鱼藤酮多克隆抗体的制备和残留分析方法

为建立鱼藤酮免疫分析方法,以鱼藤酮为原料合成4-{2-[(2R,6as,12as)-1,2,6,6a,12,12a-六羟基-2-异丙烯基-8,9-二甲氧基色烯(3,4-b)喃(2,3-h)色烯-6-亚基]肼基}-4-丁酸(简称鱼藤酮腙琥珀酸半酯,RH-HS)半抗原,通过碳化二亚胺法(EDC)、活酯法(AE)将半抗原与牛血清蛋白(BSA)偶联制备免疫原Ⅰ、Ⅱ.将免疫原分别免疫BALB/c鼠获得相应的抗体Ⅰ和抗体Ⅱ,测定抗体冻干粉效价,分别为4.10×105和8.29×105.并进行抗原抗体的竞争抑制能力、特异性和鱼藤酮的添加回收试验.结果表明:抗体Ⅰ、Ⅱ对鱼藤酮最低检测限(IC20)分别为0.440μg/mL和0.437μg/mL.两抗体对吡蚜酮、克百威的交叉反应率均小于1.1%,抗体Ⅰ对4个与鱼藤酮结构相类似化合物的交叉反应率均高于56%,较抗体Ⅱ高.鱼藤酮的免疫分析技术(EUSA)与HPLC检测手段相比,在检测线性范围和灵敏度方面,两者差异不明显;在样品前处理方面,ELISA显示出快速、简单、有效的优点.     

几种植物源活性物质对昆虫细胞的毒力测定

本文测定几种植物源活性物质对甜菜夜蛾离体培养细胞系IOZCAS-Spex-II和草地贪夜蛾离体培养细胞系Sf 9细胞增殖活性的抑制作用.试验结果表明,喜树碱、羟基喜树碱和鱼藤酮对两种昆虫细胞具有较好的抑制作用.在测定的时间(2、6、12、24、48、72 h)和剂量(1.0×10-3、1.0×10-2、1.0×10-1、1.0×100,1.0×101、1.0×102μmol/L)范围内,喜树碱、羟基喜树碱和鱼藤酮对甜菜夜蛾IOZCAS-Spex-II和草地贪夜蛾Sf 9的细胞毒性具有较好的时间和剂量依赖性,而印楝素、乌头碱、次乌头碱、伪石榴皮碱、马钱子碱和吴茱萸碱对其细胞毒性具有一定的时间效应,无明显的剂量关系.     

三唑磷酶联免疫吸附测定法中包被抗体稳定剂的研究

选择了甘油、山梨醇、聚乙二醇(PEG)、卵清蛋白(OVA)、多肽、糖、氨基酸等试剂,通过经验法和正交法相结合的手段配制了一系列稳定剂,对吸附在酶标板上的三唑磷多克隆抗体进行处理(37℃,1 h)后,再在37℃下连续贮存 7 d,利用直接竞争ELISA法对不同稳定剂处理的包被抗体免疫活性、亲合性及检测灵敏度进行检测,并与未经稳定剂处理的对照进行比较,筛选得到效果较好的稳定剂 1 (质量分数:甘油2.5%,氨基酸1.5%,蛋白胨3.0%,离子螯合剂0.1%,防腐剂0.01%)。用稳定剂 1 处理包被抗体后,4~6℃下保存半年及37℃下保存14 d的试验结果表明,抗体的活性相对保持率分别为97.8%和94.2%;其免疫活性、亲合性(I50分别为68.43和54.38 ng/mL)及灵敏度(I10分别为3.72和 3.22 ng/mL)与常规方法包被的抗体(包被好后不贮存,直接检测,I50为60.73 ng/mL,I10为 3.11 ng/mL)无明显差异;冻融试验表明,经稳定剂 1 处理的三唑磷抗体在反复冻-融次数不超过8次时其活性也是稳定的。说明筛选出的稳定剂可以显著提高三唑磷多克隆包被抗体的稳定性,可用于三唑磷ELISA试剂盒的生产。     

浅谈形态学和分子生物学在昆虫鉴定中的作用

本文简介了形态学和分子生物学技术在昆虫鉴定中的优点和不足,表明形态学鉴定在昆虫鉴定中仍起主导作用,分子生物学作为高新技术在口岸鉴定工作中起到补充作用。旨在对口岸应用这两种方法鉴定时提供参考。     

亚洲玉米螟幼虫血淋巴中酚氧化酶原的多克隆抗体制备及免疫学特性分析

为探讨酚氧化酶原(PPO)的合成转运机制,采用硫酸铵沉淀、Blue Sepharose CL-6B亲和层析和Phenyl Sepharose CL4B疏水层析等方法从亚洲玉米螟幼虫血淋巴中纯化得到PPO,纯化倍数为369.85,回收率为35.34%.采用免疫印迹和酶联免疫吸附进行纯化PPO的免疫原性及其多克隆抗体的效价测定,并以透射电镜检测PPO在亚洲玉米螟幼虫体壁和中肠中的分布.结果表明,纯化PPO蛋白全酶相对分子量约为158 kD;免疫2只鼠后所得pAb间接ELISA效价均高达10-5,其中第2号小鼠pAb效价最高为1:4.08×104;PPO多克隆抗体具有相对较高的特异性;在亚洲玉米螟5龄幼虫中肠和体壁组织中金颗粒呈团状和点状分布.     

豌豆种传花叶病毒检测鉴定

豌豆种传花叶病毒(pea seed-borne mosaic virus,PSbMV)世界广泛分布,在国外已造成豌豆大量减产,国内有仅小范围低密度分布的报道。加拿大豌豆种子中挑拣皱缩、黑斑等症状的豌豆,先采用双抗体夹心酶联免疫吸附(DAS-ELISA)方法检出PSbMV阳性,再利用反转录PCR(RT-PCR)、序列比对分析和构建系统进化树对阳性样本进行验证鉴定,结果显示与PSbMV序列相似度为99.25%,综上确定从进境豌豆种子中检出豌豆种传花叶病毒。     

氯氟氰菊酯及其代谢物的人工抗原合成与多克隆抗体的制备

于氟氰菊酯分子的酸部分连接4-氨基丁酸间隔臂(HCA1)、醇部分连接丁二酸酐间隔臂(HCB2),分别合成了两种不同的半抗原,通过碳二亚胺法将HCA1和HCB2分别与牛血清蛋白(BSA)偶联制备了人工抗原HCA1-BSA和HCB2-BSA,通过混合酸酐法将HCA1、HCAS 和HCB2分别与卵清蛋白(OVA)偶联制备包被抗原,两种人工抗原通过免疫新西兰大白兔制备相应的多克隆抗体。结果表明,用HCA1-BSA获得的多克隆抗体对氯氟氰菊酯和氯氟氰菊酸有免疫反应性(IC50值分别为33.12 和0.95 mg/L),用HCB2-BSA获得的多克隆抗体对氯氟氰菊酯几乎没有免疫反应性。     

固相抗体直接竞争EL ISA 法测定氯黄隆在土壤中的残留动态

采用包被抗体直接竞争EL ISA 法测定了氯黄隆在盆土中的残留动态和农田土壤中氯黄隆的残留量。结果表明: EL ISA 法测定土壤中氯黄隆的检测限2、45 g/ hm2 第一年麦收时中性沙质土壤和弱碱性沙壤土中氯黄隆的残留量分别为0. 24、0. 56 ng/g 和0. 30、0. 96 ng/g, 第二年分别为0. 28、0. 67 ng/g 和0. 38、1. 09 ng/g , 与平行进行的高效液相色谱法、生物测定法测定结果基本一致。     

GFP与水稻条纹病毒病害特异蛋白的融合基因在sf9昆虫细胞中的表达

 用重叠延伸PCR(overlap Polymerase Chain Reaction,overlap-PCR)方法获得了含有绿色荧光蛋白(Green fluorescence protein,GFP)和水稻条纹病毒(Rice stripe virus,RSV)病害特异性蛋白SP(Disease-specific protein)两者的融合基因(GFP-SP),并将其克隆至载体pMD18-T,得到的重组质粒pMD18-T-GFP-SP经Xba Ⅰ/Hind Ⅲ双酶切后与经相同方法处理过的杆状病毒转移载体pFastBacHTb相连接构建成重组转移质粒pFastBacHTb-GFP-SP。酶切和测序鉴定证明了其序列的正确性并且无移码现象发生。将此质粒转化入含穿梭载体Bacmid的感受态细胞DH10Bac,得到含有目的基因片段的重组杆状病毒穿梭质粒rb-GFP-SP。以之转染草地贪夜蛾(Spodoptera frugiperda,sf9)细胞,24~48h后在显微镜200倍可见光视野下观察到被感染细胞发生了细胞和细胞核变大、细胞内颗粒物增多、细胞脱落甚至裂解等一系列与正常的st9昆虫细胞形态有明显区别的变化。荧光显微镜下可见部分细胞发出清晰的绿色荧光,且大部分荧光集中在细胞质部分。这些结果表明,GFP-SP融合蛋白在st9昆虫细胞内成功表达。     

酶联免疫吸附反应(ELISA)在烟草病毒病检测中的应用

本文综述了酶联免疫吸附分析法(ELISA)在烟草病毒检测中的应用。对EIISA的基本类型,改进和提高灵敏度的方法进行了分析,并阐述了该技术的某些发展趋势。     

Engineering of a Single Chain Variable Fragment Antibody Specific for the Citrus tristeza virus and its Expression in Escherichia coli and Nicotiana tabacum

Citrus tristeza virus (CTV) is one of the most destructive citrus virus diseases in the world. The construction of an engineered antibody, EMBL accession number AJ278109, able to specifically recognize its antigen, i.e. the coat protein of CTV, directly on infected plant material without any purification or manipulation of the entire woody plant. The potential uses of this engineered antibody are discussed.     

Isolation,characterization and insect growth inhibitory activity of major turmeric constituents and their derivatives against Schistocerca gregaria (Forsk) and Dysdercus koenigii (Walk)

Curcuminoids, the major colouring constituents of Curcuma longa (turmeric) rhizome powder, comprise mainly three closely related curcumins (I, II and III). A simple method has been devised for their efficient extraction and separation. Their structures have been confirmed by ¹H NMR spectroscopy and unique mass fragmentation pattern. Curcumin‐I, the major constituent has been converted to five alkyl ether derivatives, which have been tested along with the parent compounds and other extractives for insect growth inhibitory activity against Schistocerca gregaria and Dysdercus koenigii nymphs. At 20 µg per nymph, benzene extract and dibutyl curcumin‐I were the most active (60% inhibition) against S gregaria, whereas at 50 µg per nymph these substances exhibited moderate growth‐inhibitory activity (45%) against D koenigii nymphs. At these concentrations, turmeric oil caused 50–60% nymphal mortality in both test insects. The insect control activity of most of the turmeric products was comparable to or better than that of a commercial neem formulation. © 2000 Society of Chemical Industry     

菜蛾盘绒茧蜂多分DNA病毒EP-1-like基因克隆、原核表达与多克隆抗体制备方法

本实验提取被菜蛾盘绒茧蜂寄生后24h的小菜蛾幼虫体内总RNA,反转录合成cDNA,以此为模板PCR扩增出菜蛾盘绒茧蜂多分DNA病毒(Cotesia vestalis polydnavirus,CvBV)EP-1-like基因,将其分别克隆到表达载体pET30a、pET28a中,转化宿主菌BL21(DE3),获得单克隆重组质粒pET-30a-EP1L和pET-28a-EP1L。经IPTG诱导,pET-28a-EP1L成功表达了约34.8kDa的包涵体蛋白。将诱导条件优化以后,采用割胶回收的方法纯化包涵体,将纯化后的表达蛋白免疫新西兰大耳兔制备多克隆抗体。ELISA分析表明制备的抗体效价达1:128000,Western blot检测证明抗体具有良好的免疫反应特异性。     

Insecticidal potential of Bowman-Birk proteinase inhibitors from red gram (Cajanus cajan) and black gram (Vigna mungo) against lepidopteran insect pests

The present study assessed the insecticidal potential of purified Bowman-Birk proteinase inhibitors from red gram (RgPI) and black gram (BgPI) towards lepidopteran insect pests. Both RgPI and BgPI showed remarkable inhibitory activity against midgut trypsin-like proteinases of Achaea janata. While, BgPI showed moderate to low inhibitory activity towards the midgut trypsin-like proteinases of Helicoverpa armigera, Spodoptera litura, Papilio demoleus, Amsacta albistriga, Corcyra cephalonica, Bombyx mori and Daphnis nerii, RgPI showed low to negligible inhibitory activity. The isoinhibitors of RgPI and BgPI were stable in presence of trypsin and chymotrypsin of bovine pancreatic origin, as well as larval midgut proteinases of A. janata, H. armigera and S. litura. The midgut trypsin-like proteinases of A. janata and H. armigera as well as midgut trypsin- and chymotrypsin-like proteinases of S. litura were susceptible to inhibition by RgPI and BgPI. Feeding assays displayed a dose-dependent decrease in body weight and survival rate of these larvae with increasing concentration of inhibitors supplemented in the diet. RgPI displayed significant reduction in body weight and survival rate of A. janata larvae compared with BgPI. Conversely, BgPI showed significant reduction in body weight of S. litura larvae compared with RgPI.     

小菜蛾中肠氨肽酶N2在昆虫细胞中的表达

利用昆虫细胞-杆状病毒表达系统(Bac-to-Bac baculovirus expression system)表达获得小菜蛾 Plutella xylostella(L.)中肠氨肽酶N2(aminopeptidase N,APN2)蛋白,成功建立了该蛋白在昆虫细胞中的表达体系。将对Cry1Ac毒素敏感和已产生抗性的小菜蛾中肠APN2基因插入表达载体pFAST Bac HTB中,并在草地夜蛾 Spodoptera frugiperda 细胞系(Sf9)中进行了重组表达。聚丙烯酰胺凝胶电泳(SDS-PAGE)及蛋白免疫印迹技术(Western-blotting)结果显示,在107 kDa处有1条特异性蛋白条带。研究表明,小菜蛾中肠APN2基因可在Sf9细胞中成功表达,这为继续研究其功能及抗性的关系奠定了基础。     

几种昆虫几丁质抑制剂对东亚飞蝗的毒力测定和应用评价

在室内筛选了 5种昆虫几丁质抑制剂和 1种常规杀蝗药剂对东亚飞蝗的毒效。结果表明 ,5种昆虫生长调节剂中 ,以毒饵中药剂有效含量计 ,卡死克毒效最高 ,对 4龄蝻的LC₅₀为26.5(16.5～38.0)mg/kg ,LT₅₀为4.946d ,抑太保次之,LC₅₀为284.4(133.8～449.2)mg/kg,LT₅₀为5.227d ,其余 3种毒效较低 ;但从性能价格评估 ,则以卡死克和灭幼脲较优 ,虽然致死时间较常规杀蝗剂长 ,但由于其高选择性 ,对非靶标生物影响很小 ,对环境比较安全 ,在生产上应用价值较高。常规杀蝗药剂高效氯氰菊酯对天敌和非靶标生物影响较大     

滞育七星瓢虫酰基辅酶AΔ11去饱和酶基因的克隆及表达分析

去饱和酶（Fatty acid desaturase,FAD）是生物体不饱和脂肪酸合成中的关键酶,酰基辅酶AΔ11去饱和酶基因是其中重要的一种。本试验基于七星瓢虫Coccinella septempunctata L.转录组数据,利用RT-PCR和RACE技术从滞育七星瓢虫中克隆得到酰基辅酶AΔ11去饱和酶基因全长,并命名为CsFadΔ11（GenBank登录号：MF458996）,该基因全长1447 bp,开放阅读框（ORF）1095 bp,编码364个氨基酸,预测蛋白质分子量为42.99 kD,等电点（pI）为8.87,无信号肽。氨基酸序列分析结果表明,CsFadΔ11有3个组氨酸富集区和6个跨膜结构域,与膜翅目的内华达古白蚁Zootermopsis nevadensis同源性较高。利用实时荧光定量PCR技术研究其时间表达模式,发现CsFadΔ11基因在七星瓢虫初羽化、滞育诱导10 d、滞育诱导20 d、滞育初期、滞育后期、滞育解除期以及正常发育状态下均有所表达,且在滞育早期表达量最高,其次是在滞育解除个体中。其整体表达趋势与相应时期脂积累变化情况基本相符,推测CsFadΔ11基因参与七星瓢虫脂积累调控,并进一步影响七星瓢虫滞育。     

Identification and transmission of Piper yellow mottle virus and Cucumber mosaic virus infecting black pepper (Piper nigrum) in Sri Lanka

Sri Lankan black pepper with symptoms of yellow mottle disease contained a mixture of viruses: Piper yellow mottle virus (PYMV) particles (30 × 130 nm), Cucumber mosaic virus (CMV, 30 nm diameter isometric particles), and unidentified, isometric virus-like particles (30 nm diameter). An effective purification procedure is described for PYMV. Immunosorbent and conventional electron microscopy successfully detected badnavirus particles only when at least partially purified extracts were used. PYMV was confirmed as the cause of the disease, with the other two viruses apparently playing no part in producing symptoms. PYMV was transmitted by grafting, by the insect vectors citrus mealy bug ( Planococcus citri ) and black pepper lace bug ( Diconocoris distanti ), but not by mechanical inoculation or through seeds. The CMV isolate was transmitted to indicator plants by mechanical inoculation and by the vector Aphis gossypii , but not by Myzus persicae ; but neither mechanical nor insect transmission of CMV to black pepper was successful. A sensitive polymerase chain reaction assay was developed to detect PYMV in black pepper.     

水稻条纹病毒NS2基因原核表达产物多克隆抗体制备及受侵染水稻和灰飞虱体内NS2检测

 The NS2 gene of Rice stripe virus (RSV) was amplified by RT-PCR, cloned into pGEM-T vector and sequenced. The NS2 gene was inserted into prokaryotic expression vector pET32a to produce recombinant plasmid pET32a-NS2. The recombinant plasmid was introduced into Escherichia coli strain BL21 (DE3) pLysS. SDS-PAGE and Western blot analysis confirmed that NS2 fusion protein was expressed after induction by IPTG. The recombinant NS2 protein was purified with Ni²⁺-NTA agarose affinity chromatography and the polyclonal antibody against NS2 protein was raised in rabbit. NS2 protein was successfully detected in small brown planthopper (Laodelphax striatellus) at 1:1 600 dilution of the total protein of single planthopper and in infected rice (Oryza sativa) at 1:800 dilution of 10 mg leave by dot immunobinding assay using the polyclonal antibody.