c1,峰b2。对样品Ⅰ、Ⅱ进行SDS-PAGE电泳分析表明,样品Ⅰ比其对照样品多出4条带,它们的分子量分别为：83．7kD、65．2kD、43．0kD、32．8kD;而粗VIF样品Ⅱ比其对照样品多出一个组份,并且有3个组份表达量增多,多出的组份分子量为36．0kD。对灭活指数最高的峰b2作进一步电泳分析,证明存在5条带,其中有一条带可能是对应于样品Ⅱ上多出的那个组份。基于此初步证明,感染BmNPN的家蚕的免疫血淋巴中产生了抗病毒活性物质。     

重组人肝刺激物在原核细胞中的表达与纯化

利用基因重组技术 ,构建成人肝刺激因子 (hHSS)和谷胱甘肽转移酶 (GST)融合表达载体 ,转化大肠杆菌BL 2 1(DE3 ) ,以His·Tag亲和层析纯化表达产物 ,FactorXa切割分离hHSS单体 ,并检测其生物学活性。结果显示 ,在pET 4 2a表达体系中hHSS以可溶性蛋白和包涵体两种形式存在 ,GST hHSS表达量占菌体可溶性蛋白的3 0 % ;FactorXa切割GST与hHSS之间肽腱 ,得到 3 3和 15kD两条蛋白带 ,经Western杂交证实 3 3kD条带为GST ,而 15kD条带的分子量与hHSS基因序列推测蛋白结果相符。经His·Tag再次纯化可获得hHSS单体 ,初步证实重组hHSS具有促进肝癌细胞增殖活性     

北京鸭脑钙调素的分离纯化和性质的研究

钙调素(Calmodulin,简称CaM)是一种多生理功能的调节蛋白,在脑的功能活动中有重要作用。本文采用苯基琼脂糖(phenyl-Sepharose CL 4B)层析和葡聚糖凝胶(Sephadex G-50)过滤法,从北京鸭脑中分离纯化出CaM。纯化的CaM经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和等电聚焦(IEF)电泳鉴定均为一条区带。分子量为19kD,等电点(pI)为4.15,消光系数为1.83。 对纯化的鸭脑CaM的活性和性质进行了研究。它可明显地激活牛环核苷酸磷酸二酯酶活性,在有Ca~(2+)存在的条件下,SDS-PAGE中出现电泳迁移速度的改变,紫外吸收光谱具有已知CaM特有的吸收多峰形,并观察了Ca~(2+)对荧光发射光谱的影响。其氨基酸组成中,1/3是酸性氨基酸,苯丙氨酸和酪氨酸的比例为8:2。与猪CaM和牛CaM的物理化学性质作了比较。     

用人DNA拓扑异构酶Ⅰ单克隆抗体探测此酶的不均一性

 从慢性淋巴性白血病人的周血白细胞中纯化了DNA拓扑异构酶Ⅰ,经SDS-聚丙烯酰胺凝胶电泳分析,以蛋白质染色只有一条100kD的肽链,而用此酶的单克隆抗体探测同一纯化的酶则出现100kD,90kD,83kD,80kD和74kD五条肽链,并从部分纯化的酶制剂中检测到一条34kD的小分子具有相当高的酶活性。用此抗体进一步探测了不同类型白血病人周血白细胞的DNA拓扑异构酶,发现明显的差异,不同分子量仍具有此酶的活性,说明不同细胞固有DNA拓扑异构酶Ⅰ的不均一性。     

牛脑Ca~(2+)/CaM依赖性蛋白激酶Ⅱ的纯化和鉴定

通过一系列层析法,首次从牛脑纯化得到胶凝电泳匀一的Ca~(2+)/CaM PKⅡ。凝胶过滤法测定全酶分子量为550kD,SDS-PAGE法测定亚基分子量为55kD,推测牛脑Ca~(2+)/CaM PK Ⅱ由十个相同的亚基组成。该酶活性绝对依赖于Ca~(2+)和CaM,以63kD PDE同工酶为底物,其AC_(50)分别为0.85μmol/L和0.18μmol/L;以酪蛋白为底物,其AC_(50)分别为0.22μmol/L和0.06μmol/L。牛脑Ca~(2+)/CaM PK Ⅱ旣能催化63kD PDE同工酶等多种蛋白或酶磷酸化,又能进行自身磷酸化。该酶催化63kD PDE同工酶最大磷酸参入量为1mol/mol亚基。磷酸化型63kD PDE同工酶的Ca~(2+)的AC_(50)高于非磷酸化型。     

Calmodulin and the Regulation of Succinate Dehydrogenase Activity in the Mitochondria of Zea mays

Succinate dehydrogenase (SDH) was purified by DEAE C-32 chromatography from the mitochondrial fraction of corn ( Zea mays L. ). Free calmodulin (CAM) could not be detected in the purified SDH with the method based on the ability of SDH to stimulate NAD kinase, but it still contained some CAM when measured with the ELISA method. Purified SDH could stimulate NAD kinase only after heating to release free CAM. Plain polyacrylamide gel electrophoresis (PAGE) of the pufffled SDH revealed only one peptide band, but three peptide bands were shown on SDS-PAGE, their molecular weight being 67.0 kD, 30.0 kD, 16.7 kD respectively. The 67.0 kD and 30.0 kD peptides corresponded to the large and small molecular subunit of SDH respectively. The Rf value of the 16.7 kD peptide band was identical to the standard CAM in the SDS-PAGE. From all the above evidence, the authors suggested that CAM might exert its function of SDH regulation in a binding state with the SDH molecule.     

重组人钙调素的制备及对细胞增殖作用影响的研究

The gene coding for human CaM was amplified by PCR in which pUC/hCaM3 cDNA was usd as template. After inserting the hCaM III cDNA into the expression plasmid pBV220, we constructed the hCaM3 cDNA-recombinant expression vector(hCaM3/pBV220). The recombinant plasmid was then transformed into E. coli DH5 alpha. After heat induction, a high level expression of CaM protein was obtained. SDS-PAGE analysis showed that the recombinant E. coli could express a 17 kD protein which accounted for about 20% of the total cellular protein. Western blot analysis showed that anti-CaM monoclonal antibody(McAb) specifically bound to the 17 kD band of expression product. rhCaM was purified by Phenyl-sepharose CL-4B affinity chromatography from recombinant bacterial lysate. 3-4 mg of the purified protein were obtained from 1 liter of bacterial culture. The rhCaM was able to activate NAD kinase to the same extent as the standard human brain CaM (Sigma). K562 cells and SP2/0 cells were seeded in 24-well or 96-well plate and cultured for 48 h with rhCaM and CaM-antagonist trifluoperazine(TFP). Cell proliferation rates was determined by MTT assay. There was a significant positive correlation between the concentrations of rhCaM and the cell proliferation rates. CaM-antagonist TFP had an inhibitory effect on cell proliferation rate. The inhibition could be corrected by the addition of extracellular rhCaM.     

空肠弯曲菌肠毒素理化特性的研究

经ＳＤＳＰＡＧＥ 分析发现,空肠弯曲菌细胞紧张性肠毒素（Ｃｙｔｏｔｏｎｉｃ ｅｎｔｅｒｏｔｏｘｉｎ ,ＣＥ） 的硫酸胺盐析粗提物除有一条６８ｋＤ 的带外,还有一些未分开的小分子物质,而经神经节苷脂ＧＭ１ 亲和层析后仅有６８ＫＤ 的一条带,即表明６８ｋＤ 的蛋白质为ＣＥ 的主要成分。ＣＥ 不耐热、ｐＨ 依赖和对胰酶有抗性。５６ ℃和６０ ℃加热３０ｍｉｎ 、１００ ℃加热１５ｍｉｎ 即可完全失活。其活性在ｐＨ６－０ 时最高,在ｐＨ３－０ 和９－０ 时均可使其完全丧失活性。在４ ℃保存超过３ｄ 后,其活性迅速降低。抗ＬＴ 血清能完全抑制ＣＥ 的活性。     

Calmodulin Kinase II in Pure Cultured Astrocytes

Calcium- and calmodulin-dependent protein kinase activity was studied in pure neuronal and glial cultures. The addition of calcium and calmodulin stimulated 32P incorporation into several neuronal proteins including two in the 50- and 60-kilodalton (kD) region which comigrated with purified forebrain calmodulin kinase II subunits (CaM kinase II). In mature astrocytes, CaM kinase activity was also present, and was inhibited by trifluoroperazine and diazepam. Again in homogenates of these cells, two phosphoproteins of apparent molecular masses of 50 and 60 kD comigrated with purified CaM kinase. CaM kinase activity was absent in immature mixed glia and oligodendrocytes. The presence of CaM kinase in neurons and mature astrocytes was confirmed using monoclonal antibodies specific for the 50-kD subunit of the enzyme. No immunoreactivity was observed in oligodendrocytes. The presence of CaM kinase in astrocytes suggests a more ubiquitous role of this enzyme in regulating cellular processes than was previously recognized.     

Purification of the newly found selenium-containing proteins in the arterial wall and brain of the rat

Previously several selenium-containing proteins with different subunit molecular masses (M(r)) were detected in the arterial wall and brain of rats. In continuation of this work, after labeling of rats in vivo with [(75)Se]selenite, the new selenium-containing proteins of interest were purified on a Sephadex G-200 column followed by preparative isoelectric focusing. Nuclear analytical methods (gamma-counter and gamma-detector) were applied in the detection and identification of the (75)Se-labeled proteins. The two (75)Se-containing proteins from the arterial wall migrated as 15.0- and 67.0-kDa species on SDS-PAGE gels with pI values of 4.5 and 5.1, respectively. The three (75)Se-containing proteins from brain purified to homogeneity had M(r) values of 18.0, 30.0, and 42.9 kDa and pI values of 6.3, 6.5, and 6.0, respectively. Of these proteins, the 67.0-, 42.9-, and 30.0-kDa species may be yet not characterized selenoproteins with important biological functions.     

脑缺血及复灌中胞浆50kD蛋白反磷酸化变化

目的和方法：采用蒙古沙土鼠双侧颈总动脉结扎（ＢＣＡＯ） 前脑缺血／复灌模型,通过放射性自显影,观察脑缺血及复灌时胞浆５０ ｋＤ等蛋白在有Ｃａ２ ＋／ＣａＭ 及无Ｃａ２ ＋／ＣａＭ 两种反应条件下反磷酸化（ｂａｃｋｐｈｏｓｐｈｏｒｙｌａｔｉｏｎ） 水平的变化。结果：缺血后,总蛋白激酶介导的５０ ｋＤ蛋白反磷酸化水平逐渐下降,其中,Ｃａ２＋／ＣａＭ 依赖性蛋白激酶介导的５０ ｋＤ蛋白反磷酸化水平逐渐下降,而Ｃａ２＋／ＣａＭ 非依赖性蛋白激酶介导的５０ ｋＤ 蛋白反磷酸化水平逐渐上升;复灌后,上述变化均逐渐有所恢复。结论：脑缺血时,５０ ｋＤ 蛋白在体磷酸化水平逐渐增强,蛋白激酶活性由Ｃａ２ ＋／ＣａＭ 依赖性向Ｃａ２ ＋／ＣａＭ 非依赖性转化,复灌后上述变化均有所恢复     

猪血小板外廓蛋白（profilin）的分离纯化和鉴定

本文合成了一种聚脯氨酸亲和层析凝胶,并用这种凝胶纯化了猪血小板外廓蛋白。纯化的外廓蛋白在ＳＤＳ－聚丙烯酰胺凝胶电泳中呈单一蛋白带,分子量１４ｋＤ;在体外能显著抑制肌动蛋白聚合。     

大熊猫子宫钙调素的分离纯化和性质的研究

以大熊猫子宫为材料分离纯化了钙调素(Calmodulin,CaM),经SDS-PAGE,PAGE和等电聚焦电泳鉴定,表现均一。分子量为18800道尔顿,等电点为3.6。该蛋白质分子的N-末端为封闭的。大熊猫子宫钙调素具有其它来源钙调素所特有的一些性质。对环核苷酸磷酸二酯酶有明显的激活作用,还发现对超氧化物歧化酶也有一定的激活作用。电泳行为受Ca~(2+)影响而出现特征性电泳改变,在含有Ca~(2+)的SDS凝胶电泳中,电泳速度比EGTA存在对略快,在PAGE中,有Ca~(2+)比无Ca~(2+)对电泳速度略慢。大熊猫子宫钙调素的氨基酸组成中,Phe/Tyr为8:2,可观察到钙调素特征性紫外吸收光谱。