Molecular characterization and expression of a cDNA encoding fructan:fructan 6G-fructosyltransferase from asparagus (Asparagus officinalis)

* Fructan:fructan 6G-fructosyltransferase (6G-FFT) catalyses a transfructosylation from fructooligosaccharides to C6 of the glucose residue of sucrose or fructooligosacchrides. In asparagus (Asparagus officinalis), 6G-FFT is important for the synthesis of inulin neoseries fructan. Here, we report the isolation and functional analysis of the gene encoding asparagus 6G-FFT. * A cDNA clone was isolated from asparagus cDNA library. Recombinant protein was produced by expression system of Pichia pastoris. To measure enzymatic activity, recombinant protein was incubated with sucrose, 1-kestose, 1-kestose and sucrose, or neokestose. The reaction products were detected by high performance anion-exchange chromatography. * The deduced amino acid sequence of isolated cDNA was similar to that of fructosyltransferases and vacuolar type invertases from plants. Recombinant protein mainly produced inulin neoseries fructan, such as 1F, 6G-di-beta-D-fructofuranosylsucrose and neokestose. * Recombinant protein demonstrates 6G-FFT activity, and slight fructan:fructan 1-fructosyltransferase (1-FFT) activity. The ratio of 6G-FFT activity to 1-FFT activity was calculated to be 13. The characteristics of the recombinant protein closely resemble those of the 6G-FFT from asparagus roots, except for a difference in accompanying 1-FFT activity.     

日本血吸虫Sj Cyclophilin B基因的克隆、表达及免疫保护效果分析

本研究克隆和表达了日本血吸虫Cyclophilin B(Sj CyPB)编码基因的cDNA,分析其在日本血吸虫不同发育阶段虫体的表达情况,评估该重组抗原在小鼠体内诱导的抗血吸虫免疫保护效果。本研究以日本血吸虫童虫cDNA为模板,RT-PCR扩增其基因全长cDNA,提交序列到NCBI,登录号为GQ403665。荧光实时定量PCR分析该基因在日本血吸虫不同发育阶段虫体的表达情况,构建重组表达质粒,表达纯化重组蛋白。利用Western blotting检测重组蛋白的抗原性。以重组抗原免疫小鼠,评估其对小鼠诱导的免疫保护效果。结果表明,RT-PCR获得了Sj CyPB编码基因的全长cDNA,其开放阅读框为672bp。经分析确定其为CyPs家族中的CyPB基因,命名为Sj CyPB。荧光实时定量PCR分析表明,该基因在18d童虫期表达量最高,32d次之。构建了重组表达质粒pGEX-6P-1-SjCyPB,并在大肠杆菌中成功表达,表达产物分子量为49.5kDa。Western blotting试验显示该重组蛋白具有良好的抗原性,在小鼠免疫试验中,与空白对照组比较,免疫组小鼠获得31.5%的减虫率和41.01%的肝脏减卵率。本研究获得了日本血吸虫童虫期高表达的Sj CyPB基因的全长cDNA,成功构建了Sj CyPB原核重组表达质粒,并在大肠杆菌中成功表达,证实该重组抗原在小鼠体内诱导产生了部分免疫保护效果。     

人FKBP52的基因克隆、表达及活性研究

为获得具有生物学活性的hFKBP52,来筛选新型的促神经再生药物.采用半巢式、桥联PCR及亲和层析方法,从人胎脑cDNA文库中成功扩增出hFKBP52基因,在pET28a（+）中实现了高效、可溶性的融合表达,表达量约30%.重组的蛋白质经亲和纯化至电泳纯,纯化后的hFKBP52显示出肽基脯氨基顺反异构酶活性.表明原核表达的hFKBP52具有类似于其天然蛋白质的生物学活性.     

腐皮镰孢菌壳聚糖酶的酶学性质研究及其在酿酒酵母工业菌株中的表达

摘要：【目的】本研究旨在了解腐皮镰孢菌（Fusarium solani）壳聚糖酶的基本酶学性质及其在壳寡糖生产中的应用,构建能高效分泌表达壳聚糖酶的酿酒酵母工业菌株。【方法】采用RT-PCR扩增腐皮镰孢菌壳聚糖酶的cDNA序列;通过组氨酸标签,纯化得到E. coli表达的重组壳聚糖酶,并进行基本酶学性质研究;以薄层层析、高效液相色谱等技术对该酶的酶解产物进行分析;通过马克斯克鲁维酵母（Kluyveromyces marxianus）菊粉酶信号肽（INU1A）实现壳聚糖酶在酿酒酵母工业菌株N-27中的分泌表     

N-糖酰胺酶F在大肠杆菌中的高效表达及其脱糖基化作用研究

从脑膜炎脓杆菌（Flavobacterium meningosepticum）基因组中通过PCR扩增了N-糖酰胺酶F（PNGase F）基因,经酶切后与表达载体pET28a连接,获得的重组质粒转入大肠杆菌BL21(DE3)。重组大肠杆菌经诱导表达和纯化提取后,获取大量高纯度N-糖酰胺酶F,其纯度达90%以上。试验证明,经纯化的重组N-糖酰胺酶F可以切除核糖核酸酶B、转铁蛋白和人IgG等糖蛋白上的N-糖链,具有脱糖基化作用。     

Change in lactate production in Myc-transformed cells precedes apoptosis and can be inhibited by Bcl-2 overexpression

Interleukin-18 binding protein is a novel glycoprotein that we successfully cloned and expressed. First, murine interleukin-18 binding protein was purified from the sera of mice with endotoxin shock using ligand affinity chromatography. The murine interleukin-18 binding protein cDNA was cloned after RT-PCR using mixed primer pair sequences based on partial murine interleukin-18 binding protein amino acid sequence analysis. Subsequently, human interleukin-18 binding protein cDNA was cloned from cDNA libraries of normal human liver using murine interleukin-18 binding protein cDNA as a probe. Next, we transiently expressed recombinant human and murine interleukin-18 binding proteins in COS-1 cells and purified them from culture supernatants. Both recombinant interleukin-18 binding proteins did not exhibit species specificity and prevented interleukin-18 binding to its receptor. In addition, they inhibited interleukine-18 dependent IFN-gamma production from KG-1 cells effectively. These results suggest that the interleukin-18 binding protein may possess interleukine-18 antagonist activity.     

鸽β-防御素1基因的克隆及其生物学作用鉴定

【目的】从鸽子组织中克隆鸽子β-防御素1(AvBD1)基因,在大肠杆菌中表达重组鸽子AvBD1蛋白,测定其生物学特性。【方法】应用RT-PCR法从鸽子骨髓组织中扩增鸽子AvBD1基因,采用Real-time PCR法检测该基因在鸽子组织器官中的表达分布。将该基因亚克隆到大肠杆菌原核表达载体pProEX-HTa的EcoR I和Xho I双酶切位点上,构建重组表达质粒pProEX-pigeon AvBD1,将重组质粒进行诱导表达;对该重组蛋白进行纯化,通过菌落计数法测定其体外抗菌活性与理化特性。【结果】从鸽子骨髓组织中克隆到鸽子AvBD1基因,其cDNA大小为198 bp,编码65个氨基酸,经序列相似性分析,鸽子AvBD1与鸭AvBD1氨基酸序列相似性最高(81.5%)。鸽子AvBD1主要分布于免疫系统和消化系统组织中。Tricine-SDS-PAGE电泳结果表明,重组鸽子AvBD1蛋白分子量约8.8 kD,与预期大小一致。该重组蛋白具有广谱抗菌活性,高盐浓度显著降低其抗菌活性。此外,该重组蛋白的溶血活性极低。【结论】从鸽子骨髓组织中克隆到鸽子AvBD1基因,其主要分布在机体的免疫系统和消化系统中。该重组蛋白具有广谱抗菌活性,高盐浓度显著降低其抗菌活性,且该重组蛋白的溶血活性极低。     

组织因子膜外区融合蛋白基因的表达及产物的分离纯化与活性分析

 为构建表达组织因子 (TF)膜外区的融合载体 ,制备组织因子膜外区 ,抽提人胎盘组织的总RNA,通过 RT- PCR法扩增出 TF的 c DNA克隆至 p UC1 8并测定全序列 .然后以此为模板 ,再次PCR扩增出 TF膜外区 (soluble TF,s TF) c DNA,并将其插入到谷胱甘肽巯基转移酶融合表达载体 p GEX4T- 1 ,构建了 tac启动子控制下的 GST- s TF融合蛋白的表达载体 .表达的融合蛋白经亲和层析、凝血酶切得到纯化的 s TF.表达产物经 ELISA验证 ,能特异性地与 TF抗体结合 .重新脂化后 ,该产物具有较大凝血活性 .以上说明采用融合蛋白表达系统可以大量制备组织因子膜外区 ,为研制国产重组凝血活酶试剂和研究 s TF的结构和功能创造条件 .     

大肠杆菌F18菌毛操纵子全基因克隆、表达及生物学活性

利用PCR技术以猪产肠毒素大肠杆菌F18标准菌株107/86和2134P基因组DNA为模板成功地扩增出编码F18ab和F18ac完整菌毛操纵子fed基因。将它们分别克隆入表达质粒载体pET-22b( ),结合酶切和核苷酸序列分析证明了PCR预期扩增产物的正确性。然后将克隆的重组载体DNA转化至大肠杆菌BL21(DE3),构建和筛选出分别含F18ab和F18ac完整fed基因的重组菌,经过IPTG诱导表达,在电镜下观察到上述两种重组菌能分别大量表达F18ab和F18ac菌毛。用热抽提法提纯其诱导表达的F18ab和F18ac菌毛,经SDS-PAGE电泳和考马斯亮蓝染色发现提纯后菌毛获单一分子量约为15kDa蛋白条带,免疫家兔后制备出高效价的兔抗血清,玻板凝集试验和Western blot结果表明:体外诱导表达的F18ab和F18ac菌毛具有和野生F18菌毛相同的抗原性。用表达F18ab和F18ac菌毛的上述2株重组菌分别进行小肠上皮细胞体外吸附试验和吸附抑制试验,结果表明:2株重组菌和野生菌株一样具有较强的粘附易感仔猪小肠上皮细胞的能力,而用表达F18ab和F18ac重组菌提纯的菌毛制备出兔抗血清都能有效地抑制上述重组菌或野生菌株对易感仔猪小肠上皮细胞的吸附结合。     

Expression and purification of recombinant human interleukin-18 protein using a yeast expression system

Interleukin-18 (IL-18) has been reported to exert significant immunoregulatory effects on inhibiting tumor growth through stimulating natural killer (NK) cell cytotoxicity and promoting production of several cytokines, including interferon-gamma (IFN-gamma) and granulocyte/macrophage colony-stimulating factor (GM-CSF). Therefore, IL-18 might serve as a potential therapeutic target for cancer treatment. However, the resource of this protein limits its availability for the clinical practice. The purpose of this study was to express and purify recombinant human (h) IL-18 protein using a yeast expression system. We reported here that hIL-18 gene was cloned into pPICZaC vector for expressing a recombinant hIL-18 protein using a yeast expression system. The recombinant hIL-18 protein was purified using centrifugal filter devices, hydrophobic chromatography, and anion exchange chromatography. The yield and purity of the recombinant hIL-18 reached 45.1% and 97.6%, respectively. This recombinant hIL-18 was shown to induce IFN-gamma production by human peripheral blood mononuclear cells (PBMCs) and enhance NK cell cytotoxicity synergistically with IL-2. Furthermore, these recombinant hIL-18-induced effects were the same as those by standard hIL-18. Therefore, the yeast expression system used in this study provides a useful method to produce large-scale of hIL-18 for the clinical application.     

蛇毒半胱氨酸蛋白酶抑制剂的酵母表达及其对B16细胞体外侵袭的抑制作用*

目的：建立毕赤酵母表达质粒pPICZαA-cystatin,转化酵母细胞生产重组蛋白,探讨蛇毒半胱氨酸蛋白酶抑制剂（sv-cystatin）对肿瘤侵袭的生物学作用。方法：利用PCR扩增技术从pUC18/sv-cystatin质粒中扩增sv-cystatin cDNA并克隆至酵母表达载体pPICZαA上,构建重组质粒pPICZαA-cystatin电激转化Pichia pastori酵母细胞GS115,经1%甲醇诱导获得稳定表达的重组蛋白,改良Boyden小室分析重组sv-cystatin蛋白处理对B16F1细胞体外侵袭力的影响。结果：SDS-PAGE检测和Western blot分析显示分泌表达的sv-cystatin重组蛋白相对分子量约为14 kD,摇瓶发酵每升发酵培养上清可获得16 mg的重组蛋白,经亲和层析纯化获得的sv-cystatin重组蛋白具有抑制木瓜蛋白酶的活性。改良Boyden小室实验结果显示：经0.5mg/ml浓度的重组蛋白处理的B16F1细胞穿过Matrigel的细胞数明显低于对照组(52.60±4.58,106±5.9,P<0.01) ,抑制率为50%。结论：成功实现sv-cystatin的酵母表达,初步证明sv-cystatin重组蛋白可抑制小鼠B16F1细胞体外侵袭作用。     

水稻转录因子OsBP-73靶基因的初步筛选

OsBP-73是用酵母单杂交系统,以水稻蜡质基因(Wx)的顺式作用元件为诱饵,从水稻cDNA表达文库中筛选获得的转录因子。本文以OsBP-73基因为例介绍了用"下拉"(pull-down)方法筛选转录因子靶基因的一般步骤。将含有该基因DNA结合功能域的cDNA片段构建到原核表达载体上,并在大肠杆菌中诱导表达获得其蛋白p73。用纯化后的p73蛋白通过"下拉"实验对OsBP-73靶基因进行初步筛选,获得了22个阳性克隆,为进一步研究转录因子OsBP-73参与的水稻转录调控网络提供依据。     

Development of a dual recombinant vaccine to protect small ruminants against peste-des-petits-ruminants virus and capripoxvirus infections

A recombinant capripoxvirus vaccine containing a cDNA of the peste-des-petits-ruminants virus (PPRV) fusion protein gene was constructed. A quick and efficient method was used to select a highly purified recombinant virus clone. A trial showed that a dose of this recombinant as low as 0.1 PFU protected goats against challenge with a virulent PPRV strain.     

苜蓿夜蛾中肠丝氨酸蛋白酶cDNA的克隆、序列分析及原核表达

【目的】本研究旨在从苜蓿夜蛾Heliothis viriplaca中肠克隆出丝氨酸蛋白酶（serine protease, SP）基因的cDNA序列,测定原核表达后的蛋白经纯化及复性后的活性。【方法】运用RT-PCR和cDNA末端快速扩增方法（rapid amplification of cDNA ends, RACE）克隆苜蓿夜蛾幼虫中肠丝氨酸蛋白酶cDNA全序列,用大肠杆菌Escherichia coli表达系统进行表达。重组蛋白经纯化后,利用梯度透析法进行复性,以BApNA为底物,进行活性测定。【结果】克隆获得的苜蓿夜蛾中肠丝氨酸蛋白酶基因命名为HvSP（GenBank登录号：JX866720）,该基因全长880 bp,开放阅读框长762 bp,编码254个氨基酸,推测分子量和pI值分别为26.9 kDa和9.49。由HvSP推导的氨基酸与鳞翅目昆虫SP氨基酸序列的一致性在52%~95%之间,其中与棉铃虫Helicoverpa armigera SP（GenBank登录号：CAA72962）的氨基酸序列一致性最高,达95%。成功构建重组载体pET21b-HvSP进行原核表达,Western-blot鉴定确定为目的蛋白。蛋白可溶性分析发现重组蛋白为包涵体。在Glycine-NaOH缓冲液中,当pH为10.0时,复性的重组蛋白活性达到最高,为35.74 U/mL。【结论】本研究在苜蓿夜蛾体内获得了一个新的丝氨酸蛋白酶基因,且原核表达后的重组蛋白经过变性、纯化及复性后具有活性。该结果为进一步研究丝氨酸蛋白酶在鳞翅目昆虫体内的生理功能奠定了基础。     

Caenorhabditis elegans has two genes encoding functional d-aspartate oxidases

Four cDNA clones that were annotated in the database as encoding d-amino acid oxidase (DAAO) or d-aspartate oxidase (DASPO) were isolated by RT-PCR from Caenorhabditis elegans RNA. The proteins (Y69Ap, C47Ap, F18Ep, and F20Hp) encoded by the cloned cDNAs were expressed in Escherichia coli as recombinant proteins with an N-terminal His-tag. All proteins except F20Hp were recovered in the soluble fractions. The recombinant Y69Ap has functional DAAO activity, as it can deaminate neutral and basic d-amino acids, whereas the recombinants C47Ap and F18Ep have functional DASPO activities, as they can deaminate acidic d-amino acids. Additional experiments using purified recombinant proteins revealed that Y69Ap deaminates d-Arg more efficiently than d-Ala and d-Met, and that C47Ap and F18Ep show distinct kinetic properties against d-Asp, d-Glu, and N-methyl-d-Asp. This is the first time that cDNA cloning of invertebrate DAAO and DASPO genes has been reported. In addition, our study reveals for the first time that C. elegans has at least two genes encoding functional DASPOs and one gene encoding DAAO, although it had previously been thought that organisms only bear one copy each of these genes. The two C. elegans DASPOs differ in their substrate specificities and possibly also in their subcellular localization.     

人钙调素在大肠杆菌中的表达、纯化及其活性研究

利用基因重组技术,将经PCR扩增获得的人钙调素基因(hCaMcDNA)插入质粒pBV220,构建重组表达载体hCaM/pBV220,用酶切、DNA测序、PCR扩增鉴定阳性克隆.阳性重组子在大肠杆菌DH5α中经温度诱导可高效表达CaM蛋白,经15%SDS-PAGE分析,可观察到一与CaM分子量相符(约17kD)的诱导表达条带,其表达量占菌体蛋白总量20%,并主要以可溶性形式表达.Westernblot结果证实,17kD的表达条带可与标准鼠抗人CaM单克隆抗体起特异反应.用Pheny1-SepharoseCL-4B疏水亲和层析法纯化重组菌超声上清表达产物,每1L菌液可获CaM纯品3～4mg.重组人CaM(rhCaM)与牛脑CaM的氨基酸组成基本一致.生物活性测定结果提示,rhCaM具有激活NAD激酶的活性,其激活程度与标准人脑CaM几乎一致.     

流行性乙型脑炎病毒E基因的克隆、表达及初步应用

参照GenBank中的日本乙型脑炎病毒(Japanese encephalitis virus,JEV)SA14-14株序列设计了一对特异性引物,用PCR方法从SA14-14扩增E基因全长,然后克隆到pMD18-T载体中,转化宿主菌DH5a,提取阳性克隆质粒进行双酶切鉴定,将目的片段定向克隆到pET32a( )中,转化入BL21(DE3),经IPTG诱导可表达分子量约73ka的蛋白,Western blotting试验呈阳性,表明E基因得到表达。以纯化的表达产物为核心抗原,猪抗JEV血清为一抗,HRP标记羊抗猪IgG抗体为二抗建立间接ELISA方法,并初步检测了一些血清样品,结果提示表达的蛋白具有很好的应用开发价值。     

肿瘤坏死因子相关的凋亡诱导配体(TRAIL)cDNA的克隆、表达和活性测定

肿瘤坏死因子相关的凋亡诱导配体 (TRAIL)能选择性诱导肿瘤细胞凋亡 .为利用基因工程技术获得重组TRAIL蛋白可溶性片段 (sTRAIL) ,设计 1对引物 .利用PCR技术特异性扩增出sTRAIL的cDNA ,克隆于质粒pGEM 3Zf( )的EcoRⅠ和PstⅠ位点 .经测序证明序列正确后克隆于表达质粒pBV2 2 0的EcoRⅠ和PstⅠ位点 ,转化大肠杆菌DH5α .转化菌株经温度诱导 ,SDS PAGE检测和Western印迹鉴定 ,获得重组sTRAIL的高水平非融合表达菌株 .表达量占菌体总蛋白的 2 0 % .对其表达产物进行了初步纯化 ,SDS PAGE结果显示纯度可达 90 %以上 .用L92 9细胞测定其生物学活性表明 ,重组蛋白在体外能明显诱导肿瘤细胞凋亡     

中华鲟Vtg间接竞争ELISA检测方法的建立和应用

卵黄蛋白原(Vitellogenin, Vtg)被认为是一种理想的雌激素和类雌激素标志物, 通过建立一种中华鲟Acipenser sinensis血浆Vtg水平的检测方法, 进而开发一项中华鲟性腺成熟度的诊断技术。首先通过RACE-PCR方法扩增得到中华鲟vtg基因cDNA序列, 氨基酸序列分析预测其蛋白分子量大小为196 kD。构建Vtg功能区段融合原核表达载体pET32a(+)-vtg并表达纯化重组蛋白, 并以重组蛋白免疫兔子获得多克隆抗血清, Western blotting检测显示抗血清的特异性较好。以纯化的中华鲟重组Vtg蛋白为抗原, 中华鲟Vtg多克隆抗血清为抗体, 建立了中华鲟血浆Vtg的间接竞争酶联免疫检测方法(ELISA), 标准曲线线性回归方程为y= –0.2916x+0.6794, 相关系数R2为0.9976。该方法检测的灵敏度为4.12 μg/mL, 最低检测限为0.3 μg/mL, 批内和批间变异系数分别为2.52%和3.42%。通过对不同发育时期雌性中华鲟血样检测, 表明此ELISA方法可初步用于雌性中华鲟性腺发育时期监测。