禽呼肠孤病毒σ3蛋白单克隆抗体的制备及特性鉴定

用体外表达的蛋白作为免疫原来制备特异的单克隆抗体,并对制备的抗体进行鉴定。将ARVσ3蛋白基因在体外扩增,扩增产物与载体PGEX-4T-1连接并在基因工程菌中表达,体外表达的蛋白经纯化后作为免疫原来制备特异的单克隆抗体和ELISA检测包被抗原,测定纯化后蛋白的浓度,按每鼠100μg的蛋白用量免疫BALB/c小鼠,免疫4次后取其脾细胞与骨髓瘤细胞SP2/0按5∶1进行融合。对融合后的杂交瘤细胞及时筛选,阳性孔经3次有限稀释法克隆,通过间接E-LISA方法测定其抗体效价,并通过Western Blot、Dot-ELISA、直接免疫荧光、病毒中和等方法对获得的单抗特性进行检测。结果通过纯化的病毒含量为41.5 mg/mL,应用纯化的病毒免疫BALB/c小鼠后与骨髓瘤细胞融合,通过克隆筛选成功获得1株能稳定传代并分泌抗禽呼肠孤病毒单克隆抗体的杂交瘤细胞株σ3 B6-3 K3,用其制备的腹水经间接E-LISA测定效价达105以上,并且与其他参试病毒株没有交叉反应,具有良好的特异性;病毒中和试验证明其中和能力低。应用ARVσ3蛋白制备并获得的杂交瘤细胞株σ3 B6-3 K3具有很好的特异性,不具有中和ARV的关键表位,可以用于ARV的特异性检测。     

番茄环斑病毒单克隆抗体的制备及检测

将纯化的番茄环斑病毒(Tomato ringspot virus,ToRSV)制剂免疫BALB/c小鼠,末次免疫后第3天取其脾细胞与SP2/0细胞融合,采用选择性培养基、有限稀释法克隆和间接ELISA方法进行筛选,成功获得了2株分泌ToRSV单克隆抗体的杂交瘤细胞株并分别命名为1H8、1D4.用间接ELISA方法对所获得的2个杂交瘤细胞株进行亚型鉴定均为IgG1亚类.间接ELISA效价测定结果1H8为1:105,1D4为1:106.TAS-ELISA实验结果表明此2株杂交瘤细胞所分泌的单克隆抗体均能与本研究室保存的从德国引进的番茄环斑病毒分离物、从美国ATCC引进的番茄环斑病毒分离物PV-100、PV-174、PV-239发生特异性反应,而不与同属其它3种病毒:烟草环斑病毒(Tobacco ringspot virus,TRSV)、南芥菜花叶病毒(Arabis mosaic virus,ArMV)、马铃薯黑环斑病毒(Potato black ringspot virus,PBRSV)发生反应.     

马铃薯Y病毒脉坏死株系(PVYN)外壳蛋白基因的克隆、表达及其间接ELISA方法的建立

【目的】制备马铃薯Y病毒脉坏死株系（PVY^N）多克隆抗体,建立间接ELISA法用于检测PVY^N病毒。【方法】依据PVY^N的CP基因序列设计引物,利用RT-PCR方法获得CP基因并连接构建到原核表达载体,进行原核表达,以纯化的重组蛋白作为抗原免疫新西兰大白兔,获得PVY^N的抗血清并纯化。【结果】测序结果与其他已知序列比较,PVYNCP基因的核苷酸同源性95%,推断氨基酸的同源性达98%。以纯化蛋白为抗原进行免疫,成功获得了PVY^N外壳蛋白抗血清,纯化后的IgG采用间接ELISA法检测抗原,抗体效价达1：500,使用该抗体稀释度检测感染植株,结果呈阳性。【研究结论】通过分子生物学途径获得了PVY^N的多克隆抗体,建立的间接ELISA方法可以用于PVY^N的病毒检测。     

苏云金芽胞杆菌杀虫晶体蛋白CryIA单克隆抗体的制备及在转Bt基因棉毒蛋白检测上的应用

用苏云金杆菌菌株HD-1和HD-73分别发酵培养制备了孢晶混合物，孢晶混合物经分离、纯化、电泳得到CryIA、CryIA(c)杀虫晶体蛋白。用CryIA和CryIA(c)分别免疫家兔制备了两种多抗。用CryIA免疫小鼠，经细胞杂交、融合、克隆后筛选出4个单克隆株系A4F5F11、B4E6C7、B4E6D8、B4F5G11。用CryIA(c)多抗包被，B4E6D8上清液作为夹心抗体成功地建立了双抗夹心 ELISA，并检测了中棉所30、NUCOTN33B蕾期叶片中毒蛋白的含量，结果分别为23．4ng·g-1 FW、24．7ng·g-1 FW。     

抗卡那霉素单克隆抗体细胞株的筛选及间接ELISA法的建立

采用碳二亚胺(EDC)法合成卡那霉素完全抗原,免疫BALB/c小鼠,通过杂交瘤技术获得3株能稳定分泌抗卡那霉素单克隆抗体的杂交瘤(5D7、6H8、7G4)。选择其中的5D7株抗体,经ELISA鉴定,细胞上清的效价达103以上,腹水效价达到5×104以上;建立了间接竞争ELISA法,通过优化,其检测限为0.5ng/mL,抗体IC50为3.7ng/mL,与妥布霉素交叉反应率为63.8%,与其他抗生素无交叉。可见ELISA具有快速、敏感、特异、简便等特点,适合于KAN残留的快速检测,具有较高的推广应用价值。     

抗伪狂犬病毒闽A株单克隆抗体的制备及鉴定

用纯化的猪伪狂犬病病毒闽A株(PRV-FA)免疫Balb/c鼠,取脾细胞和骨髓瘤细胞进行细胞融合,经间接ELISA筛选,获得2株能稳定分泌伪狂犬病毒单克隆抗体的杂交瘤细胞株,分别命名为4G9E9和4H9C9.这2株杂交瘤细胞株细胞培养上清和小鼠腹水效价(EusA)分别为1:6 400、1:12 800及1:12 800、1:25 600.特异性鉴定结果表明,各株单抗均不与猪瘟病毒、乙脑病毒、猪呼吸繁殖障碍综合症病毒、猪细小病毒等发生交叉反应,这2株抗PRV杂交瘤细胞株的获得为进一步建立准确快速的抗原检测方法奠定了基础.     

新城疫病毒HN蛋白单克隆抗体的制备及其抗原捕捉ELISA方法的建立

为建立一种快速的新城疫病毒病原检测方法。本研究以原核表达的重组HN蛋白免疫7周龄BALB/c雌鼠,取其脾细胞与骨髓瘤细胞SP2/0进行融合,经间接ELISA方法筛选,成功获得了1株能稳定分泌抗新城疫病毒HN蛋白的McAb杂交瘤细胞,命名为4E8,4E8亚类鉴定为重链属于IgG2a,轻链属于κ链。以多克隆抗体作为包被抗体、单克隆抗体4E8作为检测抗体,通过双抗夹心ELISA各个反应条件的优化,建立检测新城疫病毒抗原捕捉ELISA方法。该方法对EDSV、ITLV、IBV、IBDV不发生交叉反应,其敏感性比HA试验要高4倍以上,与RT-PCR相比较,符合率、敏感性和特异性分别为95.6%、93.3%、96.1%。本研究建立的NDV AC-ELISA有良好的重复性、敏感性和特异性,可应用于新城疫病毒感染的早期诊断。     

口蹄疫病毒3D聚合酶B细胞表位的筛选鉴定

以口蹄疫病毒株AFT2 RNA为模板,反转录并扩增3D聚合酶基因,PCR纯化产物与pGEM-T easy载体连接并转化JM109菌株,对经凝胶电泳、PCR和EcoRI酶切法鉴定为阳性的重组质粒进行测序,通过序列比对获得AF72 3D聚合酶的核苷酸序列和推导氨基酸序列,综合分析3D聚合酶的亲水性、可塑性、抗原指数以及表面可能性等参数,预测其潜在B细胞抗原表位并人工合成表位肽段,利用间接ELISA对潜在表位进行筛选鉴定,结果显示,表位3D2和3D4为病毒株AFT2 3D聚合酶的优势B细胞表位,该结果将为进一步的FMDV多表位疫苗研究奠定基础.     

鸭肝炎病毒单克隆抗体的制备

本实验利用弗式完全佐剂和弗式不完全佐剂,乳化病毒,制备免疫抗原,对BALB/c小鼠进行三次免疫。将免疫效价达到1：10000以上的免疫小鼠脾细胞与SP2/0细胞进行融合,通过间接ELISA方法对阳性杂交瘤细胞株进行筛选,并通过有限稀释法将呈强阳性的杂交瘤细胞亚克隆3～4次,直到杂交瘤细胞阳性率达到100%。得到阳性株命名为2E3,对阳性株细胞进行扩大培养,并将细胞注入腹腔,提取腹水,测定细胞上清及腹水的效价,分别为10×25,10×26。利用亚类鉴定试剂盒测定单抗的亚类。鉴定结果为IgG1型。     

环丙沙星单克隆抗体的制备及其免疫学特性鉴定

用EDC法将BSA和OVA分别和环丙沙星（Ciprofloxacin, CIP）偶联作为免疫原和包被原,并经紫外扫描 (UV Scan)、聚丙烯酰胺凝胶电泳（SDS-PAGE）鉴定。用合成的BSA-CIP免疫BALB/c小鼠,经过3次免疫后,用间接ELISA和阻断ELISA选择细胞融合备用鼠,选择高效价、敏感的小鼠进行抗原超强免疫;取其脾细胞应用杂交瘤技术与骨髓瘤细胞建立分泌CIP单克隆抗体的杂交瘤细胞株;用体内诱生腹水法制备CIP mAb,对CIP mAb的效价、敏感性和特异性等免疫学特性进行鉴定。聚丙烯酰胺凝胶电泳鉴定表明BSA-CIP人工抗原偶联成功;免疫的3只小鼠血清抗体效价均达到10-4;其中2号小鼠血清CIP抑制效价较高且IC50最低,达48.59μg/L,融合后筛选出Z3G12B3和Z2H8C10两株敏感特异的杂交瘤细胞,其两株细胞培养上清液效价为1:512,腹水效价为1:2.56×105,Z3G12B3株对CIP的IC50为2.47μg/L,与二氟沙星、沙拉沙星有小于0.03％的交叉反应性,与其他抑制物无交叉反应性。本试验获得了高效价、敏感、特异的抗CIP mAb,为CIP残留ELISA检测试剂盒和试纸条的建立奠定了坚实的基础。     

A note on the origin of Kalanchoë x vadensis Boom & Zeilinga (Crassulaceae)

Summary K x vadensis is a hybrid of K. blossfeldiana and K. marmorata obtained after doubling the number of chromosomes.     

Mechanisms and diversity of resistance to sorghum shoot fly,Atherigona soccata

Sorghum shoot fly, Atherigona soccata, is one of the important pests of postrainy season sorghums. Of the 90 sorghum genotypes evaluated for resistance to this pest, RHRB 12, ICSV 713, 25026, 93046 and 25027, IS 33844‐5, Giddi Maldandi and RVRT 3 exhibited resistance in postrainy season, while ICSB 463, Phule Anuradha, RHRB 19, Parbhani Moti, ICSV 705, PS 35805, IS 5480, 5622, 17726, 18368 and 34722, RVRT 1, ICSR 93031 and Dagidi Solapur showed resistance in rainy season, suggesting season‐specific expression of resistance to A. soccata. ICSB 461, ICSB 463, Phule Yasodha, M 35‐1, ICSV 700, 711, 25010, 25019 and 93089, IS 18662, Phule Vasudha, IS 18551 and 33844‐5 and Barsizoot had fewer deadhearts than plants with eggs across seasons, suggesting antibiosis as one of the resistance mechanism. Five genotypes exhibited resistance with high grain yield across seasons. Correlation, path and stepwise regression analyses indicated that leaf glossiness, seedling vigour, trichome density, oviposition and leaf sheath pigmentation were associated with the expression of resistance/susceptibility to shoot fly, and these can be used as marker traits to select and develop shoot fly‐resistant sorghums.     

Avoidance of leaf rust fungi in wild relatives of cultivated cereals

Summary Avoidance of rust fungi that was based on poor appressorium induction was previously found in Hordeum chilense. In the present study 95 accessions of Triticeae were screened for avoidance of Puccinia hordei. The percentage of appressorium formation per germinated spore ranged from 6 to 90%. On none of the 41 accessions of Aegilops, Agropyron, Elymus, Secale, Thinopyrum or Triticum studied was the rate of appressorium formation lower than 25%. Lower rates of appressorium formation were, however, found on accessions of wild barley species Hordeum brachyantherum, H. marinum, H. parodii and H. secalinum. Its implications in cereal breeding are discussed.     

Reaction of tritordeum to Fusarium culmorum and Septoria nodorum

Summary Hordeum chilense is a wild barley extensively used in wide crosses in the Triticeae. It could be a valuable source of resistance to Fusarium culmorum and Septoria nodorum. Some H. chilense x Triticum spp. amphiploids, named tritordeums, were more resistant than the parental wheat line to these diseases, others were not. Average contents of ergosterol and deoxynivalenol (DON) suggested that resistance to colonization by Fusarium was the highest for Hordeum chilense, followed by tritordeum and wheat in decreasing order. In particular, the H. chilense genotypes H7 and H17 enhanced the wheat resistance to F. culmorum in its tritordeum offsprings. Resistance to S. nodorum in tritordeum was not associated with tall plant height. There is sufficient genetic variation for resistance to F. culmorum and S. nodorum among tritordeum to allow the breeding of lines combining short straw and resistance to both diseases.     

Disease resistance in protected crops and mushrooms

Summary Cultivars of tomatoes, cucumbers, lettuce and peppers have been bred for resistance to one or more pathogens. Some tomato and cucumber cultivars have resistance to a wide range of diseases. Resistance has been transient in many cases and a succession of cultivars with new genes or new combinations of resistance genes has been necessary to maintain control. There has been a number of notable exceptions and these have included durable resistance to such pathogens asFulvia fulva and tomato mosaic virus. With lettuce the resistance situation is complicated by the occurrence of fungicide resistant pathotypes. There are no strains ofAgaricus bisporus purposely bred for disease resistance.In protected flower crops only resistance to Fusarium wilt in carnations has been purposely bred but differences in disease resistance are apparent in cultivars of many ornamental crops. This is particularly so in chrysanthemums where there are cultivars with resistance to many of the major pathogens. Similar situations occur with other flower crops and pot plants. Cultivars of some species have not been systematically investigated for resistance.The need for genetic resistance will increase with the further reduction, in the limits on pesticide use and an increasing public awareness and importance of pesticide pollution.ADAS is an executive agency of the Ministry of Agiculture, Fisheries and Food and the Welsh Office.     

Genetic constitution and diversity in four narrow endemic redwoods from the family Cupressaceae

The genetic constitution and diversity of four relictual redwoods are discussed in this review. These include monotypic genera of the family Cupressaceae: coast redwood (Sequoia sempervirens), giant sequoia (Sequoiadendron giganteum), dawn redwood (Metasequoia glyptostroboides), and alerce (Fitzroya cupressoides). All four species are narrow endemics, share a number of common phenotypic traits, including red wood, and are threatened species. Fossil history suggests that the ancestors of redwoods probably originated during the Cretaceous and Tertiary periods and flourished thereafter for millions of years. Towards the end of the Tertiary period began their decline and struggle for existence that continued during the subsequent geologic upheavals and climate changes, until the survival of the present-day redwoods in the current restricted locations in the world (USA, China, and South America). Although two species, Sequoiadendron and Metasequoia, are diploids (2n = 22), and the other two are polyploids: Fitzroya a tetraploid (2n = 4x = 44), and Sequoia a hexaploid (2n = 6x = 66); they all share the same basic chromosome number x = 11. The genome size in the hexaploid Sequoia is one of the largest (31,500 MB) in the conifers, while the genome sizes of diploid Metasequoia and Sequoiadendron are about one-third (~10,000 MB) of Sequoia. Genetic diversity in the redwoods is lower than most other gymnosperms, except in Sequoia, which seems to rank near the upper quarter of the coniferous forest trees. Genomic research is sparse in the redwoods, and should be pursued for a better understanding of their genome structure, function, and adaptive genetic diversity.     

Distribution,ecology and reproductive biology of wild tomatoes and related nightshades from the Atacama Desert region of northern Chile

Over the past 20 years, several expeditions were made to northern Chile to collect populations of wild tomatoes (Solanum chilense, S. peruvianum) and allied nightshades (S. lycopersicoides, S. sitiens), and obtain information about their geographic distribution, ecology and reproductive biology. Restricted mainly to drainages of the Andean and the coastal cordillera, populations are geographically fragmented. The two nightshade species are rare and threatened by human activities. Adaptation to extreme aridity and soil salinity are evident in S. chilense and S. sitiens (the latter exhibits several xerophytic traits not seen in the tomatoes) and to low temperatures in S. lycopersicoides and S. chilense. All tested accessions are self-incompatible, with the exception of one S. peruvianum population collected at the southern limit of its distribution. Several distinguishing reproductive traits—anther color, attachment, and dehiscence, pollen size, and flower scent—suggest S. sitiens and S. lycopersicoides attract different pollinators than S. chilense and S. peruvianum. The four Solanum spp. native or endemic to Chile provide a variety of novel traits which, through hybridization and introgression with cultivated tomato, could facilitate development of improved varieties, as well as research on a variety of basic topics, including plant-pollinator interactions, abiotic stress responses, and evolution of reproductive barriers.     

The induction in vitro of adventitious shoots in Rosa

Summary Adventitious shoots were formed on excised leaves, roots and callus of Rosa persica x xanthina and on excised leaves of R. laevigata and R. wichuraiana on culture media that included BAP and NAA as growth regulators. Shoots formed freely on freshly cultured callus of R. persica x xanthina but their production declined in successive cultures and ceased after twelve weeks. Transplantation to soil was improved by rooting plantlets in cellulose plugs in vitro and transferring plantlets to soil while still in the plugs.     

The exploration of genetic resources of Portuguese cabbage and kale for resistance to several Brassica diseases

Summary Twenty three accessions of nine Portuguese cabbage and kale land races from different geographic origins were tested at the seedling stage for resistance to several important brassica diseases. Resistance to downy mildew (Peronospora parasitica), expressed as necrosis of the cotyledon mesophyll, was found in all the accessions. Type A resistance to cabbage yellows (Fusarium oxysporum f. sp. conglutinans race 1) was present in most of the landraces. Resistance to clubroot (Plasmodiophora brassicae race 6) was found in one accession of the Portuguese tree kale. High resistance to blackleg (Leptosphaeria maculans) and white rust (Albuco candida) was not detected, although several accessions showed 20 to 30% of plants with intermediate expression of resistance. All Portuguese cole accessions were susceptible to blackrot (Xanthomonas campestris pv. campestris).     

Production and characterization of intergeneric diploid cybrids derived from symmetric fusion between Microcitrus papuana Swingle and sour orange (Citrus aurantium)

Plants were regenerated from intergeneric somatic hybridization between embryogenic protoplasts of Microcitrus papuana Swingle and leaf-derived protoplasts of sour orange (Citrus aurantium L.) via electrofusion. The regenerated plants were morphologically similar to the leaf parent in growth vigor, leaf and branch structure. FCM analysis showed that they were diploids. Simple-sequence-repeat (SSR) and cleaved-amplified-polymorphic-sequence(CAPS) were employed for hybridity characterization. SSR banding patterns of the regenerated plants were identical to the leaf parent, sour orange, indicating that they possessed nuclear component derived from sour orange. DNA amplification with chloroplast and mitochondrial universal primers, followed by restriction endonuclease digestion, revealed polymorphism between the fusion parents. Therefore, this method was used to determine the cytoplasmic compositions of the regenerated plants. Banding patterns for all the polymorphic primer/enzyme combinations of the regenerated plants were similar to those of the embryogenic parent, M. papuana, suggesting that only the cytoplasmic components derived from the embryogenic parent were present in the regenerated plants. FCM, SSR and CAPS demonstrated that intergeneric diploid cybrids have been successfully obtained by symmetric fusion. Related results concerning nuclear and cytoplasmic composition of previous diploid somatic hybrids and potential mechanism for regeneration of such kind of plants are discussed herein. This revised version was published online in July 2006 with corrections to the Cover Date.