文昌鱼胰岛素样肽的表达、纯化、鉴定及其多克隆抗体制备

为了深入研究胰岛素和胰岛素样生长因子1（IGF－1）的起源和进化以及结构与功能的关系。表达了胰岛素和IGF－1的祖先分子－－文昌鱼胰岛素样肽（ILP）。重组单链ILP的基因用化学方法合成（从cDNA推测的ILPB结构域的C端和A结构域的N端用Ala-Ala-Lys三肽连接起来,并钭B28Arg突变为Lys）,克隆到表达载体pVT102-U中,ILP在酿酒酵母中得到有效表达。发酵液经4步分离纯化,得到均一的单链ILP,经质谱测定分子量和氨基酸组成分析证明表达产物正确。通过Lys-C蛋白内切酶处理将重组单链ILP转化成双链形式。虽然双链ILP与人胰岛素受体没有结合活力。但圆二色性光谱显示它与胰岛素的结构非常相似,用表达的单链ILP免疫新西兰大白兔,获得了高滴度的多克隆抗体。     

Aromatic Polyketide Synthases (Purification,Characterization, and Antibody Development to Benzalacetone Synthase from Raspberry Fruits)

p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries (Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and G. Hrazdina [1994] Phytochemistry 35: 623-628). Benzalacetone synthase condenses one malonate with p-coumarate to form the pathway intermediate p-hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is similar to those catalyzed by chalcone and stilbene synthases. We have obtained an enzyme preparation from ripe raspberries that was preferentially enriched in benzalacetone synthase (approximately 170-fold) over chalcone synthase (approximately 14-fold) activity. This preparation was used to characterize benzalacetone synthase and to develop polyclonal antibodies in rabbits. Benzalacetone synthase showed similarity in its molecular properties to chalcone synthase but differed distinctly in its substrate specificity, response to 2-mercaptoethanol and ethylene glycol, and induction in cell-suspension cultures. The product of the enzyme, p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen Phytophthora fragariae var rubi at 250 [mu]M. We do not know whether the dual activity in the benzalacetone synthase preparation is the result of a bifunctional enzyme or is caused by contamination with chalcone synthase that was also present. The rapid induction of the enzyme in cell-suspension cultures upon addition of yeast extract and the toxicity of its product, p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the pathway may be part of a plant defense response.     

Preparation and Characterization of P700-Enriched Photosystem-I Complexes from the Thermophilic Cyanobacterium, Synechococcus sp.

P700 was enriched relative to antenna pigments by treating thethylakoid membranes from thermophilic cyanobacterium (Synechococcussp.) with diethylether. The total Chi a/P700 ratio of the membraneswas 147 but decreased to 13 after the treatment with ether whichhad been 70% saturated with water. Vitamin K₁ and carotenoidswere completely removed by this treatment. The low chlorophylla to P700 ratio was retained in photosystem I reaction-centercomplexes purified from the ether-extracted membranes with TritonX-100. The midpoint potential of P700 was considerably loweredby the ether treatment of the thylakoid membranes but was partiallyreversed by the further treatment of the extracted membraneswith Triton X-100. Photo-oxidation of P700 in the purified complexeswas extremely slow under continuous illumination, indicatingthat there is no significant leakage of electrons from the primaryelectron acceptor to other bound acceptors or O₂. The photooxidationof P700 was appreciably accerelated on addition of vitamin K₃(or K₁). (Received June 23, 1988; Accepted November 25, 1988)     

Partial Purification and Characterization of Red Chlorophyll Catabolite Reductase,a Stroma Protein Involved in Chlorophyll Breakdown

Red chlorophyll (Chl) catabolite (RCC) reductase, which catalyzes the reaction of an intermediary Chl catabolite (RCC) in the two-step cleavage reaction of pheophorbide (Pheide) a into primary fluorescent catabolites (pFCCs) during Chl breakdown, was characterized and partially purified. RCC reductase activity was present at all stages of barley leaf development and even in roots. The highest specific activity was found in senescent leaves, which were used to purify RCC reductase 1000-fold. Among the remaining three proteins, RCC reductase activity was most likely associated with a 55-kD protein. RCC reductase exhibited saturation kinetics for RCC, with an apparent Michaelis constant of 0.6 mM. The reaction depended on reduced ferredoxin and was sensitive to oxygen. Assays of purified RCC reductase with chemically synthesized RCC as a substrate yielded three different FCCs, two of which could be identified as the stereoisomeric pFCCs from canola (Brassica napus) (pFCC-1) and sweet pepper (Capsicum annuum) (pFCC-2), respectively. In the coupled reaction with Pheide a oxidase and RCC reductase, either pFCC-1 or pFCC-2 was produced, depending on the plant species employed as a source of RCC reductase. Data from 18 species suggest that the stereospecific action of RCC reductase is uniform within a plant family.     

Purification,Characterization, and Submitochondrial Localization of a 58-Kilodalton NAD(P)H Dehydrogenase

An NADH dehydrogenase activity from red beet (Beta vulgaris L.) root mitochondria was purified to a 58-kD protein doublet. An immunologically related dehydrogenase was partially purified from maize (Zea mays L. B73) mitochondria to a 58-kD protein doublet, a 45-kD protein, and a few other less prevalent proteins. Polyclonal antibodies prepared against the 58-kD protein of red beet roots were found to immunoprecipitate the NAD(P)H dehydrogenase activity. The antibodies cross-reacted to similar proteins in mitochondria from a number of plant species but not to rat liver mitochondrial proteins. The polyclonal antibodies were used in conjunction with maize mitochondrial fractionation to show that the 58-kD protein was likely part of a protein complex loosely associated with the membrane fraction. A membrane-impermeable protein cross-linking agent was used to further show that the majority of the 58-kD protein was located on the outer surface of the inner mitochondrial membrane or in the intermembrane space. Analysis of the cross-linked 58-kD NAD(P)H dehydrogenase indicated that specific proteins of 64, 48, and 45 kD were cross-linked to the 58-kD protein doublet. The NAD(P)H dehydrogenase activity was not affected by ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime] -tetraacetic acid or CaCl2, was stimulated somewhat (21%) by flavin mononucleotide, was inhibited by p-chloromercuribenzoic acid (49%) and mersalyl (40%), and was inhibited by a bud scale extract of Platanus occidentalis L. containing platanetin (61%).     

水稻中类受体蛋白激酶FERONIA-like Receptor 1的表达纯化及抗体制备

类受体蛋白激酶参与调控植物的器官发育和抗逆性,是一种重要的蛋白激酶。通过对水稻中类受体蛋白激酶家族成员FERONIA-like receptor 1 (FLR1)进行蛋白质结构预测分析,发现FLR1蛋白具有跨膜结构,其N端在细胞膜外(FLR1-BW, 1～450 aa), C端在细胞膜内(FLR1-BN, 474～892 aa)。为了得到FLR1抗体用于其功能分析,将FLR1的N端和C端分别构建到原核表达载体pET-32a、pGEX-4T-1、pET-22b、pTriEx-4上,比较FLR1-BW和FLR1-BN在不同载体和不同条件下的表达效果。结果显示:FLR1-BW在pET-32a及pGEX-4T-1中能够有效表达,最优条件为16℃/16 h、0.5 mmol/L IPTG;而FLR1-BN蛋白表达后会对大肠杆菌产生细胞毒性,但在293细胞中能够表达。进一步利用纯化的FLR1-BW蛋白制备FLR1多克隆抗体,结果显示该抗体特异识别FLR1蛋白。本研究为类受体蛋白激酶的抗体制备提供了良好的思路并为研究FLR1功能奠定了基础。     

SUA41蛋白表达和纯化及其多克隆抗体制备

旨在制备特异性SUA41多克隆抗体,为深入研究其在植物生长发育中的功能提供有力的分子生物学和生物化学的工具。PCR扩增拟南芥SUA41基因中编码280个氨基酸(401-680位氨基酸)的特异片段,经过GATEWAY的DNA重组技术构建了原核表达载体pDEST17-SUA41,用热休克法转化到E.coliBL21(DE3)star感受态细胞,以异丙基β-D-硫代半乳糖苷(IPTG)诱导表达出6×His-SUA41融合蛋白,用8 mol/L尿素缓冲液溶解包涵体并且经过水逐级去除尿素获得提纯的融合蛋白,并利用Western blotting鉴定确认。融合蛋白经Ni金属螯合柱亲和层析得以纯化,用SDS-PAGE进一步纯化。纯化的融合蛋白经过SDS-PAGE后切胶回收,免疫小白兔,制备多抗血清,然后用Western blotting进行检测,鉴定血清特异性和效价。结果显示,融合蛋白6×His-SUA41免疫兔,产生特异性的SUA41兔抗血清,可以检测到细菌和拟南芥组织中SUA41蛋白。用水提纯变性剂尿素溶解的包涵体蛋白具有可行性。制备的特异性SUA41兔抗血清效价高,能够有效地识别大肠杆菌表达的和拟南芥的SUA41蛋白。在有合适的对照情况下,该兔抗血清可以用于分析植物中SUA41蛋白的功能。     

ANKRD17蛋白抗体的制备、纯化及检测

目的：制备ANKRD17（P260）蛋白的兔多克隆抗体,以与抗原相结合的方法进行抗体的纯化,并利用纯化的抗体对该蛋白进行细胞内免疫荧光检测。方法：构建表达GST—ANKRD17C端融合蛋白的质粒,在大肠杆菌中诱导表达;制备GST—ANKRD17C端抗原融合蛋白后免疫家兔,对获得的兔多克隆抗血清进行亲和纯化;纯化后的抗体经过Western blot鉴定,用于细胞免疫荧光染色检测。结果：获得较高效价的血清抗体,并对血清抗体进行了纯化;利用纯化的抗体对ANKRD17蛋白进行了细胞内免疫荧光检测,发现改蛋白定位于细胞质中。结论：制备得到的纯化抗体为研究ANKRD17蛋白的功能打下了必要的基础。     

重组酶Exo的纯化及多克隆抗体制备

目的:纯化Exo重组酶融合蛋白并制备相应抗体。方法:用阴离子交换柱对蛋白进行初步纯化,然后用Ni-NTA介质填充的层析柱分离纯化含His标签的融合蛋白,用谷胱甘肽琼脂糖4B介质填充的层析柱分离纯化GST融合蛋白;二次纯化的蛋白利用硝酸纤维素膜结合法制备抗原蛋白并免疫实验动物。结果:ELISA结果显示血清抗体效价可达到1∶12 800,说明通过Western免疫印迹自制的多克隆抗体能特异地与Exo重组蛋白相互作用。结论:该蛋白纯化方法操作简单,制备的抗原纯度高,多克隆抗体特异性好。     

Purification and Characterization of Glutamyl-tRNA Synthetase : An Enzyme Involved in Chlorophyll Biosynthesis

Chlorophyll biosynthesis starts with the synthesis of glutamyl-tRNA (glu-tRNA) by a glutamyl-tRNA synthetase (Glu RS). The glu-tRNA is subsequently transformed to δ-aminolevulinic acid (ALA), which is a committed and regulated precursor in the chlorophyll biosynthetic pathway. The Glu RS from a green alga, Chlamydomonas reinhardtii, was purified and shown to be able to synthesize glu-tRNA and to participate in ALA synthesis in a coupled enzyme assay. Physical and chemical characterization of the purified Glu RS indicated that the enzyme had been purified to homogeneity. The purified enzyme has a native molecular weight of 60,000, an isoelectric point of 4.6, and it formed a single band of 32,500 daltons when analyzed by a silver stained denaturing gel. The N-terminal amino acid sequence of the 32,500 dalton protein was determined to be Asn-Lys-Val-Ala-Leu-Leu-Gly-Ala-Ala-Gly. The molecular weight analyses together with the unambiguous N-terminal amino acid sequence obtained from the purified enzyme suggested that the native enzyme was composed of two identical subunits. Polyclonal antibodies raised against the purified and denatured enzyme were able to inhibit the activity of the native enzyme and to interact specifically with the 32,500 dalton band on Western blots. Thus, the antibodies provided an additional linkage for the structural and functional identities of the enzyme. In vitro experiments showed that over 90% of the glu RS activity was inhibited by 5 micromolar heme, which suggested that Glu RS may be a regulated enzyme in the chlorophyll biosynthetic pathway.     

抗人DR5多克隆抗体的制备与纯化

目的:分离纯化融合蛋白GST-eDR5,免疫小鼠制备抗人DR5多克隆抗体.方法:用GST纯化试剂盒和电泳两次纯化的融合蛋白GST-eDR5免疫小鼠,制备抗CST-eDR5抗血清,然后用GST纯化得到抗人DR5抗血清.通过Westem blot、ELISA方法鉴定抗血清特异性和效价.结果:通过亲和纯化得到了高纯度的融合蛋白GST-eDR5,其浓度为0.65μg/μl.免疫产生的DR5抗血清特异性高,且效价高达1:25600,纯化后的抗人DR5抗血清不再识别GST,只识别人DR5.结论:成功制备了高特异性、高效价的抗人DR5多克隆抗体,为深入研究其对肿瘤细胞的生长抑制和凋亡作用提供了实验工具.     

Purification and Characterization of an NAD(P)H:Quinone Oxidoreductase from Glycine Max Seedlings

Abstract

An NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) was purified from Glycine max seedlings by means of chromatographic procedures. After 1371-fold purification, the enzyme showed a single band in IEF corresponding to an isoelectric point of 6.1. A single band was also found in native-PAGE both by activity staining and Coomassie brilliant blue staining. The molecular mass determined in SDS-PAGE was 21900 Da, while in HPLC gel-filtration it was 61000 Da. The NAD(P)H:quinone oxidoreductase was able to use NADH or NADPH as the electron donor. Among the artificial quinones which are reduced by this enzyme, 6-hydroxydopa- and 6-hydroxydopamine-quinone are of particular interest because of their neurotoxic effects.     

抗传染性犬肝炎病毒单克隆抗体的制备及鉴定

目的制备传染性犬肝炎病毒单克隆抗体。方法将用纯化的犬腺病毒1型（CAV-1）免疫的BALB／c小鼠脾细胞与SP2／0细胞在聚乙二醇作用下融合,通过酶联免疫吸附试验（ELISA）和免疫酶试验（1EA）筛选,以有限稀释法克隆3次,制备单克隆抗体,并对制备完成的单克隆抗体进行生物学鉴定。结果成功得到2株能稳定分泌抗CAV-1的单克隆抗体杂交瘤细胞,命名为1B、2H3,经鉴定其亚型分别为IgG2a和IgG2a,2株均为kappa链。腹水的ELISA效价可达10^7-10^8,IEA效价可达1：2560—1：5120。该单克隆抗体与CPV、CDV、FPV、CCV病毒无交叉反应。结论成功制备了抗CAV-1单克隆抗体,为进一步建立相关诊断方法奠定了基础。     

GOLPH2的原核表达、纯化、抗体制备和初步应用

目前发现GOLPH2蛋白与肝癌密切相关,将golph2基因进行克隆、表达并制备多克隆抗体,为进一步研究其功能奠定基础.应用RT-PCR技术,从人肝癌细胞系HepG2细胞中扩增得到golph2 cDNA,将其克隆到原核表达载体pET21a(+)-TRX内、转化大肠杆菌DH5a,用IPTG诱导其在大肠杆菌BL21(DE3)中表达.His-tag磁珠纯化试剂盒纯化重组蛋白GOLPH2,SDS-PAGE鉴定.将纯化的重组蛋白免疫BALB/c小鼠制备多克隆抗体,采用ELISA、Western blot方法检测抗体的灵敏度和特异性,并测定临床血清标本中GOLPH2蛋白水平.成功地构建了表达TRX-GOLPH2融合蛋白的原核表达质粒pET21a(+)-TRX-GOLPH2.并在大肠杆菌BL21(DE3)内得以高效表达,且以可溶性的形式存在.SDS-PAGE和Western blot证实,重组GOLPH2蛋白质与预期结果一致.抗血清能够特异地识别52 kD重组蛋白、73 kD细胞裂解液和血清蛋白质.成功制备了GOLPH2蛋白质和多克隆抗体,能用于后续研究.     

FTIR study of the primary electron donor of photosystem I (P700) revealing delocalization of the charge in P700(+) and localization of the triplet character in (3)P700.

The effect of global (15)N or (2)H labeling on the light-induced P700(+)/P700 FTIR difference spectra has been investigated in photosystem I samples from Synechocystis at 90 K. The small isotope-induced frequency shifts of the carbonyl modes observed in the P700(+)/P700 spectra are compared to those of isolated chlorophyll a. This comparison shows that bands at 1749 and 1733 cm(-)(1) and at 1697 and 1637 cm(-)(1), which upshift upon formation of P700(+), are candidates for the 10a-ester and 9-keto C=O groups of P700, respectively. A broad and relatively weak band peaking at 3300 cm(-)(1), which does not shift upon global labeling or (1)H-(2)H exchange, is ascribed to an electronic transition of P700(+), indicating that at least two chlorophyll a molecules (denoted P(1) and P(2)) participate in P700(+). Comparisons of the (3)P700/P700 FTIR difference spectrum at 90 K with spectra of triplet formation in isolated chlorophyll a or in RCs from photosystem II or purple bacteria identify the bands at 1733 and 1637 cm(-)(1), which downshift upon formation of (3)P700, as the 10a-ester and 9-keto C=O modes, respectively, of the half of P700 that bears the triplet (P(1)). Thus, while the P(2) carbonyls are free from interaction, both the 10a-ester and the 9-keto C=O of P(1) are hydrogen bonded and the latter group is drastically perturbed compared to chlorophyll a in solution. The Mg atoms of P(1) and P(2) appear to be five-coordinated. No localization of the triplet on the P(2) half of P700 is observed in the temperature range of 90-200 K. Upon P700 photooxidation, the 9-keto C=O bands of P(1) and P(2) upshift by almost the same amount, giving rise to the 1656(+)/1637(-) and 1717(+)/1697(-) cm(-)(1) differential signals, respectively. The relative amplitudes of these differential signals, as well as of those of the 10a-ester C=O modes, appear to be slightly dependent on sample orientation and temperature and on the organism used to generate the P700(+)/P700 spectrum. If it is assumed that the charge density on ring V of chlorophyll a, as measured by the perturbation of the 10a-ester or 9-keto C=O IR vibrations, mainly reflects the spin density on the two halves of the oxidized P700 special pair, a charge distribution ranging from 1:1 to 2:1 (in favor of P(2)) is deduced from the measurements presented here. The extreme downshift of the 9-keto C=O group of P(1), indicative of an unusually strong hydrogen bond, is discussed in relation with the models previously proposed for the PSI special pair.     

SDHB蛋白的表达、纯化及多克隆抗体制备

SDHB(succinate dehydrogenage complex,subunit B)基因可能介导呼吸链生物功能和调控细胞生长.采用PCR技术扩增出SDHB基因,并将其连接到pGEX-4T-1原核表达载体中,经酶切及测序鉴定后,转化BL21细菌,并用IPTG诱导表达融合蛋白,谷胱甘肽琼脂糖珠亲和纯化,将纯化的融合蛋白免疫新西兰大白兔制备多克隆抗体,并用Western blot检测抗体.获得了SDHB原核表达重组融合蛋白及高效价的特异性兔抗SDHB多克隆抗体,为SDHB进一步的功能研究奠定了基础.     

gcm蛋白的表达、纯化及多克隆抗体的制备

gcm(glial cells missing)是调控神经元细胞和神经胶质细胞相互转化的一个基因开关.在gcm功能缺损的突变体中,预期的神经胶质细胞发育成神经元细胞;而在gcm过表达的突变体中,预期的神经元细胞转化为神经胶质细胞.此外,gcm还调控血浆细胞发育.为了进一步研究gcm在发育中的功能,需要获得gcm蛋白并制备其抗体.根据已报道的gcm基因序列,以果蝇cDNA文库为模板进行PCR扩增得到gcm部分编码区序列,然后将其连接到pET-28a载体以获得原核表达载体.重组载体经酶切测序鉴定确认后,转化大肠杆菌(E.coli)BL21,并用IPTG诱导融合蛋白表达.采用Ni-IDA凝胶柱亲和纯化蛋白,将纯化的His-gcm融合蛋白免疫新西兰大白兔制备多克隆抗体,并用Western Blot检测抗体效价.获得的gcm原核表达重组融合蛋白及高效价的特异性兔抗gcm多克隆抗体,为gcm功能的进一步研究奠定了基础.     

Nodal蛋白的表达、纯化及多克隆抗体制备

斑马鱼心脏发育模型中Nodal编码转录因子调节心脏的左右不对称发育,为了进一步研究Nodal信号途径在心脏发育中的调控作用和心脏疾病发生的分子机制,需要获得斑马鱼Nodal蛋白并制备其抗体.采用从斑马鱼心脏组织中提取RNA,通过反转录得到心脏组织各种表达基因的cDNA为模板,PCR扩增得到Nodal部分编码区序列,然后将其连接到pET-28a载体上获得原核表达.经酶切及测序鉴定后,转化Rosseta细菌,并用IPTG诱导表达融合蛋白,Ni-IDA凝胶柱亲和纯化,将纯化的融合蛋白免疫新西兰大白兔制备多克隆抗体,并用Western blotting检测抗体.获得了Nodal原核表达重组融合蛋白及高效价的特异性兔抗Nodal多克隆抗体,为Nodal功能的进一步研究奠定了基础.     

人Ndfip1和Nedd4的原核表达,纯化和抗体制备

为了进一步从蛋白水平上检测Ndfip1(Nedd4 family interacting protein 1)和Nedd4(Neuronal precursor cell-expressed developmentally down-resulated 4)在肢带型肌营养不良(Limb-girdle muscular dystrophy,LGMD)患者的肌肉组织中的表达及功能,以正常人的脑和肾脏组织的总RNA为模板,RT-PCR扩增Ndfip1和Nedd4部分编码序列,构建原核表达重组质粒pET28b-hNdfip1和pET28b-hNedd4,转化大肠杆菌BL21后,加IPTG诱导蛋白表达,Ni-NTA层析柱纯化蛋白,免疫BALB/c小鼠制备抗体.Western blot检测显示抗体能识别人和小鼠的Ndfip1和Nedd4,小鼠各组织中Ndfip1的蛋白表达谱与文献报道的Ndrip1 mRNA表达谱不一致,暗示Ndfip1具有转录和翻译水平的调控.     

Presence of the Photoactive Reaction-Center Chlorophyll of PSI (P700) in Dark-Grown Cells of a Chlorophyll-Deficient Mutant of Chlorella kessleri

Compositions of pigments and polypeptides of pale green membranesthat had been isolated from dark-grown cells of a chlorophyll-deficientmutant of Chlorella kessleri were investigated. They containedChl a in a level corresponding to about 1% of that present inthe thylakoid membranes isolated from autotrophically grownwild-type cells and a trace amount of chlorophyllide a, butneither Chl b nor carotenoids. The polypeptide profile of themutant membranes was similar to that of membranes isolated fromwild-type cells that were grown in the dark. Neither the chlorophyll-bindingsubunits of PSI nor the apoproteins of LHCP were detected bySDS-PAGE and immunoblot analysis. However, the light-minus-darkdifference spectrum of the mutant membranes revealed the presenceof the reaction-center chlorophyll of PSI (P700) at a molarratio of 190 chlorophyll (Chl a plus Chlide a) per P700. P700was more stable than Chl a and Chlide a in the light so thatprolonged illumination led to a decline in the Chl/P700 ratioto 24. The initial rate of P700 photooxidation in the mutantmembranes was comparable to that in CP1 isolated from the dark-grownwild-type cells. Under illumination with strong light, the initialrate was decreased in parallel to the decrease in Chl/P700 ratio.The results suggest that most of Chi present in the mutant membranescan transfer excitation energy to P700. (Received March 13, 1998; Accepted August 7, 1998)