稻瘟病菌单克隆抗体2B4的研究

从230个具有ELISA阳性反应的细胞株获得了11株具有高效价的单克隆抗体, 其中2B4显示较强结合作用,并均能与分生孢子、芽管和附着胞表面结合。Western blotting分析表明,单抗2B4能与孢子芽管表面的1%SDS提取物中的15kDa的蛋白结合。免疫金定位发现该蛋白确是在病菌各个形态阶段广泛存在的分泌性蛋白。采用2B4从分生孢子cDNA表达文库中筛选获得6个阳性克隆。该克隆MP1表达的蛋白抗原与实际存在的15KDa是相一致的。其基因克隆及功能分析正在研究之中。     

稻瘟病菌单克隆抗体2B4的研究

从230个具有ELISA阳性反应的细胞株获得了11株具有高效价的单克隆抗体,其中2B4显示较强结合作用,并均能与分子孢子、芽管和附着胞表面结合。Western blotting分析表明,单抗2B4能与孢子芽管表面的1％SDS提取物中的15kDa的蛋白结合。免疫金定位发现该蛋白确是在病菌各个形态阶段广泛存在的分泌性蛋白。采用2B4从分生孢子cDNA表达文库中筛选获得6个阳性克隆。该克隆MP1表达的蛋白抗原与实际存在的15 kDa是相一致的。其基因克隆及功能分析正在研究之中。     

类似S-层蛋白的苏云金芽胞杆菌伴胞晶体蛋白基因的克隆

芽胞杆菌CTC菌株被鉴定为苏云金芽胞杆菌 ,鞭毛血清型H2 ,幕虫亚种 ;产生卵圆形伴胞晶体 ,伴胞晶体蛋白为 1 0 0kD ;测定了该蛋白的N 末端序列 ,该序列与炭疽芽胞杆菌的细胞表面S 层蛋白具 92 %～ 93%相似性 ;根据Southern杂交制作了该晶体蛋白基因ctc所在位置的限制性酶切图谱 ,分别克隆了该基因 5′和 3′端所在的 2 9kbXbaI片段和 3 1kbClaIDNA片段 ,彼此间具 0 6kb重叠 ,通过拼接获得含完整ctc基因的克隆。含该基因的大肠杆菌与表达S 层蛋白的大肠杆菌具相似生长特征。初步表明CTC菌株的伴胞晶体由细胞表面S 层蛋白组成。苏云金芽胞杆菌区别于蜡状芽胞杆菌和炭疽芽胞杆菌的唯一标准是能形成伴胞晶体 ,由于S 层是细胞表面的结构成分 ,本文对CTC菌株鉴定为苏云金芽胞杆菌以及伴胞晶体作为苏云金芽胞杆菌鉴别的唯一标准提出了质疑     

一种与核酸复合的脱落酸结合蛋白

用抗脱落酸 (Abscisicacid ,ABA)结合蛋白 (ABBP)血清作为探针筛选玉米cDNA表达文库 ,从 2 0 0 0 0 0个独立噬斑中 ,仅获得 1个编码多肽的cDNA克隆 ,它与动物的某些核酸结合蛋白基因的同源性高达 6 0 %～ 6 5 % .此外 ,获得 1 2 0个编码玉米 1 7SrRNA的cDNA克隆 .鉴于此 ,对经ABA亲和纯化所得的ABBP及其抗血清进行了复查 .发现rRNA伴随ABBP而存在 ;该抗血清除能识别ABBP外 ,还能识别rRNA ,推断某些ABBP具有与核糖核酸结合的性质 ,即能与rRNA形成复合物     

噬菌体肽库筛选抗人B7-H4中和抗体的模拟抗原表位及其免疫原性鉴定

目的:用噬菌体呈现随机12肽库筛选能与抗人B7-H4(h B7-H4)中和抗体特异性结合的模拟抗原表位肽,并用其免疫小鼠检测其免疫原性。方法:以抗h B7-H4中和抗体为靶分子,用体外生物淘洗法从噬菌体呈现随机12肽库中筛选与之结合的噬菌体克隆,用竞争性细胞ELISA鉴定阳性噬菌体克隆;化学合成候选多肽,并与钥孔血蓝蛋白或破伤风毒素偶联鉴定多肽的特异性;进一步用融合蛋白免疫小鼠检测其免疫原性和抗血清的补体依赖的细胞杀伤活性(CDC)。结果:经过3轮体外筛选后随机挑取50个阳性噬菌体克隆,其中20个克隆与抗h B7-H4抗体有较强的结合能力,DNA测序得到6组结构相似的肽序列;竞争性ELISA结果显示1号肽噬菌体能与细胞表面的h B7-H4竞争性地结合抗h B7-H4单抗;点杂交结果显示1号肽能特异性结合抗h B7-H4单抗;小鼠免疫实验结果显示1号肽融合蛋白能诱导高滴度的抗h B7-H4抗血清,并且抗血清具有补体依赖的细胞杀伤活性。结论:筛选得到能与抗h B7-H4中和抗体特异性结合的12肽模拟抗原表位序列并且具有免疫原性,为进一步开发h B7-H4相关的多肽疫苗提供了实验依据。     

类似S-层蛋白的苏云金芽胞杆菌伴胞晶体蛋白基因的克隆

芽胞杆菌CTC菌被鉴定为苏云金芽胞杆菌,鞭毛血清型H2,幕虫亚种;产生卵圆形伴胞晶体,伴胞晶体蛋白为100kD;测定了该蛋白 N－末端序列,该序列与炭疽芽胞杆菌的细胞表面S－层蛋白具92－93％相似性,根据Southern杂交制作了该晶体蛋白基因ctc所在位置的限制性酶切图谱,分别克隆了该基因5’和3’端所在2．9kb XbaI片段和3．1kb Cla I DNA片段,彼此间具0．6kb重叠,通过拼接获得含完整ctc基因的克隆,含该基因的大肠杆菌与表达S－层蛋白的大肠杆菌具相似生长特征,初步表明CTC菌株的伴胞晶体由细胞表面S－层蛋白组成,苏云金芽胞杆菌区别于蜡状芽胞杆菌和炭疽芽胞杆菌的唯一标准是能形成伴胞晶体,由于S－层是细胞表面的结构成分,本文对CTC菌株鉴定为苏云金芽胞杆菌以及伴胞晶体作为苏云金芽胞杆菌鉴别的唯一标准提出了质疑。     

稻瘟病菌单克隆抗体的研制及其对稻瘟病菌附着胞形成的影响

稻温病菌的分生孢子、芽管、附着胞的混合物作为抗原免疫BALB/c小鼠,取免疫小鼠的脾细胞与SP2/0骨髓瘤细胞在50%PEG下融合成杂交瘤细胞,用间接ELISA筛选阳性孔,获11株单克隆抗体。间接免疫荧光试验表明其中4株单克隆抗体2B4、4A1、1D1和2H4分别与孢子、芽管或附着胞有特异性结合;Western blotting分析发现2B4、4A1、1D1单克隆抗体分别与孢子、芽管表面的提取物有不同的结合带;此四株单克隆抗体均干扰稻温病菌附着胞形成,并抑制稻温病菌在叶表的致病性。     

一种新的乙型肝炎病毒表面抗原结合蛋白的筛选、表达及其生物学活性的初步研究

为了研究乙肝病毒侵染肝细胞过程中的功能蛋白 ,通过印迹免疫分析技术从人肝cDNA噬菌体表达库中筛选出一株编码乙肝表面抗原结合蛋白 (hepatitisBsurfaceantigenbindingprotein ,HBsAg BP)的cDNA克隆 .基因测序结果表明 ,该cDNA具有独立的开放阅读框架 ,编码 1个由 344个氨基酸残基构成的可溶性蛋白分子 ,属于免疫球蛋白超家族成员 .将该基因克隆到原核表达载体pTriplEx后 ,在E .coliXL1 Blue菌株中获得 4 4kD的重组蛋白 .重组蛋白经Western印迹和ELISA实验证明具有与乙肝表面抗原特异性结合的能力 .进一步经流式细胞仪实验显示 ,在纯化的重组蛋白存在的情况下 ,天然的HBsAg与肝细胞株HepG2的亲和力显著增高 .结果显示 ,该乙肝表面抗原结合蛋白可能是介导乙肝病毒对肝细胞亲和侵染的可溶性辅助受体 .     

西藏青稞4个B组醇溶蛋白基因的克隆和特征

从两份西藏青稞材料中分离克隆出4个B组醇溶蛋白基因（BH1—BH4）,DNA测序结果表明：它们均包含完整的开放阅读框。推断的氨基酸序列与先前报道的大麦B组醇溶蛋白具有相同的蛋白质基本结构。系统分析表明：它们推断的氨基酸序列与栽培大麦中的B组醇溶蛋白具有较高的相关性,与野生大麦和山羊草属的醇溶谷蛋白相似性较低。并且,在4个基因BH1—BH4中,BH1与先前报道的B组醇溶蛋白基因有较低的序列相似性,因此我们对BH1基因进行了原核表达,含该基因的表达载体在大肠杆菌中表达出相对分子质量为28．15kDa并以包涵体形式存在的蛋白,进一步对其在青稞谷粒品质改良中的潜在价值进行了探讨。     

盐芥ThDREB2B基因克隆及功能分析

利用RACE技术从抗逆模式植物盐芥中克隆获得了1个DREB(dehydration responsive element binding)类转录因子基因,命名为ThDREB2B(NCBI登录号EF653377)。结果表明:(1)ThDREB2B基因cDNA全长1 486bp,包含1个954bp的开放阅读框,编码316个氨基酸;推测编码的蛋白质分子量约36.0kD,等电点为4.81,第76～135位氨基酸构成1个AP2结构域。(2)系统进化分析表明,ThDREB2B属于DREB亚家族的A-2亚组,与拟南芥AtDREB2B基因遗传距离最近。(3)半定量RT-PCR检测显示,ThDREB2B基因在正常生长条件下低丰度表达,在低温、干旱或高盐胁迫下上调表达。(4)酵母单杂交结果表明,ThDREB2B蛋白能与DRE元件特异结合,但转录激活能力弱。推测ThDREB2B蛋白可能需要翻译后修饰以获得转录激活功能。     

Composition and organisation of extracellular matrices around germ tubes and appressoria ofColletotrichum lindemuthianum

Summary The ultrastructure and composition of the extracellular matrices (ECMs) associated with germ tubes and appressoria ofColletotrichum lindemuthianum have been examined. Flexuous fibres (fimbriae), up to 6 m long and 4–30 nm in diameter, protruded from the surface of germ tubes and appressoria. Anionic colloidal gold and lectin cytochemistry showed that ECMs of germ tubes and appressoria contain basic proteins, -D-mannose and -D-galactose residues. A monoclonal antibody, UB26, was raised to infection structures isolated from leaves ofPhaseolus vulgaris infected withC. lindemuthianum. UB26 recognised a protein epitope on two glycoproteins (M_r 133,000 and 146,000). Reductions in the M_r of these proteins after treatment with peptide-N-glycosidase and trifluoromethane sulphonic acid suggest that they carry N- and O-linked side-chains. Immunofluorescence and EM-immunogold labelling showed that glycoproteins recognised by UB26 were restricted to the ECMs around germ tubes and appressoria but fimbriae were not labelled. Unlike appressorial germ tubes formed in vitro, intracellular infection hyphae were not labelled, suggesting that the glycoproteins recognised by UB26 are not present on fungal structures formed within host cells. In liquid culture, these glycoproteins were not released into the medium, suggesting they are physically linked to the cell wall. Also, the glycoproteins were not removed from glass surfaces by ultrasonication. These results suggest that glycoproteins recognised by UB26 may be involved in the adhesion of germ tubes and appressoria to substrata. Our results show that the ECMs of germ tubes and appressoria differ markedly in structure and composition from those of conidia and intracellular hyphae, and that extracellular glycoproteins are associated with specific regions of the fungal cell surface.Abbreviations ECM extracellular matrix - BPA Bauhinia purpurea agglutinin - BSA bovine serum albumin - DIC differential interference contrast - FITC fluorescein isothiocyanate - GNL Galanthus nivalis lectin - GSI-B₄ Griffonia simplicifolia isolectin B₄ - HEPES (N-(2-hydroxyethyl)piperazine-N-(2-ethanesulphonic acid) - IIF indirect immunofluorescence - IPC isopycnic centrifugation - MAb monoclonal antibody - PEG polyethylene glycol - PBS phosphate buffered saline - PNGase peptideN-glycosidase - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - TCS tissue culture supernatant - TEM transmission electron microscopy - TFMS trifluoromethane sulphonic acid     

Molecular weight determination of two genetically linked cell surface murine antigens: ThB and Ly-6

Various murine tumor lines were screened by FACS analysis for the surface antigens ThB and Ly-6.2. Positive cell lines were used for immunoprecipitation studies. A monoclonal ThB-specific antibody immunoprecipitated a unique acidic protein of approximately 16 000 daltons from several positive tumors and from concanavalin A (Con-A) and LPS activated splenic lymphocytes. Monoclonal Ly-6.2-specific antibody was used to immunoprecipitate a 33 500 dalton protein that was shown to exist in four similarly sized forms with different basic charges. In the course of these studies, the apparent molecular weight of the surface antigen T 30, immunoprecipitated with a monoclonal T 30-specific antibody from the cell line EL4, was found to be approximately 25 000 daltons.     

Dynamics of cell wall components of Magnaporthe grisea during infectious structure development

Oligosaccharides derived from cell wall of fungal pathogens induce host primary immune responses. To understand fungal strategies circumventing the host plant immune responses, cell wall polysaccharide localization was investigated using fluorescent labels during infectious structure differentiation in the rice blast fungus Magnaporthe grisea . α-1,3-glucan was labelled only on appressoria developing on plastic surfaces, whereas it was detected on both germ tubes and appressoria on plant surfaces. Chitin, chitosan and β-1,3-glucan were detected on germ tubes and appressoria regardless of the substrate. Major polysaccharides labelled at accessible surface of infectious hyphae were α-1,3-glucan and chitosan, but after enzymatic digestion of α-1,3-glucan, β-1,3-glucan and chitin became detectable. Immunoelectron microscopic analysis showed α-1,3-glucan and β-1,3-glucan intermixed in the cell wall of infectious hyphae; however, α-1,3-glucan tended to be distributed farther from the fungal cell membrane. The fungal cell wall became more tolerant to chitinase digestion upon accumulation of α-1,3-glucan. Accumulation of α-1,3-glucan was dependent on the Mps1 MAP kinase pathway, which was activated by a plant wax derivative, 1,16-hexadecanediol. Taken together, α-1,3-glucan spatially and functionally masks β-1,3-glucan and chitin in the cell wall of infectious hyphae. Thus, a dynamic change of composition of cell wall polysaccharides occurs during plant infection in M. grisea .     

Host defence against C. albicans infections in IgH transgenic mice with V(H) derived from a natural anti-keratin antibody

Fungal infections have been increasing and life-threatening in recent years, but host immune responses, especially the humoral immunity, to fungi have not been fully understood. In the present study, we report that natural antibodies from unimmunized mice bind to Candida albicans. We established a monoclonal natural antibody, 3B4, which recognized a surface antigen located at germ tubes of C. albicans. The 3B4 antibody protected mice from C. albicans-induced death in passive immunization, by mechanisms involving suppressing germ tube formation and modulating phagocytosis. Interestingly, 3B4 also bound to a self-antigen keratin. To further study the generation and anti-C. albicans activities of natural antibodies in vivo, we constructed a mu chain transgenic mouse (TgV(H)3B4) using the V(H) gene from 3B4. TgV(H)3B4 had elevated serum anti-keratin/C. albicans IgM, and were resistant to C. albicans infections. Analyses of B cell development showed that in TgV(H)3B4, B cells secreting the anti-keratin/C. albicans antibodies were enriched in the B1 B cell compartment. Our findings reveal an important role of keratin-reactive natural antibodies in anti-C. albicans immune responses, and suggest that keratin may function in selecting B cells into the B1 B cell compartment, where natural antibodies are made to fight fungal infections.     

Detection of an elicitor on infection structures of Puccinia graminis using monoclonal antibodies

The basidiomycetous fungus Puccinia graminis f. sp. tritici causes the stem rust disease of wheat. Resistance of wheat to the fungus is often associated with the hypersensitive reaction of infected host cells. A glycoprotein isolated from germ tube cell walls of the pathogen elicits a hypersensitive-like response when injected into wheat leaves. Infection structures morphologically identical to those grown on wheat were induced in the absence of the host plant, and indirect immunofluorescence together with specific monoclonal antibodies to the elicitor was employed to locate the antigen at fungal infection structures. No binding occurred to germ tubes or appressoria. The antibodies located the antigen only at that part of the fungal infection structure that develops endophytically in nature and, moreover, only at the youngest part of this structure. In rust-infected wheat leaves, the immunolabel appeared only at haustoria, the structures thought to be involved in specific recognition between host and parasite.     

The mitogen-activated protein kinase gene MAF1 is essential for the early differentiation phase of appressorium formation in Colletotrichum lagenarium

Colletotrichum lagenarium, the causal agent of cucumber anthracnose, invades host plants by forming a specialized infection structure called an appressorium. In this fungus, the mitogen-activated protein kinase (MAPK) gene CMK1 is involved in several steps of the infection process, including appressorium formation. In this study, the goal was to investigate roles of other MAPKs in C. lagenarium. The MAPK gene MAF1, related to Saccharomyces cerevisiae MPK1 and Magnaporthe grisea MPS1, was isolated and functionally characterized. The maf1 gene replacement mutants grew normally, but there was a significant reduction in conidiation and fungal pathogenicity. The M. grisea mps1 mutant forms appressoria, but conidia of the C. lagenarium maf1 mutants produced elongated germ tubes without appressoria on both host plant and glass, on which the wild type forms appressoria, suggesting that MAF1 has an essential role in appressorium formation on inductive surfaces. On a nutrient agar, wild-type conidia produced elongated germ tubes without appressoria. The morphological phenotype of the wild type on the nutrient agar was similar to that of the maf1 mutants on inductive surfaces, suggesting repression of the MAF1-mediated appressorium differentiation on the nutrient agar. The cmk1 mutants failed to form normal appressoria but produced swollen, appressorium-like structures on inductive surfaces, which is morphologically different from the maf1 mutants. These findings suggest that MAF1 is required for the early differentiation phase of appressorium formation, whereas CMK1 is involved in the maturation of appressoria.     

Development of appressoria on conidial germ tubes of <Emphasis Type="Italic">Erysiphe</Emphasis> species

Modes of branching of appressoria on conidial germ tubes of 36 Erysiphe spp. were studied. Only unlobed appressoria, termed alobatus pattern, were seen in E. lonicerae, E. magnifica and E. symphoricarpi. Viewed from above with light or scanning electron microscopes, other species had ± irregular lobing, but from below in the plane of contact with the substrate successive dichotomous branchings at 120° were seen to produce a five-lobed appressorium within 6 h. Each division produced a temporarily dormant outward-facing lobe and an inward limb that continued growth and division to form the axis of curved, hooked, single- or double-headed symmetrical or asymmetrical structures in a helicoid cyme-like pattern. Outlines of extracellular material after removal of germinated conidia confirmed this manner of branching. After 36 h some lobes re-divided forming botryose or jigsaw patterns even extending with extra appressoria to form candelabra-like structures. Conidia developed only one true germ tube; rarely secondary unswollen tubes emerged from spare shoulders or ends. The same true germ tubes developed initially on host surfaces, where secondary tubes and/or extensions from appressorial lobes grew into colony-forming hyphae. Lobed appressoria of Neoerysphe and Phyllactinia also branched at 120°. Podosphaera xanthii exhibited a simpler branching pattern.     

Enrichment of transplantable germ cells in salmonids using a novel monoclonal antibody by magnetic‐activated cell sorting

In the fish germ cell transplantation system, only type A spermatogonia (ASGs) and oogonia are known to be incorporated into the recipient genital ridges, where they undergo gametogenesis. Therefore, high colonization efficiency can be achieved by enriching undifferentiated germ cells out of whole testicular cells. In this study, we used magnetic‐activated cell sorting (MACS) for enriching undifferentiated germ cells of rainbow trout using a monoclonal antibody that recognizes a specific antigen located on the germ cell membrane. We screened the antibodies to be used for MACS by performing immunohistochemistry on rainbow trout gonads. Two antibodies, nos. 172 and 189, showed strong signals for ASGs and oogonia. Next, we performed MACS with antibody no. 172 using gonadal cells isolated from vasa‐gfp rainbow trout showing GFP in undifferentiated germ cells. We found that GFP‐positive cells are highly enriched in antibody no. 172‐positive fractions. Finally, to examine the transplantability of MACS‐enriched cells, we intraperitoneally transplanted sorted or unsorted cells into recipient larvae. We observed that transplantability of sorted cells, particularly ovarian cells, were significantly higher than that of unsorted cells. Therefore, MACS with antibody no. 172 could enrich ASGs and oogonia and become a powerful tool to improve transplantation efficiency in salmonids.