柞蚕微孢子虫3种孢壁蛋白的提取与鉴定

在微孢子虫侵染宿主的过程中,孢壁蛋白扮演着重要的角色。分别采用沸水浴法、SDS法从柞蚕微孢子虫(Nosema per-nyi,Np)孢子中提取孢壁蛋白以及利用发芽后的孢子壳提取孢壁蛋白,对提取的孢壁蛋白进行SDS-PAGE分析和质谱鉴定。结果显示,将发芽后的孢子壳以沸水浴法提取孢壁蛋白最为有效,但操作复杂,部分孢壁蛋白损失严重。初步鉴定从发芽后的孢子壳中提取的编号为P35、P32和P29的3种蛋白属于柞蚕微孢子虫孢壁蛋白,蛋白分子质量分别为35、32、29 kD,其中孢壁蛋白P32的含量最丰富,P35和P29的含量相对较低。根据相应肽指纹图谱预测孢壁蛋白P32的一级结构类似核孔蛋白,可能与孢壁的选择透过性有关,而P35与VASA2n蛋白的一级结构类似,因此有可能与柞蚕微孢子虫的繁殖有关。     

山羊黄体孕酮分泌的特点

在山羊发情周期的第１０ｄ和第１７ｄ，分别给２只和１只山羊安置颈静脉导管和后腔静脉导管，以２０ｍｉｎ间隔连续采样２４ｈ。然后，将１只黄体中期羊卵巢摘除；给另１只黄体中期羊经静脉导管注射１５－甲基ＰＧＦ２ａ，每小时注射１次，每次２４０μｇ，连续７次。３只羊都继续采样。每次都同时采取颈静脉和后腔静脉血样，用ＲＩＡ法测定血浆孕酮（Ｐ４）水平。结果表明，山羊血浆Ｐ４水平没有昼夜节律；后腔静脉血浆Ｐ４水平有明显的波动，并高于颈静脉血浆Ｐ４水平（Ｐ＜０．０ｌ）；黄体溶解和卵巢摘除后，颈静脉和后腔静脉血浆的Ｐ４水平先后降到１ｎｇ／ｍＬ以下。由此证明，山羊Ｐ４是由黄体波动性地分泌的。     

Recombinant Equine Luteinizing Hormone Stimulates Production of Progesterone from Murine Leydig, Ovine Small Luteal, and Equine Granulosal Cells

Recombinant equine luteinizing hormone (reLH) was evaluated for its ability to stimulate production of progesterone in cell lines from three species including murine Leydig tumor (MA-10), equine granulosal, and ovine small luteal cells (SLC). The response to reLH was compared with that obtained with human chorionic gonadotropin (hCG), equine chorionic gonadotropin, ovine luteinizing hormone, and equine luteinizing hormone (eLH). Media were collected hourly for 6 hours and assayed for progesterone content through radioimmunoassay. In MA-10 cells, production of progesterone was stimulated above baseline by reLH and hCG (P < .05). Ovine SLC responded to treatment with eLH, reLH, ovine luteinizing hormone, and hCG by increasing production of progesterone above that stimulated by vehicle control (P < .05). Production of progesterone in equine granulosal cells was maximally stimulated by treatment with hCG (P < .05), followed by reLH and eLH (P < .05). In conclusion, reLH elicited a progesterone response in MA-10, ovine SLC, and equine granulosal cells. Thus, reLH stimulates the production of progesterone in cell lines from three species.     

马传染性贫血病病毒核心蛋白基因的克隆和表达

根据美国马传染性贫血病病毒（EIAV）Wyoming株基因序列,设计扩增EIAVgag和p26基因的引物,用PCR法能忠实的分别扩增出全长gag和p26基因。经用杆状病毒表达系统对所获gag和p26基因进行克隆和重组,获得了高效稳定表达Gag和p26蛋白的重组杆状病毒（rBVs）。含有gag和p26基因的重组杆状病毒感染昆虫Sf21细胞,经无血清昆虫细胞培养基增殖和纯化,每升培养物能获得2mgCag或12mgp26蛋白。     

编码EIAV核输出因子蛋白Rev的cDNA克隆和序列测定

ＥＩＡＶ在繁殖过程中涉及到多种转录因子的调节,其中Ｒｅｖ蛋白是病毒编码的转录后调节因子,决定了病毒复制由早期到晚期的过渡。Ｒｅｖ是非结构蛋白,由病毒基因组全长转录产物经多次拼接而成。本研究使用ＥＩＡＶ疫苗毒感染驴巨噬细胞培养物,在病毒繁殖早期提取细胞总ＲＮＡ,反转录后使用中国ＥＩＡＶ毒株特异引物扩增病毒基因组拼接产物。将扩增产物克隆后经过核苷酸序列分析,和与基因组全序列的比较,确定了编码Ｒｅｖ蛋白的病毒基因组转录产物的编码序列和拼接位点。     

野马鼻肺炎病毒的分离鉴定

从新疆昌吉州吉木萨尔野马饲料养繁殖中心送检的野马病料中分离到１株病毒,我们对其进行了鉴定。该病毒株在ＢＨＫ－２１细胞上连传６代出现典型的细胞病变,用适应ＢＨＫ－２１细胞的病毒接种鸡胚成纤维细胞,乳豚鼠肾原代细胞,豚鼠睾丸细胞均出现程度不同的细胞病变。     

Identification and Characterization of Pig Embryo MicroRNAs by Solexa Sequencing

MicroRNAs (miRNAs) are a class of small, non‐coding RNAs of approximately 22 nucleotides in length that regulate gene expression by binding to the 3′‐untranslated regions of target mRNAs. It is now clear that miRNAs are involved in many biological processes, including proliferation, differentiation and regulation of gene expression during early embryonic development. The miRBase 16.0 (2010) shows that there are 175, 673, 408 and 1048 annotated miRNAs for Caenorhabditis elegans, Mus musculus, Rattus norvegicus and Homo sapiens, respectively. However, there are only 211 miRNAs described for Sus scrofa. In particular, the full set of miRNAs and their expression patterns are still poorly understood in the embryo. Therefore, we combined Solexa sequencing with computational techniques to analyse the sequences and relative expression levels of S. scrofa miRNAs at embryonic day 33 (E33). Of the distinct miRNAs identified, 76 previously known miRNAs and 194 candidate miRNAs were identified in head, and 77 known miRNAs and 130 predicted candidate miRNAs were identified in organ region. Furthermore, we performed additional investigation for identifying the potential target mRNAs using PicTar and TargetScan. Concurrent function analysis suggested that highly expressed miRNAs are mostly involved in the development of nerves, cerebrum, muscle and organs. Our results provide useful information for the investigation into embryonic miRNAs of pig and provide a valuable resource for investigators interested in the regulation of embryonic development in pigs and other animals.     

编码EIAV核输出因子蛋白Rev的cDNA克隆和序列测定

EIAV在繁殖过程中涉及到多种转录因子的调节，其中Rev蛋白是病毒编码的转录后调节因子，决定了病毒复制由早期到晚期的过渡，Rev是非结构蛋白，由病毒基因组全长转录产物经多次拼接而成。本研究使用EIAV疫苗毒感染驴巨噬细胞培养物，在病毒增殖早期提取总RNA，反转录后使用EIAV特异引物扩增病毒基因组拼接产物，将扩增产物克隆后经过核苷酸序列分析，通过与基因组全序列的比较，确定了编码Rev蛋白的病毒基因组转录产物的编码序列和拼接位点。     

编码EIAV反式激活蛋白Tat的cDNA克隆和序列测定

ＥＩＡＶ在繁殖过程中的转录涉及到多种因子的调节,其中ＴＡＴ蛋白是病毒编码的反式激活因子,是病毒复制必须成分。ＴＡＴ是非结构蛋白,由病毒基因组全长转录产物经多次拼接而成。本研究使用ＥＩＡＶ疫苗毒感染驴巨噬细胞培养物,在病毒增殖早期提取细胞总ＲＮＡ,反转录后使用中国ＥＩＡＶ毒株特异引物扩增病毒基因组拼接产物。将扩增产物克隆后经过核苷酸序列分析,和与基因组全序列的比较。确定了编码ＥＩＡＶ反式激活蛋白的转录产物及阅读框架及转录后拼接位点,研究发现至少有两种拼接产物编码ＴＡＴ。     

菲菜氏温扬球虫18S rDNA基因的扩增与克隆分析

菲莱氏温扬球虫是鸭球虫病的重要病原之一,为了寻找种特异性的遗传标记,本研究采集广东省某鸭场的新鲜鸭粪,通过Sheather's蔗糖漂浮法收集球虫卵囊,经形态学鉴定为菲莱氏温扬球虫;提取该球虫DNA样品,经过PCR扩增,首次获得了该虫的18S rDNA基因部分片段;对该基因进行了克隆和测序,比较了该虫株与其他原虫的亲缘关系.结果显示,对菲莱氏温扬球虫序列与艾美耳科其他球虫关系较近,系统进化树分析属于艾美耳科的另一分支.表明18S rDNA基因在鸭菲莱氏温扬球虫的分类鉴定上是一种有效的分子标记.     

瘦素和FSH对绵羊卵泡颗粒细胞孕激素分泌的影响

试验旨在探明瘦素和卵泡刺激素(FSH)对绵羊卵泡颗粒细胞孕激素分泌的影响并建立瘦素和FSH之间的相互作用.从屠宰场收集健康母羊的卵巢,机械分离卵泡,收集卵泡颗粒细胞.在细胞培养液中加入不同浓度的FSH(0 U/mL,2.5 U/mL,5 U/mL,7.5 U/mL,10 U/mL)培养24 h和48 h,通过MTT检测...     

Physiological Concentrations of Acute-Phase Proteins and Immunoglobulins in Equine Synovial Fluid

Synovial fluid (SF) is capable of reflecting infectious, immunological, or inflammatory joint conditions in horses by altering its composition and appearance. Although plasma and SF compositions are quantitatively different, this latter compartment reflects changes in plasma macromolecules. Therefore, changes in serum immunoglobulin protein concentrations tend also to alter intracapsular levels. Therefore, it is necessary to know the physiological concentrations of proteins present in SF. The aim of this study was to determine the levels of total protein, albumin, transferrin, haptoglobin, α1-acid glycoprotein, ceruloplasmin, and immunoglobulins A and G in SF of six healthy horses. The synovial proteinogram was obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The SF proteins reached a maximum of 25% of serum concentrations, varying inversely with molecular weight of the protein, except for the ceruloplasmin.     

驴源马疱疹病毒8型的分离鉴定与生物信息学分析

本研究旨在分离1株驴源马疱疹病毒8型（Equine herpesvirus type 8,EHV-8）毒株并分析其遗传特征。从山东省聊城地区某驴场采集病驴肺脏组织,经PCR方法鉴定EHV-8的感染情况;对EHV-8感染阳性的肺组织进行研磨,经反复冻融后接种于兔肾细胞（RK-13）细胞中,盲传3代,待出现细胞病变（CPE）时收集细胞,并通过PCR、间接免疫荧光试验、透射电镜等技术对EHV-8进行鉴定。利用PCR扩增获得分离株的ORF70全基因组序列,并进行生物信息学分析。研究结果显示,经PCR鉴定获得EHV-8感染阳性的肺脏组织,病料接种易感细胞RK-13后出现典型CPE,分别收集前3代的细胞培养物,经PCR扩增,均获得与预期大小一致的ORF70基因片段,将分离获得的EHV-8毒株命名为SDLC66,序列上传GenBank,获得登录号：MW816102。经测序和序列比对发现,SDLC66毒株与AHV-3毒株（GenBank登录号：U24184.1）ORF70基因的相似性为99%,与国内的EHV-8 wh毒株（GenBank登录号：JQ343919.1）、国外EHV-8/IR/2015/40（GenBank登录号：MF431614.1）、EHV-8/IR/2003/19（GenBank登录号：MF431611.1）参考毒株的相似性最高,均为99.8%。经遗传进化树分析发现,SDLC66与EHV-8（EHV-8 wh、EHV-8/IR/2015/40和EHV-8/IR/2003/19株）在一个小分支上,亲缘关系最近;与EHV-1型参考毒株（Hong Kong/57/1984、United Kingdom/32/1982、Oxfordshire/206/2013株）序列来源于同一大分支,亲缘关系较近,与EHV-4毒株（91c1和TH20p株）亲缘关系较远。间接免疫荧光试验可见与病毒蛋白特异结合的红色荧光信号;透射电镜观察可见直径约110 nm的圆形病毒粒子,并且核衣壳外有一层亮晕,亮晕外有一层囊膜。说明本试验成功分离得到1株驴源EHV-8毒株,为进一步研究其致病性和致病机制奠定基础。     

Seasonal Changes in Luteal Progesterone Concentration and mRNA Expressions of Progesterone Synthesis-related Proteins in the Corpus Luteum of Mares

Although circulating progesterone (P(4)) levels tend to change with the season, little is known about the seasonal changes of P(4) synthesis-related proteins in the corpus luteum (CL) of mares. To examine these changes, seventy-four ovaries containing a CL were collected from Anglo-Norman mares at a local abattoir in Kumamoto, Japan (~N32°), five times during one year. The stages of the CLs were classified as early, mid and regressed by macroscopic observation of the CL and follicles. The mid CL, which had the highest P(4) concentration, was used to evaluate the seasonal changes in P(4) synthesis. The luteal P(4) concentration and mRNA expression of luteinizing hormone receptor (LHCGR) were lowest during early winter and highest during late winter. The mRNA expressions of steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) were lowest during early winter and increased during late winter. These results suggest that P(4) synthesis in the CL is affected by the seasonal changes in the mRNA expressions of P(4) synthesis-related proteins in mares.     

羊驼酪氨酸酶基因部分cDNA序列的克隆及序列分析

从3～4龄青年公羊驼背部皮肤中提取总RNA作为模板,采用RT-PCR技术扩增出TYR基因的部分Cdna序列,将其克隆进Pgem-T easy vector中,经限制酶酶切鉴定后进行测序,将测序结果与小鼠、人、猩猩等其它物种的TYR核苷酸序列进行比对,发现本研究获得部分序列为羊驼酪氨酸酶基因第三外显子,该序列比较保守.     

柞蚕微孢子虫部分孢壁蛋白的分离鉴定及孢壁蛋白8的序列分析

研究柞蚕微孢子虫的孢壁蛋白组成和结构特点,有助于解明柞蚕微孢子虫在侵染过程中与蚕体细胞的互作机制。分别采用煮沸法和Laemmli法分离提取柞蚕微孢子虫总蛋白和孢壁蛋白,回收30kD左右的蛋白条带进行液质联用离子阱电喷雾质谱分析,获得的短肽序列经Mascot在线检索工具进行蛋白同源序列比对,共鉴定出27种具有功能注释的蛋白,有3种注释为孢壁蛋白。其中获得了与家蚕微孢子虫孢壁蛋白8(NbSWP8)高度同源的柞蚕微孢子虫孢壁蛋白8(NaSWP8)的基因全长序列。序列分析表明NaSWP8与NbSWP8的氨基酸序列相似性达到90%,且均具有特征性的肝素结合基序,二者的编码基因核苷酸序列的非同义替换率和同义替换率比值(dN/dS)明显小于1,表明孢壁蛋白8基因在2种蚕类微孢子虫中受到纯化选择压力的作用,基因的功能相对稳定。     

米非司酮对早孕猕猴孕酮和雌二醇分泌的研究

为开展生殖生物学研究工作提供理想的动物模型。进行了本项试验,分析了米非司酮(ＲＵ４８６)对猕猴妊娠早期血清中孕酮(Ｐ)和雌二醇(Ｅ２)的影响。结果表明,注射ＲＵ４８６－段时间内孕酮和雌二醇基本不交,但Ｅ２/Ｐ比值有所增加,说明米非司酮干扰内分泌激素之间的平衡,随着出血的发生,孕酮、雌二醇均显著下降。这种降低与出血的发生有着明确的先后关系,从而证实了激素的变化源于蜕膜受损。     

深度测序鉴定绵羊microRNA转录组

本研究采集的Texel绵羊胎儿的背最长肌样品包括5个发育阶段:70、85、100、120和135 d。通过采用Solexa测序技术对混池样品进行了microRNA(miRNA)深度测序,获得了16532850条原始序列读数。使用ACGT101-miR v4.2分析软件对miRNA测序数据进行了深度发掘;通过与最新的哺乳动物成熟miRNA序列(miRNA数据库:miRBase v17.0)、miRNA前体序列、绵羊基因组序列(绵羊基因组数据库,2010年2月)的比对分析,对绵羊microRNA转录组数据库进行了大幅更新,pre-miRNA(miRNA前体)序列增至1529条,编码的miRNA成熟体序列增至1999条。通过实时荧光定量PCR技术对深度测序获得的5个miRNA在混池样品中进行了验证。绵羊miRNA的研究起步较晚,而miRNA的发现与鉴定将促进基因调控机制及miRNA功能的研究。     

Association of Factor V Secretion with Protein Kinase B Signaling in Platelets from Horses with Atypical Equine Thrombasthenia

Background

Two congenital bleeding diatheses have been identified in Thoroughbred horses: Glanzmann thrombasthenia (GT) and a second, novel diathesis associated with abnormal platelet function in response to collagen and thrombin stimulation.

Hypothesis/Objectives

Platelet dysfunction in horses with this second thrombasthenia results from a secretory defect.

Animals

Two affected and 6 clinically normal horses.

Methods

Ex vivo study. Washed platelets were examined for (1) expression of the αIIb‐β3 integrin; (2) fibrinogen binding capacity in response to ADP and thrombin; (3) secretion of dense and α‐granules; (4) activation of the mammalian target of rapamycin (mTOR)‐protein kinase B (AKT) signaling pathway; and (5) cellular distribution of phosphatidylinositol‐4‐phosphate‐3‐kinase, class 2B (PIK3C2B) and SH2 containing inositol‐5′‐phosphatase 1 (SHIP1).

Results

Platelets from affected horses expressed normal amounts of αIIb‐β3 integrin and bound fibrinogen normally in response to ADP, but bound 80% less fibrinogen in response to thrombin. α‐granules only released 50% as much Factor V as control platelets, but dense granules released their contents normally. Protein kinase B (AKT) phosphorylation was reduced after thrombin activation, but mTOR Complex 2 (mTORC2) and phosphoinositide‐dependent kinase 1 (PDK1) signaling were normal. SH2‐containing inositol‐5''‐phosphatase 1 (SHIP1) did not localize to the cytoskeleton of affected platelets and was decreased overall consistent with reduced AKT phosphorylation.

Conclusions and clinical significance

Defects in fibrinogen binding, granule secretion, and signal transduction are unique to this thrombasthenia, which we designate as atypical equine thrombasthenia.