蜂毒溶血肽基因的定点诱变及其在大肠杆菌中的表达

从蜜蜂毒腺中提取总ＲＮＡ,通过ＲＴ－ＰＣＲ方法扩增得到了蜂毒溶血肽前体蛋白的ｃＤＮＡ,再进一步通过定点诱变在蜂毒溶血肽序列前引入了羟胺裂解位点,构建了与β－半乳糖苷酶部分序列相融合的蜂毒溶血肽诱变蛋白表达载体,序列分析结果表明,成功地引入了目的密码子且与β－半乳糖苷酶部分序列构成正确的读码框,并在大肠杆菌中表达了诱变蛋白,为基因工程生产蜂毒溶血肽提供了新途径。     

4种胡蜂前溶血肽原基因的克隆与序列比较

从雌性亚非马蜂、额斑黄胡蜂、墨胸胡蜂、大胡蜂毒腺中抽提总RNA,通过RT-PCR方法扩增得到4种胡蜂蜂毒前溶血肽原的cDNA,再将扩增产物克隆到pGEM⑥-Teasy vector上。测序结果表明：扩增得到的片段长度均为213bp,系蜂毒前溶血肽原编码区的cDNA。经序列比较,4种胡蜂蜂毒前溶血肽原之间的氨基酸序列同源性都超过95％。亚非马蜂、额斑黄胡蜂、墨胸胡蜂、大胡蜂各自与意大利蜜蜂蜂毒前溶血肽原氨基酸序列的同源性分别为95．8％、100％、97．2％、97．2％。结果表明：蜂毒前溶血肽原一级结构序列具有很高的保守性,尽管胡蜂和蜜蜂属于膜翅目不同的总科,但它们的前溶血肽原基因却非常相似。     

蜂毒溶血肽作用机理研究进展

该文系统归纳了蜂毒溶血肽的生物学功能,分子结构与性质,溶解血细胞的机理,以及溶血和抑菌活性与结构的关系, 并结合本实验室研究着重综述了近年来国内外对蜂毒溶血肽作用机理的研究。 提出今后研究应该向定量化方向发展,应用计算技术深入阐明蜂毒肽作用的动力学过程及结构与功能的关系,为今后指导分子设计打下一定的理论基础。     

蜜蜂蜂毒主要成分与功能研究进展

蜜蜂蜂毒(honeybee venom)作为重要的蜂产品之一,其中的很多蛋白在抗炎、抗癌、抗菌、抗辐射和杀虫等方面具有很好的效果.20世纪40年代以来,国内外在蜂毒活性成分分析、作用机理、重要基因克隆和毒蛋白功能等方面进行广泛地研究,取得了重要的进展.本文的目的是总结蜜蜂蜂毒主要成分磷脂酶A2、透明质酸酶、蜂毒肽、蜂毒明肽、肥大细胞脱粒肽和镇静肽等毒蛋白的基因结构、生化特性及功能等方面的研究进展,为蜂毒基因的研究和利用提供一定的理论基础.     

蜂毒溶血肽前体蛋白cDNA的克隆及其融合蛋白的表达

从蜜蜂毒腺中提取总RNA ,通过RT PCR扩增得到了蜂毒溶血肽前体蛋白的cDNA ,将扩增产物克隆到 pT7Blu T载体上 ,再进一步将插入片段酶切并连接到 pUC1 1 8载体上 ,构建了重组质粒pUMP。DNA序列分析结果表明 ,克隆得到的cDNA序列与所发表序列完全相同 ,且与 β 半乳糖苷酶部分序列构成正确的读码框。含重组质粒 pUMP的大肠杆菌DH5α表达了与β 半乳糖苷酶部分序列融合的蜂毒溶血肽前体蛋白     

中华蜜蜂和意大利蜜蜂蜂毒前溶血肽原蛋白cDNA的克隆与序列分析

分别从中华蜜蜂Apis cerana cerana和意大利蜜蜂Apis mellifera工蜂毒腺中抽提总RNA,通过RT-PCR方法扩增,各得到了蜂毒前溶血肽原蛋白的cDNA,再将扩增产物克隆到pGEM-Teasy载体上,进行测序和序列分析。结果表明,所扩增到的这两个片段长度均为213 bp,均为编码蜂毒前溶血肽原的cDNA,并分别推导出两者所编码的氨基酸序列。经序列比较,中华蜜蜂前溶血肽原与意大利蜜蜂、印度蜜蜂Apis cerana indica前溶血肽原的同源性都为97%。所报道的中华蜜蜂蜂毒前溶血肽原的核苷酸序列的GenBank登录号为AF487907。     

小麦肽免疫活性及抗氧化作用的研究

本文旨在研究Alcalase水解小麦蛋白产物中免疫活性组分对小鼠免疫功能和抗氧化功能的调节作用及相互关系.实验结果表明,小麦活性肽可提高小鼠抗体生成细胞含量、血清溶血素水平、脾细胞增殖能力以及腹腔巨噬细胞的吞噬能力;小麦活性肽还可提高小鼠血清清除DPPH和清除羟自由基的能力,增强小鼠肝脏超氧化物歧化酶活性、过氧化氢酶活力和总抗氧化能力,降低丙二醛含量.且该组分调节机体抗氧化的能力与其免疫调节作用具有一定的相关性.     

26肽蜂毒素与基因变构IL-2嵌合蛋白的原核表达、纯化及活性测定

在E .coliDH5α中表达26肽蜂毒素与基因变构人白细胞介素2的嵌合体蛋白 ,并进行初步纯化和抗原性检测。利用剪接式重叠延伸 (splicedoverlapextension ,SOE)技术在26肽蜂毒素与基因变构IL-2的基因之间导入四个易形成空间结构的氨基酸残基组成的连接肽 (Gly4Ser)₃,构建嵌合体蛋白的基因,克隆到原核表达载体pGEX4T-2中,重组质粒转化宿主菌DH5α,经IPTG诱导表达目的融合蛋白,纯化蛋白经Thrombin酶切后进行SDS-PAGE与Western-blot鉴定。结果表明重组蛋白有良好的抗原性 。     

MMP-2结合肽-蜂毒素杂合基因的表达纯化及活性测定

为开发抗癌导向多肽药物,用从噬菌体十二肽库中筛选的与MMP-2具有高度亲和性的十二肽与蜂毒素连接,采用基因工程方法在大肠杆菌中进行了高效表达;并分别通过亲和层析、肠激酶酶切、凝胶层析获得高纯度的多肽(相对分子质量45000),经体外抑瘤作用测定该融合蛋白具有明显抑制肿瘤细胞的生长。该研究对肿瘤的导向治疗和临床应用提供了一定的帮助。     

胶原蛋白肽增强小鼠免疫力的实验研究

目的 探讨用生物酶提取的胶原蛋白肽增强小鼠免疫力的作用及相关机制.方法 80只清洁级昆明小鼠被随机分成正常对照组、胶原蛋白肽低剂量组[3 g/(kg·bw)]、中剂量组[7.5 g/(kg·bw)]和高剂量组[12 g/(kg·bw)],每日给药2次,连续2周.检测免疫器官重量、抗体生成细胞数(Jerne改良玻片法)、半数溶血值(HC50)的测定及小鼠腹腔巨噬细胞吞噬鸡红细胞实验.结果 高剂量组可提高脾脏指数、胸腺指数、半数溶血值及吞噬细胞吞噬率和吞噬指数、可增加抗体生成细胞数;中剂量组可提高胸腺指数、增加抗体生成细胞数、提高小鼠半数溶血值及吞噬细胞吞噬率和吞噬指数;低剂量组可增加抗体生成细胞数、提高吞噬细胞的吞噬率和吞噬指数.结论 三种剂量胶原蛋白肽对正常小鼠的细胞免疫、体液免疫均有显著的增强作用,高剂量组效果最强.     

Bee venom melittin is a potent toxin for reducing the threshold for calcium-induced calcium release in human and equine skeletal muscle.

The modulation of Ca2+ release by synthetic bee venom melittin was examined in equine and human terminal cisternae-containing fractions. Melittin (0.1 microM) decreased the threshold of Ca(2+)-induced Ca2+ release by 20% in equine muscle and by 36% in human muscle. If terminal cisternae fractions were first preloaded with Ca2+ to greater than about 75% of the threshold of Ca(2+)-induced Ca2+ release and then melittin added, an immediate and sustained release of Ca2+ occurred in preparations from both species. Addition of melittin after a Ca2+ preload of < 50% of the threshold of Ca(2+)-induced Ca2+ release did not elicit sustained Ca2+ release. Ruthenium red (10 microM) antagonized all effects of melittin on Ca2+ release. Melittin (0.1-10 microM) did not affect [3H]ryanodine binding. Melittin (0.1 microM) slightly (10%) inhibited the Ca2+ pump and this action was not antagonized by ruthenium red. These findings suggest that melittin may be an important new probe of the Ca(2+)-modulated Ca2+ release process that does not act at the ryanodine binding site.     

Conformational and thermodynamic properties of peptide binding to the human S100P protein

S100P is a member of the S100 subfamily of calcium-binding proteins that are believed to be associated with various diseases, and in particular deregulation of S100P expression has been documented for prostate and breast cancer. Previously, we characterized the effects of metal binding on the conformational properties of S100P and proposed that S100P could function as a Ca2+ conformational switch. In this study we used fluorescence and CD spectroscopies and isothermal titration calorimetry to characterize the target-recognition properties of S100P using a model peptide, melittin. Based on these experimental data we show that S100P and melittin can interact in a Ca2+-dependent and -independent manner. Ca2+-independent binding occurs with low affinity (Kd approximately 0.2 mM), has a stoichiometry of four melittin molecules per S100P dimer and is presumably driven by favorable electrostatic interactions between the acidic protein and the basic peptide. In contrast, Ca2+-dependent binding of melittin to S100P occurs with high affinity (Kd approximately 5 microM) has a stoichiometry of two molecules of melittin per S100P dimer, appears to have positive cooperativity, and is driven by hydrophobic interactions. Furthermore, Ca2+-dependent S100P-melittin complex formation is accompanied by significant conformational changes: Melittin, otherwise unstructured in solution, adopts a helical conformation upon interaction with Ca2+-S100P. These results support a model for the Ca2+-dependent conformational switch in S100P for functional target recognition.     

Static and kinetic studies of calmodulin and melittin complex.

Ca2+ binding to calmodulin triggers conformational change of the protein which induces exposure of hydrophobic surfaces. Melittin has been believed to bind to Ca(2+)-bound calmodulin through the exposed hydrophobic surfaces. However, tryptophan fluorescence measurements and gel chromatography experiments with the melittin-calmodulin system revealed that melittin bound to calmodulin at zero salt concentration even in the absence of Ca2+; addition of salt removed melittin from Ca(2+)-free calmodulin. This means not only the hydrophobic interaction but also the electrostatic interaction contributes to the melittin-calmodulin binding. The fluorescence stopped-flow studies of the dissociation reaction of melittin-calmodulin complex revealed that Ca2+ removal from the complex induced a conformational change of calmodulin, resulting in reduction of the hydrophobic interaction between melittin and calmodulin, but the electrostatic interaction kept melittin still bound to calmodulin for a subsecond lag period, after which melittin dissociated from calmodulin. The fluorescence stopped-flow experiments on the dissociation reaction of complex of melittin and tryptic fragment(s) of calmodulin revealed that the lag period of the melittin dissociation reaction was attributable to the interaction between the C-terminal half of calmodulin and the C-terminal region of melittin.     

Phospholipase A2-independent Ca2+ entry and subsequent apoptosis induced by melittin in human MG63 osteosarcoma cells

Melittin, a peptide from bee venom, is thought to be a phospholipase A(2) activator and Ca(2+) influx inducer that can evoke cell death in different cell types. However, the effect of melittin on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and viability has not been explored in human osteoblast-like cells. This study examined whether melittin altered [Ca(2+)](i) and killed cells in MG63 human osteosarcoma cells. [Ca(2+)](i) changes and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Melittin at concentrations above 0.075 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was abolished by removing extracellular Ca(2+). Melittin-induced Ca(2+) entry was confirmed by Mn(2+) quenching of fura-2 fluorescence at 360 nm excitation wavelength which was Ca(2+)-insensitive. The melittin-induced Ca(2+) influx was unchanged by modulation of protein kinase-C activity with phorbol 12-myristate 13-acetate (PMA) and GF 109203X, or inhibition of phospholipase A(2) with AACOCF(3) and aristolochic acid; but was substantially inhibited by blocking L-type Ca(2+) channels. At concentrations of 0.5 microM and 1 microM, melittin killed 33% and 45% of cells, respectively, via inducing apoptosis. Lower concentrations of melittin failed to kill cells. The cytotoxic effect of 1 microM melittin was completely reversed by pre-chelating cytosolic Ca(2+) with BAPTA. Taken together, these data showed that in MG63 cells, melittin induced a [Ca(2+)](i) increase by causing Ca(2+) entry through L-type Ca(2+) channels in a manner independent of protein kinase-C and phospholipase A(2) activity; and this [Ca(2+)](i) increase subsequently caused apoptosis.     

Divalent cation-sensitive pores formed by natural and synthetic melittin and by Triton X-100

Leakage of ions and low-molecular-weight metabolites from Lettre cells is induced by synthetic melittin, as effectively as by melittin isolated from bee venom; in each case leakage is inhibited by Ca2+, Zn2+ or H+. Inhibition of leakage by divalent cations is reversible in that Lettre cells incubated with melittin (or with Triton X-100) in the presence of inhibitory amounts of Zn2+, when freed of Zn2+ by EGTA or by centrifugation, begin to leak (in Zn2(+)-sensitive manner). Electrorotation of Lettre cells is altered by melittin, compatible with membrane permeabilization; melittin plus Zn2+ does not alter electrorotation until Zn2+ (and unbound melittin) are removed. Melittin or Triton X-100 added to calcein-loaded liposomes induces leakage of calcein; divalent cations inhibit. Energy transfer between liposome-associated melittin and 2-, 7- or 12-(9-anthroyloxy)stearate (AS) is maximal with 12-AS; addition of Zn2+ has little effect. Circular dichroism spectra of melittin plus liposomes are unaffected by Zn2+. These results show that the formation of divalent cation-sensitive pores is not dependent on the presence of endogenous membrane proteins and that the action of divalent cations is not by displacement of melittin (or Triton) from the lipid bilayer.     

Melittin inhibition of the gastric (H+ + K+) ATPase and photoaffinity labeling with [125I]azidosalicylyl melittin

Melittin is a 26-amino acid amphipathic polypeptide toxin from bee venom which forms anion-selective ion channels in bilayers and biological membranes under the influence of membrane potential. Melittin has been shown to interact with a number of membrane proteins. We found that melittin inhibited K+-stimulated ATP hydrolysis by the (H+ + K+) ATPase in parietal cell apical membrane vesicles derived from histamine-stimulated rabbit gastric mucosa with a KIapp of 0.5 micron. Melittin also inhibited K+-stimulated p-nitrophenyl hydrolysis activity which is associated with the gastric (H+ + K+) ATPase in a dose-dependent manner with a KIapp of 0.95 micron. ATP-driven, K+-dependent H+ transport was inhibited over this same concentration range, even in the absence of a membrane potential. Melittin did not appear to increase the H+ leak from vesicle with preformed H+ gradients when the H+ pump was arrested by Mg2+ chelation, but all possible membrane perturbation effects were difficult to rule out. However, the data suggest that melittin exerts its inhibitory effect through interaction with the (H+ + K+) ATPase. In order to determine whether direct interactions between the (H+ + K+) ATPase and melittin occurred, a radioactive derivative of melittin, [125I]azidosalicylyl melittin, was prepared and photoreacted with sealed rabbit gastric membranes and highly purified hog gastric membrane containing the (H+ + K+) ATPase. In the purified hog preparation only a 95,000-Da band, the (H+ + K+) ATPase was labeled, while in the rabbit preparation a 95,000-Da band and one other membrane protein of 70,000 Da were labeled with this reagent. Label incorporation into the (H+ + K+) ATPase and the 70,000-Da band was greatly reduced by addition of excess unlabeled melittin, suggesting specificity of the interaction. Label incorporation occurred in the absence of ATP or added salts and was not reduced by SCH28080 (a K+ site inhibitor) suggesting that the melittin binding site was distinct from the luminal K+ site of action of SCH28080.     

Melittin exerts multiple effects on the release of free fatty acids from L1210 cells: lack of selective activation of phospholipase A2 by melittin

Melittin is known as a phospholipase A2 (PLA2) activator, but the selectivity of its effect on PLA2 is uncertain. We examined the selectivity of melittin effect on the release of free fatty acids (FFAs) from L1210 cells using various inhibitors. A systemic lipid analysis by HPLC and GLC revealed that melittin induced release of various FFAs including saturated, monounsaturated, and polyunsaturated FFAs. Various PLA2 inhibitors examined exerted only minimal effects on the melittin-induced arachidonic acid (AA) and palmitic acid (PAL) releases. Specific inhibitors of phosphatidylinositol-phospholipase C (U73122) and diacylglycerol lipase (RHC80267) exerted significant inhibitory effects on both AA and PAL releases. These results suggest that melittin-induced FFA release is most likely due to multiple participations of various types of lipases. Since BAPTA/AM, an intracellular Ca2+ chelator, did not influence the FFA release, the Ca2+ influxed by melittin appeared not to be a key factor for the FFA release. The mimicking of the melittin-induced FFA release by digitonin, a membrane-permeabilizing agent, implies that the membrane-perturbing action of melittin is likely the cause of the FFA release. Melittin also induced release of multiple FFAs from other cell lines including P388D1 and HL60. The rapid melittin-stimulated phospholipase D (PLD) observed in L1210 cells appeared not directly related to the steady release of FFA, as indicated by the fact that the PLD was not blocked by RHC80267. In view of melittin's multiple effects on the composition of cellular lipids, we conclude that melittin does neither exclusively release any single FFA nor selectively activate PLA2 in L1210 cells. The problem of using melittin as a PLA2 activator is discussed.     

Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles

Melittin, a cationic, amphiphilic polypeptide, has been reported to inhibit the ATPase activity of the catalytic portions of the mitochondrial (MF1) and chloroplast (CF1) ATP synthases. Gledhill and Walker [J.R. Gledhill, J.E. Walker. Inhibition sites in F1-ATPase from bovine heart mitochondria, Biochem. J. 386 (2005) 591-598.] suggested that melittin bound to the same site on MF1 as IF1, the endogenous inhibitor polypeptide. We have studied the inhibition of the ATPase activity of CF1 and of F1 from Escherichia coli (ECF1) by melittin and the cationic detergent, cetyltrimethylammonium bromide (CTAB). The Ca2+- and Mg2+-ATPase activities of CF1 deficient in its inhibitory epsilon subunit (CF1-epsilon) are sensitive to inhibition by melittin and by CTAB. The inhibition of Ca2+-ATPase activity by CTAB is irreversible. The Ca2+-ATPase activity of F1 from E. coli (ECF1) is inhibited by melittin and the detergent, but Mg2+-ATPase activity is much less sensitive to both reagents. The addition of CTAB or melittin to a solution of CF1-epsilon or ECF1 caused a large increase in the fluorescence of the hydrophobic probe, N-phenyl-1-naphthylamine, indicating that the detergent and melittin cause at least partial dissociation of the enzymes. ATP partially protects CF1-epsilon from inhibition by CTAB. We also show that ATP can cause the aggregation of melittin. This result complicates the interpretation of experiments in which ATP is shown to protect enzyme activity from inhibition by melittin. It is concluded that melittin and CTAB cause at least partial dissociation of the alpha/beta heterohexamer.     

Interaction of melittin with ion channels of excitable membranes

The effect of the neurotoxin melittin on the activation of ion channels of excitable membrane, the plasmalemma of Characeae algae cells, isolated membrane patches of neurons of mollusc L. stagnalis and Vero cells was studied by the method of intracellular perfusion and the patch-clamp technique in inside-out configuration. It was shown that melittin disturbs the conductivity of plasmalemma and modifieds Ca(2+)-channels of plant membrane. The leakage current that appears by the action of melittin can be restored by substituting calmodulin for melittin. Melittin modifies K(+)-channels of animal cell membrane by disrupting the phospholipid matrix and forms conductive structures in the membrane by interacting with channel proteins, which is evidenced by the appearance of additional ion channels.     

Protection of cells against membrane damage by haemolytic agents: divalent cations and protons act at the extracellular side of the plasma membrane

The protective effect of Ca2+, Zn2+ and H+ against membrane damage induced by different haemolytic agents has been studied by measuring monovalent cation leakage and haemolysis of erythrocytes, and phosphoryl[3H]choline and adenine nucleotide leakage from Lettre cells prelabelled with [3H]choline. The protective effect of Ca2+ and Zn2+ on erythrocytes damaged by Staphylococcus aureus alpha-toxin, Sendai virus or melittin is unaffected by the addition of A23187, even though this ionophore greatly increases the uptake of 45Ca2+ or 65Zn2+. The same result has been found for the protective effect of Zn2+ on Lettre cells damaged by S. aureus alpha-toxin, Sendai virus, melittin or Triton X-100. Leakage of phosphoryl[3H]choline from prelabelled Lettre cells is inhibited if extracellular pH is lowered; lowering the intracellular pH without affecting the extracellular pH, affords little protection. It is concluded that Ca2+, Zn2+ and H+ protect cells against membrane damage induced by haemolytic agents by an action at the extracellular side of the plasma membrane.