牛源犬新孢子虫NcSRS2-NcGRA7融合基因的克隆与生物信息学分析

为了解牛源犬新孢子虫NcSRS2-NcGRA7融合基因的生物学特性,本试验提取牛源犬新孢子虫基因组DNA,应用PCR技术扩增犬新孢子虫表面蛋白基因NcSRS2和致密颗粒蛋白基因NcGRA7,SOE-PCR技术拼接NcSRS2和NcGRA7基因,构建NcSRS2-NcGRA7融合基因重组克隆质粒,并进行PCR鉴定、双酶切鉴定及生物信息学分析。结果,NcSRS2基因扩增片段大小为1 061 bp,NcGRA7基因扩增片段大小为364 bp,NcSRS2-NcGRA7融合基因扩增片段大小为1 482 bp;获得重组克隆质粒pMD18-NcSRS2-NcGRA7经PCR鉴定、双酶切鉴定正确;测序分析表明,与Gen-Bank中已发表的美国株犬新孢子虫(AF061249、AF176649)核苷酸序列同源性为99%;经DNAman等软件分析,预测NcSRS2-NcGRA7融合蛋白抗原指数较高,融合蛋白二级结构以α-螺旋和β-折叠为主,三级结构中2种蛋白独立折叠,并借助Linker互相连接,功能互不影响。本试验为牛源犬新孢子虫NcSRS2-NcGRA7融合蛋白的免疫学研究奠定了基础。     

校企合作共建高职畜牧专业教学资源库的研究与实践

专业教学资源库建设是促进教学改革、提高教学质量的重要切入点,是借助网络信息技术平台,实现优质教学资源共享,为教师教学、学生和社会人员自主学习提供系统资源的综合工程.针对校企合作教学资源库建设研究与实践,探讨了高职畜牧专业教学资源库建设的背景与定位、建设目标与思路、主要建设内容以及问题与思考.     

Tree Planting in an Urbanizing South Africa / BOOMAANPLANTING IN VERSTEDELIKENDE SUID-AFRIKA

The title of this paper might well read “Tree planting in an urbanizing and industrializing South Africa”, because in modern South Africa industry is prominent in the urban scene. The importance of trees in the urban landscape is stressed. Purposes for which trees are used in commercial, industrial and residential areas are listed. An account is given of tree planting in boulevards and avenues, state and civic centres, car parks, bus termini, recreational parks, botanical gardens and arboreta, zoological gardens, sports enclosures and children's playgrounds. The role of trees in industrial areas is discussed and the vegetating of mine dumps described. Passing reference is made to the uses of trees on the sea front. Tree planting for economic purposes by civic authorities is briefly touched upon. The value of trees in private gardens is underlined.     

影响两系杂交粳稻制种纯度的主要原因及其控制对策

两系杂交粳稻制种中,光温敏核不育系育性转换起点温度偏高、自交结实、制种基地选择和季节安排不妥以及栽培管理措施不当是影响制种纯度的主要原因。因此,选育育性转换起点温度低的光温敏核不育系,通过技术手段强化除杂,根据不育系育性转换温光特性选择合适的制种基地并合理安排制种季节,改进栽培管理措施和严格制种的田间纯度检测,是控制两系杂交粳稻制种纯度的重要对策。     

Spontaneous Malignant T Cell Lymphoma in a Young Male Common Marmoset (Callithrix jacchus)

We histopathologically and immunohistochemically investigated a case of malignant lymphoma that spontaneously developed in a male common marmoset at two years of age. Beginning at two years four months of age, the animal had an enlargement of the submandibular and inguinal lymph nodes, small subcutaneous nodules near the right breast and an approximately fivefold increase in peripheral lymphocyte count compared with the previous examination value. The postmortem findings at two years eight months of age showed lymphadenopathy with enlargement of the thymus and spleen. Small- to intermediate-sized neoplastic lymphocytes had diffusely proliferated in the enlarged nodes. The neoplastic cells were pleomorphic and had irregularly shaped nuclei. The nuclear chromatin staining revealed hyperchromatism in the small-sized cells, and the intermediate-sized cells exhibited vesicular staining. An immunohistochemical examination indicated that the neoplastic lymphocytes were positive for CD3 and negative for CD20, thus suggesting that they had originated from T cells. In addition, the proliferation of high endothelial venules and reactive epithelioid histiocytes was observed. Scattered tingible body-laden macrophages were infrequently detected. Neoplastic lymphocytes were also observed in the thymus, spleen, heart, lungs, liver, kidneys, adrenal glands and femoral and sternal bone marrow. This malignant lymphoma in a young male common marmoset was considered to fit the category of “peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS)” according to the new WHO system of classification.     

柔嫩艾美耳球虫钙依赖蛋白激酶3在293T细胞中的表达研究

为构建柔嫩艾美耳球虫钙依赖蛋白激酶3（Eimeda tenella calcium-dependent protein kinases3,EtCDPK3）基因的重组质粒pCAGGS-EtCDPK3,并转染293T细胞进行表达,以柔嫩艾美耳球虫孢子化卵囊cDNA为模板,经PCR扩增其含完整开放阅读框的序列,将其克隆至pGEM．Teasy载体,构建pGEM—Teasy-EtCDPK3重组质粒,双酶切回收目的片段后与相应酶切的真核表达载体pCAGGS连接,构建真核重组表达质粒pCAGGS-EtCDPK3。该重组质粒经酶切和测序鉴定正确后转染293T细胞进行表达,分别用免疫印迹和间接免疫荧光鉴定EtCDPK3基因的表达情况。结果显示所构建的真核重组表达质粒pCAGGS—EtCDPK3经过双酶切鉴定,可见1条大小约为1302by的目的条带,测序结果与实验室已获得的EtCDPK3序列完全一致;Western blot结果可见大小约为49kDa的目的蛋白条带,间接免疫荧光实验可以检测到红色荧光。这些研究结果表明已成功构建了EtCDPK3的真核重组表达质粒pCAGGS-EtCDPK3,并在真核细胞中获得表达,为深入研究EtCDPK3的功能和制备DNA疫苗奠定了基础。     

苹果褪绿叶斑病毒的分子杂交检测

[目的]探索检测苹果褪绿叶斑病毒的技术体系。[方法]利用CTAB法提取感病苹果叶片总RNA,以总RNA为模板,利用特异性引物,建立利用cDNA探针检测苹果褪绿叶斑病毒的技术体系。[结果]扩增出了预期片段,经克隆、测序证明该片段为苹果褪绿叶斑病毒的特异性片段。以带有特异片段的质粒为模板,利用PER技术制备出地高辛标记的cDNA探针。[结论]以质粒为模板可高效制备大量探针。地高辛标记探针为非放射性标记探针,安全性较好,灵敏度较高,具有重要的应用价值。     

Lipocalin核糖体展示文库的构建及雌二醇特异性模拟抗体的筛选

[目的]构建Lipocalin核糖体展示文库,并筛选雌二醇特异性模拟抗体。[方法]以Lipocalin蛋白家族中的BBP(胆汁三烯结合蛋白)为模板,设计包含16个随机突变位点的引物,合成BBP基因文库;引入核糖体展示所必需的全部组件,采用重叠PCR技术将BBP库与核糖体展示组件进行拼接,构建核糖体展示Anticalin模拟抗体库;再将构建的Anticalin文库进行体外转录与翻译,产生模拟抗体-核糖体-mRNA三联复合体文库。以小分子抗原雌二醇为靶标,采用固相亲和方法筛选抗雌二醇抗体,通过改变Mg2+浓度将模拟抗体-核糖体-mRNA三联复合体解离,得到与模拟抗体对应的mRNA,经过RT-PCR得到筛选后的DNA文库并重新引入核糖体展示元件进行下一轮淘筛。[结果]通过5轮生物淘筛,模拟抗体得到富集,获得高特异性抗雌二醇抗体。其中有一株克隆的亲和力为54 mmol/L(Kon=4.975 6×104个/M·S;Koff=0.002 7个/S)。IC50与LOD值分别为50和0.071 ng/ml。[结论]获得的雌二醇模拟抗体可以作为抗体并用于检测动物体内雌二醇的残留。