Trypsin Inhibitor from Poecilanthe parviflora Seeds: Purification, Characterization, and Activity Against Pest Proteases

Plants synthesize a variety of molecules, including proteinaceous proteinase inhibitors, to defend themselves of being attacked by insects. In this work, a novel trypsin inhibitor (PPTI) was purified from the seeds of the native Brazilian tree Poecilanthe parviflora (Benth) (Papilioinodeae, Leguminosae) by gel filtration chromatography on a Sephadex G-100 followed by Superdex G75 chromatography (FPLC), Sepharose 4B-Trypsin column, and fractionated by reversed-phase HPLC on a C-18 column. SDS-PAGE showed that PPTI consisted of a single polypeptide chain with molecular mass of about 16 kDa. The dissociation constant of 1.0 x 10(-7) M was obtained with bovine trypsin. PPTI was stable over a wide range of temperature and pH and in the presence of DTT. The N-terminal sequence of the PPTI showed a high degree of homology with other Kunitz-type inhibitors. Trypsin-like activity in midguts of larval Diatraea saccharalis, Anagasta kuehniella, Spodoptera frugiperda, and Corcyra cephalonica were substantially inhibited by PPTI.     

Kunitz型丝氨酸蛋白酶抑制剂IsKuI-1的原核表达、纯化及活性研究

目的:原核表达并制备重组蜱kunitz型丝氨酸蛋白酶抑制剂IsKuI-1,检测其抗凝血及抑制蛋白酶活性。方法:构建pET32a-sumo/IsKuI-1原核表达质粒,并转入到E. coli BL21(DE3)中,用IPTG诱导表达。表达产物经Ni-NTA亲和层析,在层析柱上用SUMO蛋白酶切割融合伴侣,纯化后得到重组目的多肽rIsKuI-1。用PT及aPTT法检测重组目的多肽的抗凝活性,发色底物法检测rIsKuI-1对丝氨酸蛋白酶的抑制活性。结果:用原核表达系统获得了rIsKuI-1,其无延长PT及aPTT活性,对人中性粒细胞弹性蛋白酶具有较好的抑制活性(IC50=1.83μM),且特异性强。结论:IsKuI-1是一种活性较好的人NE抑制剂。因此为进一步探讨rIsKuI-1的生物学功能及其作为新药开发应用奠定了基础。     

Molecular Cloning of cDNA That Encodes Chymotrypsin Inhibitor ECI from Erythrina variegata Seeds and Its Expression in Escherichia coli

Synthetic oligonucleotides representing all possible sequences of the N-terminal and internal amino acid sequences of the chymotrypsin inhibitor ECI from Erythrina variegata seeds were used to generate a probe specific for ECI-related sequences by the polymerase chain reaction on the E. variegata genomic DNA. A lambda phage cDNA library constructed from poly(A⁺) RNA from maturing seeds was screened with the ECI gene thus obtained as a probe and characterized by DNA sequencing. The cloned ECI cDNA comprised 737 nucleotides and one open reading frame that encoded a polypeptide chain of 203 amino acids including a signal peptide composed of 24 amino acids. An expression plasmid was designed for export of the recombinant inhibitor into the periplasm. For this purpose, the cDNA fragment encoding matured ECI was ligated into the NcoI and BamHI sites following the pel B signal sequence in the expression vector pET-22b and expressed in Escherichia coli BL21 (DE3). However, this attempt failed as the recombinant inhibitor caused the formation of inclusion bodies in E. coli cells as a heterologous preprotein (SR-ECI), with the pel B upstream leader. SR-ECI was made soluble and renatured by refolding and reoxidation, and subsequently processed with pronase to give rise to recombinant ECI (R-ECI) that had an extra methionine residue attached to the N-terminal amino acid of ECI. Purified R-ECI inhibited chymotrypsin almost as strongly as authentic ECI.     

Expression of Winged Bean Basic Agglutinin in Spodoptera frugiperda Insect Cell Expression System

In this paper we report the successful expression of the winged bean basic agglutinin (WBA I) in insect cells infected with a recombinant baculovirus carrying the WBA I gene and its characterization in terms of its carbohydrate binding properties. The expressed protein appears to have a lower molecular weight than the native counterpart which is consistent with the lack of glycosylation of the former. Moreover, the expressed protein maintains its dimeric nature. Hence, a role for glycosylation in modulation of dimerization of WBA I is ruled out unlike Erythrina corallodendron (EcorL). Despite this the protein is active, with its sugar specificity unaltered.     

Purification and Characterization of the Acidic Lectin from Winged Bean Seeds

Winged bean acidic lectin was purified by DEAE-Sephadex A-50 and affinity chromatography on N-acetylgalactosamine-agarose gel. The purified lectin was a glycoprotein homogeneous on polyacrylamide gel electrophoresis, isoelectric focusing, and gel filtration. The molecular weight of the lectin was 52,000 by gel filtration, and SDS-polyacrylamide gel electrophoresis gave a single component of molecular weight of 27,000. Its isoelectric point was 5.5. The acidic lectin was rich in acidic amino acids, and contained 2mol of methionine but no cystine. It also agglutinated both trypsinized and untreated human erythrocytes (types A, B, AB and O), but not rabbit erythrocytes. The hemagglutination was inhibited by d-galactose and related sugars. Modification of the acidic lectin with N-bromosuccinimide caused a concomitant loss of the hemagglutinating activity with oxidation of tryptophan residue. The acidic lectin was immunologically different from the purified winged bean basic lectin by double immunodiffusion using antiserum raised against the basic lectin.     

鹰嘴豆种子胰蛋白酶抑制剂的分离纯化与鉴定

为了寻找具有药物作用的天然胰蛋白酶抑制物,采用硫酸铵分级沉淀、离子交换层析(DEAE-纤维素52)及Sephadex G-100凝胶层析等方法, 从鹰嘴豆种子中分离出一种鹰嘴豆胰蛋白酶抑制剂（CPTI）. 研究表明：CPTI对胰蛋白酶有较强的抑制作用,抑制率达80%,而对胰凝乳蛋白酶抑制作用较弱,抑制率为32%, 对胃蛋白酶、木瓜蛋白酶及枯草杆菌蛋白酶均无抑制作用; 用SDS-PAGE测得CPTI近似分子质量为25.7 kD; CPTI具有较高的热稳定性,在100 ℃下加热60 min,对胰蛋白酶活性仍保持78%抑制率; Lineveaer-Burk作图得知该抑制剂属竞争性抑制类型. 动力学测定显示,来自鹰嘴豆中的CPTI对胰蛋白酶的抑制作用常数(Ki)为3.99×10^-7 mol/L.     

人vasostatin的克隆、表达、纯化及活性检测

从成人肝脏cDNA文库中,PCR扩增得到人vasostatin基因编码区序列,将此序列插入原核表达载体pQE30进行表达,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)测定表明产物以包涵体形式存在,表达量占菌体总蛋白量的50%以上.包涵体洗涤后溶于8 mol/L尿素溶液,在变性条件下通过镍-氨三乙酸(Ni-NTA)金属螯合亲和层析柱进行纯化后,再经透析进行复性.N端氨基酸序列、分子质量、等电点等理化指标的测定结果与理论值相符.用内皮细胞增殖试验、内皮细胞迁移试验以及鸡胚尿囊膜血管生成试验等方法进行活性检测,证实复性的表达产物具有抑制内皮细胞增殖和迁移、抑制鸡胚尿囊膜血管生成的功能.     

牦牛白细胞介素2(IL2)基因cDNA 的分子克隆和表达研究

目的克隆牦牛免疫基因并研究其免疫特性。方法以伴刀豆球蛋白A（ConA）激活的体外培养的牦牛血液淋巴细胞,提取激活淋巴细胞的总RNA,用RT—PCR方法,从中克隆出白细胞介素2cDNA,连接到T载体上测序,并亚克隆到pC,EX4T-1表达质粒,在大肠杆菌进行了重组表达,纯化融合表达YIL2产物,MTT比色法测定其体外刺激牦牛血液淋巴母细胞增殖的免疫活性。结果YIL2cDNA序列分析显示：在其编码区442位点的一个碱基发生突变（由C突变为T）,从而导致终止密码子（TAA）出现,使YIL2蛋白表达提前终止,与其它牛IL2蛋白相比少了8个氨基酸。MTT比色法测定结果表明重组牦牛IL2蛋白体外能显著促进牦牛淋巴母细胞的增殖。结论本实验成功克隆了牦牛IL2基因,其原核表达产物具有显著的免疫活性,这为研制新型免疫增强剂来提高牦牛对各种疾病的抵抗力,增强牦牛疫苗的免疫保护率奠定了基础。     

丝氨酸蛋白酶抑制剂Ea的表达纯化与活性分析

Ea是一种植物来源的丝氨酸蛋白酶抑制剂,分子量为18kD。利用其与丝氨酸蛋白酶家族成员的结合特性,可用于丝氨酸蛋白酶的结构与功能研究,也可作为亲和层析的配体而用于丝氨酸蛋白酶的纯化。将Ea基因插入大肠杆菌表达载体pET11a,在BL21(DE3)菌中以包涵体形式表达出重组蛋白质,表达量可占菌体蛋白质总量的30%。将包涵体变性、复性,得到具有天然抑制活性的rEa。经两步纯化所得rEa的纯度达到967%以上。活性分析表明,rEa对胰蛋白酶和人组织型纤溶酶原激活剂均有抑制作用。制备成rEaSepharose亲和柱可有效结合胰蛋白酶。     

人TIMP—3cDNA的克隆,表达及其抗血管生成作用

从人新鲜的胎盘组织中提取总ＲＮＡ,以ＲＴ－ＰＣＲ法获取了人组织金属蛋白酶抑制－３成熟蛋白的ｃＤＮＡ。序列分析表明,ＴＩＭＰ－３成熟蛋白含有１８８个氨基酸残基,其中１２个半胱氨酸残基在ＴＩＭＰ家族中高度保守。将人ＴＩＭＰ－３ｃＤＮＡ插入含７启动子的质粒ｐＥＴ－２４构建表达质粒ｐＥＴ－ＴＩＭＰ３,转化大肠杆菌ＢＬ２１,筛选表达菌株ＢＬＴＩＭＰ３。     

人Mn—SOD cDNA的克隆及高效表达

用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）,以人肝细胞株（Ｌ０２）总ＲＮＡ为模板,扩增了人锰超氧化物歧化酶（ｈＭｎ－ＳＯＤ）的ｃＤＮＡ。重组到Ｔ７启动子控制下的表达载体ｐＥＴ－２４ａ（＋）中,构建表爱质粒ｐＥＴ－ＭｎＳＯＤ,并转化大肠杆菌ＢＬ２１（ＤＥ３）。ＳＤＳ－ＰＡＧＥ及蛋白质印迹分析表明,经１ｍｍｏｌ／Ｌ异丙基硫代－β－Ｄ－半乳糖苷（ＩＰＴＧ）诱导后,可高效表达一分子量为２２ｋＤ的蛋白质,与抗人     

小鼠canstatin C端片段基因的克隆及其在大肠杆菌中的表达

为了构建小鼠canstatinC端片段的原核表达载体并在大肠杆菌中表达。以小鼠肝脏组织总RNA为模板,通过RT-PCR扩增小鼠canstatinC端片段(mCan-C)基因,克隆到pMD18-T载体中并进行序列分析。将mCan-C基因定向克隆于原核表达载体pET30a(+)中,构建表达载体pET／mCan-C,转化大肠杆菌BL21(DE3),IPTG诱导表达。结果表明,小鼠canstatinC端片段的cDNA长度为399bp,含有1个终止密码,编码132个氨基酸,与已知的人canstatinC端片段氨基酸的同源性为61％。IPTG诱导mCan-C在大肠杆菌E．coliBL21中表达,表达量约占菌体总蛋白量的28％,重组蛋白主要以包涵体形式存在。首次克隆了小鼠canstatinC端片段的cDNA,IPTG诱导mCan-C在大肠杆菌E．coliBL21中高效表达。小鼠canstatinC端片段的cDNA序列已收入GenBank,接受号为:AY502947。     

大鼠肝脏F抗原基因的克隆、表达及产物纯化

肝脏F抗原是一种新型的能直接反映肝损伤程度的血清学标志,它是1968年由美国学者Fravi从小鼠肝中分离出来的[1].F抗原是存在于哺乳动物肝细胞质中的一种水溶性不稳定的单链蛋白质,相对分子量为43kD[2],正常肝组织中的F抗原含量是血清中的1370倍,只有肝细胞被破坏时,F抗原才释放     

人铜锌超氧化物歧化酶cDNA的克隆,测序及表达

用逆转录聚合酶链反应（ＲＴＰＣＲ）,以人胎肝组织总ＲＮＡ为模板,扩增了人铜锌超氧化物歧化酶（ｈＣｕ,ＺｎＳＯＤ）的ｃＤＮＡ,并进行序列分析,将该ｈＣｕ,ＺｎＳＯＤｃＤＮＡ重组到Ｔ７启动子控制下的分泌型表达载体ｐＥＴ２２ｂ（＋）中,构建表达质粒ｐＥＴＳＯＤ,并转化大肠杆菌ＢＬ２１（ＤＥ３）。ＳＤＳＰＡＧＥ及蛋白质印迹分析表明,经１ｍｍｏｌ／Ｌ异丙基硫代βＤ半乳糖苷（ＩＰＴＧ）诱导后,可高效表达一分子量为１９ｋＤ的蛋白质,与抗人ＳＯＤ多抗有特异的免疫反应,表达量约为菌体总蛋白质的３０％,具有特异性ＳＯＤ酶活性,酶活力可达１７９７ｕ／ｍｌ培基。     

番茄GDP-D-甘露糖焦磷酸化酶cDNA的克隆、表达及定位

GDP-D-甘露糖焦磷酸化酶催化GDP-D-甘露糖的合成,是植物抗坏血酸生物合成途径中上游的关键酶。以马铃薯GDP-D-甘露糖焦磷酸化酶cDNA序列为信息探针,在GenBank dbEST数据库中找到65条高度同源的番茄EST序列,通过序列拼接及RACE-PCR得到了番茄该基因的全长cDNA序列,命名为LeGMP。LeGMP与马铃薯GDP-D-甘露糖焦磷酸化酶cDNA序列一致率为96％,推导的氨基酸序列与马铃薯、烟草、紫苜蓿、拟南芥的GDP-D-甘露糖焦磷酸化酶基因的一致率分别为99％、97％、91％、89％。经Northern杂交分析,LeGMP在番茄根、茎、叶、花、果实中都有表达,但表达水平有差异。利用75个番茄远缘杂交重组系（IL系）将LeGMP定位在番茄第3染色体上的D区段（3-D）。     

棉花GhDHAR2基因克隆、功能序列分析及原核表达

通过RT-PCR方法从棉花纤维组织中克隆得到脱氢抗坏血酸还原酶基因GhDHAR2的cDNA,该基因开放阅读框为639 bp,编码212个氨基酸的蛋白质。同源性序列对比分析显示,GhDHAR2蛋白具有较高的保守性,具有典型的功能结构域,包括GST-N家族和GST-C-DHAR家族的功能结构域;进化树分析显示GhDHAR2和拟南芥AtDHAR2在进化关系上较近。将GhDHAR2基因连接到原核表达载体pET-28a中,将重组载体pET28a-GhDHAR2转入到表达菌株BL21(DE3)中,通过IPTG诱导表达出重组GhDHAR2蛋白,SDS-PAGE凝胶电泳分析显示重组蛋白大小约为28 kD,诱导表达的重组蛋白具有较高的DHAR活性。首次克隆了棉花GhDHAR2基因,通过结构域分析其可能的作用,并成功进行蛋白体外表达及酶活性分析。     

萌发绿豆中水解大豆胰蛋白酶抑制子蛋白酶的纯化及固定化

大豆是豆类植物中最早发现存在蛋白酶抑制子的 ,由于其存在影响了豆类的利用价值 ,因此研究人员一直在寻找着解决办法。采用加热处理方法不能彻底钝化豆类蛋白的蛋白酶抑制子活性 ,且豆类蛋白的含硫氨基酸主要存在于各类蛋白酶抑制子中 ,从豆类蛋白中除去抑制子蛋白将大大降低其营养效价。本研究的目的是试图寻找一种可在常温下降解豆类胰蛋白酶抑制子的蛋白酶 ,从而钝化豆类的胰蛋白酶抑制活性。在前期工作中 ,我们发现枯草杆菌蛋白酶 (Ｓｕｂ ｔｉｌｉｓｉｎ)可在在常温下降解花生及大豆胰蛋白酶抑制剂[1] ,近期我们的研究表明 ,Ａｌｃａ…     

非洲绿猴层黏连蛋白受体的克隆、表达及纯化

目的:克隆、表达非洲绿猴层黏连蛋白受体(LR),对该受体蛋白进行纯化、复性研究。方法:采用RT-PCR从Vero-E6细胞总RNA中扩增非洲绿猴LR的cDNA序列,克隆到pGEM-TEasy载体上;将LR编码区插入到表达载体pET22b( ),转化大肠杆菌BL21(DE3);用IPTG进行诱导表达;用镍亲和柱进行亲和纯化;采用分部透析方法进行复性。结果:非洲绿猴LR基因序列与人LR的基因编码序列有16个碱基差异,但蛋白序列只有1个氨基酸的改变。LR蛋白以包涵体形式表达。采用分部透析方法可使部分蛋白得到复性。结论:克隆了首个非洲绿猴的相对分子质量为37000的LR的基因,与人LR基因序列有16个碱基差异,但其中15处都为同义突变,所以二者的蛋白质序列只有1个氨基酸改变,即293位D→E,提示LR在进化中具有很大的保守性。     

Cloning, Expression, and Mapping of GDP-D-mannose Pyrophosphorylase cDNA from Tomato (Lycopersicon esculentum)

GDP-D-mannose pyrophosphorylase (GMP, EC 2.7.7.22) catalyzes the synthesis of GDP-D-mannose and represents the first committed step in plant ascorbic acid biosynthesis. Using potato GMP cDNA sequence as a querying probe, 65 highly homologous tomato ESTs were obtained from dbEST of GenBank and the putative cDNA sequence of tomato GMP was assembled. The full-length GMP cDNA of tomato was cloned by RACE-PCR with primers designed according to the assembled cDNA sequence. The full-length cDNA sequence contained a complete open reading frame (ORF) of 1 086 bp, which encoded 361 amino acid residues. This gene was designated as LeGMP (GenBank accession No. AY605668). Homology analysis of LeGMP showed a 96% identity with potato GMP and the deduced amino acid showed 99%, 97%, 91% and 89% homology with GMP from potato, tobacco, alfalfa and Arabidopsis thaliana, respectively. Northern blot analysis showed that LeGMP was constitutively expressed in roots, stems, leaves, flowers and fruits of tomato; but the expression levels varied.LeGMP was mapped to 3-D using 75 tomato introgression lines (ILs), each containing a single homozygous RFLP-defined chromosome segment from the green-fruited species Lycopersicon pennellii.