大鼠Shh-N融合蛋白的表达、纯化及功能研究

为了获得重组Sonic hedgehog N端蛋白（Shh-N）并研究其功能,应用PCR技术扩增Shh-N cDNA,然后克隆至原核表达质粒中,转化E.coli后获得表达菌株, 经1 mmol/L IPTG诱导高效表达出带有His-tag的融合蛋白,其中大部分为可溶性蛋白,少量为包涵体.用His-tag特异性结合树脂纯化可溶性融合蛋白,经SDS-聚丙烯酰胺凝胶电泳鉴定为单一区带,凝胶自动扫描分析表明,Shh-N的纯度达85%以上.纯化后的Shh-N在成纤维生长因子8(FGF₈)的协同作用下,能诱导神经前体细胞（NPC）向酪氨酸羟化酶阳性神经元（TH⁺）发育.在此基础上可以诱导不同类型的人类干细胞定向分化为多巴胺(DA)神经元,从而为临床治疗帕金森病（PD）提供充足的供体细胞.     

β-半乳糖苷酶和阿拉伯糖异构酶共表达一步法催化乳糖到塔格糖

[目的] 构建一株以廉价原料乳糖为底物合成塔格糖的重组菌株,实现一步法高效生物合成稀有糖——塔格糖。[方法] 从Escherichia coli K-12基因组中,PCR扩增出阿拉伯糖异构酶araA和β-半乳糖苷酶lacZ基因,以SD-AS为连接子,利用pET28a-1载体串联表达于Escherichia coli BL21（DE3）,获得重组菌E.coli BL21/pET28a-araA-lacZ,对重组菌全细胞催化合成塔格糖的条件进行了工艺优化与放大研究。[结果] araA和lacZ基因在E.coli BL21中同时高效表达,在最优条件（pH 8.0、温度50℃、5 mmol/L Mn²⁺、添加0.5 mol/L硼酸和0.1% SDS）下,E.coli BL21/pET28a-araA-lacZ全细胞转化100 g/L乳糖,合成塔格糖最高产量达24.03±2.03 g/L,乳糖到塔格糖的摩尔转化率为45.67%,随着底物乳糖浓度的提高,塔格糖产量呈不同程度的提高,当投加500 g/L底物乳糖时,全细胞合成塔格糖产量最高达83.81±1.38 g/L。[结论] 通过2个关键靶酶的编码基因araA和lacZ在E.coli BL21细胞中进行共表达,实现了以重组菌全细胞为催化剂转化廉价底物乳糖,一步法高效合成稀有糖塔格糖,该研究为生物法制备低能量的功能性稀有糖奠定了较好的研究基础。     

植物防御素PD5基因在毕赤酵母中的分泌表达*

将植物防御素PD5基因克隆到载体pPIC9的XholI和EcoRI位点之间,得到重组载体pPIC9/PD5。pPIC9/PDF5经BglII线性化,电转化法转入Pichia pastorisGS115,MD和MM平板筛选表型为His⁺Mut^s的转化子,PCR鉴定阳性转化子。转化子D24用BMGY培养至OD₆₀₀为6.0时,经BMM诱导,上清液用Tricien-SDS-PA     

睾酮假单胞菌3α-羟类固醇脱氢酶基因的质粒载体构建及表达

从土壤中分离睾酮假单胞菌,提取其基因组DNA,PCR扩增3α-羟类固醇脱氢酶（3α-hsd）基因,将扩增产物用NdeⅠ/BamHⅠ消化,切下目的基因片段克隆到质粒pET-15b中构建重组pET-15b.将重组pET-15b转化入E.coli DH5α中,经酶谱分析和测序,鉴定出正确的重组质粒pET-15b.将重组pET-15b转化入宿主菌E.coli BL21(DE3) pLysS中,用硫代半乳糖苷(IPTG)进行诱导表达.提取细菌总蛋白质进行SDS-聚丙烯酰胺凝胶电泳(PAGE)分析并测定酶活性.粗提物中酶活性高达2.45×10⁵ U/L.利用重组蛋白中的6个组氨酸(His)组成的“标签”进行亲和层析,经一步金属螯合亲和层析纯化后,重组蛋白在SDS-PAGE上呈现出均一的单一条带,回收率达68%.活性和纯度均较高的目的蛋白3α-HSD的获得,为血清总胆汁酸酶循环法测定奠定了基础.     

鸭源致病性大肠杆菌Ⅰ型菌毛pilA基因的原核表达及重组蛋白对强毒攻击的免疫保护作用

根据GenBank中人源大肠杆菌pilA基因序列,用OLIGO6.0设计PCR引物,从鸭源致病性大肠杆菌GH1.2中扩增到pilA基因并将其克隆至pMD18-T载体,经PCR、酶切和DNA测序鉴定后,将鸭源致病性大肠杆菌pilA基因正向插入原核表达载体pET-32a（+）的BamHⅠ和HindⅢ位点间,成功构建了重组表达质粒pET-32a-pilA。重组表达质粒pET-32a-pilA转化表达宿主菌BL21（DE3）,用IPTG诱导,表达出了大小约为36kD的pilA重组蛋白。表达产物用镍柱亲和层析纯化,与等量弗氏佐剂混合制备pilA重组蛋白疫苗,分别在1日龄、8日龄时两次对雏鸭进行免疫,二免后2周测定鸭血清中的ELISA抗体效价,并以10⁹PFU同源菌株GH1.2攻毒,根据攻毒后鸭的死亡率、E.coli分离率和各组织器官的病变等级来判定pilA重组蛋白的免疫保护效果。结果pilA重组蛋白免疫鸭的血清中ELISA抗体效价为1∶12800,全菌灭活苗免疫组的血清ELISA抗体效价为1∶200;同源菌株攻毒后,pilA重组蛋白免疫保护组鸭的死亡率、E.coli分离率和各组织器官的病变程度均比攻毒对照组下降且差异显著或极显著,与全菌灭活苗免组比较差异不显著。表明pilA重组蛋白对同源菌株GH1.2的感染具有一定的保护效果     

重组人血管内皮细胞生长因子121 cDNA的基因克隆、表达和鉴定

应用RT-PCR方法,扩增人VEGF₁₂₁ cDNA基因片段,与酵母表达载体pPIC9K重组,获得表达质粒p9KVEGF₁₂₁.该质粒转化毕赤酵母菌GS115,用G418-YPD平板筛选高拷贝转化子,PCR鉴定VEGF₁₂₁ cDNA与酵母染色体整合状态,高拷贝转化子用甲醇诱导表达.工程菌用5 L发酵罐发酵,表达产物r-hVEGF₁₂₁占培养液中总蛋白量70%以上.纯化产物促进牛毛细血管内皮(BCE)细胞增殖,并强烈促进血管通透.     

异子蓬PEPC基因原核表达及其重组菌在非生物胁迫下的耐受力解析

该研究在Escherichia coli Transetta（DE3）中表达了C₄盐生植物异子蓬的磷酸烯醇式丙酮酸羧化酶基因（PEPC）,并进行了酶学特性及非生物胁迫响应分析,以期初步阐明PEPC基因在非生物胁迫下的生物学功能,为异子蓬PEPC的非生物胁迫的抗性生理研究提供参考依据。基于5′-RACE技术获得异子蓬PEPC全长基因（GenBank登录号为KP985714）,构建了E.coli Transetta::pGEX-4T-1-PEPC重组菌株。通过分光光度法和酶联免疫吸附技术（ELISA）分别测定了PEPC重组蛋白的酶活和含量,并检测E.coli Transetta::pGEX-4T-1-PEPC重组菌株在非生物胁迫下的耐受性。生物信息学分析发现,该PEPC基因的cDNA全长为2 901 bp,编码966个氨基酸,与甜菜（Beta vulgaris）PEPC的氨基酸序列一致性达90%,具有PEPC的典型保守结构域（PEPcase）以及VlTAHPTQsiRR和VMIGYSDSgKDAG活性位点;PEPC蛋白属PEPC-1型的不含信号肽的非分泌型亲水蛋白。Western blot结果显示,融合GST的PEPC蛋白的分子量约130~140 kD;在37 ℃用0.8 mmol/L IPTG诱导E.coli Transetta::pGEX-4T-1-PEPC重组菌株显示出更高的PEPC酶活及含量,而且E.coli Transetta::pGEX-4T-1-PEPC重组菌在200~800 mmol/L NaCl、5%~20% 聚乙二醇（PEG） 6 000、25 ℃~52 ℃温度范围、50~400 μmol/L甲基紫精和pH 3.0~11.0的非生物胁迫下生长均明显优于对照。研究表明,异子蓬PEPC基因的表达提高了E.coli耐受非生物胁迫的能力。     

鸭源致病性大肠杆菌Ⅰ型菌毛pilA基因的原核表达及重组蛋白对强毒攻击的免疫保护作用

根据GenBank中人源大肠杆菌pilA基因序列,用OLIGO6.0设计PCR引物,从鸭源致病性大肠杆菌GH1.2中扩增到pilA基因并将其克隆至pMD18-T载体,经PCR、酶切和DNA测序鉴定后,将鸭源致病性大肠杆菌pilA基因正向插入原核表达载体pET-32a（+）的BamHⅠ和HindⅢ位点间,成功构建了重组表达质粒pET-32a-pilA。重组表达质粒pET-32a-pilA转化表达宿主菌BL21（DE3）,用IPTG诱导,表达出了大小约为36kD的pilA重组蛋白。表达产物用镍柱亲和层析纯化,与等量弗氏佐剂混合制备pilA重组蛋白疫苗,分别在1日龄、8日龄时两次对雏鸭进行免疫,二免后2周测定鸭血清中的ELISA抗体效价,并以10⁹PFU同源菌株GH1.2攻毒,根据攻毒后鸭的死亡率、E.coli分离率和各组织器官的病变等级来判定pilA重组蛋白的免疫保护效果。结果pilA重组蛋白免疫鸭的血清中ELISA抗体效价为1∶12800,全菌灭活苗免疫组的血清ELISA抗体效价为1∶200;同源菌株攻毒后,pilA重组蛋白免疫保护组鸭的死亡率、E.coli分离率和各组织器官的病变程度均比攻毒对照组下降且差异显著或极显著,与全菌灭活苗免组比较差异不显著。表明pilA重组蛋白对同源菌株GH1.2的感染具有一定的保护效果     

重组刺桐胰蛋白酶抑制剂a的制备及其在tPA突变体纯化中的应用

将基因工程菌株E.coliBL21(DE3) pET22b-mETIa高密度发酵,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导,重组刺桐胰蛋白酶抑制剂a(rETIa)蛋白在E.coli中得到较高水平表达,表达量占菌体总蛋白的40%以上.经菌体破碎、包涵体变性、复性,二步柱层析纯化得到电泳纯的rETIa蛋白.测得rETIa对t-PA突变体(NTA)的抑制平衡常数K_i为8.72×10^－8 mol/L.据此利用纯化的rETIa蛋白制备rETIa-Sepharose 4B亲和层析柱.直接一步纯化NTA复性液,纯化的NTA纯度达90 %以上,收率为96.2 %,纯化倍数为13.2,比活为(565.7±71.3) U/μg.     

番茄转录因子基因SlWRKY6的克隆与原核表达分析

该研究基于番茄基因组数据库SGN(Sol Genomic Network)信息,利用RT PCR从栽培番茄‘M82’(Solanum lycopersicum)中成功克隆到番茄SlWRKY6基因(登录号：Solyc02g080890),通过qRT PCR方法和原核表达初步验证其生物学功能。结果表明：(1)生物信息学分析显示,番茄SlWRKY6基因ORF全长1 653 bp,编码550个氨基酸,其蛋白结构含有1个WRKYGQK保守结构域和C₂H₂锌指结构域,属于IIb类;其基因启动子上游1 500 bp含有多个激素响应元件和非生物胁迫响应元件。(2)进化树分析显示,SlWRKY6与潘那利番茄SpWRKY31 X1(NP_001352691.1)的相似性最高,且定位于细胞核内。(3)qRT PCR结果显示,SlWRKY6基因在番茄根、茎、叶中均有表达,在叶中的表达量最高,且受盐和干旱诱导表达。(4)SDS PAGE及Western blot结果显示,pET 30a SlWRKY6重组蛋白的大小约66 kDa,与预期大小一致。(5)原核表达分析显示,重组菌E. coli BL21∷pET 30a SlWRKY6在不同浓度盐(NaCl)和干旱(Mannitol)胁迫下生长速度显著低于对照菌E. coli BL21∷pET 30a,且在400 mmol/L NaCl、800 mmol/L甘露醇胁迫条件下最为显著;滴板实验初步验证SlWRKY6转录因子能提高重组菌E. coli BL21∷pET 30a SlWRKY6在ABA和pH 9(NaOH)胁迫的耐受性;在400 mmol/L NaCl、pH 5(HCl)、800 mmol/L甘露醇胁迫条件下耐受能力降低。研究表明,SlWRKY6转录因子可能通过参与ABA途径来响应非生物胁迫。     

Calponin binds G-actin and F-actin with similar affinity

Calponins are actin-binding proteins that are implicated in the regulation of actomyosin. Calponin binds filamentous actin (F-actin) through two distinct sites ABS1 and ABS2, with an affinity in the low micromolar range. We report that smooth muscle calponin binds monomeric actin with a similar affinity (K(d) of 0.15 microM). We show that the arrangement of binding is similar to that of F-actin by a number of criteria, most notably that the distance between Cys273 on calponin and Cys374 of actin is 29A when measured by fluorescent resonance energy transfer, the same distance as previously reported for F-actin.     

Calponin in Non-Muscle Cells

Calponin is an actin filament-associated regulatory protein expressed in smooth muscle and non-muscle cells. Calponin is an inhibitor of the actin-activated myosin ATPase. Three isoforms of calponin have been found in the vertebrates. Whereas the role of calponin in regulating smooth muscle contractility has been extensively investigated, the function and regulation of calponin in non-muscle cells is much less understood. Based on recent progresses in the field, this review focuses on the studies of calponin in non-muscle cells, especially its regulation by cytoskeleton tension and function in cell motility. The ongoing research has demonstrated that calponin plays a regulatory role in non-muscle cell motility. Therefore, non-muscle calponin is an attractive target for the control of cell proliferation, migration and phagocytosis, and the treatment of cancer metastasis.     

碱性调宁蛋白的研究进展

碱性调宁蛋白是一个首先从鸡砂囊和牛主动脉中分离出的相对分子质量为34×103的碱性蛋白。它在平滑肌中特异表达,结合钙调蛋白,肌动蛋白,肌球蛋白,抑制肌球蛋白的ATP酶活性,参与平滑肌收缩、细胞信号转导、维持细胞骨架、抑制细胞增生等。     

Calponin and tropomyosin interactions.

The interaction between chicken gizzard calponin and tropomyosin was examined using viscosity, light scattering, electron microscopy and affinity chromatography. At neutral pH, 10 mM NaCl and in the absence of Mg2+, calponin induced tropomyosin filaments to form paracrystals thus decreasing the viscosity while increasing dramatically the light scattering of the tropomyosin solution. Electron micrographs of the uranyl acetate stained calponin-tropomyosin complex showed the presence of spindle shaped paracrystals with regular striation patterns and repeating units of about 400 A. Under similar conditions, smooth muscle caldesmon also induced tropomyosin to form paracrystals. To localize the calponin-binding site on tropomyosin, binding of fragments of tropomyosin, generated by chemical and mutational means, to a calponin-affinity column was studied. The COOH-terminal tropomyosin fragment Cn1B(142-281) and the NH2-terminal fragment CSM-beta(1/8/12-227) bound to a calponin-affinity column with an affinity similar to that of intact tropomyosin; while the NH2-terminal fragment, Cn1A(11-127), did not bind, indicating that the calponin-binding site(s) resides within residues 142-227 of tropomyosin. To determine the involvement in calponin binding of the area around Cys-190 of tropomyosin, fragments with cleavage sites near or at Cys-190 were used. Thus, while fragments Cy2(190-284) and CSM-beta(1/8/12-200) bound weakly to the calponin-affinity column, fragment Cy1(1-189) did not. These results demonstrate that calponin binds to tropomyosin between residues 142 and 227, and that the integrity of the region around Cys-190 of tropomyosin is important for strong interaction between the two proteins.     

Calponin蛋白家族在细胞骨架重构中的作用

Calponin蛋白家族包括calponin(CaP)和平滑肌(SM)22α,此家族的重要结构特征是由单一CH结构域和CLR结构域组成。CaP和SM22α属于肌动蛋白细胞骨架结合蛋白,可通过与F-肌动蛋白相互作用来调节肌动蛋白细胞骨架重构,影响细胞的生物学行为,进而影响相关疾病的发生与发展。     

Mechanism of Calponin Stabilization of Cross-Linked Actin Networks

The actin-binding protein calponin has been previously implicated in actin cytoskeletal regulation and is thought to act as an actin stabilizer, but the mechanism of its function is poorly understood. To investigate this underlying physical mechanism, we studied an in vitro model system of cross-linked actin using bulk rheology. Networks with basic calponin exhibited a delayed onset of strain stiffening (10.0% without calponin, 14.9% with calponin) and were able to withstand a higher maximal strain before failing (35% without calponin, 56% with calponin). Using fluorescence microscopy to study the mechanics of single actin filaments, we found that calponin increased the flexibility of actin filaments, evident as a decrease in persistence length from 17.6 μm without to 7.7 μm with calponin. Our data are consistent with current models of affine strain behavior in semiflexible polymer networks, and suggest that calponin stabilization of actin networks can be explained purely by changes in single-filament mechanics. We propose a model in which calponin stabilizes actin networks against shear through a reduction of persistence length of individual filaments.     

Calponin: biological, chemical and structural properties

Calponin distribution in smooth muscle cells and its properties in experiments in vitro are described. A comparison of these properties with those of other regulatory proteins and of proteins presumed to play this role suggest that calponin has another, yet unknown function. On the basis of existing experimental and theoretical data, an attempt has been made to quantitatively estimate the content of structural elements of the polypeptide chain and their localization. With consideration of the structural calponin domains, a general model of the secondary structure of all known calponin sequences is suggested. Based on the known roentgenographic data of the CH-domain of other proteins a possible organization of the calponin molecule hydrophobic core has been proposed.     

绵羊钙调蛋白调节基因Calponin h1 的分子克隆

在一项研究中我们发现雌激素体在胚胎发育后期对绵羊子宫平滑肌Calponin (CaP) 基因的活动有明显上调作用,而CaP一直被作为观察其他基因表达水平变化的基准参照基因（Reference Gene）。迄今为止, 绵羊CaP尚未完整克隆,为进一步了解其结构和功能,根据人、小鼠和家猪的同源保守区序列设计锚定寡核苷酸引物,通过5′-RACE及3′-RACE方法克隆了绵羊子宫平滑肌组织全长CaP h1 cDNA (GenBank登录号： AY327118), 在cDNA序列的基础上, 又通过PCR-SSP方法获得了CaP h1基因除内含子1、2之外的其余4个内含子全部序列 （GenBank登录号分别为：AY771807,AY771808, AY771809, AY771810） 。DNA序列测定和分析表明,绵羊子宫平滑肌CaP h1 cDNA全长1499bp, 编码297个氨基酸,5′-UTR及3′-UTR分别为79bp和529bp。CaP h1基因组DNA的克隆和序列分析表明,绵羊CaP全长约8kb,由 7个外显子和6个内含子组成。 同源序列比较发现,该基因外显子在不同物种间相对保守;与人类、野猪、小鼠、大鼠和鸡Calponin mRNA同源性分别为88％、92％、81％、79％和81％,但不同物种间内含子存在较大差异(>50%)。本研究填补了绵羊CaP基因分子克隆的空白,为进一步研究该基因的功能及子宫平滑肌收缩的调节机理奠定了基础。