Structure of complement receptor 2 in complex with its C3d ligand

Complement receptor 2 (CR2/CD21) is an important receptor that amplifies B lymphocyte activation by bridging the innate and adaptive immune systems. CR2 ligands include complement C3d and Epstein-Barr virus glycoprotein 350/220. We describe the x-ray structure of this CR2 domain in complex with C3d at 2.0 angstroms. The structure reveals extensive main chain interactions between C3d and only one short consensus repeat (SCR) of CR2 and substantial SCR side-side packing. These results provide a detailed understanding of receptor-ligand interactions in this protein family and reveal potential target sites for molecular drug design.     

Structure of dual function iron regulatory protein 1 complexed with ferritin IRE-RNA

Iron regulatory protein 1 (IRP1) binds iron-responsive elements (IREs) in messenger RNAs (mRNAs), to repress translation or degradation, or binds an iron-sulfur cluster, to become a cytosolic aconitase enzyme. The 2.8 angstrom resolution crystal structure of the IRP1:ferritin H IRE complex shows an open protein conformation compared with that of cytosolic aconitase. The extended, L-shaped IRP1 molecule embraces the IRE stem-loop through interactions at two sites separated by approximately 30 angstroms, each involving about a dozen protein:RNA bonds. Extensive conformational changes related to binding the IRE or an iron-sulfur cluster explain the alternate functions of IRP1 as an mRNA regulator or enzyme.     

Structure of a eukaryotic CLC transporter defines an intermediate state in the transport cycle

CLC proteins transport chloride (Cl(-)) ions across cell membranes to control the electrical potential of muscle cells, transfer electrolytes across epithelia, and control the pH and electrolyte composition of intracellular organelles. Some members of this protein family are Cl(-) ion channels, whereas others are secondary active transporters that exchange Cl(-) ions and protons (H(+)) with a 2:1 stoichiometry. We have determined the structure of a eukaryotic CLC transporter at 3.5 angstrom resolution. Cytoplasmic cystathionine beta-synthase (CBS) domains are strategically positioned to regulate the ion-transport pathway, and many disease-causing mutations in human CLCs reside on the CBS-transmembrane interface. Comparison with prokaryotic CLC shows that a gating glutamate residue changes conformation and suggests a basis for 2:1 Cl(-)/H(+) exchange and a simple mechanistic connection between CLC channels and transporters.     

His标签的水稻硅转运蛋白表达载体的构建及在巴斯德毕赤酵母中的表达

以水稻根系c DNA为模板,PCR扩增含His标签的Lsi1基因开放阅读框,并将该基因克隆到巴斯德毕赤酵母表达载体p PIC9k中,PCR及测序验证重组质粒p PIC9k-Lsi1目的基因序列.将验证正确的重组质粒p PIC9k-Lsi1通过SacⅠ酶切线性化,电转到毕赤酵母GS115感受态细胞中,通过MD/MM板筛选出甲醇利用快型的酵母转化子,再通过不同浓度梯度的G418平板筛选多拷贝转化菌株.提取酵母基因组,PCR验证正确的菌株即为工程菌(GS115/p PIC9k-Lsi1).用1%甲醇诱导该工程菌72 h后,收集上清,经SDS-PAGE检测到位于32.8 ku处的目的蛋白即为Si转运蛋白(Os Lsi1).     

猪γ干扰素基因真核重组表达质粒的构建及活性测定

以含有全长猪γ干扰素基因的质粒为模板,通过PCR方法扩增猪γ干扰素并引入酶切位点,酶切后与PCI真核表达载体连接在一起,测序后经比较,所扩增序列同Genebank报道的IFN-γ序列同源性为99·8%.将重组质粒转染PK细胞,检测所转染的pIFN-γ的抗病毒活性.试验结果表明,转染pIFN-γ试验组与正常细胞接毒对照组比较有显著差异,说明所构建的重组质粒转染后具有抗病毒作用.     

耐碱Mn-SOD基因克隆与真核表达载体的构建

以高产耐碱Mn-SOD产生菌Bacillussp.110-2总DNA为模板,通过PCR扩增得到耐碱Mn-SOD基因,编码一个完整的开放阅读框,共202个氨基酸,含18个碱性氨基酸,包括12个赖氨酸和6个精氨酸。与地衣芽孢杆菌同源性为99%,与极端耐碱芽孢杆菌(Bacillus halodurans)同源性为78.7%。克隆片段酶切后,插入到巴斯德毕赤酵母表达载体pPIC9K中,获得毕赤酵母真核表达载体pPIC9K-SOD。     

Crystal structure of the eukaryotic ribosome

Crystal structures of prokaryotic ribosomes have described in detail the universally conserved core of the translation mechanism. However, many facets of the translation process in eukaryotes are not shared with prokaryotes. The crystal structure of the yeast 80S ribosome determined at 4.15 angstrom resolution reveals the higher complexity of eukaryotic ribosomes, which are 40% larger than their bacterial counterparts. Our model shows how eukaryote-specific elements considerably expand the network of interactions within the ribosome and provides insights into eukaryote-specific features of protein synthesis. Our crystals capture the ribosome in the ratcheted state, which is essential for translocation of mRNA and transfer RNA (tRNA), and in which the small ribosomal subunit has rotated with respect to the large subunit. We describe the conformational changes in both ribosomal subunits that are involved in ratcheting and their implications in coordination between the two associated subunits and in mRNA and tRNA translocation.     

Structure of human urokinase plasminogen activator in complex with its receptor

The urokinase plasminogen activator binds to its cellular receptor with high affinity and initiates signaling cascades that are implicated in pathological processes including tumor growth, metastasis, and inflammation. We report the crystal structure at 1.9 angstroms of the urokinase receptor complexed with the urokinase amino-terminal fragment and an antibody against the receptor. The three domains of urokinase receptor form a concave shape with a central cone-shaped cavity where the urokinase fragment inserts. The structure provides insight into the flexibility of the urokinase receptor that enables its interaction with a wide variety of ligands and a basis for the design of urokinase-urokinase receptor antagonists.     

3-1E/mChIL-15融合基因构建及在真核细胞中的表达

将鸡堆形艾美尔球虫(E.acervulina)子孢子和裂殖子表面抗原3-1E基因片段与鸡白细胞介素15成熟蛋白基因片段(mChIL15)通过四个柔性氨基酸SPGS连接,构建并鉴定真核表达质粒pcDNA3.1/3-1E-linker-mChIL-15。表达质粒纯化后应用磷酸钙法体外转染293T细胞,通过间接免疫荧光和免疫组织化学方法对重组质粒的体外瞬时表达进行检测。结果表明,成功构建了融合基因3-1E-linker-mChIL-15,转染后30h可检测到融合基因编码的融合蛋白在293T细胞中的瞬时表达。研究为鸡艾美尔球虫基因工程疫苗进一步研制及应用奠定了基础。     

猪瘟病毒囊膜蛋白E2基因真核分泌型表达载体的构建及其表达

【目的】构建猪瘟病毒囊膜蛋白(E2 protein)基因的真核表达载体,并将其在猪脐静脉血管内皮细胞中进行表达。【方法】采用PCR技术扩增出E2蛋白全长基因(E2qc)序列和去除跨膜基因(E2sh)序列,并将其克隆入真核表达载体pSecTag2(A)中,构建pSecTag-E2qc和pSecTag-E2sh表达载体,分别进行PCR、双酶切及测序鉴定。用脂质体法将阳性克隆瞬时转染猪脐静脉血管内皮细胞,对转染细胞进行RT-PCR检测目的基因的转录情况,同时对转染细胞及细胞上清液进行SDS-PAGE和Western-blot分析,以检测目的蛋白的表达情况。【结果】成功克隆了E2蛋白基因全长序列1119bp和去除E2蛋白跨膜基因序列999bp的目的基因。构建的表达载体经PCR、双酶切法及测序鉴定均无误。转染后细胞的RT-PCR结果显示,目的基因被成功转录。转染后细胞及细胞上清液的SDS-PAGE和Western-blot结果显示,目的基因被成功表达。【结论】成功构建了猪瘟病毒E2蛋白的真核表达载体,转染猪脐静脉血管内皮细胞后,分泌表达了猪瘟病毒E2重组蛋白。     

药用植物及其提取物的营养调控作用

随着畜牧业的发展和社会的进步,人们崇尚自然、追求健康意识不断增强,抗生素作为饲料添加剂的安全性引起了广泛的争议。药用植物及其提取物以其来自天然、无(或小)毒副作用、无污染和无残留等独特优点,在饲料、畜牧业中作为抗生素的替代品,发挥营养、保健、提高免疫力、抗应激、抗氧化和改善畜产品品质等多方面的应用越来越受到人们的重视。文章以东北农业大学动物营养研究所几年来在药用植物及其提取物对动物生长性能、内分泌、免疫功能、抗应激、抗氧化、改善代谢、提高肉质等方面的初步研究为主,介绍了国内近年来对药用植物及其提取物的营养调控作用的研究成果。     

GPC3-siRNA真核质粒载体的构建及Huh-7细胞转染

目的探讨GPC3-siRNA真核质粒载体构建并转染Huh-7细胞的可行性。方法构建真核质粒载体pcDNA3.1＋GPC3和GPC3-siRNA-1633,采用脂质体法瞬时转染至Huh-7细胞中。MTT法检测转染后Huh-7细胞的细胞活性,荧光定量PCR、Western blot分别检测GPC3 mRNA和蛋白表达。结果 GPC3-siRNA-1633基因和GPC3真核质粒载体转染Huh-7细胞后细胞存活率高,转染GPC3-siRNA-1633的Huh-7细胞出现GPC3 mRNA和蛋白表达下调。结论 GPC3-siRNA-1633可成功转染至Huh-7细胞,并抑制GPC3表达。     

猪FATP1基因5'调控区多态性及其与脂肪性状的相关分析

脂肪酸转运蛋白1(Fatty acid transport protein 1,FATP1)是脂肪酸转运蛋白家族(FATPs)的一员,具有促进脂肪细胞吸收利用长链和极长链脂肪酸的功能,在脂肪沉积过程中发挥着重要的作用.采用直接测序法寻找FATP1基因5'调控区+64～-723 bp片段的单核苷酸多态性,在-586 bp处发现了一个T→C突变SNP(单核苷酸多态性)座位,利用PCR-RFLP的方法检测它在3个试验猪群体中的基因型,发现该座位在皮特兰和金皮F2代猪群中存在多态性,而在金华猪中仅表现为FATP1g-586T FATP1g-586T基因型.建立混合模型方程组分析该座位对188头金皮F2代猪脂肪性状的效应,结果显示,猪FATP1基因第-586座位的不同基因型与肩背膘厚、6～7肋背膘厚、最后一根肋骨处背膘厚、平均背膘厚和板油重显著相关(P＜0.05),表明FATP1基因5'调控区的第-586位上的T→C突变对猪脂肪沉积有一定的影响.     

大鳞副泥鳅生长激素基因cDNA克隆及其真核表达载体的构建

从脑垂体中提取总RNA,用RT-PCR方法克隆大鳞副泥鳅生长激素基因(pGH)cDNA。结果表明,克隆到的pGH的开放阅读框包括633 bp,编码210个氨基酸,其中包括22个氨基酸的信号肽和188个氨基酸的成熟肽,GenBank注册号为DQ350432。把pGH成熟肽的cDNA插入真核表达载体pPIC3.5K,PCR技术、酶切和测序证明重组子中确实定向插入了pGH成熟肽片段;将重组的pPIC3.5K-pGH用SalⅠ酶切后,转化巴斯德毕赤酵母GS115,PCR技术筛选证明pGH已经整合到酵母染色体上。从而成功克隆大鳞副泥鳅生长激素基因cDNA,并构建了胞内真核表达载体pPIC3.5K-pGH。     

牛白细胞介素-17真核表达载体的构建及在牛乳腺上皮细胞中的表达

【目的】在体外分离培养牛乳腺上皮原代细胞,并构建牛白细胞介素-17(bIL-17)基因真核表达质粒,观察重组质粒在牛乳腺上皮细胞的表达.【方法】提取牛脾脏细胞总RNA,通过RT-PCR扩增bIL-17,将bIL-17连入pMD19-T进行测序,测序成功后将bIL-17插入含有增强型绿色荧光蛋白报告基因的真核表达质粒pEGFP-N3上,构建真核表达质粒pEGFP-N3-bIL-17.用脂质体法将pEGFP-N3-bIL-17质粒转染于牛乳腺上皮细胞,荧光显微镜观察绿色荧光蛋白在细胞中的表达,RT-PCR检测bIL-17基因在细胞内的转录,定量ELISA检测bIL-17蛋白在细胞内的表达情况.【结果和结论】成功构建具有绿色荧光和新霉素抗性的双选择标记的牛白细胞介素-17真核表达载体,并可在牛乳腺上皮细胞成功表达.     

青草沙水库浮游植物群落结构及其与环境因子的关系

自2011年1月、4-12月逐月对上海市水源地——青草沙水库的浮游植物群落结构进行调查和分析。本研究共检出浮游植物8门99属223种,细胞丰度变化范围为86.37×104~2 594.63×104cell/L,全年优势种为小环藻、卷曲鱼腥藻、微小色球藻、针晶蓝纤维藻、球衣藻、啮蚀隐藻,偶有咸水和半咸水种检出。蓝藻密度在全年占据优势,优势类群季节演替明显,表现为:春季为色球藻属、蓝纤维藻属,夏季为微囊藻属,秋季为卷曲鱼腥藻、鞘丝藻属。典范对应分析(CCA)结果表明浮游植物群落结构与水温、悬浮物含量和浮游动物的牧食作用相关。