八倍体小冰麦及其亲本种子醇溶蛋白和高分子量麦谷蛋白亚基的研究

对５个八倍体小冰麦种子醇溶蛋白和高分子量麦谷蛋白亚基的电泳谱带进行了分析,结果表明：八倍体小冰麦中１和中２的电泳谱带基本相同,中３、中４、中５的电泳谱带基本相同,但完全不同于中１和中２的类型。八倍体小冰麦中１和中２同天蓝冰草（Ａｇｒｏｐｙｒｏｎｉｎｔｅｒｍｅｄｉｕｍ（Ｈｏｓｔ）Ｐ．Ｂ．＝Ｅｌｙｔｒｉｇｉａｉｎｔｅｒｍｅｄｉａ（Ｈｏｓｔ）Ｎｅｖｓｋｉ＝Ｔｈｉｎｏｐｙｒｕｍｉｎｔｅｒｍｅｄｉｕｍ（Ｈｏｓｔ）ＢａｒｋｗａｒｔｈａｎｄＤｅｗｅｙ）在高分子量麦谷蛋白亚基上存在一条相同的谱带,在醇溶蛋白谱带上出现了小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）和冰草均没有的带型。中３、中４、中５在醇溶蛋白谱带上具有一条冰草×染色体组的特征谱带,其基因表达程度同冰草类似。从５个八倍体小冰麦种子醇溶蛋白和高分子量麦谷蛋白亚基的电泳图谱结果,分析了八倍体小冰麦染色体组构成及亲本来源,并探讨了八倍体小冰麦在优质麦育种过程中的价值。     

Development of Panel of Monoclonal Antibodies Specific to Urochordate Cell Surface Antigens

Monoclonal antibodies are an important tool in the study of botryllid ascidians’ immunology and developmental biology. Here we describe the development of a panel of 38 monoclonal antibodies that are specific to Botryllus schlosseri (Ascidiacea; subfamily Botryllinae) cell surface antigens. Many of these hybridomas recognize (by enzyme-linked immunosorbent assay and immunohistochemistry) epitopes of Botrylloides subpopulations (SP) II and III from the Mediterranean coast of Israel and show, on blood cell smear assays, reactions with subsets of Botryllus circulating blood cells. Fluorescence-activated cell sorting analyses using antibodies positive for botryllid tissues revealed up to 3.6% positive cells. ELISA screenings were performed with 64 new monoclonal antibodies on 5 different individual botryllid ascidian colonies (B. schlosseri, Botrylloides). The positive antibodies in this panel identified a large number of different antigenic determinants, some of which distinguish Botryllus versus Botrylloides colonies, and other, different colonies within these two species, or different cell types within tissues, embryos, and buds of individual colonies. Only 21 monoclonal antibodies tested positive with all colonies. Cross-reactivity with at least one Botrylloides colony was recorded in 49 hybridomas that identified Botryllus cells. This wide panel of monoclonal antibodies is the first such detailed set of monoclonals available for studies on botryllid ascidians.     

Characterization of Antigenic Polypeptides Recognized by Monoclonal Antibodies Specific to the Myoplasm of Eggs of the Ascidian Ciona intestinalis

The determinants responsible for the differentiation of ascidian larval muscle cells are thought to be contained within the egg myoplasm. To analyze the macromolecules composing the myoplasm, several hybridoma cell lines which secrete monoclonal antibodies specific to myoplasmic components of Ciona eggs have been established (17). In the present investigation, seven of these myoplasm-specific antigens were characterized according to their molecular features and distribution patterns within the egg cytoplasm. Four of the seven antigenic polypeptides were shown to be components of the cortical cytoplasm, two were related to mitochondria, and one is likely to be a yolk protein. An antigen recognized by IIG6B2 antibody, which inhibited muscle development when injected into fertilized eggs, was a single polypeptide of relative molecular mass about 40,000 and isoelectric point about 5. The antigen was designated myoplasmin-C1 after its characteristic localization. The IIF9E9 antigen was a single 35-kDa polypeptide related to mitochondria and was thus designated myoplasmin-M1. The other five antibodies recognized two or more spots by immunoblotting analysis using two-dimensional gel electrophoresis. All of these myoplasm-specific antigens, except for the IIH10D6 antigen, are likely to be produced by the oocyte itself. Synthesis of IIH10D6 antigen seems to be associated with test cells.     

重金属锌单克隆抗体的制备及鉴定

通过双功能螯合剂S-20-(4-异硫氰苄基)-二乙烯三胺五乙酸(p-SCN-Bn-DTPA)将锌离子(Zn2+)分别与载体蛋白匙孔血蓝蛋白(keyhole limpet hemocyanin,KLH)和牛血清白蛋白(bovine serum albumin,BSA)偶联.通过二喹啉甲酸(bicinchoninic acid,BCA)法测抗原蛋白浓度,对抗原、KLH和BSA分别进行紫外分光光度计扫描,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)进行定性鉴定,利用石墨炉原子吸收分光光度法检测抗原中Zn2+含量等,成功获得了免疫抗原Zn-DTPA-KLH和检测抗原Zn-DTPA-BSA、DTPA-BSA.用Zn-DTPA-KLH免疫BALB/c小鼠,通过细胞融合,极限稀释法亚克隆,间接酶联免疫吸附法(enzyme-linked immuno sorbent assay,ELISA)筛选,获得了1株稳定分泌抗重金属锌抗体的杂交瘤细胞株(Z1A5).Z1A5染色体数目在100以上,所分泌的抗体为IgM亚类,轻链为kappa型,腹水型抗体效价高达1∶51 200.本研究为锌离子残留免疫学检测方法的建立提供了物质及技术基础,对提高风险评估工作的效率和质量,保障食品安全有重要现实意义.     

Systematic Analysis of Pericarp Starch Accumulation and Degradation during Wheat Caryopsis Development

Although wheat (Triticum aestivum L.) pericarp starch granule (PSG) has been well-studied, our knowledge of its features and mechanism of accumulation and degradation during pericarp growth is poor. In the present study, developing wheat caryopses were collected and starch granules were extracted from their pericarp to investigate the morphological and structural characteristics of PSGs using microscopy, X-ray diffraction and Fourier transform infrared spectroscopy techniques. Relative gene expression levels of ADP-glucose pyrophosphorylase (APGase), granule-bound starch synthase II (GBSS II), and α-amylase (AMY) were quantified by quantitative real-time polymerase chain reaction. PSGs presented as single or multiple starch granules and were synthesized both in the amyloplast and chloroplast in the pericarp. PSG degradation occurred in the mesocarp, beginning at 6 days after anthesis. Amylose contents in PSGs were lower and relative degrees of crystallinity were higher at later stages of development than at earlier stages. Short-range ordered structures in the external regions of PSGs showed no differences in the developing pericarp. When hydrolyzed by α-amylase, PSGs at various developmental stages showed high degrees of enzymolysis. Expression levels of AGPase, GBSS II, and AMY were closely related to starch synthesis and degradation. These results help elucidate the mechanisms of accumulation and degradation as well as the functions of PSG during wheat caryopsis development.     

Development of Rabbit Monoclonal Antibodies for Detection of Alpha-Dystroglycan in Normal and Dystrophic Tissue

Alpha-dystroglycan requires a rare O-mannose glycan modification to form its binding epitope for extracellular matrix proteins such as laminin. This functional glycan is disrupted in a cohort of muscular dystrophies, the secondary dystroglycanopathies, and is abnormal in some metastatic cancers. The most commonly used reagent for detection of alpha-dystroglycan is mouse monoclonal antibody IIH6, but it requires the functional O-mannose structure for recognition. Therefore, the ability to detect alpha-dystroglycan protein in disease states where it lacks the full O-mannose glycan has been limited. To overcome this hurdle, rabbit monoclonal antibodies against the alpha-dystroglycan C-terminus were generated. The new antibodies, named 5–2, 29–5, and 45–3, detect alpha-dystroglycan from mouse, rat and pig skeletal muscle by Western blot and immunofluorescence. In a mouse model of fukutin-deficient dystroglycanopathy, all antibodies detected low molecular weight alpha-dystroglycan in disease samples demonstrating a loss of functional glycosylation. Alternately, in a porcine model of Becker muscular dystrophy, relative abundance of alpha-dystroglycan was decreased, consistent with a reduction in expression of the dystrophin-glycoprotein complex in affected muscle. Therefore, these new rabbit monoclonal antibodies are suitable reagents for alpha-dystroglycan core protein detection and will enhance dystroglycan-related studies.     

A Comparison of Seed and Seedling Phytochrome in Avena sativa L. using Monoclonal Antibodies

Hilton, J.R. and Thomas, B. 1985. A comparison of seed and seedlingphytochrome in Avena saliva L.using monoclonal antibodies.—J.exp. Bot. 36: 1937–1946.The kinetics of phytochrome accumulationduring imbibition and germination of seeds of Avena saliva L.cv. Saladin has been determined in vivo by spectrophotometryand in extracts by using Enzyme–Linked Immunosorbent Assay(EL1SA). In vivo measurements show two phases of phytochromeincrease. The first occurs during the initial 2 h of imbibitionand is associated with the hydration of the seed proteins; thesecond larger increase begins after 16 h, due probably to denovo synthesis. An ELISA using monoclonal antibodies purifiedfrom dark grown Avena seedlings detected only the second increasein phytochrome content. Mixing experiments indicate that theinability to detect phytochrome by ELISA during the first 16h is not due to the presence of inhibitors in the extracts.It is concluded that pre–existent seed phytochrome isantigenically dissimilar to seedling phytochrome. These twopools of phytochrome are stable and unstable respectively withregard to Pfr destruction. Key words: Immunology, phytochrome, seed     

Development of ELISA for Detection of Mercury Based on Specific Monoclonal Antibodies Against Mercury-Chelate

Immunoassays for heavy metals offer an alternative approach to traditional techniques for detection of mercury. In this study, a mercury-chelate was prepared with 1-(4-aminobenzyl) ethylenediamine-N,N,N′,N′-tetraacetic acid (aminobenzyl-EDTA). The resulting complex was linked to keyhole limpet hemocyanin (KLH) or bovine serum albumin via the amino group and used as the immunizing antigen or detection antigen, respectively. BALB/c mice were immunized with KLH-aminobenzyl-EDTA-Hg and spleen cells from BALB/C mice were fused with Sp2/0 cells. One cell line (5F7) produced monoclonal antibodies with preferential selectivity and sensitivity for aminobenzyl-EDTA-Hg. This cell line had an affinity constant of 4.31?×?10⁹ L/mol and its cross-reactivity (CR) with other metals was <2%. The antibody was used for competitive indirect ELISA (CI-ELISA) for Hg²⁺ measurements. The detection range was 0.087–790.4 μg/L and the lower limit of detection was 0.042 μg/L. The concentrations of mercury in environmental water samples obtained by CI-ELISA correlated well with graphite furnace atomic absorption spectrometry (GFAAS), and the mean recovery was 88.82% to 104.64%. These results indicate that this method could be used for monitoring mercury of water.     

Production and Characterization of Two Monoclonal Antibodies Specific for Plasmopara halstedii

Sunflower downy mildew, caused by the fungus Plasmopara halstedii, is a potentially devastating disease. We produced two monoclonal antibodies (MAbs) (12C9 and 18E2) by immunizing mice with a partially purified extract of P. halstedii race 1. Both MAbs detected in enzyme-linked immunosorbent assay (ELISA) all races of P. halstedii present in France. No cross-reactions were observed with Plasmopara viticola or with other fungi commonly associated with sunflowers. Both MAbs recognized the same three fungal antigens with molecular masses of 68, 140, and 192 kDa. However, the epitopes on the fungal antigens were distinct and repetitive. Seed homogenates from infected plants were incubated in wells coated with MAb 18E2. This resulted in the trapping of P. halstedii antigens that were identified with biotinylated MAb 12C9. No reactions were seen with seed homogenates from healthy plants. Thus, our results suggest that these MAbs might be used to develop a sandwich ELISA detection system for P. halstedii in infected seeds.     

Serodiagnosis of Pepper Veinal Mottle Virus in West Africa using Specific Monoclonal Antibodies in DAS-ELISA

In order to develop a reliable diagnostic test for pepper veinal mottle virus (PVMV), a panel of monoclonal antibodies (mAbs), raised to a Nigerian isolate (PVMW-Ni), was evaluated by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) on infected pepper samples from seven West African countries. PVMV from different geographical locations was serologically very homogeneous and no significant epitope differences were detected even with a very sensitive double-mAb sandwich ELISA. This ELISA format was optimized with mAb 6C12 and its application in a diagnostic test resulted in the identification of PVMV in Burkina Faso, Togo and Sénégal where it had not been previously reported.     

Accumulation of Prolamines and Glutelins during Rice Seed Development: a Quantitative Evaluation

The levels of prolamines and glutelins, the storage proteinsof rice, were quantified during seed development by immunoblotanalysis. Although both storage proteins were first detectedin 10 day old seeds and their amounts steadily increased throughoutseed development, the relative proportions of glutelins andprolamines were not constant. The molar ratio of glutelins toprolamines was 1.7 in 10 day old seeds and this ratio steadilydecreased to 1.2 in 25 day old seeds due to the increased synthesisand accumulation of prolamines specifically during the latterstages of seed development. In vivo pulse chase labelling studiesconfirmed that the rate of prolamine synthesis as compared tothat evident for glutelin increased during the latter half ofseed development and that protein turnover was not the basisfor the differences in accumulation patterns of these storageproteins. These results indicate that the storage proteins exhibitdifferent temporal accumulation patterns during seed developmentand, moreover, demonstrate that prolamines comprise a much largerproportion of the total storage protein fraction than previouslyrecognized. (Received August 31, 1992; Accepted November 27, 1992)     

单克隆抗体ND-1-量子点荧光探针对大肠癌细胞的特异成像研究

目的制备抗人大肠癌单克隆抗体ND-1的量子点荧光探针,实现对大肠癌细胞的靶向成像。方法采用共价偶联方法,以1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)和N-羟基硫代琥珀酰亚胺(NHS)为缩合剂,通过在反应体系中加入不同摩尔比例的单克隆抗体ND-1和游离量子点QD605进行条件优化,制备偶联产物ND-1-QD605荧光探针;利用荧光光谱扫描技术对ND-1-QD605进行光学特性表征,并检测其抗光漂白能力;利用免疫荧光方法检测ND-1-QD605对大肠癌细胞的靶向结合能力。结果在量子点QD605与单克隆抗体ND-1摩尔比1:40条件下,可实现二者的高效偶联;荧光光谱分析显示ND-1-QD605保留了游离量子点QD605优良的荧光特性;在激发光照射1h内,ND-1-QD605荧光强度未发生明显改变;荧光显微镜观察可见该探针能够与表达有相应抗原LEA的人大肠癌CCL187细胞特异性结合,呈现高灵敏度、特异性荧光成像。结论制备的单克隆抗体ND-1的量子点荧光探针具有大肠癌细胞靶向成像能力,有望为大肠癌的体内靶向成像研究和临床诊断提供新方法。     

专一识别脱落酸甲酯的单克隆抗体的制备与应用

专一识别２－顺（Ｓ）ＡＢＡ甲酯的单克隆抗体来源于以ＡＢＡ分子中的１－ＣＯＯＨ为偶联位点合成的免疫原。它与游离态ＡＢＡ和结合态ＡＢＡ葡萄糖酯的交叉反应仅分别为１％与３．５％,而与ＡＢＡ类似物,如２－顺－黄质醛、紫黄质以及ＡＢＡ的２－反式异构体和（Ｒ）－对映体则无交叉反应。利用该抗体建立的高度灵敏和精确的ＡＢＡｍｅ酶联免疫测定法,其检测线性范围为０．０４８～１．５２ｐｍｏｌ。通过ＡＢＡｍｅＥＬＩＳＡ和ＧＡ１＋３ＥＬＩＳＡ分析可知羊蹄叶片衰老与内源ＧＡ１＋３／ＡＢＡ比值的下降有关。     

Monoclonal Antibodies Specific for Non-Derivatized Gibberellins I. Preparation of Monoclonal Antibodies against GA4 and Their Use in Immunoaffinity Column Chromatography

Cell lines producing monoclonal antibodies specific for non-derivatizedgibberellins (GAs) were prepared from spleen cells of a mouseimmunized with an immunogen that carried 16-substituted GA₄as a hapten. The conditions for association and dissociationof antibody-hapten complexes were examined to determine themost effective procedure for immunoaffinity chromatography.The final procedure adopted was proven to be very effectiveby its application to the analysis of GAs in the anthers ofrice and immature seeds of Phaseolus vulgaris ⁴Present address: Department of Agricultural Chemistry, UtsunomiyaUniversity, Mine-machi 350, Utsunomiya, 321 Japan (Received August 8, 1990; Accepted March 7, 1991)     

Wheat Seed Carboxypeptidase and Joint Action on Gliadin of Proteases from Dry and Germinating Seeds

A carboxypeptidase preparation, homogeneous according to polyacrylamidegel electrophoresis and ultracentrifugation, was obtained fromwheat seeds. The isolation procedure included (NH₄)₂SO₄ fractionation,gel-filtration on Sepharose-6B and affinity chromatography onCABS-Sepharose. Mr of the enzyme determined by gel-filtrationwas 126 000. The enzyme consisted of two non-identical subunitsof Mr 60 000 and 63 000. The pl of the carboxypeptidase was5.7. The inhibitory analysis revealed that the isolated enzymeis a serine carboxypeptidase. The carboxypeptidase preferentiallyhydrolysed N-substituted dipeptides with aromatic amino acidresidues at the C-terminus and showed weak hydrolysing activitytowards gliadin. The combined action of carboxypeptidase andaspartic proteinase from dry wheat seeds led to an increasein the degree of proteolysis of the storage protein comparedto that resulting from the sum of the action of individual enzymes.The cysteine proteinase from germinated wheat seeds caused completedegradation both of gliadin, which had first been treated withproteases of dry seeds (aspartic proteinase plus carboxypeptidase),and of untreated gliadin. However, when gliadin had first beenhydrolysed with dry seed proteases, the rate of its proteolysiswith cysteine proteinase increased 3–4 times comparedto the non-hydrolysed gliadin. The data indicate the importanceof preliminary modification of gliadin with dry wheat proteases,which, apparently, enhances the supply of nutritive substancesto the embryo in the course of seed germination. Key words: Wheat, gliadin, carboxypeptidase, proteinases, proteolysis     

Monoclonal Antibodies Specific for Xanthomonas campestris Bacteria Pathogenic on Wheat and other Small Grains, in Comparison with Polyclonal Antisera

4 hybridoma cell lines (named F1-AA9-D9, F1-AB3-B6, F1-BC7-C1 and F2-CA7-F11) secreting monoclonal antibodies to Xanthomonas campestris pv. undulosa were produced by fusing splenocytes from immunized Lou rats with IR983F myeloma cells. Whole cells were used both as immunogen and as antigen in ELISA and indirect immunofluorescence tests.
The monoclonal antibodies produced reacted positively with X. c. pv. undulosa (38 strains), pv. translucens (3), pv. hordei (3), pv. cerealis (2) and pv. secalis (1).
Strains from other pathovars ( X. c. pv. arrhenatheri, pv. graminis, pv. manihotis, pv. oryzicola, pv. poae and pv. pruni ) and from other species ( X axonopodis, X. ampelina ) and genus ( Pseudomonas, Erwinia, Clavibacter , wheat saprophytic strains) gave a negative reaction. In comparison, seven polyclonal rabbit antisera showed to be less specific: they reacted with unrelated X. campestris pathovars as well as with Pseudomonas strains. Nevertheless, the use of phenol-treated cells in Ouchterlony double immunodiffusion could reduce the effect of cross-reaction for antisera.
The detection of X. c. pv. undulosa by indirect immunofluorescence on infected wheat seed lots has already been applied with success.     

抗新型隐球菌荚膜相关蛋白CAP10单克隆抗体的制备及鉴定

目的:以新型隐球菌荚膜相关蛋白CAP10为靶抗原,制备并鉴定特异性抗CAP10的单克隆抗体。方法:用纯化的重组CAP10免疫BALB/c小鼠,血清抗体效价达到适当水平时进行细胞融合;经多次亚克隆筛选出分泌特异性抗体的细胞株,制备单抗腹水并进行抗体效价测定及亚类鉴定。结果:获得11株能稳定分泌抗新型隐球菌荚膜相关蛋白CAP10的单克隆抗体杂交瘤细胞株,抗体效价高且抗原特异性强。结论:获得了针对新型隐球菌荚膜相关蛋白CAP10的单克隆抗体,为深入研究CAP10蛋白的功能,以及临床新型隐球菌的检测和血清型分析奠定了基础。