应用脉冲场凝胶电泳技术制备鲫鱼全基因组DNA

为了研究用脉冲场凝胶电泳技术制备高分子质量基因组DNA时的最佳条件,试验采用鲫鱼为材料,将肌肉组织研磨过滤获得细胞悬液,包被于低熔点琼脂糖包中,用蛋白酶K消化胶块后进行脉冲电泳。结果表明:采用1%琼脂糖凝胶,电泳温度为14℃,电压为4 V/cm,起始和终止脉冲时间为1~40 s,脉冲角度为120°,冷凝泵流量为70 L/h,电泳时间为18 h,能获得1 000.0 kb的基因组DNA,满足基因组文库构建的需要。     

维生素E缺乏致雏鸡脾脏淋巴细胞凋亡

用低维生素E(VE)和添加不饱和脂肪酸的日粮饲喂雏鸡建立VE缺乏雏鸡模型.采用吖啶橙和溴化乙锭双荧光染色法及透射电镜,观察雏鸡脾脏淋巴细胞凋亡的形态学变化和超微结构变化;用DNA片段化定量分析及琼脂糖凝胶电泳法检测雏鸡脾脏凋亡淋巴细胞DNA的损伤.结果,VE缺乏雏鸡脾脏淋巴细胞的形态和超微结构均发生了凋亡变化,出现典型的DNA梯形带.DNA片段化的程度明显也高于对照组,组间差异极显著(P<0.01).表明VE缺乏可致雏鸡脾脏淋巴细胞凋亡.     

水泡性口炎病毒诱导BHK-21细胞的凋亡

采用HE、Giemsa染色、透射电镜以及DNA琼脂糖凝胶电泳等研究了水泡性口炎病毒（VSV）诱导BHK-21细胞凋亡的过程。结果显示：VSV感染BHK-21细胞后,光镜下可见细胞圆缩,细胞器固缩、核仁消失、染色质凝聚和核碎裂、凋亡小体出现;电镜下观察到染色质聚集形成典型的新月形,胞浆中充满大量空泡,细胞核因染色质凝聚也发生了空泡化;1％的琼脂糖凝胶电泳出现180-200bp整倍数的DNA梯形条带。结果表明,VSV诱导BHK-21细胞凋亡是其致细胞病变的主要表现形式之一。     

South-Western blot mapping方法的建立及其应用于锥虫细胞核中DNA结合蛋白的观察

South-Westerm blot mapping是一种结合Western blotting和Southern blotting某些特点的方法.本文介绍用其成功地观察到锥虫核蛋白中DNA结合蛋白的情况,并对一个分子量在40000左右、于较严谨条件下与DNA结合的核蛋白进行了特性鉴定.该蛋白等量地存在于锥虫的前循环期和血液期,对双链DNA有较大的亲和力,并能与酵母菌复制起始片段结合.本文还介绍了锥虫细胞核的提取技术和核蛋白的制备技术.     

基因打靶用于哺乳动物体细胞核移植技术研究进展

哺乳动物体细胞核移植技术以其供核效率高而在动物克隆技术中占有明显的优势；基因打靶技术通过外源DNA与染色体DNA间的重组可达到定点修饰，改造基因组的目的，它与体细胞核移植技术相结合具有巨大的技术优势和广阔的应用前景。将为畜牧生产和医学生物学的研究带来前所未有的价值。     

中国斗鸡高分子质量DNA的高效制备

采集斗鸡翅静脉血液,加ACD抗凝,离心收集血细胞,经细胞计数后,用低融点琼脂糖包埋血细胞,蛋白酶K消化,经脉冲电泳分离,获得了较纯净的720 kb以上的DNA.对大片段DNA进行限制性酶切,回收特定大小的DNA片段.利用该方法制备的高分子质量DNA,经酶切、回收的特定大小DNA片段可用于后续细菌人工染色体(BAC)文库的构建和基因组分析.     

绿色荧光蛋白在鸡胚成纤维细胞中的表达

应用阳离子脂质体介导法,将含绿色荧光蛋白(GFP)基因的质粒pEGFP-C1转染到培养至第二代的鸡胚成纤维细胞(CEF)中,同时使用活体DNA染料Hoechst3334(2H342)对CEF细胞核荧光染色,通过荧光倒置显微镜和RT-PCR方法检测GFP的表达产物。在荧光倒置显微镜下可见CEF的胞质和核均呈现绿色荧光,且细胞核的绿色荧光强度强于细胞质,在H342作用下,细胞核呈现蓝色荧光;转染细胞中转录产物经RT-PCR扩增后,在凝胶上可见与GFP基因片段分子量大小一致的条带。可见,GFP基因可以被转染至CEF中,并在CEF中表达。     

无包埋体对虾病毒（NOSV）的纯化和随机文库构建

通过超薄切片和负染电镜观察,从患暴发性传染病死亡的中国对虾体组织中,发现一种无包埋体对虾病毒（ＮＯＳＶ）或称中国对虾类杆状病毒（ＰｃＢＬＶ）。对其用不连续蔗糖梯度离心纯化,观察了其核衣壳的负染结构：核衣壳平均大小为６２ｎｍ×３１４ｎｍ,由蛋白壳粒环堆砌而成,每３层环形成１个重复单位,重复单位之间有较大空隙;核衣壳一端较平,另一端近于帽状。提取纯化病毒的核酸,电泳后用低熔点琼脂糖回收,用ＥｃｏＲＩ酶切,克隆于ｐＵＣ１８质粒中,共获得１６个阳性克隆质粒,克隆片段总长度约５３．４５ｋｂ。     

家兔全血基因组DNA提取方法

以肝素钠抗凝的皖系长毛兔全血为材料,以常规的酚氯仿抽提法为基础,采用改进的方法提取基因组DNA.结果表明,提取的基因组DNA纯度高,用琼脂糖凝胶电泳检测基因组DNA,主带清晰、无拖尾现象.     

猪繁殖与呼吸综合征病毒非结构蛋白2分段真核表达及其细胞定位分析

为探讨非结构蛋白2(non-structural protein 2,NSP2)在猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)复制过程中的作用及机制,本研究构建真核重组质粒pc DNA 3.1-5'Flag-NSP2-147-323,并瞬时转染至HEK293T中,同时设置完整NSP2基因序列重组质粒作为对照,转染48 h后分别用不同的抗体进行间接免疫荧光监测。结果显示,转染对照组中细胞核周围可见特异性荧光,而转染真核重组质粒pc DNA 3.1-5'Flag-NSP2-147-323后,不仅在细胞核周围,在细胞核内也可以观察到特异性荧光。本研究成功构建真核重组质粒pc DNA 3.1-5'Flag-NSP2-147-323,并可在HEK 293T细胞中正确表达,为进一步研究NSP2在PRRSV复制过程中的作用提供依据和基础。     

流式细胞术分析苜蓿相对DNA含量的样品处理方法

为制备用于流式细胞仪分析的高纯度苜蓿(Medicago sativa)细胞核悬液,筛选最适合流式细胞仪分析的苜蓿细胞核提取方法,选用江苏地方品种淮阴苜蓿为供试材料,分别采用酶解法和机械解离法提取苜蓿细胞核悬液;用5种细胞核分离缓冲液及两种混合酶解液制备样品,根据各自优缺点筛选最适宜的苜蓿细胞核提取法。在机械法所用的5种细胞核提取缓冲液中,LB01缓冲液提取效果最好,提取的细胞核形态及内部结构完整,获得的细胞核量多。利用两种混合酶液制备苜蓿细胞核悬液,均出现倍性明显的峰,混合酶液Ⅰ制备的单细胞核悬液优于混合酶液Ⅱ,且酶解法制备细胞核悬液,可获得数量更多更完整的原生质体。但利用酶解法制备细胞核原生质体,其悬浮液中杂质多,需时较长。结果表明,LB01缓冲液为最适合制备苜蓿细胞核悬液的提取缓冲液。     

Comparative autoradiographic study of parasite-host-cell cyclical relationship in lymphoblastoid cell lines infected with Theileria annulata and Theileria parva in vitro

Cyclical interactions between intracellular schizonts of the Ankara and Hissar strains of Theileria annulata and the Muguga strain of T. parva and the parasitised host lymphoblasts have been studied autoradiographically by following the incorporation of (3H) thymidine into parasite and host cell nuclei, and also by quantitating the number of schizont nuclei per lymphoblast, at various stages and phases of host cell cycle. The synthesis of DNA by Theileria schizonts and the parasitised host lymphoblasts was found to be asynchronous and to occur at different phases of the host cell interphase stage. While the lymphoblast nuclear DNA incorporates (3H) thymidine during the S phase, schizont nuclei were labelled during the G2 phase of the host lymphoblast interphase stage. The replication of schizont nuclei took place before the metaphase stage of host cell cycle, viz, prometaphase, so that the mean schizont nuclear number at host prometaphase and at anaphase--telophase was consistently more or less double the mean nuclear number at interphase.     

Inhibitory effects of long-wave ultraviolet radiation of isolated chicken liver nuclei on the Mg2+-dependent transition of chromatin structure

Since the turbidity of nuclear suspensions is known to be correlated with the nuclear morphology, the effects of long-wave ultraviolet (UVA) radiation on Mg2+-dependent structural transition of chromatin in isolated chicken liver nuclei were monitored by measuring the relative turbidity of nuclear suspensions. UVA radiation of the nuclei inhibited the Mg2+-dependent change in relative turbidity of nuclear suspensions in a UVA dose-dependent manner under aerobic conditions but not under N2 conditions. No inhibitory effect of UVA radiation on the change in relative turbidity was observed in the presence of 50 mM NaN3, which scavenges singlet oxygen (1O2) and hydroxyl radicals (*OH). In contrast, 100 mM dimethyl sulfoxide, which primarily scavenges *OH, did not show the inhibitory effect of UVA radiation. The amounts of DNA-protein crosslinks increased with UVA dose under aerobic conditions but not under N2 conditions. The present study showed that UVA radiation of isolated nuclei inhibited the Mg2+-dependent unfolding of condensed chromatin and that O2 is likely to be involved in this process. Furthermore, the formation of DNA-protein crosslinks may contribute to the inhibition.     

Lead exposure induces pycnosis and enucleation of peripheral erythrocytes in the domestic fowl

Lead (Pb) shot were administered orally to young chickens to determine the effects on red blood cells (RBCs). The concentrations of Pb in the blood of young chickens rose rapidly after Pb administration and were maintained at high levels for several days. The number of RBCs with pycnotic nuclei, reticulocytes and enucleated RBCs increased concurrently. Pycnotic nuclei were surrounded by enlarged nucleolemmal cisternae, which sometimes opened to the extracellular space. Gel electrophoresis showed that the presence of pycnotic nuclei was not associated with DNA fragmentation typical of apoptosis. It was concluded that exposure to lead shot changes nuclear morphology in the peripheral blood of domestic fowl.     

低分子质量左聚糖对人体肠道菌群调节作用

采用三氯乙酸(trichloroacetic acid,TCA)法除去Paenibacillus bovis sp.nov BD3526来源的高分子质量左聚糖中蛋白质的同时制备得到低分子质量左聚糖,并用离子交换层析法对低分子质量左聚糖进行分离纯化,用凝胶渗透色谱(gel permeation chromatography,GPC)法测定纯化后样品的分子质量及其分布,用核磁共振(nuclear magnetic resonance,NMR)仪和傅里叶变换红外光谱(fourier transform infrared spectrometer,FT-IR)表征其化学结构,用原子力显微镜(atomic force microscope,AFM)表征其在水溶液中的构象,最后采用荷兰应用科学研究(The Netherlands Organization For Applied Scientific Research,TNO)组织建立的体外筛选平台评价其对人体肠道菌群的调节作用.体外实验结果表明,此低分子质量左聚糖能促进双歧杆菌特别是婴儿双歧杆菌和长双歧杆菌的增殖,其促进作用优于低聚果糖(fructooligosaccharide,FOS).     

多枝赖草的C-分带与核型分析

通过对多枝赖草的根尖染色体进行常规制片分析和利用BSG方法对其染色体进行C-分带研究,确定多枝赖草的染色体核型组成为2n=4x=28=18m 10sm,确认多枝赖草的标准C-分带带型为2n=28=5CT 2CTI 2CT 1CTI 1CTI 1C 1T 1TI。     

Nuclear Morphometric Analysis of Leydig Cells of Male Pubertal Rats Exposed In Utero to Di(n-butyl) Phthalate

We recently reported that prenatal rat exposure to di(n-butyl) phthalate (DBP) induced Leydig cell (LC) hyperplasia after nine weeks (wks) of age, yet the number of LCs was similar to that of the vehicle group until seven weeks. Nuclear pleomorphism of hyperplastic LCs is common and is considered to be continuous progressive degeneration. Thus, computer-assisted image cell nuclear analysis of LCs was performed on 5- and 7-wk-old Sprague-Dawley (SD) rats whose dams had been administered DBP (i.g.) at 100 mg/kg/day or vehicle (corn oil) on gestation day 12 to 21. The results of the 5-wk-old DBP group were similar to those of the vehicle group; LC nuclei of the 7-wk-old DBP group showed normal ploidy and similar amounts of DNA. However, the size, elongation and peripheral chromatin aggregation parameters were significantly higher, and the reticular chromatin distribution and isolated chromatin aggregation parameters were significantly lower compared with the vehicle group. The present study quantitatively demonstrated nuclear morphological alterations in rat LCs at 7 wks old (puberty) due to the prenatal DBP administration before apparent LC hyperplasia developed.     

鸭疫里默氏杆菌实时荧光定量PCR快速检测方法的建立与应用研究

根据GenBank登录的鸭疫里默氏杆菌外膜蛋白(OmpA)基因保守区序列,设计引物和探针,建立了直接检测鸭疫里默氏杆菌的实时荧光定量PCR快速方法。10倍梯度稀释RA菌株制备DNA模板测定方法的灵敏度,结果可在8.0×103CFU/mL浓度下特异地检测出目的菌,最低能检测到RA的DNA模板为8.0拷贝/μL;对鸭源大肠杆菌、多杀性巴氏杆菌、沙门氏菌、变形杆菌、金黄色葡萄球菌进行检测,结果均为阴性,建立的实时荧光定时PCR方法特异性强。用RA菌株人工感染雏鸭,感染后定时采集咽喉拭子、泄殖腔拭子、心血、肝脏、肺脏、脑,分别用实时荧光定量PCR检测,对脏器进行病原菌分离。结果表明,1/10半数致死量接种组,感染后8 h从心血、肝脏、脑组织检测到RA核酸,16 h后全部试样呈阳性;24 h首先从肝脏分离到RA,心血、肝脏、肺脏、脑组织均分离出RA。心血和脏器RA实时荧光定量PCR阳性检出率为63.8%(51/80),RA分离率为28.8%(23/80)。10个半数致死量接种组,1 h即可从咽喉拭子、心血和肝检测到RA核酸,5 h全部样品为阳性;5 h从肺和脑分离到RA。RA人工感染鸭的心血、肝脏、肺脏、脑实时荧光定量PCR阳性检出率为86.3%(69/80),RA分离率为60%(48/80)。用加热裂解法提取样品RA DNA只需30 min,建立的实时荧光定量PCR检测RA只需2 h,适用于RA的快速诊断、流行病学调查、种鸭引进隔离检疫、活鸭及其产品国内市场和进出口检验检疫。     

人工三倍体桑品种丰田2号的育成

将鲁桑系二倍体桑品种桐乡青用秋水仙碱诱导成四倍体,以此为母本与广东桑二倍体伦教109号为父本杂交育成人工三倍体品种丰田2号。与二倍体桑品种荷叶白相比,其单位面积产叶量提高36.9%;桑叶养蚕的万蚕茧层量提高10.8%,种茧育单蛾良卵数提高18.3%;中抗桑疫病,农艺性状好,表现出优良三倍体优质高产的典型特性。