Cloning and expression of a sialic acid-binding lectin from the snail Cepaea hortensis

Highly degenerated gene-specific oligonucleotide primers (GSPs) were constructed from the amino acid sequence of tryptic fragments produced from the purified sialic acid-specific lectin of the garden snail Cepaea hortensis. From the albumin glands, the total RNA or the mRNA was prepared. Combination of a universal primer with the GSPs delivered gene-specific fragments of about 650, 620 and 280 bp by polymerase chain reaction (PCR). These fragments were cloned into the vector pDrive (Qiagen) and sequenced. The resulting cDNA sequence consisted of 744 bp, including an open reading frame of 480 bp. The encoded protein consists of 159 amino acids, including the putative signal sequence peptide. The mature protein should comprise 141 amino acid residues with a calculated molecular mass of 15,529 Da. The expression of the recombinant lectin in Escherichia coli resulted in a soluble protein reacting specifically with rabbit antiserum raised against the native lectin.     

Purification and molecular cloning of a new galactose-specific lectin from <Emphasis Type="Italic">Bauhinia variegata</Emphasis> seeds

A new galactose-specific lectin was purified from seeds of a Caesalpinoideae plant, Bauhinia variegata, by affinity chromatography on lactose-agarose. Protein extracts haemagglutinated rabbit and human erythrocytes (native and treated with proteolytic enzymes), showing preference for rabbit blood treated with papain and trypsin. Among various carbohydrates tested, the lectin was best inhibited by D-galactose and its derivatives, especially lactose. SDS-PAGE showed that the lectin, named BVL, has a pattern similar to other lectins isolated from the same genus, Bauhinia purpurea agglutinin (BPA). The molecular mass of BVL subunit is 32 871 Da, determined by MALDI-TOF spectrometry. DNA extracted from B. variegata young leaves and primers designed according to the B. purpurea lectin were used to generate specific fragments which were cloned and sequenced, revealing two distinct isoforms. The bvl gene sequence comprised an open reading frame of 876 base pairs which encodes a protein of 291 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to have 263 amino acid residues and 28 963 Da in size.     

兔多杀性巴氏杆菌C51-3株黏附蛋白的表达、纯化及其抗原性检测

应用PCR从兔多杀性巴氏杆菌C51-3株基因组DNA中扩增出编码36 kD黏附蛋白的cp36基因, 将其克隆到pMD18-T载体并对插入片段进行测序。以重组质粒pMD18-cp36为模板, 用PCR扩增得到编码信号肽除外的成熟黏附蛋白基因cpm36, 并克隆到原核表达质粒pQE30中, 得到重组质粒pQE30-cpm36, 转化大肠杆菌M15, 在IPTG诱导下表达融合蛋白CPM36, 经Ni2+-NTA亲和层析纯化。DNA测序结果表明cp36基因片段大小为1032 bp, 与已报道的16个血清型多杀性巴氏杆菌cp36基因的核苷酸序列比较, 同源性在76.9%~100%之间。SDS-PAGE结果显示, 表达分子量约为37 kD的带有6×His标签的CPM36蛋白, 与预期分子量相符。Western blotting结果表明, 抗重组蛋白抗体分别能与CPM36蛋白和多杀性巴氏杆菌36 kD蛋白发生特异性反应, 证明原核表达蛋白具有抗原性, 为进一步开展多杀性巴氏杆菌免疫保护性抗原的研究奠定了基础。     

SUA41蛋白表达和纯化及其多克隆抗体制备

旨在制备特异性SUA41多克隆抗体,为深入研究其在植物生长发育中的功能提供有力的分子生物学和生物化学的工具。PCR扩增拟南芥SUA41基因中编码280个氨基酸(401-680位氨基酸)的特异片段,经过GATEWAY的DNA重组技术构建了原核表达载体pDEST17-SUA41,用热休克法转化到E.coliBL21(DE3)star感受态细胞,以异丙基β-D-硫代半乳糖苷(IPTG)诱导表达出6×His-SUA41融合蛋白,用8 mol/L尿素缓冲液溶解包涵体并且经过水逐级去除尿素获得提纯的融合蛋白,并利用Western blotting鉴定确认。融合蛋白经Ni金属螯合柱亲和层析得以纯化,用SDS-PAGE进一步纯化。纯化的融合蛋白经过SDS-PAGE后切胶回收,免疫小白兔,制备多抗血清,然后用Western blotting进行检测,鉴定血清特异性和效价。结果显示,融合蛋白6×His-SUA41免疫兔,产生特异性的SUA41兔抗血清,可以检测到细菌和拟南芥组织中SUA41蛋白。用水提纯变性剂尿素溶解的包涵体蛋白具有可行性。制备的特异性SUA41兔抗血清效价高,能够有效地识别大肠杆菌表达的和拟南芥的SUA41蛋白。在有合适的对照情况下,该兔抗血清可以用于分析植物中SUA41蛋白的功能。     

A normal mucin-binding lectin from the sponge Craniella australiensis

A lectin, Craniella australiensis (CAL), was isolated from sponge C. australiensis by ion-exchange on DEAE-Sephacel and purified by gel filtration on Sephadex G-150 and HPLC on DEAE-5PW. The purified lectin was a trimeric protein as revealed by SDS-PAGE and MALDI-TOF analysis. SDS-PAGE showed that the CAL protein had a molecular mass of 54 kDa, and consisted of three 18 kDa subunits. Gel filtration of purified lectin on Sephadex G-200 indicates that it exists as a 54 kDa protein in its native state. The amino acid composition was rich in Thr and Glx. CAL was found to agglutinate native and trypsinized human A, B erythrocytes, and agglutinate native erythrocytes of mouse, sheep, rabbit and chicken, and trypsinized erythrocytes of sheep and rabbit. The hemagglutination activity was inhibited by glycoproteins such as PSM and asialo-PSM, but not by any of the monosaccharides tested. The activity was stable between 20 and 70 degrees C. Significant CAL activity was observed between pH 5 and 8. The lectin reaction is independent of the presence of divalent cations Ca2+ and Mg2+. The sequence of N-terminal residues of CAL was determined as TSSCQSIVVE. The lectin showed a potent mitogenic response towards BALB/c splenocytes.     

Cloning and Sequencing of a Lectin Protein Gene from the Roots of Sophora flavescens (in English)

A lectin protein（SFL） with molecular weight about 32 kD which markedly agglutinated rabbit and human red blood cells was purified from the roots of Sophora flavescens Ait. This protein, and apparently inhibited the growth of Fusarium vasinfectum Atk., Gibberella saubinetii (Mont.) Sacc., and Piricularia oryzae Cav. A set of degenerate PCR primer was synthesized according to the N-terminal sequence of the purified protein. The full-length cDNA coding the lectin was cloned by RT-PCR and 5′-RACE and sequenced (GenBank AF285121). The deduced amino acid sequence indicates that a preprotein with 284 amino acid residues is firstly translated and then processed to a mature protein with 254 amino acids. A N-Glycosylation site is the Asn 182 residue.      

Molecular characterization of a new lectin from the marine alga Ulva pertusa

A new lectin, named UPL1, was purified from a green alga Ulvapertusa by an affinitychromatography on the bovine-thyroglobulin-Sepharose 4B column. The molecular mass of the algal lectinwas about 23 kD by SDS-PAGE, and it specifically agglutinated rabbit erythrocytes. The hemagglutinatingactivity for rabbit erythrocytes could be inhibited by bovine thyroglobulin and N-acetyl-D-glucosamine. Thelectin UPL1 required divalent cations for maintenance of its biological activity, and was heat-stable, and hadhigher activity within pH 6-8. The N-terminal amino acid sequence of the purified lectin was determined(P83209) and a set of degenerate primers were designed. The full-length cDNA of the lectin was cloned byrapid amplification ofcDNA ends (RACE) method (AY433960). Sequence analysis of upll indicated it was! 084 bp long, and encoded a premature protein of 203 amino acids. The N-terminal sequence of the matureUPL1 polypeptide started at amino acid 54 of the deduced sequence from the cDNA, indicating 53 aminoacids lost due to posttranslational modification. The primary structure of the Ulva pertusa lectin did not showamino acid sequence similarity with known plant and animal lectins. Hence, this protein may be the paradigmof a novel lectin family.     

凋亡抑制因子Survivin在大肠杆菌TB1中的表达纯化及鉴定

 本文旨在克隆凋亡抑制因子Survivin基因,并在大肠杆菌中进行可溶性表达与初步纯化. 采用RT-PCR法,扩增人凋亡抑制因子survivin cDNA,并克隆入原核表达载体pMAL p2X中,转化TB1大肠杆菌感受态细胞.经0.3 mmol/L IPTG诱导2 h后,收集菌体蛋白,进行SDS-PAGE、ELISA及Western 印迹鉴定. 实验获得凋亡抑制因子survivin编码区cDNA,以构建的原核表达载体pMAL-p2X survivin转化菌株后,可表达凋亡抑制因子survivin和麦芽糖结合蛋白(MBP)的融合蛋白,相对分子质量(Mr) 为58 000.并成功利用Factor Xa将融合蛋白裂解开.ELISA和Western 印迹表明,融合蛋白能与抗凋亡抑制因子survivin单克隆抗体特异性结合.获得的凋亡抑制因子survivin全长cDNA可在大肠杆菌TB1中以MBP survivin融合蛋白的形式表达,成功地将survivin目的蛋白和MBP蛋白分离,为深入研究survivin的结构和功能奠定了基础.     

Isolation and characterization of a full-length cDNA encoding the 55-kDa rabbit zona pellucida protein.

A full-length cDNA (rc55) encoding the major rabbit zona pellucida (ZP) glycoprotein (55 kDa) has been cloned and sequenced. A lambda gt11 expression library was constructed using poly(A)+ mRNA isolated from sexually immature rabbit ovaries which contain large numbers of developing follicles. The rc55 cDNA was identified using affinity purified polyclonal antibodies specific to ZP antigens which are shared among mammalian species. The deduced amino acid sequence of the full-length rc55 clone was matched to the NH2-terminal 25-amino acid sequence obtained for this protein. The predicted amino acid sequence consists of 540 amino acids including a putative signal peptide of 18-24 residues and six potential N-glycosylation sites. The cDNA hybridizes to a 2000-base species of mRNA from rabbit ovary which is not detected in other rabbit tissues. The message is present early in ovarian follicular development and is approximately 600-fold greater in sexually immature as compared with sexually mature rabbit ovaries. This cDNA was expressed as a cro-beta-galactosidase fusion protein using the pEX expression vector. Antibodies against native rabbit ZP, affinity-purified on the recombinant 55-kDa ZP protein, were found to recognize the native rabbit ZP glycoprotein, indicating partial conservation of native epitopes in the expressed recombinant protein.     

BjαIT的表达、抗血清制备及scFv基因库的构建

根据成熟昆虫神经毒素BjαIT的氨基酸序列,人工合成毒素基因,并克隆至大肠杆菌表达载体pPET-30a（＋）。在IPTG的诱导下,神经毒素在大肠杆菌中融合表达,表达产物经镍亲和层析纯化,纯化蛋白免疫BALB／c小鼠,制备了特异性较高的抗血清,抗体滴度达1：512,000。提取免疫小鼠脾细胞总RNA,通过RT-PCR分别扩增重链可变区（VH）及轻链可变区（VL）基因片段,在连接肽linker的连接下成功构建了包含抗BjαIT的全长scFv基因库。本研究为新型蝎昆虫神经毒素BjαIT的检测奠定了基础。     

Isolation and partial characterisation of galactose-specific lectins from African yam beans, Sphenostyles stenocarpa Harms.

A new galactose-specific lectin was isolated from African yam bean (Sphenostyles stenocarpa Harms) by affinity chromatography on galactose-Sepharose 4B. SDS-PAGE analysis resulted in four polypeptide bands of approximately 27, 29, 32 and 34 kDa, respectively. Based on the analysis of carbohydrate content and native PAGE, it is likely that the Sphenostyles lectin is a tetrameric glycoprotein with M(r) of approximately 122 kDa. N-terminal protein sequencing of purified lectins from four different Sphenostyles accessions shows that the four polypeptides have largely identical amino acid sequences. The sequences contain the conserved consensus sequence F-F-LILG characteristic of legume lectins, as well as Phaseolus vulgaris proteins in the arcelin-alpha-amylase inhibitor gene family. The lectin agglutinates both rabbit and human erythrocytes, but with a preference for blood types A and O. Using Western blotting, the lectin was shown to accumulate rapidly during seed development, but levels dropped slightly as seeds attained maturity. This is the first time a lectin has been purified from the genus Sphenostyles. The new lectin was assigned the abbreviation LECp.SphSte.se.Hga1.     

禽多杀性巴氏杆菌P1059成熟黏附蛋白的原核表达、纯化和抗原性检测

目的：建立rCPM36在大肠杆菌中的表达体系,纯化表达产物并检测其抗原性。方法：运用PCR方法从禽多杀性巴氏杆菌国际标准株P1059基因组中扩增出编码36kDa成熟黏附蛋白的cpm36基因,构建原核表达载体pQE30-cpm36,转化到大肠杆菌M15中并诱导表达目的蛋白,用镍离子螯合层析柱纯化目的蛋白及制备其抗体,Western印迹分析其抗原性。结果：SDS-PAGE结果显示目标蛋白以可溶性形式表达在大肠杆菌M15细胞质中,其相对分子质量为37kDa,Western印迹结果表明表达蛋白具有良好的抗原性。结论：成功构建出原核表达载体并实现了目的蛋白表达,用镍离子螯合层析柱纯化得到具有抗原性的蛋白,为进一步开展禽多杀性巴氏杆菌黏附因子和保护性抗原的研究奠定基础。     

Chemical and physicochemical characterization of the sialic acid-specific lectin from Cepaea hortensis

A sialic acid-specific lectin was isolated from the albumin glands of the garden snail Cepaea hortensis by affinity chromatography on fetuin-Sepharose following gel filtration on Superdex 200. The purified native lectin showed a molecular mass of about 95 kDa by gel filtration and 100 kDa by SDS electrophoresis. It was cleaved by boiling in buffer containing SDS in three serological identical bands corresponding to molecular masses of about 24, 20 and 16 kDa, respectively. From these three fragments, only the 24- and the 20-kDa bands were found to be glycosylated. Only the three sugars mannose, galactose and N-acetylglucosamine could be detected in a molar ratio of 3:8.6:2. The oligosaccharide moieties seem to be N- and partially O-glycosidic bound. Isoelectric focusing (IEF) of the purified lectin revealed a heterogeneous pattern with bands in the pH range of 4.3-5.0. Isolated bands of different isoelectric points showed in SDS electrophoresis the same three fragments with molecular masses of 24, 20 or 16 kDa. The heterogeneity of the lectin was revealed either by IEF or amino acid sequencing of internal tryptic peptides.     

A study on renin binding protein (RnBP) in the human kidney

We purified, from human kidney, a protein that reacts with rabbit anti-porcine kidney renin binding protein (RnBP) antiserum by trapping with porcine kidney renin. The purified preparation showed a single protein peak on gel filtration by high performance liquid chromatography (HPLC) and two protein bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The latter two kinds of protein were identified as the porcine renin and human kidney protein from their electrophoretic mobilities and reactivity toward rabbit anti-porcine kidney renin and RnBP antisera. The molecular weights of the purified preparation and the human kidney protein were estimated to be 56,000 by HPLC and 43,000 by SDS-PAGE, respectively. The specific activity of porcine renin in the purified preparation was 8.6 mg angiotensin I per mg of protein per h at 37 degrees C and pH 6.5. This specific activity was about one-fifth that of free porcine renin. Therefore, it is suggested from the reactivity toward the anti-porcine RnBP antiserum and inhibitory action toward porcine renin that the human kidney protein is RnBP and that the human RnBP is purified as a complex with porcine renin.     

海芋凝集素cDNA的分子克隆及其性质预测

用cDNA末端快速扩增-聚合酶链式反应（RACE-PCR）方法克隆了海芋（Alocasia macrorrhiza）凝集素的全长cDNA（GenBank检索号DQ340864）,并用多种生物信息学工具对其性质进行了预测。根据来源于天南星科其他植物的凝集素和类似蛋白的保守区的DNA序列,设计了几个海芋凝集素基因aml特异引物（GSP）。用RNeasy试剂盒从海芋块茎中提取出总RNA,并以此为模板,用SMART^TM RACE cDNA扩增试剂盒提供的经特殊设计的通用引物以及不同的基因特异引物,分别获得海芋凝集素5′-和3′-RACE-PCR扩增片段。这些PCR产物经0．8％琼脂糖凝胶纯化后,分别与T克隆载体pMD 18-T相连,筛选获得阳性克隆并提取质粒,经双酶切和特异引物的PCR验证无误后,进行序列分析。从5′-和3′-RACE-PCR测序结果拼接出全长海芋凝集素cDNA序列,并用新设计自5′-RACE-PCR 5′末端的引物GSP7进行全长3′-RACE-PCR反应,获得全长海芋凝集素cDNA克隆并再次测序验证。这一新克隆的海芋凝集素cDNA的长度为1124核苷酸,分析表明它是一个编码270个氨基酸残基的蛋白质,其等电点为pH 5．7,相对分子量为29．7kD。同源性分析结果表明,海芋凝集素与其他来源于天南星科的甘露糖凝集素以及相似蛋白具有高度同源性。在海芋凝集素序列中发现了2个B型凝集素功能区域和3个甘露糖的结合位点。综合上述信息,认为这一新克隆的海芋凝集素cDNA是一个编码甘露糖识别凝集素的基因序列。     

创伤弧菌vvhA基因的克隆、原核表达系统的构建及鉴定

目的 克隆创伤弧菌（Vibrio vulnificus,Vv）溶细胞素基因（υυhA）,构建原核表达系统并鉴定其表达产物的免疫性.方法 采用PCR技术从Vv GTC333和WZ01株DNA中扩增全长υυhA基因,T-A克隆后测定其核苷酸序列.采用pET32a质粒构建vvhA基因原核表达载体,在E coli BL21（DE3）宿主菌中用不同浓度的IPTG诱导目的重组蛋白rVvhA表达,采用Ni-NTA亲和层析法提纯rVvhA,SDS-PAGE检测表达和提纯效果.采用兔抗Vv全菌抗体的Western Blot和兔抗rVvhA血清的免疫扩散试验鉴定其免疫反应性和免疫原性.结果 所克隆的vvhA基因核苷酸序列与GeneBank公布的同源性分别为96.09%和98.26%.在0.5 mmol/L IPTG诱导下,rVvhA产量可占细菌总蛋白的18%.提纯的rVvhA经SDS-PAGE后仅显示单一的蛋白条带.重组蛋白rVvhA能与兔抗Vv全菌抗体发生特异性结合,免疫家兔可获得高效价抗体.结论 该研究成功地构建了创伤弧菌υυhA基因高效原核表达系统,所表达的rVvhA具有良好的免疫原性和免疫反应性,可作为Vv免疫检测试剂盒及疫苗的抗原.     

禽多杀性巴氏杆菌C48-3株成熟黏附蛋白的表达及其免疫原性检测

目的：在大肠杆菌原核表达系统中高效表达禽多杀性巴氏杆菌成熟黏附蛋白Cpm39,并检测其免疫原性。方法：通过BamHⅠ和SalⅠ双酶切重组载体pMD18-cpm39获得cpm39基因片段,将该基因片段克隆至表达载体pMAL-p2X上,构建表达质粒pMAL-p2X-cpm39,转化至大肠杆菌BL21（DE3）,在IPTG诱导下表达融合蛋白,用禽多杀性巴氏杆菌C48-3株Cp39天然粘附蛋白的免疫血清经Western印迹检测其免疫原性。结果：SDS-PAGE结果显示表达的融合蛋白相对分子质量为78×103,与预期结果相符,而Western印迹结果表明诱导表达的融合蛋白MBP-Cpm39能与Cp39天然黏附蛋白抗体发生特异性反应。结论：构建了表达质粒pMAL-p2X-cpm39,获得了具有免疫原性的重组融合蛋白,为进一步研究禽多杀性巴氏杆菌成熟黏附蛋白的免疫保护功能奠定了基础。     

猪流感病毒抗原表位基因表达载体构建与免疫原性分析

根据猪流感病毒血凝素蛋白基因(Heamuglutinine, HA)的核苷酸序列, 设计、筛选HA蛋白氨基酸序列的主要表位多肽4个, 将4个片段以柔性连接串联成模拟蛋白, 核苷酸约为300 bp, 体外扩增该模拟蛋白基因, 插入到原核表达载体pET30a(+)中, 转染宿主菌诱导表达, 结果获得分子量为20 kD的表达蛋白, 该蛋白可与抗His-tag抗体、抗猪流感病毒H1N1、H3N2亚型高免血清发生免疫学反应。纯化后免疫小鼠, ELISA及血凝抑制(Heamuglutinine inhibitor, HI)试验检测, 小鼠产生针对多肽抗原的血清抗体, 同时还可检测到H1N1、H3N2亚型SIV血凝抗体。流氏细胞仪检测免疫组外周血淋巴细胞高于对照组, 说明该模拟蛋白具有与H1N1、H3N2亚型猪流感病毒相似的免疫原性及反应原性, 为H1N1、H3N2血清亚型猪流感病毒疫苗研制提供了新手段。     

锥虫早老素蛋白亲水区的克隆和表达纯化

目的体外表达锥虫早老素蛋白亲水区肽段,以制备抗血清用于功能研究。方法根据锥虫早老素蛋白二级结构特性,设计引物分别扩增N端及C端片段的亲水区肽段基因,装入原核表达载体进行表达,并通过变性纯化方法获得足够量表达蛋白,制备兔抗血清。结果成功扩增并克隆锥虫早老素蛋白亲水区片段L2及L7．并分别采用变性磁珠法和变性树脂法进行大量蛋白产物纯化,浓缩纯化产物制备兔抗血清经Westem blot杂交验证,出现目的蛋白大小阳性条带。结论成功表达锥虫早老素蛋推测N端及C端亲水肽段,并成功制备抗血清．可用于锥虫早老素蛋白功能分析。     

牛免疫缺陷病毒基质蛋白和衣壳蛋白的可溶性表达

基质蛋白和衣壳蛋白是BIV的主要结构蛋白,在病毒感染及整个复制周期中起重要作用。本文采用 pTXB系统在大肠杆菌中表达出融合状态的牛免疫缺陷病毒 BIV基质蛋白 MA及衣壳蛋白 CA,经几丁质亲合、自剪切纯化后,获得不含融合片段的纯化产物。每克湿菌体MA产量可达毫克级,CA表达量达十毫克级。用原核表达获得的高纯度CA蛋白免疫大白兔获得的抗血清,经Western Blot 分析显示能够与病毒颗粒的 CA蛋白发生特异反应,证实表达产物具有良好的免疫原性和反应原性,可用于制备相应抗体,为研究 BIV相应基因表达变化,进行体外蛋白质相互作用试验提供工具。