稻曲病菌毒素的活性测定、抗体制备与细胞定位

稻曲病菌在PD液体培养基中生长良好,并能产生对植物细胞具有高度生物抑制活性的毒素。生物学活性测定表明,用100％的甲醇能提取稻曲病菌液体培养物中的粗毒素。粗毒素对小麦胚根胚芽的生长有强烈的抑制作用。把毒素主要成分Ustiloxin A和BSA偶联后,制备了抗血清,ELISA检测表明用两种偶联剂偶联所制备的抗体效价分别为1：20000和1：6000。进一步的免疫胶体金标记分析表明,所制备的抗体能与茵丝中分泌的毒素特异性结合,说明所获得的抗体是特异性的。     

抗金黄色葡萄球菌卵黄抗体的制备及活性研究

目的:制备金黄色葡萄球菌特异性卵黄抗体并考察其活性。方法:采用灭活的金黄色葡萄球菌免疫产蛋母鸡,通过水稀释法、硫酸铵和硫酸钠盐析及凝胶过滤法分离、纯化抗体。抗体效价测定及体外抑菌试验分别采用酶联免疫吸附法(ELISA)和液体培养基比浊法。结果:纯化所得抗体纯度达95.10%,回收率为20.08%。抗体对金黄色葡萄球菌具有特异性,效价最高可达1:6400。体外抑菌试验表明,抗体抑菌活随IgY浓度的增加而增强,当特异性IgY的浓度为10mg/ml时,能完全抑制细菌生长。结论:抗体制备及纯化所述方法简便、可行,所制抗体具有特异性和抑菌活性。     

稻曲球及稻曲病菌菌落微结构的SEM观察

本文对不同培养条件下稻曲病菌菌落及稻曲球的微结构进行了扫描电镜比较研究。在PS培养液里进行液体培养时,稻曲病菌很少产生分生孢子和厚垣孢子,只有培养后期漂浮在培养液表面的菌落可以产生大量的厚垣孢子。病原菌在进行PSA固体培养时,大部分菌株在培养后期产生大量的成堆分布的厚垣孢子,少部分菌株在菌落上产生散生的厚垣孢子。说明暴露于空气有助于稻曲病菌产生厚垣孢子。在煮熟的带壳谷粒上稻曲病菌的生长明显比在去壳上的要慢得多。微结构分析表明,稻曲球表面是一层密集的厚垣孢子,菌丝与稻粒的胚乳层界限分明,大部分稻曲球中部有大块的发育良好的胚乳,并充满密集的淀粉粒。说明稻曲病菌可能在开花灌浆后开始侵染,而且至少后期是腐生的。     

稻曲球及稻曲病菌菌落微结构的SEM观察

本文对不同培养条件下稻曲病菌菌落及稻曲球的微结构进行了扫描电镜比较研究。在PS培养液里进行液体培养时,稻曲病菌很少产生分生孢子和厚垣孢子,只有培养后期漂浮在培养液表面的菌落可以产生大量的厚垣孢子。病原菌在进行PSA固体培养时,大部分菌株在培养后期产生大量的成堆分布的厚垣孢子,少部分菌株在菌落上产生散生的厚垣孢子。说明暴露于空气有助于稻曲病菌产生厚垣孢子。在煮熟的带壳谷粒上稻曲病菌的生长明显比在去壳上的要慢得多。微结构分析表明,稻曲球表面是一层密集的厚垣孢子,菌丝与稻粒的胚乳层界限分明,大部分稻曲球中部有大块的发育良好的胚乳,并充满密集的淀粉粒。说明稻曲病菌可能在开花灌浆后开始侵染,而且至少后期是腐生的。     

靶向金纳米棒对隐球菌活性影响的体外实验研究

目的：设计对隐球菌荚膜特异性标记的靶向金纳米棒,研究靶向金纳米棒的体外光热作用对隐球菌活性的影响。方法晶种生长法制备金纳米棒,偶联隐球菌荚膜抗体,检测表征,与隐球菌体外孵育,近红外激光照射,检测隐球菌活性变化。结果成功制备与荚膜抗体偶联的金纳米棒,体外近红外照射后,隐球菌活性较未偶联抗体的金纳米棒组显著降低。结论靶向性金纳米棒显著增强了近红外激光对隐球菌的光热效应,可用于治疗隐球菌感染的新尝试。     

植物苯丙氨酸解氨酶的研究——Ⅲ.玉米小斑病菌Helminthosporium maydis T小种和大斑病菌Helminthosporium turcicum毒素对苯丙氨酸解氨酶(PAL)的刺激作用

玉米小斑病菌T小种能产生致病毒素,毒素对感病品系叶肉细胞原生质体能产生强烈的毒害作用,并刺激PAL活性增高。T小种毒素对T细胞质雄性不育系有强烈的选择特异性,仅在高浓度时才对正常细胞质保持系有轻微毒害。玉米大斑病菌产生的毒素其作用与小斑病菌T小种毒素相反,即对正常细胞质保持系有选择特异性,但强度较弱。红光对T细胞质雄性不育系和正常细胞质保持系玉米植株刺激PAL活性增高的程度无甚差异。毒素和红光刺激玉米PAL活性增高的机理是不同的。PAL活性的增高与玉米抗病性成负相关。     

纳米磁珠偶联CFP-10特异性单抗富集结核分枝杆菌方法的建立

目的:初步建立基于纳米磁珠偶联培养滤液蛋白10(CFP-10)特异性单抗富集结核分枝杆菌的方法。方法:大肠杆菌表达CFP-10抗原,采用鼠杂交瘤技术制备相应的CFP-10单抗,用NHS修饰的纳米磁珠偶联单抗,用以捕获结核分枝杆菌并进行抗酸染色检测。结果:制备的CFP-10单抗效价为1∶640000;免疫荧光检测显示CFP-10抗体偶联到磁珠上,抗酸染色验证了磁珠偶联的单抗能有效地捕获富集结核分枝杆菌。结论:建立了纳米磁珠偶联CFP-10特异性单抗有效捕获富集结核分枝杆菌的方法,为提高抗酸染色方法的阳性检出率奠定了基础。     

玉米大斑病菌Ht—毒素的萃取及其致病活性

采用体外(in vitro)固体和液体培养玉米大斑病菌——大斑病长晨蠕孢(Helminthosporium turcicum),培养物经有机溶剂萃取后减压蒸发获得了Ht-粗毒素,最后用种子根伸长抑制,离体根冠细胞死亡和离体叶片致萎等生物测定法,对玉米大斑病菌的体外代谢产物进行了致病活性和毒性测定,筛选出乙腈等有机溶剂,可以用于Ht-毒素的萃取,为Ht-毒素的进一步提纯和结构分析奠定了基础。     

除草剂2,4-D特异性多克隆抗体的制备及鉴定

目的是制备特异性的2,4-D多克隆抗体.用活性酯和混合酸酐法将半抗原2,4-D分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)共价偶联;以2,4-D-BSA作免疫原免疫两只新西兰白兔;以2,4-D-OVA包被96孔酶标板,用间接酶联免疫吸附分析(ELISA)法检测抗血清效价和竞争性ELISA法检测2,4-D.结果显示2,4-D与BSA和OVA的偶联比率分别为32∶1和18∶1,抗血清效价＞6.4×105,在优化条件下2,4-D的最低检测限达37ng/mL.实验成功地制备了2,4-D特异性多克隆抗体,为其ELISA方法的建立提供了条件.     

稻曲病菌厚垣孢子壁多糖的提取方法优选

探究稻曲病菌Ustiloginoidea virens (Cooke) Takahashi厚垣孢子壁多糖的最佳提取方法,为孢壁多糖含量和组成的研究提供基础.采用5种方法提取该病菌黑色厚垣孢子壁多糖,用苯酚-硫酸法测定多糖含量.经研究比较,最佳提取方法为复合酶-热水浸提-sevag法,最佳提取条件是复合酶量4％,pH 4,浸提温度70℃,浸提时间120 min,物料比1:75(V/V);在优选的方法和条件下,测定稻曲病菌黑色厚垣孢子壁粗多糖相对得率21.2％,多糖含量72 3％;黄色厚垣孢子壁粗多糖相对得率17.5％,多糖含量66.7％,前者明显高于后者.研究表明复合酶-热水浸提-sevag法的工艺简单、可行,适宜稻曲病菌厚垣孢子壁多糖的测定.     

棉铃虫中肠钙粘蛋白、氨肽酶N及碱性磷酸酯酶的抗血清对Cry1Ac和Cry2Aa毒力的影响

【目的】Cry1A和Cry2A类Bt蛋白通过特异性地与昆虫中肠上的受体蛋白结合而发挥杀虫作用,现已广泛应用于转基因抗虫作物。本研究旨在进一步明确Cry2A类蛋白的作用机制和Cry1A受体蛋白在Cry2A发挥毒力中的作用。【方法】本研究首先提取了棉铃虫Helicoverpa armigera的BBMV,制备了钙粘蛋白(CAD)、氨肽酶N(APN)和碱性磷酸酯酶(ALP)3种受体蛋白的抗体和抗血清;然后,利用Western blot检测BBMV上这3种受体蛋白后,利用抗体封闭技术比较了敏感棉铃虫和Cry1Ac抗性棉铃虫(BtR)中3种受体蛋白的抗血清对Cry1Ac和Cry2Aa毒力的影响。【结果】对敏感品系棉铃虫,这3种已知的Cry1Ac受体蛋白抗血清显著地降低了Cry1Ac和Cry2Aa的毒力。其中APN抗血清对Cry1Ac毒力的影响最大,棉铃虫幼虫的死亡率降低了84.44%;ALP抗血清对Cry2Aa的毒力影响最大,棉铃虫幼虫死亡率比对照降低了71.04%。Cry1Ac对Cry1Ac抗性棉铃虫(BtR)的毒力显著降低,Cry2Aa的毒性也减弱。在Cry1Ac抗性棉铃虫(BtR)中,3种受体抗血清对Cry1Ac的影响比在敏感棉铃虫中的影响小,尤其是CAD和APN抗血清对Cry1Ac毒力的抑制率显著低于在敏感棉铃虫中的抑制作用;CAD和ALP抗血清对Cry2Aa毒力的影响与在敏感棉铃虫中的影响差异不显著,但APN抗血清可以显著降低Cry2Aa对Cry1Ac抗性棉铃虫(BtR)的毒力。【结论】棉铃虫CAD,APN和ALP不仅参与了Cry1Ac的杀虫过程,也对Cry2Aa毒力有一定的影响,而且这3种蛋白可能与棉铃虫对Cry1Ac和Cry2Aa产生抗性及交互抗性相关。     

昆虫病原线虫共生菌杀虫毒素研究进展

对昆虫病原线虫共生菌杀虫毒素的种类、与口服毒性有关的杀虫毒素以及口服毒性与杀虫毒素基因的关系等研究进展进行了综述,并对未来的研究方向提出了作者的见解。     

Total synthesis and biological evaluation of ustiloxin natural products and two analogs

Synthetic investigations of ustiloxin natural products are described. The first total synthesis of ustiloxin F was completed in 15 steps via ethynyl aziridine ring-opening by a phenol derivative. The results of biological tests of synthetic ustiloxins D and F, and two analogs, O-Me-ustiloxin D and 6-Ile-ustiloxin, demonstrated that the free hydroxyl group ortho to the ether linkage is critical for activity and variations at the Val/Ala site produce changes in the biological activity suggesting the need for further perturbations at this site to more extensively study the tubulin binding.     

Analysis of structural proteins and products of cell-free translation of vaccinia virus of mRNA using mono-specific antibodies

The monospecific antiserums to the major proteins p12, p19, p20, p42, p61 of the vaccinia virus coat were obtained and analyzed. The dynamics of the proteins accumulation in the infected cells has been studied. Products of the cell-free translation of the total viral mRNA precipitated by the obtained antiserums were identified. The p20 protein has been found to be a result of p12 protein reversible oligomerization under the conditions of electrophoresis. The antigenic relation of p12 and p20 proteins to a p42 protein, also a p12 oligomer, has been demonstrated. The possibility is discussed to use the obtained antiserums for mapping of the genes corresponding to structural proteins in the genome of the vaccinia virus.     

Recognition of antigenic differences among neurons using antiserums to clonal neural retina hybrid cells.

Antiserums prepared against hybrid cell strains formed between freshly isolated rodent neural retina cells and a human fibroblast cell line W.I. 18, VA-2 recognize cell surface antigens that are 1) restricted to the neural retina, 2) present in both embryonic and adult retina, and 3) localized to groups of cells within the retina. Normal segregants (i.e., those lines that had retained rodent chromosomes and lost human chromosomes) were used as immunogens in rabbits to produce the antiserums. Antiserums against both whole cells and purified plasma membranes were adsorbed with human parent VA-2 cells and nonneural and neural rodent tissue to remove cross-reacting specificities. All six antiserums studied continued to react with embryonic retina after brain cross-reactivity was removed.     

圆弧青霉菌毒素-青霉酸人工抗原的合成与鉴定

为建立圆弧青霉毒素-青霉酸的免疫学检测方法, 研究了青霉酸(PA)的人工抗原合成。通过碳二亚胺法将青霉酸(PA)分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)联结, 得到青霉酸人工抗原PA-BSA和PA-OVA。采用紫外扫描光谱法、SDS-PAGE和动物免疫试验对合成的抗原进行鉴定。结果显示联结后的人工抗原特征性吸收峰出现偏移, PA与BSA的偶联比为23.2:1, PA与OVA的偶联比为10.4:1。以PA-BSA为免疫抗原免疫小鼠, PA-OVA为包被抗原, 采用间接ELISA检测抗血清, 其效价达到1:12 800。表明青霉酸的人工抗原已合成, 为建立有效的免疫检测方法提供了基础。     

圆弧青霉菌毒素-青霉酸人工抗原的合成与鉴定

为建立圆弧青霉毒素-青霉酸的免疫学检测方法, 研究了青霉酸(PA)的人工抗原合成.通过碳二亚胺法将青霉酸(PA)分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)联结, 得到青霉酸人工抗原PA-BSA和PA-OVA.采用紫外扫描光谱法、SDS-PAGE和动物免疫试验对合成的抗原进行鉴定.结果显示联结后的人工抗原特征性吸收峰出现偏移, PA与BSA的偶联比为23.2:1, PA与OVA的偶联比为10.4:1.以PA-BSA为免疫抗原免疫小鼠, PA-OVA为包被抗原, 采用间接ELISA检测抗血清, 其效价达到1:12 800.表明青霉酸的人工抗原已合成, 为建立有效的免疫检测方法提供了基础.     

Induction of high level of specific antibody response to the neutralizing epitope ELDKWA on HIV-1 gp41 by peptide-vaccine

The monoclonal antibody 2F5 recognizing the neutralizing epitope ELDKWA on the C-domain could neutralize 90% of the investigated HIV-1 isolates. Low levels of ELDKWA-epitope-specific antibodies were observed in HIV-1-infected individuals. To induce high levels of antibodies to ELDKW-epitope, C-domain peptide (P2) was conjugated with a carrier peptide (KGGG)(7)-K (K/G). P2-K/G-conjugate induced high level of antibodies in mice by titer 1:25,600 to ELDKWA-epitope. P2-K/G-BSA-conjugate induced antibody response to ELDKWA-epitope (1:320-6400) in mice. The ELDKWA-epitope-specific antibodies of 19.8 and 34.6 microg/per milliliter serum were isolated from two rabbit antiserums (1:25,600). The levels of ELDKWA-epitope-specific antibodies induced in rabbits were greater than 1 microg/ml, a level considered to confer long-term protection. These results demonstrate the potential role of the C-domain peptide of gp41 to develop an effective ELDKWA-based epitope/peptide-vaccine against HIV-1.     

Surface immunoglobulins on mouse myeloma cells.

Plasma cells from 22 different transplantable mouse myelomas (PCT) were tested by 14 different class-specific and type-specific anti-immunoglobulin antiserums for cytotoxicity effects using a trypan blue-exclusion method. Seven IgF,κ tumors were all sensitive to anti-IgF and anti-κ antiserums. None of the other antiserums showed a cytotoxic effect. Five IgG,κ and four IgH,κ tumors were lysed by anti-κ serums, but not by anti-IgG or anti-IgH or the others. One of two IgA,κ tumors was lysed by anti-κ, but neither was lysed by anti-IgA or the other serums.One IgM,λ tumor was lysed by anti-IgM and anti-λ. Two λ Bence Jones tumors were not lysed by anti-λ, but both of these and the IgM,λ tumor were lysed by anti-κ. One κ Bence Jones tumor was lysed only by anti-κ serum.Only the IgF tumors and an IgM tumor were lysed by the appropriate anti-heavy chain serum, and $\text{21}\text{22}$ lines had κ surface determinants.