猪圆环病毒2型－细小病毒－伪狂犬重组病毒免疫小鼠试验研究

为了研究表达猪2型圆环病毒ORF2基因和猪细小病毒VP2基因的重组伪狂犬病毒疫苗株SA215(D)的疫苗价值,对该病毒株安全性、免疫原性、保护力及对PRV Fa株定值进行了研究。研究结果表明重组病毒SA215（D）毒力显著低于PRV Fa强毒株,可以抵御伪狂犬病强毒Fa株的攻击,并能抵御其在小鼠体内的定植;抗体检测显示SA215（D）免疫小鼠后第2周抗PRV、PCV2和PPV的抗体水平均很低,加强免疫后第4周抗体水平大幅上升,且显著高于阴性对照组,而与阳性对照组无明显差异,表明SA215（D）可诱导小鼠产生体液免疫反应。淋巴细胞增殖试验显示SA215（D）可诱导小鼠产生较强的淋巴细胞增殖反应,与亲本株SA215组相比,差异显著(0.01     

Isolation and identification of canine parvovirus serotype 2a and its VP2 protein expression in transgenic tobacco

A strain of canine parvovirus (CPV) was isolated from feces of an ill puppy in an animal hospital in Wuhan, China. It was designated as CPV/WH02/06. This isolate was identified as serotype CPV-2a by the hemagglutination test, CPV Ag detection strip, electron microscopy, and PCR. The vp2 gene was cloned and sequenced and assigned GenBank accession number EU377537. A 1242 bp segment of the 5' region of the vp2 gene was cloned and inserted into the binary vector pBI121 and used for Agrobacterium-mediated tobacco transformation. Transgenic tobacco plants were selected on MS medium supplemented with 100 μg/mL kanamycin and 100 μg/mL timentin. Integration of the vp2 gene into the tobacco genome was confirmed by PCR using T1 progeny plants, and the expression of the VP2 protein was confirmed by Western blotting.     

检测PCV2、PPV、PRV疫苗株与野毒株的多重PCR方法

本文建立了一种同时检测猪圆环病毒2型(PCV2)、细小病毒(PPV)、及伪狂犬病毒(PRV)疫苗株与野毒株的多重PCR方法.根据GenBank上发表的PCV2、PPV和PRV gB、gE基因序列,针对各自保守区各设计一对特异性引物,用这四对引物对同一样品中的PCV2、PPV和PRV gB、gE进行检测,结果可同时扩增出269bp(PCV2)、581bp(PPV)、372bP(PRV gB)及147bp(PRV gE)四条特异性片段.对JEV、PRRSRV、大肠杆菌和双蒸水的PCR扩增结果均为阴性;敏感性测定结果表明,该多重PCR能检出10pg PCV2、PPV和PRV gB、gE检测敏感度分别为10-6.2、10-3.8、10-5.8TCID50的模板.该方法的建立对临床上进行这三种疾病的鉴别诊断和混合感染的检测具有重要意义.     

I型鸭肝炎病毒VP1、3D基因克隆及其在大肠杆菌中的表达

根据GenBank中的I型鸭肝炎病毒全基因序列设计了扩增I型鸭肝炎病毒VP1、3D基因的引物, 用该特异性表达引物从I型鸭肝炎病毒cDNA模板中扩增得到目的基因VP1、3D, 用相同的限制性内切酶酶切目的基因和表达载体pET32a后构建重组表达载体, 转化宿主BL21(DE3), 用不同浓度的IPTG诱导VP1、3D基因的表达, 收集菌液进行SDS-PAGE电泳, Western-blotting分析蛋白免疫原性。结果表明, VP1、3D在大肠杆菌中表达量较高, 表达产物的分子量约为48 kD、68 kD, 并能被兔抗DHV-1血清所识别。I型鸭肝炎病毒VP1、3D蛋白在大肠杆菌中表达产物具有免疫原性。     

A preliminary analysis of antineoplastic activity of parvovirus MVMp NS—1 proteins

Human gastric cancer MKN-45 cells were transfected with pULB 3238,a plasmid carrying MVMp MS-1 gene with its original P4 promoter replaced by the glucocorticoid inducible promoter MMTV-LTR.After the integration and expression of NS-1 gene,some of the transfectants died,while others remained alive,but the growth features of survived cells were changed.For further study on the antineoplastic function of parvoviral NS-1 protein in vivo,transgenic mice carrying NS-1 genes were established by conventional method.Among 4 founders,one of them was found to be able to transmit the transgene to around 50% of their offsprings.RT-PCR was performed to indicate the expression of NS-1 gene in transgenic mice and its mRNA appeared in a variety of tissues.The expression of integrated NS-1 gene may correlate with the decreased incidence of tumor induced in vivo by chemical carcinogens.     

Ultrastructure des cellules de Leydig et des cellules de Sertoli au cours du cycle testiculaire du Canard Pékin

Résumé L'ultrastructure des cellules de Leydig et des cellules de Sertoli du testicule du Canard Pékin a été étudiée au cours de la phase printanière du cycle sexuel, soit de janvier à juillet. Parallèlement on a effectué chez les mêemes animaux la recherche histochimique de la ⁵-3 -hydroxystéroïdedeshydrogénase (⁵-3 -HSDH) ainsi que le dosage, par chromatographie en phase gazeuse des stéroïdes androgènes dans le plasma veineux périphérique et dans le testicule.Les cellules de Leydig du Canard possèdent les organites cytoplasmiques spécifiques des cellules stéroïdogènes (reticulum lisse, mitochondries à crêtes tubulaires) ainsi que d'autres structures souvent rencontrées dans ce type cellulaire (microfilaments, vacuoles, granules denses). Les cellules de Sertoli contiennent un reticulum agranulaire moins développé que celui des cellules de Leydig et, très rarement, des mitochondries à crêtes tubulaires. Ces divers organites cytoplasmiques subissent un cycle saisonnier. La différenciation du reticulum lisse et des crêtes mitochondriales tubulaires commence en janvier et atteint son optimum en mars. Leur régression s'amorce en avril; d'abord accompagnée de structures dégénératives transitoires; elle conduit à la dispartion totale de ces organites en mait. Aucun indice de nécrose n'est observé dans ces cellules. Histochimiquement, une activité ⁵-3 -HSDH est présente dans les cellules de Leydig et, à un degré moindre, dans les tubes séminifères. Son intensité varie au cours du cycle.La confrontation de l'étude morphologique avec les résultats des dosages hormonaux montre qu'il existe une bonne corrélation entre le développement puis la régression du reticulum lisse et des crêtes tubulaires des mitochondries ainsi que des critères histochimiques de la ⁵-3 -HSDH d'une part et l'évolution de la testostérone plasmatique et testiculaire d'autre part. De plus on observe une augmentation du rapport testostérone/⁴-androstènedione testiculaire parallèlement au développement des organites cytoplasmiques. Ces organites semblent donc bien impliqués dans la synthèse et la sécrétion de la testostérone chez le Canard.

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Ultrastructure of Leydig and Sertoli cells in the testicular cycle of the Pekin duckBiochemical and cytoenzymological correlations

Summary Leydig and Sertoli cells of the testis of the Pekin duck were studied ultrastructurally during the spring phase of the sexual cycle, from January to July. Simultaneously, in the same animals, ⁵-3 -hydroxysteroiddehydrogenase (⁵-3 -HSDH) activity was ascertained histochemically and androgenic steroids of the plasma and testes were assayed by gas-liquid chromatography.The Leydig cells of the duck possess cytoplasmic organelles specific to steroidogenic cells (smooth reticulum, tubular mitochondria) as well as other structures often found in this cell type (microfilaments, vacuoles, denses bodies). The Sertoli cells contain an agranular reticulum that is less developed than that of the Leydig cells, and rarely show mitochondria with tubular cristae. These various cytoplasmic organelles undergo a seasonal cycle. The differentiation of the smooth reticulum and the mitochondrial tubular cristae begins in January and reaches a maximum in March. They begin to regress in April, at first with transitory degenerative structures, and then by total disappearance of these organelles by May. No indication of necrosis is observed in the cells. Histochemically ⁵-3 -HSDH activity is present in the Leydig cells, and to a slightly lesser degree in the seminiferous tubules. The intensity varies during the cycle.The comparison of the results of the morphological study with the hormone assays shows that a good correlation exists with the development and regression of the smooth endoplasmic reticulum and tubular cristae in the mitochondria, as well as the histochemical criteria of the ⁵-3 -HSDH on one hand, and the levels of plasma and testicular testosterone on the other hand. In addition there is an increase in the ratio of testicular testosterone to ⁴-androstenedione which parallels the development of the cytoplasmic organelles. These organelles thus seem to be implicated in the synthesis and secretion of testosterone in the duck.

Nous tenons à remercier très vivement Mme G. Collenot qui nous a initiées aux techniques d'histoenzymologie et nous a très gentiment permis de faire cette partie de nos recherches dans son laboratoire. Nous remerçions également M. Claude Pennarun, photographe, pour son excellente collaboration.     

异羟基洋地黄毒甙元(Digoxigenin)标记的探针在乙型肝炎病毒核酸杂交中的应用

应用异羟基洋地黄毒甙元标记的探针,检测了人和鸭的血清及肝脏中的乙型肝炎病毒核酸,并与~(32)P标记的同位素探针做了比较。结果证明,该探针的特异性和敏感性与同位素探针一致(0.2pg)。它可用于各种核酸杂交试验,如打点杂交、Southern和Northern转印杂交试验等。恰当地从标本中提取待测核酸,是应用该探针的重要条件。     

贵州省石阡鸳鸯湖国家湿地公园越冬鸳鸯数量、分布及日行为初探

为了解贵州省石阡鸳鸯湖国家湿地公园越冬鸳鸯Aix galericulata的种群密度、日行为特征及人为干扰对其的影响,于2017年10月—2018年3月,采用直接计数法对鸳鸯数量、性比及人为干扰下的种群密度进行了22次调查。越冬中期,种群数量稳定在755只±17只,性比为1.14∶1,各条河沟的数量之间的差异具有高度统计学意义(X^2=84.041,df=5,P<0.01),即大河、小河与坝坎为鸳鸯分布及活动的主要区域;种群密度与人为干扰次数呈极显著负相关(R=-0.799,n=90,P<0.01),在人为干扰次数为1次±1次的区域,种群密度为42只/hm^2±3只/hm^2～53只/hm^2±14只/hm^2,当人为干扰次数达到7次±3次时,几乎没有鸳鸯活动。同时采用瞬时扫描法对鸳鸯的8种越冬行为进行了34 d(442 h)的观察,共记录各行为136 933次。取食、休息和修整为鸳鸯的主要行为;除了修整,鸳鸯在保育区与非保育区的各行为时间分配差异有高度统计学意义(P<0.01);越冬日行为节律极具规律性,取食高峰在07∶00—08∶00、14∶00—15∶00和17∶00—18∶00;休息高峰在13∶00—14∶00和15∶00—16∶00;飞行高峰在06∶00—07∶00(飞回湖区)和18∶00—19∶00(飞往夜栖地);修整在各时间段发生均较为频繁。     

Evidence for activation of Amh gene expression by steroidogenic factor 1

The anti-Müllerian hormone gene (Amh) is responsible for regression in males of the Müllerian ducts. The molecular mechanism of regulation of chicken Amh expression is poorly understood. To investigate the regulation of chicken Amh expression, we have cloned Amh cDNAs from quail and duck as well as the promoter regions of the gene from chicken, quail, and duck. The expression patterns of Amh during embryonic development in these three species were found to be similar, suggesting that the regulatory mechanisms of Amh expression are conserved. The sequence of the proximal promoter of Amh contains a putative binding site for steroidogenic factor 1 (SF1), the protein product of which can up-regulate Amh in mammals. We showed here that SF1 is able to activate the chicken Amh promoter and binds to its putative SF1 binding site. These results suggest that SF1 plays a role in regulation of Amh expression in avian species.     

北京鸭线粒体基因组全序列测定和分析

线粒体DNA作为遗传标记,已在家鸡(Gallus gallus)和家鹅(Anser anser)的研究中取得了重大进展,而对家鸭(Anas platyrhychos domesticus)的研究却很少.本研究参照近源物种线粒体基因组序列设计15对引物,通过PCR扩增、测序、拼接,获得北京鸭(A.platyrhychos)线粒体基因组全序列,初步分析其特点和各基因的定位.结果显示,北京鸭线粒体基因组全长16 604 bp,碱基组成为29.19%A、22.20%T、15.80%G、32.81%C,包含13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个非编码控制区(D-loop),基因组成及排列顺序与其他鸟类相似.基于线粒体D-loop区全序列,用N-J法构建了7种雁形目鸟类系统进化树,结果表明,北京鸭与绿头鸭(A.platyrhychos)系统进化关系较近.