降钙素基因相关肽家族受体及其受体活性修饰蛋白

降钙素基因相关肽家族是一类多功能的激素家族 ,参与人体的多种生物学功能 ,与多种疾病有关。降钙素基因相关肽受体包括降钙素受体 (CTR)和降钙素受体样受体 (CRLR) ,CTR可以独自与降钙素结合 ,而CRLR必须与一组称作受体活性修饰蛋白 (RAMPs)的蛋白质共同作用才能发挥生物学功能。综述CTR的研究概况及CRLR与RAMPs相互作用的机制和表达调控 ,以期为人们设计新型药物提供参考。     

金黄色葡萄球菌B型肠毒素受体拮抗剂的原核表达、纯化及活性初步分析

目的:利用大肠杆菌表达葡萄球菌肠毒素B(SEB)的受体拮抗剂蛋白,检测其与SEB的结合能力,为今后利用其进行SEB快速检测奠定基础.方法:首先确定SEB受体拮抗荆的基因序列,然后将SEB受体拮抗剂质粒转化大肠杆菌BL21(DE3),利用IPTG诱导表达获得蛋白,产物经镍柱纯化后,ELISA鉴定其与SEB结合能力.结果:该质粒在大肠杆菌中获得表达,表达产物可与SEB特异性结合,两者结合能力可达ng/mL.结论:本研究成功对SEB受体拮抗剂进行了原核表达、纯化及初步活性分析.     

牙鲆甲状腺激素受体TRαA的原核表达和多克隆抗体的制备及应用

为研究牙鲆甲状腺激素受体TRαA在牙鲆变态发育过程中的调控作用,将TRαA基因克隆插入融合表达栽体pET30a,并在大肠杆菌Escherichia coli DE3(BL21)中进行诱导表达.表达菌株经1 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导4 h后,重组蛋白TRaA表达并形成包涵体.SDS-PAGE和Western blotting检测鉴定表达产物.包涵体经变性后在His-Bind树脂进行亲和层析纯化,柱上复性法对重组蛋白复性,获得纯度较高的目的蛋白,蛋白复性的效果良好.用纯化后的目的蛋白免疫新西兰家兔制备多克隆抗体.Dot blotting检测抗体效价达1:200 000,检测证明抗体特异性良好.此外,通过染色质免疫沉淀技术鉴定了在活体细胞中多克隆抗体与TRαA的特异性结合.表明了甲状腺激素通过其受体在体内参与碱性磷酸酶(ALP)基因的转录调控.     

脑药物转运载体——抗转铁蛋白受体单链抗体的克隆表达及鉴定

为构建特异性的脑药物转运载体 ,分段合成了抗大鼠转铁蛋白受体的单链抗体基因 (Ox2 6 scfv) .经重叠PCR拼接成完整片段 ,克隆入pUC19载体中 ,测序正确后克隆到大肠杆菌表达载体pET 15b E .tag上 .IPTG诱导 ,表达产物分子量为 2 9kD ,约占菌体总蛋白量的 4 0 % .包涵体经 6mol L盐酸胍变性后 ,过SephacrylS 30 0HR分子筛柱复性蛋白 .免疫酶染色实验表明 ,该单链抗体能与转铁蛋白受体特异性结合 ,为建立以转铁蛋白受体为介导的血脑屏障转运载体打下了基础     

牙鲆甲状腺激素受体TRaA的原核表达和多克隆抗体的制备及应用

为研究牙鲆甲状腺激素受体TRαA在牙鲆变态发育过程中的调控作用,将TRαA基因克隆插入融合表达载体pET30a,并在大肠杆菌Escherichia coli DE3(BL21)中进行诱导表达。表达菌株经1mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导4h后,重组蛋白TRαA表达并形成包涵体。SDS-PAGE和Western blotting检测鉴定表达产物。包涵体经变性后在His-Bind树脂进行亲和层析纯化,柱上复性法对重组蛋白复性,获得纯度较高的目的蛋白,蛋白复性的效果良好。用纯化后的目的蛋白免疫新西兰家兔制备多克隆抗体。Dotblotting检测抗体效价达1:200000,检测证明抗体特异性良好。此外,通过染色质免疫沉淀技术鉴定了在活体细胞中多克隆抗体与TRαA的特异性结合,表明了甲状腺激素通过其受体在体内参与碱性磷酸酶(ALP)基因的转录调控。     

rhM-CSFsR在大肠杆菌中的表达及其配基结合活性分析

从人胎盘中提取总RNA,利用RT-PCR技术扩增出巨噬细胞集落刺激因子受体(M-CSFR)胞外具有结合活性区域的cDNA,经平端连接将其克隆到原核表达载体pET-28a的His-Tag下游,转化大肠杆菌BL21(DE3)后,经IPTG诱导,重组的人可溶型M-CSFR(rhM-CSFsR)在宿主菌中获得高效表达,表达量约占菌体总蛋白的38%.重组蛋白经Ni2+组氨酸结合树脂螯合层析柱纯化,获得了纯化的rhM-CSFsR,经SDS-PAGE显示为单一区带,其表观分子量为34kD.用酶联免疫吸附分析(enzyme-linkedimmunosorbentassay,ELISA)证明rhM-CSFsR有明显的M-CSF专一结合活性,Kd值为7.8nmol/L,只有一个M-CSF结合位点.本实验结果显示原核表达的rhM-CSFsR具有明显的配基结合活性,提示M-CSFR的糖基化程度对于其配基结合活性不是必不可少的,为深入进行rhM-CSFsR的生物学功能及其临床意义的研究打下了良好的基础     

成纤维细胞生长因子受体3胞外配体结合区的重组表达及其与A型肉毒毒素重链C端的相互作用

目的:实现成纤维细胞生长因子受体3(FGFR3)胞外配体结合区FGFR3D23的表达与纯化,并鉴定其与A型肉毒毒素重链C端(Bo NT/AHc)的相互作用。方法:全基因合成的FGFR3D23基因片段经酶切连入p TIG(+)质粒,转化大肠杆菌BL21(DE3)后IPTG诱导表达,获得的包含体经Ni2+柱纯化,透析复性后通过pull-down及ELISA检测纯化蛋白与Bo NT/AHc的相互作用。结果与结论:SDS-PAGE分析结果显示得到相对分子质量约27×103的蛋白特异表达条带,表达量为1.39 mg/L,包含体占73.4%;纯化和复性得到的目的蛋白纯度约96%,复性率约3%;pull-down及ELI-SA实验结果证明FGFR3D23可以与Bo NT/AHc特异性地相互作用。     

肾炎致病原重组受体相关蛋白的表达及纯化

用pGEX载体系统体外构建了Heymann肾炎致病原受体相关蛋白(RAP)重组表达质粒,经IPTG诱导,该质粒表达的融合蛋白在大肠杆菌中得到了高效表达,其表达量达39．4％,经GST-Sephrose 4B亲和层析,得到了高度纯化,其诱导产生的抗体经蛋白质印迹法分析证明能识别肾皮质天然抗原44ku受体相关蛋白.RAP表达及纯化的成功为研究致病原病理性表型提供了有利条件．     

抗Her-2-scFv-SEC2融合免疫毒素的构建和功能研究

用基因工程方法,将金黄色葡萄球菌肠毒素 C2 与抗人表皮生长因子受体 HER-2 单链抗体 scFv-B1,以一连接短肽连接,构建融合免疫毒素 B-L-SEC2,并用改进的新型表达载体 pASK75-EX,在大肠杆菌 BL21(ED3)中表达. 以不溶性包涵体形式表达的目的蛋白经变性后以镍离子螯和层析纯化,并以透析法进行复性. 流式细胞术和 MTT 实验结果表明,纯化复性的融合免疫毒素 B-L-SEC2,在体外具有与 HER-2 过表达的靶细胞 SK-Br-3 特异性结合的活性,并对该细胞产生显著的特异性生长抑制作用.     

重组人可溶性BAFF蛋白的原核表达、纯化及鉴定

目的:在大肠杆菌中表达人B细胞活化因子可溶性胞外域134~285(rhsBAFF134-285)蛋白,并进行纯化与鉴定。方法:重组表达质粒pET-32ammrhsBAFF在大肠杆菌Rosetta-gami B(DE3)中于16℃下进行IPTG诱导表达。经His亲和层析纯化得到融合蛋白后进行TEV蛋白酶酶切切除Trx融合标签,进而纯化得到rhsBAFF目的蛋白,SDS-PAGE、Western Blot和ELISA鉴定纯化产物。结果:Trx-rhsBAFF融合蛋白相对分子量约31000,16℃表达时主要以包涵体形式表达,上清中也有部分表达。融合蛋白纯化后经酶切再纯化得到相对分子量约17000、纯度95%以上的rhsBAFF目的蛋白,鉴定可被小鼠抗rhBAFF单克隆抗体和小鼠抗rhBAFF多抗血清特异性识别。结论:成功原核表达并纯化得到rhsBAFF蛋白,为进一步开发用于研究人类自身免疫性疾病的BAFF检测试剂盒奠定基础。     

Role of the N-terminal domain of the calcitonin receptor-like receptor in ligand binding

Calcitonin receptor-like receptor (CRLR) is a seven-transmembrane (7-TM) domain class B G protein-coupled receptor (GPCR) which requires coexpression of different receptor activity modifying proteins (RAMP) to become a functional calcitonin gene-related peptide (CGRP) receptor or an adrenomedullin (AM) receptor. The N-terminal (Nt) extracellular region of class B GPCRs in ligand binding has been reported for receptors such as glucagon and parathyroid hormone. We hypothesize that the Nt-domain of CRLR (Nt-CRLR) is an autonomously folded unit possessing a well-defined structure and is involved in ligand binding and specificity. To obtain structural and functional information on the Nt-CRLR, we cloned and expressed the Nt-CRLR as a fusion protein in Escherichia coli. Overexpressed protein formed an inclusion body, which was refolded and purified, resulting in a soluble monomeric protein. Far-UV CD and fluorescence spectra of Nt-CRLR showed characteristics of a folded protein. The ability of Nt-CRLR to bind CGRP and AM independent of RAMPs was determined by studying inhibition of (125)I-CGRP and (125)I-AM binding to pregnant rat uterine membrane in the presence of Nt-CRLR protein. We observe that Nt-CRLR inhibits (125)I-CGRP and (125)I-AM binding to rat uterus in a dose-dependent fashion (IC(50) = 0.25 and 0.29 muM, respectively). Taken together, our data provide evidence that Nt-CRLR is structured and further that a significant part of the binding affinity comes from binding to the Nt-domain.     

Characterization of the parathyrin receptor in renal plasma membranes by labelled hormone and labelled antibody binding techniques.

The parathyrin receptor in renal cortex has been investigated by studying the binding of 125I-labelled parathyrin, or of unlabelled parathyrin detected with 125I-labelled antibodies, to a partially purified plasma membrane fraction. The kinetics of hormone uptake demonstrated a biphasic response in both systems at 22 degrees C but this phenomenon was not detectable at 37 degrees C. Specific displacement of lactoperoxidase labelled 125I-labelled parathyrin occurred with 8 ng unlabelled bovine parathyrin. The apparent affinity constant was 2.3-10(8) M(-1) and the apparent binding capacity of the membranes 1.25 pmol/mg protein. Using the labelled antibody technique the receptor showed maximal binding at pH 7.0-7.5. As little as 80 pg bovine parathyrin produced a significant increase in binding of labelled anti-bovine parathyrin antibody and saturation of binding sites was demonstrated at 2.5 pmol/mg protein. Oxidized hormone showed undetectable binding. Treatment of membranes with phospholipases A or D, or Trypsin greatly reduced subsequent hormone binding. Prior incubation of membranes with 1-34 synthetic parathyrin decreased the binding of intact hormone whereas gastrin, insulin and glucagon had no effect. Growth hormone and calcitonin slightly increased parathyrin binding.     

一种新型融合蛋白(RGD)3/tTF的基因表达与活性分析

为了发展一种新型的融合蛋白(RGD)3/tTF用于肿瘤血管的选择性栓塞治疗,利用PCR技术重组(RGD)3/tTF融合基因,克隆于pET22b( )载体,表达于E.coliBL21(DE3)。用镍柱纯化融合蛋白。凝血实验与FⅩ活化实验检测融合蛋白tTF组分的活性。间接ELISA分析(RGD)3/tTF与αvβ3的特异结合能力。pET22b( )/(RGD)3/tTF重组质粒成功获得并表达于E.coliBL21(DE3)。纯化蛋白(RGD)3/tTF能有效诱发血液凝固,活化FⅩ。(RGD)3/tTF与αvβ3的特异结合能力比RGD/tTF提高了32%。新型融合蛋白(RGD)3/tTF已在E.coli系统成功表达,表达蛋白保持tTF的活性并显示比RGD/tTF更高的与αvβ3的结合能力。     

Expression and purification of the hormone binding domain of the Drosophila ecdysone and ultraspiracle receptors

Escherichia coli vectors were constructed for the production of a protein complex that mimics the native ecdysone receptor (EcR) isolated from Drosophila. The two steroid receptors, ultraspiracle (USP) and EcR, were expressed as truncations, retaining primarily the hormone binding domains. The recombinant receptor complex was able to mimic the pharmacology of the native receptor with respect to both synthetic and natural agonists. USP and EcR fusion proteins could be expressed in separate cell lines and then recombined following isolation to yield a ligand binding preparation with a dissociation constant (K(D)) for Ponasterone A of 1.5 nM and a total yield of 1.9 pmol ligand binding sites/mg protein. Alternatively, the simultaneous coexpression of both receptors increased yields by several orders of magnitude to 6 nmol ligand binding sites/mg protein with a K(D) of 0.6 nM. Chromatographic analysis under native conditions showed that EcR, when expressed alone, migrated as a variety of complexes, mostly coming out in the void volume as denatured, insoluble, aggregate. In contrast, purified extracts of coexpressed EcR and USP eluted as a single peak with a mobility indicating a heterodimer. The majority of the coexpressed fusion receptors, following purification, formed functional steroid binding sites. A detailed scheme is provided for the expression and isolation of milligram quantities of highly purified receptor dimer.     

Agonist-promoted internalization of a ternary complex between calcitonin receptor-like receptor, receptor activity-modifying protein 1 (RAMP1), and beta-arrestin

The calcitonin receptor-like receptor (CRLR) is a seven-transmembrane domain (7TM) protein that requires the receptor activity-modifying protein 1 (RAMP1) to be expressed at the cell surface as a functional calcitonin gene-related peptide (CGRP) receptor. Although dimerization between the two molecules is well established, very little is known concerning the trafficking of this heterodimer upon receptor activation. Also, the subcellular localization and biochemical state of this ubiquitously expressed protein, in the absence of CRLR, remains poorly characterized. Here we report that when expressed alone RAMP1 is retained inside the cells where it is found in the endoplasmic reticulum and the Golgi predominantly as a disulfide-linked homodimer. In contrast, when expressed with CRLR, it is targeted to the cell surface as a 1:1 heterodimer with the 7TM protein. Although heterodimer formation does not involve intermolecular disulfide bonds, RAMP-CRLR association promotes the formation of intramolecular disulfide bonds within RAMP1. CGRP binding and receptor activation lead to the phosphorylation of CRLR and the internalization of the receptor as a stable complex. The internalization was found to be both dynamin- and beta-arrestin-dependent, indicating that the formation of a ternary complex between CRLR, RAMP1, and beta-arrestin leads to clathrin-coated pit-mediated endocytosis. These results therefore indicate that although atypical by its heterodimeric composition and its targeting to the plasma membrane, the CGRP receptor shares endocytotic mechanisms that are common to most classical 7TM receptors.     

The effects of C-terminal truncation of receptor activity modifying proteins on the induction of amylin receptor phenotype from human CTb receptors

Receptor activity modifying proteins (RAMPs) interact with calcitonin receptors to produce novel amylin receptor phenotypes. We have recently demonstrated that the short intracellular C-terminus of RAMPs plays a key role in the function of amylin receptors derived from the CTa calcitonin receptor through the use of chimeric RAMPs and RAMPs that are truncated at the C-terminus [15, Udawela M, Christopoulos G, Morfis M, Christopoulos A, Ye S, Tilakaratne N, Sexton PM. A critical role for the short intracellular C terminus in receptor activity modifying protein function. Mol Pharmacol 2006;70:1750-60., 18, Udawela M, Christopoulos G, Tilakaratne N, Christopoulos A, Albiston A, Sexton PM. Distinct receptor activity-modifying protein domains differentially modulate interaction with calcitonin receptors. Mol Pharmacol 2006;69:1984-89.]. The calcitonin receptor in humans is expressed as two major alternatively spliced isoforms termed CTa and CTb. Relatively little is known about how alternate splicing of the receptor affects the interaction between calcitonin receptors and RAMPs. We have examined the effect of RAMP truncation, through use of mutant constructs that delete the last 8 amino acids of each of the 3 known human RAMPs, and characterised these for interaction with CTb receptors through co-expression in COS-7 cells. As seen with the CTa receptor isoform, RAMP truncation caused a marked loss in induction of AMYb receptor phenotypes as characterised by (125)I-rat amylin radioligand binding assays and cAMP accumulation assays; the latter as a marker of receptor signalling. The effect was most pronounced for RAMP1 and RAMP2 deletion mutants, but attenuated responses were also observed with co-expressed RAMP3 deletion mutants. These data support a direct role for the RAMP C-terminus in the interaction of RAMP/calcitonin receptor complexes with intracellular accessory proteins involved in signalling and/or receptor trafficking.     

Adrenomedullin peptide: gene expression of adrenomedullin, its receptors and receptor activity modifying proteins, and receptor binding in rat testis--actions on testosterone secretion

Adrenomedullin (ADM) has been shown to be present in the human and rat male reproductive systems. This study demonstrates the expression of ADM in the rat testis and its effect on the secretion of testosterone. Whole testicular extracts had 5.43 +/- 0.42 fmol of immunoreactive ADM per milligram of protein and 84 +/- 8 fg of ADM mRNA per picogram of Actb (beta-actin) mRNA. Immunocytochemical studies showed positive ADM immunostaining in the Leydig cells and in the Sertoli cells. Gel filtration chromatography of testicular extracts showed two peaks, with the predominant one eluting at the position of the ADM precursor. Furthermore, the testis was shown to coexpress mRNAs encoding the calcitonin receptor-like receptor and receptor activity modifying protein 1 (Ramp1), Ramp2, and Ramp3. These account for the specific binding of ADM to the testis, which was partially inhibited by human ADM (22-52) and by human calcitonin gene-related peptide (8-37), the ADM and calcitonin gene-related peptide receptor antagonists, respectively. Administration of ADM to testicular blocks in vitro resulted in a dose-dependent inhibition of hCG-stimulated release of testosterone, which was abolished by the administration of ADM (22-52). Our results suggest a paracrine effect of ADM on testicular steroidogenesis.     

PDGF受体结合域与乙肝病毒核心抗原的融合表达

化学合成血小板源性生长因子受体结合域１３肽基因,并与乙肝病毒核心抗原基因５′端融合,序列分析表明化学合成的１３肽基因及融合后基因的阅读框架正确．将融合基因亚克隆于ｔａｃ启动子控制的ｐＥＴ３ａ表达质粒中并于大肠杆菌中表达．表达产物经ＥＬＩＳＡ、ＷｅｓｔｒｅｎＢｌｏｔ鉴定表明,融合蛋白已被表达,其单位分子量与推算值一致．电镜观察证明所表达的融合蛋白能形成颗粒．     

Adrenomedullin receptor is found exclusively in noradrenaline-secreting cells of the rat adrenal medulla

Adrenomedullin, originally identified in the adrenal medulla, has binding sites in the adrenal gland; however, its role in the adrenal medulla is unclear. This study was designed to characterise adrenomedullin binding sites in the rat adrenal medulla, using ligand binding studies, immunocytochemistry, and mRNA analysis. A single population of specific adrenomedullin receptors was identified in adrenal medullary homogenates. 125I-Adrenomedullin was displaced only by adrenomedullin1-50 and not by calcitonin gene-related peptide or amylin at concentrations up to 100 nmol/L. The receptor K(D) was 3.64 nmol/L with a receptor density of 570 fmol/mg of protein. Analysis of mRNA revealed that the genes encoding both the putative adrenomedullin receptors, termed calcitonin receptor-like receptor (CRLR) and L1, were expressed in the rat adrenal medulla. Dual-colour indirect-labelled immunofluorescence was used to localise phenylethanolamine N-methyltransferase (PNMT) and the adrenomedullin receptor in the same section. PNMT is the enzyme that converts noradrenaline to adrenaline and is not expressed in noradrenaline-secreting cells. These studies revealed that both CRLR and L1 were expressed only in cells that did not express PNMT, suggesting that adrenomedullin receptors are only found in noradrenaline-secreting cells. Further evidence to support this conclusion was provided by the demonstration of colocalisation of adrenomedullin receptors with dopamine beta-hydroxylase, confirming the presence of the receptors in medullary chromaffin cells. Taken together, these data suggest that adrenomedullin acts through a specific adrenomedullin receptor in the rat adrenal medulla. RT-PCR and northern blot analysis revealed greater abundance of mRNA for L1 than for CRLR, possibly suggesting that L1 may be the major adrenomedullin receptor expressed in this tissue. As it has been reported that adrenomedullin is synthesised predominantly by adrenaline-secreting cells, it appears likely that adrenomedullin is a paracrine regulator in the adrenal medulla.