斑点免疫金银染色技术检测鸡传染性法氏囊病毒的研究

本研究成功建立了检测鸡传染性法氏囊病毒（ＩＢＤＶ）抗原的斑点—免疫金银染色技术（Ｄｏｔ－ＩＧＳＳ），并确定了操作流程的最佳试验条件。应用该法对纯化ＩＢＤＶ抗原的最低检出量为０．１９５３ｎｇ／点，其敏感性为Ｄｏｔ－ＥＬＩＳＡ的８倍。特异性阻断试验和交叉反应试验证明Ｄｏｔ－ＩＧＳＳ具有较高的特异性。Ｄｏｔ－ＩＧＳＳ和Ｄｏｔ－ＥＬＩＳＡ对５４份自然感染法氏囊样品检测的阳性率为９６．３％和９２．５％（χ２＝３．１１７９，Ｐ＞０．０５），统计学分析两者无显著差异。研究表明本法具有敏感、特异性强、经济、安全等优点，可用于ＩＢＤＶ感染的特异诊断。本研究为免疫金技术应用于畜禽传染病的诊断和研究打下了基础     

甘薯羽状斑驳病毒外壳蛋白基因在大肠杆菌中的表达及特异抗血清的制备

根据已报道的甘薯羽状斑驳病毒（ＳＰＦＭＶ）外壳蛋白（ＣＰ）基因的核苷酸序列合成引物,利用ＲＴ－ＰＣＲ的方法获得ＣＰ基因后,再将其克隆到原核表达载体ｐＥＴ３０ａ（＋）中。ＳＤＳ－ＰＡＧＥ分析表明,经ＩＰＴＧ诱导,ＣＰ基因在大肠杆菌ＢＬ２１（ＤＥ３）ｐＬｙｓＳ中获得了高效表达。以含表达产物的凝胶为抗原,免疫家兔,制备了ＳＰＦＭＶ外壳蛋白的特异性抗血清。Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ和点免疫（Ｄｏｔｂｏ     

定量测定甲胺磷残留的间接竞争ELISA的建立和初步应用

以水溶性碳化二亚胺法将甲胺磷（ＭＴＰ）分子偶联牛血清白蛋白（ＢＳＡ）上,用此合成的免疫原复合物制备了特异性的兔抗甲磷多抗血清。抗血清的ＥＬＩＳＡ效价为１：１２８００－１;５１２００,抗血清与乙酰甲胺磷的交叉反应率为１０．５％,与供试的其它９种有机磷农药的交叉反应率均小于２．８％,用同样的方法合成了甲胺磷－卵清蛋白复合物作为包被抗原,建立了适于环境中甲胺磷残留分析的间接竞争ＥＬＩＳＡ工程程序,在明确     

生长因子对卵母细胞体外成熟及早期胚胎体外发育的影响

为了探讨生长因子（ＥＧＦ，ＰＤＧＦ）对牛卵母细胞体外成熟与早期胚胎体外发育的影响，本研究进行了两个实验。实验１：在卵母细胞体外成熟（ＩＶＭ）中，将卵母细胞平均随机的放入表皮生长因子浓度为０，２．５，２５，５０μｇ／Ｌ的成熟培养液和对照组中。实验２：在早期胚胎体外培养（ＩＶＣ）过程中，将受精卵平均随机分配入５个不同培养液的处理组：（１）ＴＣＭ－１９９＋１０％阉公牛血清（ＳＳ）；（２）ＴＣＭ－１９９＋ＥＧＦ＋ＰＤＧＦ；（３）ＴＣＭ－１９９＋ＥＧＦ；（４）ＴＣＭ－１９９＋ＰＤＧＦ；（５）ＴＣＭ－１９９＋无生长因子、其中ＥＧＦ：５０μｇ／Ｌ，ＰＤＧＦ：０．１μｇ／Ｌ。２，３，４，５组有０．１％ＰＶＡ和０．３％ＢＳＡ，并与颗粒细胞单层协同培养。卵母细胞的体外成熟（ＩＶＭ）、体外受精（ＩＶＦ）和早期胚胎体外培养（ＩＶＣ）的方法与Ｌｕ等（１９８７）报道的相同。结果表明：浓度为５０μｇ／Ｌ的处理组分裂率与对照组无差异（８９．０％，９２．４％，Ｐ＞０．０５），而浓度为０，２．５，２５μｇ／Ｌ的处理组分裂率均显著低于对照组（７８．６％，８２．３％，８４．３％，Ｐ＜０．０５），在囊胚率和第７ｄ一级胚胎率上各组均无差异。ＥＧＦ在体     

免疫印迹分析试验感染肝片吸虫和大片吸虫绵羊的IgG应答

免疫印迹(Westernblot)分析试验感染肝片吸虫和大片吸虫绵羊的抗片形吸虫分泌捧泄产物(FhESP和FgESP)IgG应答，结果表明，FhESP中分子质量在17．1～21．6ku之间的4条主要蛋白带仅被肝片吸虫感染以后(0WPI以后)的血清识别且不被对照组血清识别；FgESP中分子质量在19．0～71．1ku之间的6条主要蛋白带仅被大片吸虫感染以后(0WPI以后)的血清识别且不被对照组血清识别。     

固氮粪产碱菌（Alcaligenes faecalis）胞外多糖的分离纯化及成分分析

固氨粪产碱菌野生型Ａ１５０１菌株及其胞外多糖（ＥＰＳ）突变株Ｅｘｏ＋＋（Ａ１５３２）和ＥｘＯ－（Ａ１５３１）,以及ｎｉｆＡ和ｎｔｒＣ－ｎｉｆＡ转化子（Ａ１５１３和Ａ１５２３）的ＥＰＳ,由高分子量（２５０ｋＤ左右）组分（ＥＰＳＨ）和低分子量小于４０ｋＤ组分（ＥＰＳＬ）组成。两个组分中均含有葡萄糖、半乳糖、果糖和戊糖,其主要成分葡萄糖和半乳糖的分子摩尔比：Ａ１５０１－ＥＰＳＨ为２：１、ＥＰＳＬ为３：１;Ａ１５３１－ＥＰＳＨ为２：１、ＥＰＳＬ为１：１;Ａ１５３２－ＥＰＳＨ为８：１、ＥＰＳＬ为５：１;Ａ１５１３－ＥＰＳＨ和ＥＰＳＬ均为４：１;Ａ１５２３－ＥＰＳＨ为４：１、ＥＰＳＬ为８：１。高分子量ＥＰＳ中还含有多肤,其氨基酸组分在各突变株和转化子之间差异明显,而ＥＰＳＬ中仅含有痕量多肽。     

苄嘧黄隆及甲黄隆的气相色谱分析方法研究

本文研究了苄嘧黄隆和甲黄隆的气相色谱分析方法。采用（１）５％ＯＶ－１０１／Ｃｈｒｏ－ｍｏｓｏｒｂＧＡＷＤＭＣＳ（０．２５～０．１８ｍｍ）；（２）５％新戊二醇己二酸聚酯／ＣｈｒｏｍｏｓｏｒｂＧＡＷＤＭ－ＣＳ（０．２５～０．１８ｍｍ）的两条色谱柱，邻苯二甲酸二戊酯为内标，氢火焰离子化检测器，标准偏差（ＳＤ）≤０．０６４％，变异系数（ＣＶ）≤２．１％，平均回收率为９６．７％～９８．８％。     

离体诱发甘蓝型油菜长角果和矮秆突变体

本试验对ＥＭＳ诱变甘蓝型油菜（Ｂｒａｓｓｉｃａｎａｐｕｓ）小孢子胚状体培养物的有效浓度和适宜时间进行了初步探索。用０．２％和０．２５％ＥＭＳ处理３～５ｈ，９２．Ｂ１０品系出现了长角果和矮秆突变体，Ｄ０８３中产生了窄叶和皱叶不育株。     

黄瓜同种异体嫁接组合形成过程中特异蛋白质的产生

用ＰＡＧ等电聚焦、ＳＤＳ－ＰＡＧＥ梯度电泳和双向电泳分析了黄瓜同种异体嫁接组合不同发育时期的全蛋白变化。结果表明：与愈伤处理比较，嫁接后第２天到第１０天嫁接组合的不同发育时期，稳定地出现三种新合成的特异蛋白质；通过分子量（ＭＷ）和等电点（ｐＩ）的分析表明这三种特异蛋白质的分子量：等电点分别为：１７．６ｋＤ：４．８５，１５．２ｋＤ：６．２６，２０．６ｋＤ：７．６。最后，对高等植物的嫁接亲和性机理问题     

细粒棘球蚴生发层细胞系可溶性抗原的免疫学及SDS-PAGE分析

本研究报道了细粒棘球蚴生发展细胞可溶性抗原的免疫学研究结果及ＳＤＳ－ＰＡＧＥ结果，将原头节可溶性抗原，生发展可溶性抗原，囊液抗原免疫Ｂａｌｂ／ｃ小鼠制备抗血清并用原头节人工感染Ｂａｌｂ／ｃ小鼠制备阳性鼠血清，再用生发展细胞系可溶性抗原以间接ＥＬＩＳＡ法进行检测以观察该抗原的反应原性；用ｐＡｇ，ｇＡｇ，ＳＨＦ以及生发展分泌抗在分别对曾反种过细胞系细胞的Ｂａｌｂ／ｃ小鼠的血清进行检测，以了解细胞系抗原     

Prokaryotic Expression of Mouse Sox2 Gene and Preparation of Its Polyclonal Antibody

Sox2, one of genes which express in embryonic stem (ES) cells, plays an important role in selfrenewal of ES cells, and is used to make induced pluripotent stem (iPS) cells. In this study, we subcloned mouse (Mus muscules) full-length Sox2 cDNA and inserted Sox2 into pET-41 a expression vector. Then the recombinant vector was transformed into Escherichia coli BL21 (DE3) cells to express Sox2 fusion protein. The expressed products were identified by Western blot using anti-GST antibody. The result indicated that the size of Sox2 fusion protein was in 61 kD. And the protein was recovered from the SDS-PAGE. And we used the sox2 fusion protein to prepare polyclonal antibody. The Dot blotting result showed that the anti-Sox2 antiserum had 1:12,800 titers. The antibody will be useful for studying the function of Sox2 and its role in ES cell self-renewal.     

筛选抗片形吸虫病绵羊品种的研究进展

本文综述了抗片形吸虫病绵羊品种的研究概况 ,通过红细胞压积、红细胞平均体积、红细胞平均血红蛋白浓度、血清谷氨酸脱氢酶、γ 谷氨酰胺转移酶、天冬氨酸转氨酶等血液学参数 ,囊蚴感染成活率的测定等方法来筛选抗片形吸虫病的绵羊品种 ,研究证明印度尼西亚细尾绵羊对大片吸虫感染具有较高的抵抗力 ,并概述了其抗虫感染的机制及杀虫作用时间。印度尼西亚细尾绵羊对大片吸虫的抵抗力包括先天性抵抗力和获得性抵抗力 ,杀虫时间集中在感染后 3-4周和 1 5-2 1周。未发现对肝片吸虫感染有高抵抗力的品种。     

Role of root exudates on the sorption of arsenate by ferrihydrite

Iron oxy‐hydroxides in soil are known to have a large affinity for arsenate (As(V)) inorganic species. At the soil–root interface such mineral components are embedded by mucilaginous material that is secreted from continuously growing root cap cells. In order to determine the role of plant mucilages in As(V) sorption by iron oxy‐hydroxides, we layered a calcium (Ca)‐polygalacturonate network (CaPGA) on to amorphous iron (Fe) (III) hydroxide (ferrihydrite, Fh) particles. The scanning electron micrographs of the CaPGA network coating the ferrihydrite (Fh–CaPGA) show a regular structure with a honeycomb‐like pattern where interlacing fibrils form a porous system. The FT‐IR spectra of Fh–CaPGA suggest that CaPGA fibrils are retained by the surface Fe(III) nuclei of Fh through electrostatic interactions. The sorption experiments carried out at pH 4.3 and 5.8 indicated a smaller amount of As(V) sorbed by Fh–CaPGA than by Fh alone, being less after 3 and 24 hours of reaction by about 70 and 30%, respectively. The sorption of As(V) by Fh was also studied in the presence of caffeic acid (CAF), an important root exudate. Simultaneous sorption kinetics show that As(V) sorption by Fh is almost independent of CAF concentration, indicating a greater affinity of arsenate ions towards the Fh surfaces. However, the amount of As(V) sorbed by the Fh coated by CaPGA, in the presence of 0.25, 0.5 and 1.0 mm CAF, is markedly smaller by about 20, 27 and 40%, respectively, than that found in the As(V)–CAF‐Fh ternary systems. This is caused mainly by redox reactions involving CAF and the surface Fe(III) nuclei of Fh leading to the formation of CAF oxidation products which prevent As(V) sorption.     

不同毛管配置对水氮分布和机采棉根系生长的影响

通过田间试验,研究不同滴灌配置对机采棉根系生长、水氮运移和氮肥利用率的影响。设置3种滴灌毛管配置方式:(1)内嵌式滴灌毛管+夹管(EB);(2)内嵌式毛管+侧管(ES);(3)迷宫式毛管+侧管(LS);施氮(N)量均为300 kg/hm~2;同时,以ES处理不施氮肥为对照(CK)。结果表明:滴灌施肥24 h后,土壤水分及硝态氮均主要分布在0—40 cm土层。LS和EB处理水分和硝态氮在作物行下方的根区含量高,ES处理硝态氮分布向宽行偏移。90%以上棉花根系分布在0—30 cm土层,但EB处理根系分布更浅,其超过80%根系分布在15 cm以内土层;ES处理与LS、EB处理相比,根干物质量分别显著降低31.7%和25.5%;ES处理根长密度、根表面积、根体积显著高于LS和EB处理。LS处理显著增加产量和氮肥利用率,较ES处理分别增加9.4%和18.0%;EB处理产量和氮肥利用率也较ES处理分别增加6.5%和8.5%。机采棉使用迷宫式滴灌毛管并在侧管铺设毛管,水分和硝态氮分布与根系分布相匹配,能显著促进棉花根系生长,增加氮吸收量并提高产量和水氮利用效率。     

草莓果实特异表达基因annfaf 在E. coli 中的表达及抗血清的制备

摘要：将草莓?穴Fragaria ananassa Duch. ?雪果实中特异表达的膜联蛋白基因annfaf的cDNA连接重组到pET-30a质粒中，构建了融合和非融合蛋白表达载体。annfaf 基因转化大肠杆菌(E. coli )后以包含体形式得到了高效表达，同时研究了annfaf基因表达的影响因素。实验结果表明, 最佳培养温度是37 ℃ ，培养时间为4 h，当培养液中加入利福霉素时可明显提高表达量中目的蛋白的含量。SDS-PAGE检测表明该蛋白分子量为35 kD，与目的蛋白相同。以表达的蛋白作抗原免疫家兔，制备了抗Annfaf蛋白的抗血清。ELISA检测及Western印迹表明，制备的抗血清可与其免疫抗原发生特异的免疫学反应，且具有较高的效价。该实验结果为进一步研究Annfaf蛋白在草莓果实细胞中的定位及其生理功能奠定了基础。     

玉米叶绿体铁氧还蛋白1的原核表达及部分性质分析

叶绿体铁氧还蛋白(Fd)通过活性中心的铁硫簇传递还原力,在各种氧化还原途径中起重要作用.本研究中,氨基酸序列比对显示玉米中5种Fd的叶绿体导肽同源性很低,而去除导肽的成熟蛋白氨基酸序列具有很高的同源性.采用RT-PCR技术从玉米幼叶总RNA中克隆了编码成熟Fd1的基因.并分别插入pQE 80和p28SUMO表达载体,转...     

Field Evaluation of Ammonium Sulfate versus Two Fertilizer Products Containing Ammonium Sulfate and Elemental Sulfur on Soybeans

ABSTRACT

Two separate field trials comparing three different commercial sulfur (S) fertilizers were conducted in 2016 and 2017, respectively, to evaluate their initial agronomic effectiveness in increasing soybean grain yield. The S compounds of these products were elemental S (ES) and ammonium sulfate (AS). The fertilizer sources were (1) AS, (2) granular MAP-10 S (5% ES + 5% AS-S) and (3) bulk-blend (bentonite-ES) + (AS) containing 25% ES + 25% AS-S. Rates of total S applied were 0, 5.6, 11.2, 22.4 and 33.6 kg S ha^?1. A significant S response in grain yield was observed with all S sources. The grain yield was significantly higher with AS than with the other two S sources at S rates ≤22.4 kg S ha^?1 of total S applied but equal for all sources above this rate. All data points for the three S sources followed the same S response function based on a quadratic plateau model when the grain yield was plotted versus AS-S rate applied. This suggests that there was no significant ES oxidation from MAP-10S and (ES) + (AS) products to provide soybean crop growth to maturity. The experimentally observed AS-S rate at which the maximum grain yield was attained in the 2 years of fieldwork was 11.2 kg S ha^?1 while the calculated average AS-S rate based on a quadratic plateau model was 13.4 kg S ha^?1.

Abbreviations: AS: ammonium sulfate; ES: elemental S; MAP: monoammonium phosphate; DAP: diammonium phosphate; TSP: triple superphosphate.     

Scorpion toxins modify phytopathogenic fungus physiology. A possible source of new fungicides

Seven toxins (F1-F7) were purified from Tityus discrepans scorpion venom on a C18 HPLC column. The compounds were fungitoxic on Macrophomina phaseolina. The molecular masses of F1-F7 were (Da) 1061.1, 7328.8, 7288.3, 7268.5, 7104.6, 6924.6, and 6823.3, respectively. It is not known if F1 is a small peptide or some other kind of organic molecule. Compounds F2-F7 were peptides. The most potent was F7, with a minimal inhibition concentration of 0.4 μg/μL and a concentration for 50% inhibition of 0.13 μg/μL. Fungal esterase activity was abolished by F2, F3, and F5 and inhibited by 89, 60, 58, and 54% by F4, F6, F7, and F1, respectively. F1, F2, F5, and F7 induced an increase on hyphae chitin wall and septum thickness. Peptides F3-F6 induced efflux of the fluorescent dye Na-CoroNa Red complex from hyphae. Only F5 and F6 were inhibited by the prokaryote sodium channel blockers amiloride and mibefradil. Gas chromatography-mass spectrometry analysis suggested that F1, F5, F6, and F7 altered sterol biosynthesis either by inhibiting ergosterol biosynthesis or by producing ergosterol analogues. The peptides affect M. phaseolina viability by three mechanisms: decreasing esterase activity, altering Na(+) membrane permeability, and altering wall sterol biosynthesis. It seems that interfering with sterol synthesis is an important mechanism behind the effect of the fungicideal toxins. However, the antifungal effects at short times are indicative of a direct esterase inhibition, which, with the increased membrane leakiness to Na(+), makes the fungus inviable.     

水稻草矮病毒基因组vRNA3 NS3基因的克隆、序列分析及原核表达

摘要: 根据RGSV(rice grassy stunt virus )-IR分离物的RNA3序列设计引物，采用RT-PCR技术扩增出RGSV-SX分离物的NS3基因，并进行序列测定及原核表达。结果表明，NS3基因由588个核苷酸组成，编码22.9 kD蛋白。构建了可在E. coli DH5α中表达的质粒pGTNS3，经IPTG诱导，SDS-PAGE分离纯化得到分子量为49.0 kD的GST-NS3融合蛋白，并制备抗血清。应用Western blot 分析寄主水稻(Oryza sativa)和介体昆虫体内NS3基因表达产物，仅在感病水稻植株中检测到NS3蛋白，而在提纯病毒、介体昆虫体内则未检测到。     

香蕉束顶病毒海口分离物核穿梭蛋白（NSP）的纯化及抗血清制备

本研究以本实验室保存的含重组载体pDEST17-NSP的原核表达菌株E．coliBL21（DE3）为材料，于25℃、0．1mmol／LIPTG条件下诱导4h，集菌后超声波破碎，获得以包涵体形式表达的约20kD的融合蛋白。实验结果表明，将沉淀的融合蛋白溶于含6mol／L尿素的Binding Buffer中，再经Ni^2＋-NTA亲和层析纯化后，可获得高纯度的融合蛋白。将纯化融合蛋白经12％SDS-PAGE电泳，切胶回收目的带，液氮研磨并按1：1（W／V）混合佐剂，4次免疫家兔，获得BBTV病毒核穿梭蛋白的特异性抗血清。以融合蛋白作抗原，间接ELISA法测定其抗血清效价为1：5000。田间检测样品的最佳抗血清工作浓度为1：500。Western Blot鉴定结果表明抗血清能与目的蛋白特异性结合。本研究的结果将为下一步NSP基因转录调控和蛋白功能研究奠定一定基础。