利用抗原结合多肽嫁接抗体技术制备抗hCG单域抗体

本研究旨在人绒毛膜促性腺激素(hCG)的结合多肽的基础上应用嫁接抗体技术制备抗hCG单域抗体,简化单域抗体制备过程,提高多肽生化稳定性。利用单域抗体通用骨架(cAbBCII10),以hCG结合多肽取代互补决定区CDR1或CDR3,合成cAb BCII10嫁接抗体全基因序列并与sfGFP基因序列融合后,插入到带有His标签的原核表达载体pET30a(+)中,成功构建了pET30a-(His6)-cAbBCII10-CDR1/hCGBP1-sfGFP与pET30a-(His6)-cAbBCII10-CDR3/hCGBP3-sfGFP融合蛋白表达质粒。将重组质粒转化大肠杆菌BL21(DE3),用IPTG诱导表达融合蛋白,得到高表达量的可溶性融合蛋白。利用Ni-NTA亲和柱纯化得到纯蛋白,应用SDS-PAGE鉴定纯化的蛋白为正确表达的目标蛋白。通过抗原抗体结合实验,发现hCG结合多肽嫁接到单域抗体通用骨架的互补决定区CDR1或CDR3后都有抗原结合活性,具有相似的抗体滴度,且嫁接到CDR3后的抗原结合活性比CDR1要高(2–3倍)。嫁接抗体基本保留了所用单域抗体框架较为稳定的生化特性,具有一定的热稳定性和较好的碱耐受性,同时,所接入的hCG结合片段对hCG具有较特异的结合活性,为进一步优化抗原结合多肽嫁接抗体技术制备抗hCG单域抗体提供了可靠的实验基础     

应用RAD多肽展示体系制备抗hCG类抗体分子

应用基于激烈火球菌Pyrococcus furiosus重组酶RadA的ATP酶结构域(RAD骨架)的多肽展示体系,通过嫁接人绒毛膜促性腺激素(hCG)结合多肽,制备抗hCG类抗体分子。通过合成hCG结合多肽插入RAD多肽展示位点的类抗体基因,成功构建了pET30a-RAD/hCGBP-sfGFP原核表达载体,在大肠杆菌中诱导蛋白表达,分离、纯化获得类抗体蛋白,通过亲和吸附-GFP荧光检测方法测定类抗体对hCG的结合活性,并与应用单域抗体通用骨架制备的嫁接抗体比较活性差异。结果显示,RAD类抗体分子对hCG分子具有较高的亲和性和特异性,显著优于单域嫁接抗体,并与商业单克隆抗体的活性相当;同时,利用RAD多肽展示骨架制备的抗hCG类抗体,具有较高的生化稳定性,是一种具有应用潜力的抗体替代分子。     

亲和层析法纯化人绒毛膜促性腺激素

本文介绍了一种应用抗hCG β亚单位单克隆抗体亲和层析提纯hCG的方法。结果表明,该法具有简便、快速、经济、产品活性高等优点。hCG回收率为96％,免疫学活性17000IU/mg蛋白,生物学活性(小鼠子宫称重法)达到10 000U/mg蛋白以上。     

正常人卵巢绒毛膜促性腺激素(hCG)受体的研究

23例正常卵巢制备之细胞膜,可与绒毛膜促性腺激素(hCG)发生特异性结合,最大结合率为13.2±2.24％,Scatchard作图分析,得一直线。23例正常卵巢之受体量为O.66±0.142×10~(-10)M/μg膜蛋白,Kd值为10.16±5.5×10~(-9)M。     

人绒毛膜促性腺激素β亚基和绵羊垂体促性腺激素共有α亚基组成的单链多肽的生物合成

采用重叠PCR方法,将人绒毛膜促性腺激素β亚基(hCGβ)cDNA3'端与绵羊垂体促性腺激素共有α亚基(oLHα)cDNA 5'端串联起来,构建了hCGβ-oLHα嵌合cDNA。将嵌合cDNA克隆入核型多角体病毒(AcNPV)表达载体pVL1393得到表达型质粒pVL1393-hCGβ-oLHα,并与BaculoGold~(TM)线性化AcNPV基因组DNA共转染昆虫细胞Sf9,筛选得到重组病毒AcNPV-hCGβ-oLHα。以扩增后的重组病毒感染昆虫细胞进行表达,并进一步经偶联抗hCGβ单抗亲和层析柱纯化得到hCGβ-oLHα单链多肽。SDS-PAGE银染和免疫印迹分析结果表明表达产物在非还原条件下表观分子量约40.5kD,还原条件下约为38.0kD,这说明表达产物具有链内二硫链及次级构象。竞争抑制~(125)I-hCGβ与抗体结合的检测结果表明其与hCGβ抗体结合活性较天然hCG弱,但较天然hCGβ略有增加。由此可见单链多肽作为抗hCG夏合抗原避孕疫苗的靶抗原,具有潜在的应用前景。     

分子佐剂C3d3增强hCGβ蛋白疫苗免疫原性及其抗血清中和hCG生物学活性的作用

本文观察分子佐剂C3d3增强hCGβ蛋白避孕疫苗体液免疫效力的能力。采用分子生物学技术以phCMV1为载体分别构建分泌型、带有6个组氨酸纯化标签的真核表达质粒phCMV1-6His-hCGβ-C3d3和phCMV1-6His-hCGβ,在CHO 细胞中获得重组蛋白的稳定、高效表达,并用镍柱和凝胶过滤层析对其进行分离、纯化。分别用hCGβ-C3d3融合蛋白、hCGβ+弗氏佐剂和单用hCGβ免疫生育期雌性BALB/c 小鼠,共免疫两次,间隔4周。ELISA 测定血清中抗hCGβ抗体滴度,并对各组小鼠产生的抗血清中和hCG 生物学活性的能力进行比较。结果表明hCGβ单独免疫组在加强免疫后才见抗体生成,其抗体滴度比hCGβ-C3d3融合蛋白免疫组低1995倍,C3d3的佐剂能力是弗氏佐剂的10倍(初次免疫)-32倍(再次免疫),并且hCGβ-C3d3融合蛋白免疫小鼠产生的抗血清具有很强的中和hCG 生物学活性的作用。实验证明通过分子佐剂C3d3可以大幅提高机体对hCGβ的体液免疫应答能力。     

分子佐剂C3d3增强hCGβ蛋白疫苗免疫原性及其抗血清中和hCG生物学活性的作用

本文观察分子佐剂C3d3增强hCGβ蛋白避孕疫苗体液免疫效力的能力。采用分子生物学技术以phCMV1为载体分别构建分泌型、带有6个组氨酸纯化标签的真核表达质粒phCMV1-6His-hCGβ-C3d3和phCMV1-6His-hCGβ,在CHO细胞中获得重组蛋白的稳定、高效表达,并用镍柱和凝胶过滤层析对其进行分离、纯化。分别用hCGβ-C3d3融合蛋白、hCGβ 弗氏佐剂和单用hCGβ免疫生育期雌性BALB／c小鼠,共免疫两次,间隔4周。ELISA测定血清中抗hCGβ抗体滴度,并对各组小鼠产生的抗血清中和hCG生物学活性的能力进行比较。结果表明hCGβ单独免疫组在加强免疫后才见抗体生成,其抗体滴度比hCGβ-C3d3融合蛋白免疫组低1995倍,C3d3的佐剂能力是弗氏佐剂的10倍(初次免疫)-32倍(再次免疫),并且hCGβ-C3d3融合蛋白免疫小鼠产生的抗血清具有很强的中和hCG生物学活性的作用。实验证明通过分子佐剂C3d3可以大幅提高机体对hCGβ的体液免疫应答能力。     

LbCpf1基因的原核表达、纯化与体外切割检测

目的:为获得具有体外切割活性的LbCpf1蛋白。方法:将毛螺菌科细菌ND2006(Lachnospiraceae bacterium ND2006)的LbCpf1基因编码区连接至pHis*6(IV),构建原核表达质粒CRISPR-LbCpf1-6*His。将该重组质粒转化BL21(DE3)感受态细胞,IPTG诱导目的蛋白表达,经镍柱亲和层析纯化、透析除盐和凝胶电泳检测等步骤获得重组蛋白,进行体外切割试验鉴定重组蛋白切割活性。结果:双酶切鉴定和测序结果表明成功构建重组质粒CRISPR-LbCpf1-6*His,经转化后获得含有重组质粒CRISPR-LbCpf1-6*His的BL21(DE3)蛋白表达菌株。将菌株接种于37℃,160 r/min,IPTG终浓度为0.5 mmol/L的条件下诱导5 h,最终镍柱纯化除盐后的LbCpf1蛋白终浓度可达400 ng/μl,在体外适宜条件下,该重组蛋白可与成熟的crRNA结合切断标靶DNA。结论:获得的高纯度LbCpf1蛋白具有体外切割活性,可用于后续基因编辑研究。     

LbCpf1基因的原核表达、纯化与体外切割检测 *

目的:为获得具有体外切割活性的LbCpf1蛋白。方法: 将毛螺菌科细菌ND2006(Lachnospiraceae bacterium ND2006)的LbCpf1基因编码区连接至pHis*6(IV),构建原核表达质粒CRISPR-LbCpf1-6*His。将该重组质粒转化BL21(DE3)感受态细胞,IPTG诱导目的蛋白表达,经镍柱亲和层析纯化、透析除盐和凝胶电泳检测等步骤获得重组蛋白,进行体外切割试验鉴定重组蛋白切割活性。结果: 双酶切鉴定和测序结果表明成功构建重组质粒CRISPR-LbCpf1-6*His,经转化后获得含有重组质粒CRISPR-LbCpf1-6*His的BL21(DE3)蛋白表达菌株。将菌株接种于37 ℃,160 r/min,IPTG终浓度为0.5 mmol/L的条件下诱导5 h,最终镍柱纯化除盐后的LbCpf1蛋白终浓度可达400 ng/μl,在体外适宜条件下,该重组蛋白可与成熟的crRNA结合切断标靶DNA。结论: 获得的高纯度LbCpf1蛋白具有体外切割活性,可用于后续基因编辑研究。     

家蝇肽聚糖识别蛋白(PGRP-SA)的克隆表达及细菌结合研究

对家蝇PGRP-SA基因进行克隆表达以及研究其重组蛋白与细菌结合能力。从构建的家蝇(Musca domestica)幼虫cDNA质粒文库中筛选到PGRP-SA基因,以cDNA质粒为模板设计引物,通过PCR扩增,获得PGRP-SA基因完整编码序列。运用生物信息学方法对该基因及其编码蛋白进行预测和分析。构建pET-28a(+)-PGRP-SA重组质粒,转化到大肠杆菌BL21(DE3)中进行诱导表达及蛋白纯化。利用半定量RT-PCR检测PGRP-SA在家蝇3龄幼虫不同组织中的表达量差异。PGRP-SA重组蛋白进行微生物结合实验。结果表明,PGRP-SA基因ORF全长615 bp,编码204个氨基酸,理论分子量22.8 k D,等电点9.11,具有保守的PGRP结构域。成功构建了pET-28a(+)-PGRP-SA重组质粒,蛋白经IPTG诱导后在大肠杆菌中获得表达,经亲和层析柱纯化获得目的蛋白,利用Western blot检测证明纯化蛋白与预期大小相符。PGRP-SA在家蝇3龄幼虫血淋巴、脂肪体、前肠、中肠、气管、马氏管都有表达,血淋巴组织中表达量最高,后肠无表达,由此说明PGRP-SA基因的表达具有一定的组织性。PGRP-SA重组蛋白能与金黄色葡萄球菌和大肠杆菌结合,与白色念珠菌不能结合。成功表达及纯化家蝇PGRP-SA蛋白,证实家蝇PGRP-SA能与金黄色葡萄球菌和大肠杆菌结合。     

Bactericidal substances in cytoplasm of leucocytes. An attempt to separate lysozyme by gel filtration

A substance with a bactericidal effect onEscherichia coli was isolated from the cytoplasm of polymorphonuclear leucocytes. It consists of a mixture of different active substances and therefore a gel filtration using s Sephadex G-100 column was used to separate lysozyme from other active substances. The extract was separated by this procedure into five peaks, out of which peak I. possesed a significant bactericidal activity, peak II. and III. had a weak one, whereas the lysozyme activity was present in peak III., IV. and V.     

Purification and properties of a fibrinolytic enzyme from Bacillus subtilis

A fibrinolytic enzyme obtained from B. subtilis was purified, using DEAE-cellulose column chromatography, and gel filtration on Sephadex G-100. The preparation was homogeneous as tested by gel filtration on Sephadex G-200, and disc electrophoresis. The molecular weight of this enzyme was 29.400 estimated by gel filtration on Sephadex G-100. The optimum pH for enzyme activity was 7.2 Copper ions significantly increased enzyme activity, while Zn++ and Mn++ caused marked inhibition.     

Purification and partial characterization of a lectin from the fresh leaves of Kalanchoe crenata (Andr.) Haw

A haemagglutinating protein from the saline extracts of Kalanchoe crenata leaves, which agglutinate all human blood types, was purified to homogeneity by ion-exchange chromatography on a DEAE-Cellulose column followed by gel filtration on a Sephadex G-100 column. The purified protein showed one band, both in non-denaturing PAGE and SDS-PAGE. The M(r) that was determined by SDS-PAGE was 44,000 Da and that estimated from gel filtration was 47,000. Treatment of the haemagglutinating protein with 5 mM EDTA diminished the haemagglutinating activity to 50% of the original level. The addition of divalent cations, 10 mM Mg(2+), 10 mM Mn(2+), or 10mM Ba(2+), totally restored and enhanced the activity. The protein showed maximum activity over the 3-7 pH range and was heat-resistant. It was also a glycoprotein containing about 1.5% carbohydrate.     

Purification of the phosphofructokinase regulatory factors

In this paper, the existence and purification of two species of phosphofructokinase regulatory factor activity are reported. The purification procedure included liver homogenization and ultracentrifugation, a 93 degrees C heat step on the supernate, precipitation with ammonium sulfate, DEAE-cellulose column chromatography, and Sephadex G-75 (fine) chromatography. Two discrete regions of factor activity were eluted from the DEAE-cellulose column with a 0 to 0.5 M linear NaCl gradient. The lesser anionic fraction was not significantly retarded by DEAE-cellulose at pH 7.6, and was referred to as factor A. The more anionic form, factor B, eluted at about 0.2 M NaCl. The presence of two active fractions was confirmed by separation of factor activity (prior to DEAE-cellulose chromatography) into two discrete species by preparative isoelectric focusing on granulated gel. The isoelectric points were approximately 7.0 for factor B and 8.5 for factor A. Factor A and factor B exhibited quite different elution volumes, i.e., apparent molecular weights, when applied to a Sephadex G-75 column. Rechromatography on a Sephadex G-75 column was used for further purification and estimation of native molecular weight. The gel filtration method yielded a molecular weight of 13,800 +/- 1,800 for factor A. Factor A activity eluted as a symmetrical protein peak of constant specific activity, suggesting a homogeneous preparation. For factor B, the absorption at 280 nm and activity profile did not directly overlap. When the peak absorbance at 280 nm was considered, a molecular weight range of 39,000 +/- 4,000 was found, and on the basis of activity the molecular weight range was 36,000 +/- 4,000. After the final Sephadex G-75 chromatographic step, sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis of each SDS-treated factor preparation indicated that factor A, after visualization by silver staining, was homogeneous, with a subunit molecular weight of approximately 12,000. The factor B preparation consisted of two major polypeptides (11,000 and 18,000). The data appeared to support the conclusions that factor B was a dimer of the 18,000-Da subunit, and that the major contaminant was a tetramer of the 11,000-Da subunit.     

枯草杆菌BS-98分泌的抗真菌蛋白的分离纯化及其部分性质的研究

拮抗菌（Bacillussubtilis）BS-98菌株在BPY液体培养基中产生的蛋白质经硫酸铵分级盐析、SephsdexG-100柱层析、DEAE-纤维素柱层析和SephadexG－100柱再层析后,分离纯化得到的抗菌蛋白在电泳中显现出三条带。经初步纯化的抗菌蛋白对热稳定,对蛋白酶部分敏感。抑菌谱测定表明,抗菌蛋白对芦笋茎枯病菌、小麦赤霉病菌、棉花枯萎病菌、棉花黄萎病菌、灰霉菌、立枯丝核菌等病原真菌及黄瓜角斑病菌都具有强烈的抑制作用。     

Insulin-like substance and insulin-degrading complex in hemolysates of human erythrocytes

Human erythrocyte lysate was fractionated on various gel filtration media and immunoreactive insulin, insulinase and the influence of individual fractions of the insulin-degrading activity were determined. The hemolysate was shown to contain a complex of substances including an insulin-like substance, insulinase, protease inhibitor and insulinase activator. The insulin-like substance eluted from a Sephadex G-50 column in the same manner as native insulin, and its concentration exceeded the plasma level. Insulinase (Mr 100,000) degraded insulin to the trichloroacetic acid soluble fragments but did not degrade protein or glycoprotein hormones from human pituitaries. Insulinase was inhibited by low temperature, aprotinin and by a newly discovered protease inhibitor from erythrocytes which also inhibits serine proteases--trypsin and chymotrypsin. Another newly discovered substance eluted from a Sephadex G-100 column in the region of low molecular weight substances and showed an insulinase activating activity. The elution patterns of the protease inhibitor and insulinase activator suggest the possibility of the presence of more than one inhibiting and activating factor. The experimental results suggest that the insulin-degrading complex plays a role of a regulator of plasma insulin level. The nonpancreatic origin of the insulin-like substance is also possible.     

Stimulatory action of follicular fluid components on maturation of granulosa cells from small porcine follicles

Follicular fluid obtained from large (6-12 mm) porcine follicles (LFF) was investigated to determine its stimulatory activity on progesterone secretion and on follicle stimulating hormone (FSH) induction of 125I-human chorionic gonadotropin (hCG)-luteinizing hormone (LH) binding sites in porcine granulosa cells in a 4-day culture. Incubation of granulosa cells harvested from small porcine follicles (1-2 mm) with 50% LFF led to stimulation of LH/hCG binding sites and progesterone secretion. After partial purification of pooled LFF or proteins precipitated with 90% ethanol on Sephadex G-100 the greatest stimulatory activity was found in the second protein peak eluted from the column. Chromatography of part of the active fraction on DEAE Sephacel using a continuous gradient of NH4HCO3 yielded seven protein fractions. The second fraction, which eluted early, contained the majority of the stimulatory activity which was purified about 32-fold compared to native LFF. In contrast, addition of follicular fluid recovered from small porcine follicles inhibited FSH induction of LH/hCG binding sites and progesterone secretion. It can be concluded, that maturation of granulosa cells from small follicles may be enhanced by protein(s) present in LFF, but not in fluid recovered from less mature follicles.     

Polypeptide antibiotic 26a from Bacillus subtilis. II. Isolation and purification procedures.

Both the analytical and preparative methods by which the preparations of 26a bydrochloride salt with a high antibacterial activity and 20--30% recovery have been obtained from the fermentation fluids of Bacillus subtilis are presented. On an industrial scale the antibiotic can be yielded by absorption of bioactivity on Amberlite CG-50I column and precipitation with picric acid of crude substance from active elutes as adduct which was divided on equilibrated CM--cellulose and finally purified by gel filtration on Sephadex G-25 column. The purified preparation gave a single band by SDS-polyacrylamide gel electrophoresis and one ninhydrin-positive spot by thin-layer chromatography on silica gel G plates corresponding to single zones of bioactivity on bioautograms, and moved as a single peak of almost constant antibacterial activity on Sephadex G-75, G-100 and G-200 columns. The potency of the purest preparations, lot Sephadex G-25, was 6,500--7,000 arbitrary units/mg, and were characterized as follows: purification factor, 57; purity of 98--100% by densitometer scans of SDS-polyacrylamide gels; MIC for Sarcina lutea by twofold agar dilution assay, 0.306 microgram/ml.     

Subcellular distribution of enkephalin precursor proteins in rat dental pulp and the usefulness as a substrate for enkephalin-producing enzymes

The subcellular distribution of enkephalin (EK) precursor proteins was investigated to clarify the intracellular site of biosynthesis of EK in rat dental pulp tissue. The contents of met-EK-like peptides in nuclear, microsomal, and supernatant fractions of the pulp tissue were markedly increased after sequential digestion with trypsin and carboxypeptidase B, indicating the enrichment of the precursors in these fractions. Sephadex G-100 gel filtration showed a common peak of the precursor proteins in the homogenate and its microsomal and supernatant fractions, and the molecular weight was determined to be about 58,000 by SDS polyacrylamide gel electrophoresis. Both the partially purified precursor protein from the supernatant fraction and N alpha-benzoyl-DL-arginine-beta-naphthylamide (BANA) were used as substrates for a lysosomal enzyme separated by Sephadex G-75 gel filtration. The major peak of EK-producing activity of the enzyme was identical with that of BANA-degrading activity of the enzyme. These results demonstrate the EK-producing activity of lysosomal proteinase, and also indicate the usefulness of the two substances as substrates for the enzyme.     

Purification and characterization of fish surface mucin

Fish surface mucin from Pampus argenteus was extracted with different organic solvents and the residue passed through Sephadex G-200. The major peak was purified by DEAE-Cellulose chromatography and five fractions were obtained. Carbohydrate and protein contents showed that major peak is a glycoprotein. Rechromatography of this component on the Sephadex G-200 column gave a single peak, with an estimated minimal molecular weight of 6.9 X 10(5). Analysis of individual sugar components revealed the presence of galactose, glucose, mannose, arabinose, N-acetyl glucosamine, N-acetyl galactosamine and sialic acid. The most represented amino acids are threonine, serine, proline, glutamic acid and glycine. The N-terminal amino acid end was blocked. Nearly 47% of sulphate was acid labile. Sialic acid and fucose were released rapidly by mild acid hydrolysis. The presence of blood group-A activity suggests that some kind of terminal alpha-Gal-NAC may be present.