双抗体夹心ELISA检测HIV-1 gp41抗原方法的建立

目的:建立检测HIV-1gp41抗原的双抗体夹心ELISA,并探讨其临床应用的可行性。方法:用饱和硫酸铵(SAS)纯化抗HIV-1gp41-5单克隆抗体(mAb),用HRP标记后建立双抗体夹心ELISA法,对其灵敏度及特异性进行检测,并用该方法对40份HIV-1阳性血清进行了检测。结果:用mAbE12(5μg/mL)为包被抗体,2H6为酶标记抗体(1∶900)建立了双抗体夹心ELISA法,检测gp41-5多肽的灵敏度是100pg/mL。对HIV-1阳性血清中gp41抗原的检出率为67.5%(27/40)。结论:建立了特异性强、灵敏度良好的检测HIV-1gp41抗原的双抗体夹心ELISA法。     

人参皂苷Rb1转化菌株筛选及转化产物分析

【背景】许多微生物能够对皂苷类化合物进行生物转化,因此,通过微生物对皂苷类化合物不同位置结构的修饰能获得高活性的皂苷成分。【目的】从分离纯化的菌株中筛选能将人参皂苷Rb₁转化为药理活性较高的稀有人参皂苷。【方法】从三七根际土壤及三七茎中分离纯化了36株真菌,首先利用产β-葡萄糖苷酶的方法对菌株进行皂苷转化活性初筛,再以人参皂苷Rb₁为底物进行皂苷转化活性复筛,通过薄层色谱(thinlayerchromatography,TLC)、高效液相色谱(high performance liquid chromatography, HPLC)和质谱(mass spectrometry, MS)等方法对转化产物进行分析。【结果】筛选出一株对人参皂苷Rb₁具有较高转化活性的菌株F17,通过形态学观察及对内转录间隔区(internaltranscribedspacer,ITS)序列分析,菌株F17被鉴定为拟盘多毛孢属菌(Pestalotiopsis biciliata)。P. biciliata可将人参皂苷Rb₁转...     

PRRSV-N蛋白单抗制备与直接免疫荧光方法建立

为了建立一种快速的PRRSV诊断方法,本研究以纯化的PRRSV-TJ-PGEX-6P-N重组蛋白处理抗原免疫BALB/C小鼠,利用淋巴瘤细胞融合技术,包被带有His标签的PRRSV-N蛋白,通过间接ELISA方法筛选到两株可以稳定分泌的N蛋白特异性单克隆抗体,命名为N-13、N-29。通过间接ELISA方法,WB与IFA方法对其进行鉴定,并测定其亲和常数。腹水效价分别为1:4 000与1:5 000,选择腹水效价高的N-13进行纯化标记,并对标记抗体的特异性、敏感性与保存期进行鉴定;通过DFA条件的摸索,建立了一种敏感的DFA诊断方法,并对其和PRRSV分离鉴定常用的CPE观察法进行比较。结果表明该DFA方法结果准确,可以作为实验室病毒分离鉴定和猪生产中该疾病诊断的有效手段。     

毛细管电泳技术在蛋白质翻译后修饰中的应用

蛋白质中翻译后修饰蛋白的鉴定是蛋白质组学研究的主要内容之一。毛细管电泳技术由于其高分辨能力、低样品上样量、易操作性和较少的分析时间等特点,迅速发展成为一种重要的分离技术。通过毛细管电泳与质谱连用,可以得到许多关于蛋白质鉴定、纯化和结构改变方面的十分有价值的信息。对毛细管电泳技术进行了简单介绍,并且就其在蛋白质磷酸化和蛋白质糖基化研究中的应用进行了综述。     

酪丝亮肽荧光标记物的合成及其在肿瘤治疗靶点研究中的应用

酪丝亮肽是一种具有抗肿瘤活性的小分子三肽,它可以诱导造成肝癌细胞发生凋亡坏死,从而杀伤肿瘤细胞,但是酪丝亮肽在肝癌细胞的亚细胞定位尚不十分明确．为了达到对酪丝亮肽进行示踪进而观察其亚细胞定位的目的,使用荧光物质（5（6）－羧基叫甲基罗丹明琥珀酰业胺酯,5（6）－TAMRASE）对酪丝亮肽进行丫标记,应用非变性聚丙烯酰胺凝胶电泳、毛细管电泳和荧光分光光度法对标记酪丝亮肽进行纯化和鉴定．并在激光扫描共聚焦显微镜下观察了荧光标记酪丝亮肽在人肝癌BEL－7402细胞中的分布．结果显示,合成的酪丝亮肽荧光标记物性质稳定,标记的酪丝亮肽在人肝癌BEL－7402细胞的胞浆中呈聚集分布．     

UreB蛋白B细胞抗原表位快速筛选与鉴定

以幽门螺旋杆菌(Helicobacterpylori,Hp)主要抗原蛋白尿素酶B(ureaseB,UreB)为靶蛋白,建立一种新的B细胞抗原表位筛选与鉴定方法.运用Fmoc固相肽合成法合成11条HpUreB蛋白的单表位抗原肽片段,在其氨基端标记FITC荧光素,应用荧光偏振方法(fluorescencepolarization,FP)快速鉴定这些肽片段的抗原性,并通过FP法在大规模样品中快速筛选相应抗体滴度高、分布人群广的优势抗原表位肽.结果表明,合成的11条UreB蛋白线性抗原肽中,10条具有较强的抗原性,其中No.2、No.5和No.11抗原肽相应的特异性抗体在感染Hp的人群中分布较广,抗体滴度较高,为UreB的优势抗原表位肽.对抗原表位进行多参数综合分析与设计,通过FP技术快速鉴定抗原肽,并筛选优势抗原表位肽,对于疾病的抗原表位谱研究具有重要的意义,同时在疾病的诊断、分型及治疗中具有重要的应用前景.     

天花粉蛋白胰酶肽图谱的毛细管区带电泳法分析

目的:以天花粉蛋白胰蛋白酶解肽段为测定对象,用毛细管区带电泳法(CZE)研究天花粉蛋白的肽图谱分离条件。方法:采用未涂层石英毛细管(长50cm,内径75μm,有效长度42cm),以50mmol/L磷酸盐和150mmol/L三氟乙酸溶液为运行缓冲液,在25℃、pH2.0和压力为3447.4Pa(×10s)的条件下进样,以12kV恒压电泳分离,检测波长214nm。结果:运用CZE也能较好地对天花粉蛋白进行肽图谱分离,在缓冲体系中加入离子对试剂三氟乙酸,可极大地改善多肽的峰形和分辨率;同时运用反相高效液相色谱(RP-HPLC)技术,也很好地鉴定了部分肽段在CZE和RP-HPLC肽图谱中的对应关系。结论:与传统的RP-HPLC分析天然或重组蛋白肽图谱相比,CZE也不失为一种鉴定蛋白肽图谱的有效、快速和简单的方法。     

重组牛乳源金黄色葡萄球菌GapC蛋白优势B细胞抗原表位的预测和筛选

旨在表达牛乳源金黄色葡萄球菌(Staphylococcus aureus)GapC蛋白并对其B细胞抗原表位进行预测与鉴定，本研究利用实验室分离鉴定的S. aureus分离株15119扩增GapC基因并构建重组表达质粒pET-28a-GapC，诱导纯化得到分子量为44 kD重组蛋白GapC，以此免疫新西兰大白兔，获得特异多克隆抗体。利用生物信息学方法，对GapC蛋白的二级及三级结构进行分析，预测其B细胞抗原表位，并利用特异性抗体对筛选的表位进行鉴定。结果表明，GapC蛋白具有良好的免疫原性，筛选出7个线性B细胞抗原表位，利用兔抗重组GapC蛋白多克隆抗体鉴定得到了PL 5(²²¹ IPEIDGKLDGGAQRVP²³⁶)多肽和PL 7(²⁶⁴KNASNESFGYTEDEIVSSDVVGM²⁸⁶)2个优势B细胞表位。本研究成功制备了GapC蛋白，预测并鉴定了2个优势抗原表位，为其嵌合表位疫苗的开发提供了技术支持。     

DNA分析与扩增

931549利用毛细管系列电泳进~DNA.]II序[英3/Huang,X.C.…,Ahal.Chem.一1992,64(8)一.2149~2154[译自DBA,1992,11(23),92—12849] 一种DNA测序方法利用了毛细管系列电泳,双色荧光检测和双染料标记。在一系列毛细管上分离Sanger DNA测序片段,再用双色激光激发的confocal荧光扫描器进行桂上检测。在单个毛细管里分离出4套DNA测序片段,然后用双编码法进行区别,其中每套片段用两个有特定比率的染料标记的引物标记。这样就有可能筛选出电泳迁移率相同的染料l关键的是甩不同染料标记的DNA片段之间不应该出现电泳迁移的差异,而且染料具…     

用鞘磷脂作为标志分子鉴定脂筏

脂筏是质膜双层中富含鞘脂、胆固醇及特殊蛋白质的质膜微区.对其功能的研究,首先要对其进行分离和鉴定.常利用密度梯度超速离心将其分离,然后以脂筏中富含的神经节苷脂GM₁作为标志分子,利用荧光或生物素标记的霍乱毒素-B亚基进行亲和标记来鉴定脂筏.但这一鉴定方法操作复杂、费时、易对环境造成污染,所用关键试剂霍乱毒素不易获得,再加上一些组织GM₁含量甚微或不含GM₁,使其应用受到局限.为建立一个特异性高又对各种组织广泛适应的脂筏鉴定方法.对两种细胞系脂筏的脂类组分进行了分析.结果发现,可用鞘磷脂作为脂筏的特异性标志分子,采用高效薄层层析技术对脂筏进行鉴定.     

Identification of a high-affinity binding protein for a hepta-beta-glucoside phytoalexin elicitor in soybean.

A putative receptor protein for a hepta-beta-glucoside phytoalexin elicitor was identified by photoaffinity labeling of detergent-solubilized proteins from soybean root membranes. Incubation of partially purified beta-glucan-binding proteins with a photolabile 125I-labeled 2-(4-azidophenyl)ethyl-amino conjugate of the heptaglucoside elicitor, followed by irradiation with ultraviolet light (366 nm) resulted in specific labeling of a 70-kDa band in SDS/PAGE. Half-maximal inhibition of the 125I-labeling of the protein band by underivatized hepta-beta-glucoside was achieved by 15 nM heptaglucoside. Analysis of the affinity of radiolabel incorporation into the protein by ligand-saturation experiments, gave an apparent Kd value of 3 nM, in full agreement with the results from radioligand-binding studies. Good correlation was also observed between the amount of radiolabel incorporated into the protein and the binding activity of the fractions obtained at different stages in the purification of heptaglucoside-binding activity. Photoaffinity labeling of proteins purified by glucan-affinity chromatography showed the 70-kDa band as the main component along with weak 125I-labeling of a 100-kDa band. The 70-kDa band was also the major protein visualized by silver staining after SDS/PAGE of this fraction, suggesting that it is the predominant form of the heptaglucoside-binding proteins in detergent-solubilized soybean membranes.     

Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. II. Site of labeling with iodoacetamide and its fluorescent derivative

Iodoacetamide (IAA) and its fluorescent derivative, 5-(2-iodoacetamidoethyl) amino-naphthalene-1-sulfonate (IAEDANS) specifically bind to a site on the C-terminal half of sarcoplasmic reticulum (SR) Ca2+,Mg2+-ATPase. The location of this specific binding site was identified. SR membranes were treated with 150 microM [14C]IAA at pH 7.0 and 30 degrees C. One mole of IAA per mole of ATPase was bound in 6 h without affecting the Ca2+-transport activity. [14C]IAA-labeled SR membranes were cleaved with BrCN, and 14C-labeled peptide fragments were separated by Sephadex LH-60 chromatography and then digested further with trypsin. A radioactive peptide (Ala-Cys 674-Cys-Phe-Ala-Arg) was purified by Sephadex LH-20 chromatography and C18 reversed phase HPLC (Cys denotes the [14C]IAA-binding site). IAEDANS-labeling was carried out by reacting SR membranes with 50 microM IAEDANS for 5 h, at pH 7.0 and 30 degrees C. A fluorescent peptide was successfully purified by the same procedures as for the IAA-labeled peptide, and the amino acid sequence analysis of this peptide revealed that the IAEDANS labeling site was identical with the IAA binding site.     

Purification of a reconstitutively active K+/H+ antiporter from rat liver mitochondria

We describe purification of three different states of the 82-kDa K+/H+ antiporter from rat liver mitochondria. The denatured 82-kDa protein, identified by its selective labeling with [14C]dicyclohexylcarbodiimide (DCCD), was purified by preparative two-dimensional gel electrophoresis. This purified product was used to raise and immunopurify monospecific polyclonal antibodies. Western blot analysis showed that the [14C] DCCD-labeled 82-kDa protein is not a DCCD-crosslinked product. The native, [14C]DCCD-labeled, 82-kDa protein was purified by (NH4)2SO4 fractionation and column chromatography, using 14C labeling and gel electrophoresis to track the protein. The native, non-DCCD-labeled 82-kDa protein was purified by similar procedures, using immunopurified antibodies to track the protein. DCCD binding had no effect on chromatographic behavior of the antiporter protein. This protocol resulted in purification of the 82-kDa protein to apparent homogeneity. The purified, native 82-kDa protein was reconstituted into proteoliposomes and assayed for K+ transport with the new fluorescent probe, PBFI. K+ transport was electroneutral and was inhibited by DCCD, Mg2+, and timolol. The turnover number for K+ transport was about 1000 s-1, very similar to the value previously estimated in intact mitochondria.     

A novel fusion partner for enhanced secretion of recombinant proteins in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Expressing proteins with fusion partners improves yield and simplifies the purification process. We developed a novel fusion partner to improve the secretion of heterologous proteins that are otherwise poorly excreted in yeast. The VOA1 (YGR106C) gene of Saccharomyces cerevisiae encodes a subunit of vacuolar ATPase. We found that C-terminally truncated Voa1p was highly secreted into the culture medium, even when fused with rarely secreted heterologous proteins such as human interleukin-2 (hIL-2). Deletion mapping of C-terminally truncated Voa1p, identified a hydrophilic 28-amino acid peptide (HL peptide) that was responsible for the enhanced secretion of target protein. A purification tag and a protease cleavage site were added to use HL peptide as a multi-purpose fusion partner. The utility of this system was tested via the expression and purification of various heterologous proteins. In many cases, the yield of target proteins fused with the peptide was significantly increased, and fusion proteins could be directly purified with affinity chromatography. The fusion partner was removed by in vitro processing, and intact proteins were purified by re-application of samples to affinity chromatography.     

Purification of the surface cAMP receptor in Dictyostelium

We have previously identified and demonstrated reversible ligand-induced modification of the major cell surface cAMP receptor in Dictyostelium discoideum. The receptor, or a subunit of it, has been purified to homogeneity by hydroxylapatite chromatography followed by two-dimensional preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purification was monitored by following 32Pi incorporated by photoaffinity labeling with 8-azido-[32P]cAMP or by in vivo labeling with 32Pi. Two interconvertible forms of the receptor, designated R (Mr 40,000) and D (Mr 43,000), co-purified. Two-dimensional peptide maps of independently purified and 125I-iodinated R and D forms of the receptor were nearly identical but did have several distinct peptides. The estimated 6000-fold purification required is consistent with the number of cell surface binding sites assuming there are not multiple binding sites/polypeptide. In the accompanying article we report the generation of a monospecific polyclonal antiserum which has helped to further elucidate the physical properties and developmental regulation of the cAMP receptor.     

The purification of a detergent-soluble glucose-6-phosphatase from rat liver.

A highly active and soluble glucose-6-phosphatase has been purified to near homogeneity from rat liver. Successful purification has been initiated by covalent labeling of the enzyme in native rat liver microsomes with pyridoxal 5'-phosphate and NaBH4, followed by solubilization of the microsomes with Triton X-100, chromatography on phenyl-Sepharose, hydroxyapatite, DEAE-Sephacel and a second chromatography step on hydroxyapatite. The final enzyme preparation obtained was approximately 700-fold purified over the activity of starting microsomes. As judged by SDS/PAGE the purified glucose-6-phosphatase is composed of a single protein with a molecular mass of 35 kDa. The present work demonstrates that the purified glucose-6-phosphatase must be arranged in the native microsomal membrane so that it is accessible to pyridoxal 5'-phosphate from the cytoplasmic side.     

Induction of anti B-cell malignance CTL response by subfamily-shared peptides derived from variable domain of immunoglobulin heavy chain

The variable domain of immunoglobulin heavy chain (Ig HV) is well-characterized tumor associated antigen expressed in B-cell malignancies, which may function as a T-cell target. However, T-cell epitopes derived from shared framework regions (FRs) of each IgHV subfamily capable of inducing cytotoxic T lymphocytes (CTLs) against the B-cell malignancy, have not been identified. Using the specific PCR primers of seven IgHV gene subfamilies, we amplified the IgHV gene rearrangement for 108 cases of B-cell acute lymphoblastic leukemia (B-ALL) patients. The IgHV gene rearrangement fragments of B-ALL patients were directly sequenced then classified into seven different subfamilies. The T-cell epitopes encoded by the IgHV gene in the B-ALL patients were predicted by SYFPEITHI and BIMAS programs and compared with those from 56 representative germline IgHV sequences in the genebank. For the HLA-A*0201 locus, we found 1 or 2 top score shared epitopes from each subfamily and got 12 epitopes altogether. Results showed that ten of them were in the FRs. Using an antigen-specific T-cell expansion system, we generated the peptide-special CTLs in vitro, which were capable of killing B lymphoma cell lines that belonged to the same IgHV subfamily in a peptide-specific and HLA-restricted manner. Furthermore, we proved that the cytotoxicity of CTLs was IgHV subfamily-specific. These data indicate possible immunotherapy approaches for B-cell malignances patients based on IgHV gene subfamilies.     

Delta 6-desaturase activity in liver microsomes of rats fed diets enriched with cholesterol and/or omega 3 fatty acids.

A glycoprotein antigen was purified from human brain by immunoaffinity chromatography using the 44D10-monoclonal IgG, and its chemical nature was investigated. The yield of antigen was estimated at 91% and a 4340-fold purification was obtained relative to the white-matter homogenate. The antigen preparation from brain was further purified by preparative SDS/polyacrylamide-gel electrophoresis (PAGE) to obtain a glycoprotein with an Mr of 80,000 consisting of a single polypeptide. Amino acid analyses revealed a composition which was high in acidic and neutral amino acids, and low in basic residues. The presence of both glucosamine and galactosamine suggested that the glycoprotein contained both N- and O-linked glycans. Neutral sugar analyses showed that fucose, galactose and mannose were present. An assay for sialic acid determined that there were approximately 20 mol of sialic acid per mol of glycoprotein. Chemical cleavage of oligosaccharides by trifluoromethanesulphonic acid followed by SDS/PAGE showed that carbohydrate accounted for 25,000 of the 80,000-Mr glycoprotein.     

Purification and identification of adipogenesis inhibitory peptide from black soybean protein hydrolysate

Adipogenesis inhibitory peptide was isolated and identified from black soybean (Rhynchosia volubilis Lour.) hydrolysate. An adipogenesis inhibitor was purified using consecutive methods including: ultrafiltration (MWCO; 3 and 10kDa), gel filtration chromatography (Superdex Peptide 10/300 GL column), and reverse-phase high-performance liquid chromatography (microBondapak C(18) column). Also, the adipogenesis inhibition effect of the purified peptide was measured by observation of droplet of 3T3-L1 adipocyte by Oil Red O staining in the highest active fraction in each step. The peptide was shown to inhibit the differentiation of the 3T3-L1 pre-adipocyte, which was confirmed by morphological study. The adipogenesis inhibitory peptide was purified 71.43-fold from black soybean hydrolysate throughout a five-step purification procedure. The adipogenesis inhibitor was identified to be a tripeptide, Ile-Gln-Asn, having an IC(50) value of 0.014 mg protein/ml. Furthermore, the synthetic tripeptide (Ile-Gln-Asn) exhibited the similar adipogenesis effects to the purified peptide. Thus, these results showed the potential anti-obesity effect of the purified peptide through control of adiposity.