绵羊体外成熟卵母细胞的电激活及其在体外孤雌发育的研究

首先对绵羊卵母细胞收集及其体外成熟（ＩＶＭ）条件进行了摸索,然后对影响电刺激激活ＩＶＭ卵母细胞的因素进行了研究,观察激活后卵母细胞在体外的发育能力。结果表明,剥离法比注射器吸卵法所获得的卵母细胞成熟率高,激活更加正常。剥离法收集的卵母细胞体外成熟培养２７小时后,以含０．１ｍｍｏｌ／Ｌ ＣａＣｌ２、０．１ｍｍｏｌ／Ｌ ＭｇＳＯ４和１０ｍｍｏｌ／Ｌ组氨酸的０．２８ｍｏｌ／Ｌ肌醇（ｉｎｏｓｉｔｏｌ）作基     

Codon modification for the DNA sequence of a single-chain Fv antibody against clenbuterol and expression in Pichia pastoris

The expression efficiency was improved for the recombinant single-chain variable fragment (scFv) against clenbuterol (CBL) obtained from mouse and expressed in the methylotrophic yeast Pichia pastoris GS115, by redesigning and synthesizing the DNA sequence encoding for CBL-scFv based on the codon bias of P. pastoris. The codons enco4ding 124 amino acids were optimized, in which a total of 156 nucleotides were changed, and the G+C ratio was simultaneously decreased from 53 to 47.2 %. Under the optimized expression conditions, the yield of the recombinant CBL-scFv (41 kDa) antibodies was 0.223 g L^–1 in shake culture. Compared to the non-optimized control, the expression level of the optimized recombinant CBL-scFv based on preferred codons in P. pastoris demonstrated a 2.35-fold higher yield. Furthermore, the recombinant CBL-scFv was purified by Ni-NTA column chromatography, and the purity was 95 %. The purified CBL-scFv showed good CBL recognition by a competitive indirect enzyme-linked immunoassay. The average concentration required for 50 % inhibition of binding and the limit of detection for the assay were 5.82 and 0.77 ng mL^–1, respectively.     

慢性间歇低氧对幼鼠脑区p38MAPK的影响

目的：研究慢性间歇低氧对幼鼠部分脑区p38MAPK的影响。方法：SPF级健康雄性SD幼鼠（3-4周龄）50只,随机分为5组（n=10）：间歇低氧2周组（2IH组）、间歇低氧4周组（4IH组）、间歇低氧4周后恢复组（4F组）、对照2周组（2C组）和对照4周组（4C组）。建立慢性间歇低氧幼鼠模型,以RT-PCR法和Westemblot法分别测幼鼠海马及前额叶皮层p38MAPKmRNA和磷酸化p38MAPK（p-p38）蛋白的表达。结果：21H、4IH和4F组幼鼠海马、前额叶皮层的p38MAPK mRNA及p-p38蛋白均明显高于相应对照组（P均〈0．05）。结论：慢性间歇低氧可激活幼鼠部分脑区D38MAPK。     

12产地甘草中18α-甘草酸及18β-甘草酸的含量分析

目的:分析不同产地甘草中18α-甘草酸及18β-甘草酸的含量差异,为不同产地甘草的质量评价提供参考。方法:采集7个省区12个不同产地的甘草种子,栽培于北京中医药大学药草园,一年后以其中180株甘草作为实验材料,利用内转录间隔区(ITS)序列鉴定其基原,采用高效液相色谱(HPLC)法测定其18α-甘草酸及18β-甘草酸含量,并分析其差异性及相关性。结果:ITS鉴定结果显示180株甘草样品均为乌拉尔甘草。HPLC分析结果显示,18α-甘草酸的标准曲线为y=6×10-7x-0.0029(R2=0.9982),在0.0111~0.2214μg范围内线性良好;18β-甘草酸的标准曲线为y=1×10-6x+0.0164(R2=0.9999),在0.2256~4.5120μg范围内线性良好。12个产地中山西应县甘草样品的18α-甘草酸及18β-甘草酸含量均最高,新疆尼勒克县甘草样品的18α-甘草酸及18β-甘草酸含量均最低,且所有样品中18α-甘草酸与18β-甘草酸的含量均存在显著相关关系。结论:不同产地甘草质量差异显著,本实验结果可为不同产地甘草的质量评价提供参考。     

220例成人麻疹临床特征分析

本文旨在回顾性分析2003年1月～2008年12月上海交通大学附属第六人民医院收治的220例成人麻疹患者临床资料,探讨成人麻疹患者临床特征。220例患者(男124例,女96例),平均年龄(34.6±4.2)岁,外来人员占70.91%,发病集中于4、5月,血清麻疹IgM抗体阳性率为93.18%,183例(83.18%)热峰高于39℃,86例(39.09%)并发麻疹病毒性肝炎,31例(14.09%)并发腹泻。麻疹发病年龄后移,具有较重的病毒血症以及肝脏损害等临床特征。     

半滑舌鳎雌性特异AFLP标记CseF783的克隆及其在遗传性别鉴定中的应用

半滑舌鳎性别控制和全雌育种等研究领域中迫切需要一种能够快速鉴定鱼类个体遗传性别的有效方法。文章采用AFLP技术, 利用选择性引物组合(E-ACT/M-CAA)从半滑舌鳎中筛选到一条雌性特异的AFLP标记。对该标记进行二次PCR扩增、琼脂糖凝胶回收、克隆、测序。分析表明, 序列全长为791 bp, 与GenBank中的序列无同源性。以该雌性特异AFLP标记DNA序列为模板, 设计了一对特异的PCR引物, 成功地将其转化为SCAR(Sequence characterized amplified regions)标记, 并在100尾已知性别的半滑舌鳎个体(雌雄各50尾)中进行验证, 结果表明, 该SCAR标记在所有雌性个体中均扩增得到一条长度为324 bp的DNA条带, 而在49尾雄性个体中均扩增不到该DNA条带(有1尾雄性个体例外), 证明该SCAR标记是雌性特异的, 并可用于半滑舌鳎个体遗传性别鉴定。随后, 利用该SCAR标记检测了3日龄半滑舌鳎幼苗, 结果表明, 雌性个体比例为41.7%。     

Loop-mediated isothermal amplification for the rapid detection of Haemophilus parasuis

Haemophilus parasuis infection is of considerable economic importance in the swine industry due to the high costs associated with treatment and loss of animals all over the world. In the present study, loop-mediated isothermal amplification (LAMP) is described for the rapid and specific detection of this species. A primer set derived from the inf B gene of H. parasuis was used to validate the assay using 15 H. parasuis reference strains, 39 clinical isolates, 75 positive samples, and 18 other pathogens. The results indicated that positive reactions were confirmed for all H. parasuis strains and specimens by LAMP after 45 min reaction at 65 °C in a water bath, and no cross-reactivity was observed from other non-H. parasuis strains. The detection limit of the conventional PCR was 25 copies, while that of the LAMP was five copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. LAMP is likely to be more suitable as a routine diagnostic tool, especially in clinics without complicated equipment such as thermal cycling machines and electrophoresis apparatus. In these scenarios, the H. parasuis LAMP assay has the potential for field diagnosis.     

番茄1-氨基环丙烷羧酸(ACC)合成酶基因的反义RNA-核酶嵌合DNA序列的构建

根据番茄ACC合成酶基因（LE—ACC2）DNA序列,以番茄(LycopersiconesculentumMill)果实的总DNA为模板,利用PCR技术扩增得到预期大小的该基因编码区内部分DNA序列,插入到质粒载体pGEM—3zf(＋)的BamHⅠ和HindⅢ位点之间后转化E．coliDH—5α,可选出重组子pRE,经酶切,PCR及DNA序列分析证明克隆成功;将pRE上的目的DNA序列以反义方式构建到我室已合成并克隆的含核酶DNA序列的重组质粒pRⅠ的BamHⅠ和HindⅢ之间,构成含有反义RNA-核酶嵌合DNA序列的重组质粒pREⅠ,经酶切及序列分析,结果与预期一致.     

Whirly转录因子研究进展

Whirly蛋白是广泛存在于植物细胞内的一种转录因子。它既能与单链DNA结合,也能与RNA结合,无论在细胞核还是在质体内都有着广泛而复杂的生物学功能。本文概述了Whirly蛋白的结构、种类、分布及其作用机制,并重点讨论了其在细胞核及质体内的功能,最后对Whirly蛋白研究中需要解决的问题做了展望。     

Effects of simulated drought stress on carotenoid contents and expression of related genes in carrot taproots

Carotenoids are liposoluble pigments found in plant chromoplasts that are responsible for the yellow, orange, and red colors of carrot taproots. Drought is one of the main stress factors affecting carrot growth. Carotenoids play important roles in drought resistance in higher plants. In the present work, the carotenoid contents in three different-colored carrot cultivars, ‘Kurodagosun’ (orange), ‘Benhongjinshi’ (red), and ‘Qitouhuang’ (yellow), were determined by ultra-high-performance liquid chromatography (UPLC) after 15% polyethylene glycol (PEG) 6000 treatment. Real-time fluorescence quantitative PCR (RT-qPCR) was then used to determine the expression levels of carotenoid synthesis- and degradation-related genes. Increases in β-carotene content in ‘Qitouhuang’ taproots under drought stress were found to be related to the expression levels of DcPSY2 and DcLCYB. Increases in lutein and decreases in α-carotene content in ‘Qitouhuang’ and ‘Kurodagosun’ under PEG treatment may be related to the expression levels of DcCYP97A3, DcCHXE, and DcCHXB1. The expression levels of DcNCED1 and DcNCED2 in the three cultivars significantly increased, thus suggesting that NCED genes could respond to drought stress. Analysis of the growth status and carotenoid contents of carrots under PEG treatment indicated that the orange cultivar ‘Kurodagosun’ has better adaptability to drought stress than the other cultivars and that β-carotene and lutein may be involved in the stress resistance process of carrot.