人可溶性血管内皮细胞生长因子受体Flt-1基因在链霉菌中的克隆表达

应用RTPCR技术,从人脐静脉内皮细胞中扩增出编码人可溶性血管内皮细胞生长因子 (VEGF)受体Flt1胞外区前四个结构域的基因片段,亚克隆至pUCl8质粒进行测序,将目的基因片段连接至链霉菌表达载体pSGLgpp,获得重组质粒pSGLgppF,将其转化至Streptomyces lividans TK24, 获得基因工程菌株Sreptomyces lividans (pSGLgppF),对其培养上清液进行SDSPAGE及Western blot分析,结果 显示,在636kD处有特异性条带出现,表明sFLT1在链霉菌中获得了成功表达,受体配基结合实验显示表达产物与VEGF可特异性结合,表明其具有配基结合生物活性。     

苏云金杆菌vip3A基因的克隆、表达及杀虫活性分析

用全长PCR方法从野生型苏云金杆菌（Bacillus thuringiensis,Bt）菌株S184中克隆了2.3 kb大小的vip3A基因并进行了序列分析。将vip3AS184基因插入表达载体pQE30构建了表达质粒pOTP,转化大肠杆菌M15,转化子经1mmol/L IPTG诱导后可表达89 kD大小的Vip3A-S184蛋白,并得到Western blot证实。蛋白可溶性试验表明,目的蛋白中约有19%是可溶的,用透射电镜观察到大多数蛋白是以包涵体形式存在的。因此,可以在自然条件下进行目的蛋白的纯化和对家兔进行免疫制备多克隆抗体,用于苏云金杆菌Vip3A蛋白表达的检测。利用IPTG进行诱导培养的菌液对甜菜夜蛾（Spodoptera exigua）、斜纹夜蛾(S.litura)和棉铃虫(Helicoverpa armigera)等3种害虫的初孵幼虫进行生物测定,结果表明,Vip3AS184蛋白对夜蛾科害虫具有较高的杀虫活性。     

猪β干扰素在毕赤酵母中的分泌表达及其对伪狂犬病毒的抑制作用

了研制高活性的重组猪β干扰素,对PoIFNβ成熟蛋白第3、7和164位的3个氨基酸密码子进行毕赤酵母偏嗜性改造并构建了酵母表达载体pPICZαAPIB。pPICZαAPIB经SacⅠ酶切线性化后电转化导入毕赤酵母菌株X33。多株PCR鉴定为阳性的酵母转化子经甲醇诱导发酵分泌表达了PoIFNβ, 其中B1株酵母的PoIFNβ产量最高,约为2.5×10.5U/mL,其表达量约为60μg/mL,比活为4.17×10.6U/mg。将发酵上清液用PEG20000浓缩后进行SDSPAGE和Western blot检测,结果表明表达产物是分子量约为28kDa和25kDa蛋白的混合物,两者均可与PoIFNβ阳性抗血清发生特异反应。表达产物比PoIFNβ理论推导分子量（约20.8kDa）大,推测可能是表达产物发生了不同程度的糖基化。重组PoIFNβ对伪狂犬病毒在细胞中增殖可呈现抑制作用,并且rPoIFNβ对伪狂犬病毒在MDBK细胞上早期增殖的抑制效果最为明显。     

大肠杆菌-分枝杆菌穿梭表达质粒pBCG-2100的构建及应用

利用分子生物学方法,构建了大肠杆菌分枝杆菌(E.coliMycobacterium)穿梭表达质粒pBCG-2100,研究了编码日本血吸虫中国大陆株谷胱甘肽S转移酶(Glutathione Stransferase,GST)抗原基因在卡介苗(Bacillus Calmette Guerin,BCG)中的表达。以含人结核杆菌热休克蛋白(Heat shock protein,hsp)70基因全长序列的质粒pMT-70为模板,扩增出hsp70启动子,测序选出无错配的启动子,将其定向克隆入E.coliMycobacterium穿梭质粒pBCG-2000中,构建成E.coliMycobacterium穿梭表达质粒pBCG-2100。再将编码GST的cDNA按正确的阅读框顺序,克隆到pBCG-2100中hsp70启动子的下游,得到分枝杆菌表达质粒pBCG-GST。将pBCG-GST电转化入BCG中,筛选出重组BCG疫苗,经热诱导后所表达的重组GST(rGST)抗原,为可溶性蛋白,经纯化后,在SDS-PAGE上分子量为26kD处可见明显的表达蛋白带,其表达量占BCG菌体总蛋白的13%。Western blot提示rGST能与抗GST的抗体反应。     

点状产气单胞菌脯氨酰内肽酶基因的克隆与表达

用活性筛选法从产气单胞菌点状亚种ST7833 (Aeromonas puctata subsp.puctata ST7833)的基因组中克隆了脯氨酰内肽酶 (Prolyl Endopeptidase,简称apPEP)的基因,测定了含有PEP基因的33kb DNA片段的序列,第202092bp编码了690个氨基酸组成的脯氨酰内肽酶,经检索是一种新的PEP基因。并构建了一株组成性高效表达PEP的基因工程菌BL21/pGEMPEP。BL21/pGEMPEP在 YH培养基中apPEP的表达量占菌体总蛋白的30%左右,活力是野生菌的112倍,表达产物主要为可溶性的胞内蛋白,约5%分泌到胞外。非还原SDSPAGE显示为单体,分子量为76kD,与基因序列预测的分子量一致。试管培养后纯化得到了纯度大于90%的重组脯氨酰内肽酶,比活力为67U/mg。     

杀鞘翅目苏云金芽孢杆菌新菌株及其杀虫剂的研究

从中国土壤中分离出2株杀鞘翅目昆虫的苏云金芽孢杆菌(Bacillus thuringiensis) YM03及SHQ11-10。YM03的血清型为H8a8b,SHQ1110的H血清型未知。二菌株皆产近菱形的薄扁伴孢晶体,分别含68～70kD和65kD的晶体蛋白质。毒力生物测定证明对柳蓝叶甲(Plagiodera versicolora)及马铃薯甲虫(Leptinotarsa decemlineata)有高毒效。发酵性能良好。YM03粉剂田间防治马铃薯甲虫有高效。稀释400倍喷雾,防治效果达94.6%。     

Qβ RNA 复制酶中亚基的共表达对β亚基溶解性的影响

在体外RNA和蛋白质合成及自复制系统的研究中,Qβ RNA复制酶作为以RNA为模板的RNA聚合酶,是比较重要的应用酶种之一。该酶由4个亚基组成,其中只有 β亚基是由病毒基因编码,而其他3个亚基都是宿主蛋白。利用普通表达载体合成Qβ复制酶时,得到的β亚基几乎都是不溶性蛋白,从而影响了Qβ复制酶的活性和产率。为尝试提高β亚基的溶解性,构建含有β亚基基因的表达质粒pBAD 33rep,同时利用pET21a(+)为表达载体表达其他3个亚基进行共表达研究。不同亚基组合的共表达结果通过SDSPAGE分析表明,当β亚基与EFTuTs亚基共表达时,溶解度有一定的提高,而且可溶性部分也具有复制酶活性。通过调节共表达诱导物浓度,相对增强可溶性β亚基的表达,可溶性Qβ复制酶酶量得到相应的提高。     

苏云金芽孢杆菌vip3A基因的检测及保守性分析

Vip3A蛋白是苏云金芽孢杆菌（Bacillus thuringiensis,Bt）在营养期分泌的一类新型杀虫蛋白。用PCR方法从114个Bt菌株和41个Bt标准菌株中筛选到39株即约25%的菌株含有vip3A基因。利用所制备的Vip3A蛋白的多克隆抗体对以上含有vip3A基因的Bt菌株进行Western印迹分析,发现多数PCR反应为阳性的菌株都产生89 kD大小的蛋白,其中有4株没有Vip3A蛋白的表达。从以上菌株中挑选2个对夜蛾科害虫具有较高和较低毒力的菌株,即S101和611,并分别进行vip3A基因的克隆和测序,再与GenBank上所登录的其它6个全长vip3A基因和2个已报道的但未登录GenBank的vip3A基因进行核苷酸和氨基酸序列比较,结果表明,vip3A是一个极其保守的基因。将以上所克隆的2个vip3A基因即vip3AS101和vip3A611分别插入表达载体pQE30构建了表达质粒pOTP-S101和pOTP-611,转化到大肠杆菌M15,经1 mmol/L IPTG诱导后均表达89 kD大小的Vip3A蛋白。蛋白可溶性试验表明,Vip3A-S101和Vip3A-611分别有48%和35%的蛋白是可溶的。将Vip3A-S101和Vip3A-611蛋白和已报道的Vip3A-S184蛋白对初孵斜纹夜蛾 (Spodoptera litura) 幼虫进行生物测定,结果表明,3个Vip3A蛋白对斜纹夜蛾幼虫毒力没有显著性差异,这说明了Vip3A个别氨基酸的变化对蛋白的杀虫活性没有影响。     

三种不同羊肚菌菌丝体的生长，蛋白质合成和酯酶同功酶的组成

黑脉羊肚菌(Morchella angustieeps),尖顶羊肚菌(M．Conwa)和羊肚菌(M．Esculenta)三种不同的羊肚菌在相同的培养液及培养条件下,菌丝体产量、总蛋白及可溶性蛋白质的含量无显著的差异;同一个种在含不同浓度麦芽糖、蛋白胨和维生素Bl的培养基上生长时,总蛋白和可溶性蛋白质的含量无显著的差异,但菌丝体的产量的差异较大,可溶性蛋白质的种类和数量,以及酯酶同功酶的组成也有一定的差异。     

苏云金芽胞杆菌营养期杀虫蛋白基因的克隆及表达分析

选择本实验室分离的苏云金芽胞杆菌李氏亚种 (subsp. Leesis) 菌株YBT833、鲇泽亚种(subsp.Aizawai) 菌株YBT-1416和库斯塔克亚种(subsp. Kurstaki)菌株YBT1535为出发菌株,以营养期杀虫蛋白基因PCR扩增的特异片段为探针,进行总DNA酶切片段的Southern杂交定位。结果显示3株菌株的营养期杀虫蛋白基因,均位于经XbaI完全消化的4～5kb大小的DNA 片段上。将该区域DNA片段回收后克隆到pUC19载体,建立了3个较基因组文库小的亚基因组文库。通过菌落原位杂交筛选和酶切鉴定分别得到3个相应的营养期杀虫蛋白基因vip83、vip14和vip15,并对其测序。DNA序列比较发现基因vip83与已知营养期杀虫蛋白基因存在5个差异碱基。将vip83、vip14基因亚克隆到苏云金芽胞杆菌大肠杆菌穿梭载体pHT315, 分别得到重组质粒pBMB8901和pBMB8902。将它们电转化到vip^-的B.t.受体菌BMB171和4Q7,获得了相应的工程菌BMB8901-171,BMB8902-171,BMB8901-4Q7和BMB8902-4Q7。SDS-PAGE电泳检测均有88kD大小的蛋白表达。生物测定结果亦表明了,营养期杀虫蛋白Vip83和Vip14对鳞翅目棉铃虫、小菜蛾和甜菜夜蛾的三龄幼虫均有一定的杀虫活性;其中对小菜蛾的毒力最高,LC₅₀值分别为28.6,31.6,45.4和37.6μL/mL。该结果为构建高效广谱工程菌提供了实际材料和理论依据。      

纳米盘技术在临床医学领域研究新进展

膜蛋白功能广泛,参与多种细胞活动,如细胞增殖分化、信号转导、物质运输等,近年来一直是生物医学领域研究热点之一.膜蛋白天然构象的稳定是维持其生物活性的关键因素,新型纳米材料纳米盘技术采用两亲膜支架蛋白在水相中稳定磷脂分子,进而自组装形成类似于天然磷脂双分子膜环境的盘状结构,为膜蛋白的研究提供了理想平台.与传统拟膜技术相比,纳米盘具有可溶性强、稳定性佳、尺寸可控、生物相容性高、半衰期长等优点,同时可精准设计选择性靶向,应用优势巨大.本文介绍了纳米盘技术在膜蛋白结构与功能研究中的应用,并重点综述了其在临床医学领域中的研究新进展,包括纳米盘作为疏水性药物、抗肿瘤靶向治疗药物的运输载体,具有高载药率、药物可控释、靶向功能的运载能力;作为小分子蛋白的拟膜环境对目标蛋白的亲和固定性和作为高密度脂蛋白的有效补充在心血管疾病中清除胆固醇的高效性和可控性.综上,纳米盘技术能够为未来膜蛋白相关研究以及其他临床疾病的诊断与治疗提供新方法与新思路.     

融合标签技术在膜蛋白结构研究中的应用

膜蛋白高级结构的研究包括不同的层次,即膜蛋白拓扑学结构的研究、利用核磁共振技术和蛋白质晶体衍射技术对三维结构的研究,以及膜蛋白复合体的研究。在研究过程中,如果能够基于膜蛋白的拓扑学结构预测,选择合适的蛋白质或多肽融合标签,利用基因融合技术在基因水平上对膜蛋白进行改造,可以产生含有融合标签的重组膜蛋自,不仅具有原有膜蛋白的功能活性,还具有融合标签所特有的生理生化特性,将会极大地促进膜蛋白结构和功能的研究。我们就目前膜蛋白结构研究中所涉及的融合标签技术及其应用策略和所取得的进展做一简述。     

A membrane preparation that contains proteins characteristic of the rough endoplasmic reticulum

We describe a procedure for disassembling rat liver rough microsomes, which allows the purification of the rough endoplasmic reticulum (ER) membrane. Membrane-bound ribosomes and adsorbed proteins are first detached by washing rough microsomes with 5 mM Na-pyrophosphate. In a second step, the vesicle membrane is opened by digitonin, with concomitant release of the luminal content. The purification is monitored at each step by electron microscopy, and by assaying chemical constituents (protein, phospholipid, RNA) and marker enzymes for the main subcellular organelles. The final membrane preparation is representative of the ER, since it contains 24.1% of the liver glucose 6-phosphatase with a relative specific activity of 14.2. Contaminants represent less than 5% of its protein content. SDS-polyacrylamide gel electrophoresis, followed by immunoblot analysis, reveals that the ribophorins I and II, two established markers of the rough (d) domain are still present in the final membrane preparation. It also contains the docking protein (or signal recognition particle receptor) and protein disulfide isomerase, and has conserved the functional capacity to remove co- and post-translationally the signal peptide of pre-secretory proteins. The membrane preparation is suitable for studies on the polypeptide composition of the d domain.     

Tellurite effect on ATPase activity of contractile membrane protein.

The effect of tellurite on ATPase activity of the contractile membrane protein in human erythrocytes was studied. Tellurite, even at a concentration of 0.01 mM, inhibited 25 per cent of the saponin-stimulated ATPase activity of the contractile membrane protein; the inhibition increased with increasing tellurite concentration, and was reversible. On the other hand, in erythrocytes preincubated with tellurite, the ATPase activity of the membrane contractile protein, non-stimulated by saponin, increased, and the increase was further enhanced by washing the erythrocytes. The behaviour is analogous to the tellurite effect on erythrocyte morphology: incubation of erythrocytes with tellurite caused morphological changes which were more pronounced after removing the tellurite by washing. The complex effect of tellurite is discussed.     

Magic-angle spinning solid-state NMR of a 144 kDa membrane protein complex: E. coli cytochrome bo3 oxidase

Recent progress in magic-angle spinning (MAS) solid-state NMR (SSNMR) has enabled multidimensional studies of large, macroscopically unoriented membrane proteins with associated lipids, without the requirement of solubility that limits other structural techniques. Here we present initial sample preparation and SSNMR studies of a 144 kDa integral membrane protein, E. coli cytochrome bo₃ oxidase. The optimized protocol for expression and purification yields ∼5 mg of the enzymatically active, uniformly ¹³C,¹⁵N-enriched membrane protein complex from each liter of growth medium. The preparation retains endogenous lipids and yields spectra of high sensitivity and resolution, consistent with a folded, homogenous protein. Line widths of isolated signals are less than 0.5 ppm, with a large number of individual resonances resolved in the 2D and 3D spectra. The ¹³C chemical shifts, assigned by amino acid type, are consistent with the secondary structure previously observed by diffraction methods. Although the structure is predominantly helical, the percentage of non-helical signals varies among residue types; these percentages agree well between the NMR and diffraction data. Samples show minimal evidence of degradation after several weeks of NMR data acquisition. Use of a triple resonance scroll resonator probe further improves sample stability and enables higher power decoupling, higher duty cycles and more advanced 3D experiments to be performed. These initial results in cytochrome bo₃ oxidase demonstrate that multidimensional MAS SSNMR techniques have sufficient sensitivity and resolution to interrogate selected parts of a very large uniformly ¹³C,¹⁵N-labeled membrane protein. Heather L. Frericks and Lai Lai Yap contributed equally to this work.     

The major outer membrane protein of Fusobacterium nucleatum (FomA) folds and inserts into lipid bilayers via parallel folding pathways

Membrane protein insertion and folding was studied for the major outer membrane protein of Fusobacterium nucleatum (FomA), which is a voltage-dependent general diffusion porin. The transmembrane domain of FomA forms a beta-barrel that is predicted to consist of 14 beta-strands. Here, unfolded FomA is shown to insert and fold spontaneously and quantitatively into phospholipid bilayers upon dilution of the denaturant urea, which was shown previously only for outer membrane protein A (OmpA) of Escherichia coli. Folding of FomA is demonstrated by circular dichroism and fluorescence spectroscopy, by SDS-polyacrylamide gel electrophoresis, and by single-channel recordings. Refolded FomA had a single-channel conductance of 1.1 nS at 1 M KCl, in agreement with the conductance of FomA isolated from membranes in native form. In contrast to OmpA, which forms a smaller eight-stranded beta-barrel domain, folding kinetics of the larger FomA were slower and provided evidence for parallel folding pathways of FomA into lipid bilayers. Two pathways were observed independent of membrane thickness with two different lipid bilayers, which were either composed of dicapryl phosphatidylcholine or dioleoyl phosphatidylcholine. This is the first observation of parallel membrane insertion and folding pathways of a beta-barrel membrane protein from an unfolded state in urea into lipid bilayers. The kinetics of both folding pathways depended on the chain length of the lipid and on temperature with estimated activation energies of 19 kJ/mol (dicapryl phosphatidylcholine) and 70 kJ/mol (dioleoyl phosphatidylcholine) for the faster pathways.     

Parallel topology of genetically fused EmrE homodimers

EmrE is a small H+-coupled multidrug transporter in Escherichia coli. Claims have been made for an antiparallel topology of this homodimeric protein. However, our own biochemical studies performed with detergent-solubilized purified protein support a parallel topology of the protomers. We developed an alternative approach to constrain the relative topology of the protomers within the dimer so that their activity can be assayed also in vivo before biochemical handling. Tandem EmrE was built with two identical monomers genetically fused tail to head (C-terminus of the first to N-terminus of the second monomer) with hydrophilic linkers of varying length. All the constructs conferred resistance to ethidium by actively removing it from the cytoplasm. The purified proteins bound substrate and transported methyl viologen into proteoliposomes by a proton-dependent mechanism. A tandem where one of the essential glutamates was replaced with glutamine transported only monovalent substrates and displayed a modified stoichiometry. The results support a parallel topology of the protomers in the functional dimer. The implications regarding insertion and evolution of membrane proteins are discussed.     

红细胞膜带4.2蛋白的结构与功能

带4.2蛋白是一种重要的红细胞膜蛋白,与红细胞的形态、可变形性及携氧功能有至关重要的联系。它通过与带3蛋白(阴离子通道蛋白)、锚蛋白结合,稳定的连接在细胞膜的内表面,连接着膜骨架网架结构与细胞膜,是膜骨架与脂质双分子层连接的重要纽带。带4.2蛋白的缺失会引起球形或椭圆形红细胞增多症及不同程度的溶血性贫血,严重的情况需要摘除脾脏来进行治疗。近年来研究认为,带4.2蛋白在维持细胞膜骨架的完整性和稳定性方面扮演了重要角色。现对带4.2蛋白结构及功能的研究状况进行综述。     

小麦类脱水素的表达、纯化及多克隆抗体的制备

脱水素在胚胎发育后期累积,外源脱落酸(ABA)、低温、干旱和其他一些环境条件下能诱导脱水素的产生,尽管植物在脱水条件下脱水素广泛存在于细胞中,但其生化功能仍不清楚.为研究小麦在不同时期脱水素基因的表达情况和生物学功能及抗体制备,以小麦幼芽为材料,经干旱胁迫处理后,提取总RNA,通过RT-PCR得到小麦类脱水素基因片段(WZY1-1),再连接至克隆载体PUCM-T,并成功构建重组表达质粒PET-32a( )-wzy1-1,将阳性重组质粒转化于受体菌BL21(DE3)感受态细胞中,经IPTG诱导表达,进行表达产物的聚丙烯酰胺凝胶电泳(SDS-PAGE)检测.结果表明,表达蛋白位于37ku处,小麦类脱水素基因获得高效表达.表达蛋白经Ni2 琼脂糖凝胶亲和层析和透析袋电洗脱法纯化后,对兔子进行免疫,制备的抗血清通过ELISA检测到较高的多克隆抗体效价.蛋白质印迹结果显示,利用纯化的蛋白质制备的兔抗血清可以很好地和所表达的蛋白质带特异性结合,且郑引1号小麦幼苗进行干旱处理,提取粗蛋白,SDS-PAGE,蛋白质印迹检测显示,在分子质量28ku处出现特异的蛋白质条带,这说明所制备的抗血清可以与小麦叶片所表达的dehydrin蛋白特异性结合,证明其具有良好的免疫原性.     

Estimating the length of transmembrane helices using Z-coordinate predictions

Zpred2 is an improved version of ZPRED, a predictor for the Z-coordinates of alpha-helical membrane proteins, that is, the distance of the residues from the center of the membrane. Using principal component analysis and a set of neural networks, Zpred2 analyzes data extracted from the amino acid sequence, the predicted topology, and evolutionary profiles. Zpred2 achieves an average accuracy error of 2.18 A (2.17 A when an independent test set is used), an improvement by 15% compared to the previous version. We show that this accuracy is sufficient to enable the predictions of helix lengths with a correlation coefficient of 0.41. As a comparison, two state-of-the-art HMM-based topology prediction methods manage to predict the helix lengths with a correlation coefficient of less than 0.1. In addition, we applied Zpred2 to two other problems, the re-entrant region identification and model validation. Re-entrants were able to be detected with a certain consistency, but not better than with previous approaches, while incorrect models as well as mispredicted helices of transmembrane proteins could be distinguished based on the Z-coordinate predictions.