In this study,we investigated the epidemic situation and drug resistance of cow mastitis pathogens. The following aspects were contained:isolation,identification and drug sensitive test of pathogenic bactcria of cow mastitis. The results of i-dentification test showed that the mainly pathogen were Staphylococci and Streptococci(87.1%),followed by all kinds of bacil-lus(12.9%). The drug sensitivity test showed that most pathogens were resistant to common mastitis drugs.

以武汉某公司的猪-蔬菜种养循环示范基地为研究对象,研究猪-蔬菜种养循环生态农业的生产应用.通过产品品质测定、土壤检测和水体检测,分析猪-蔬菜种养循环模式对提高蔬菜品质和产量、改善土壤质量、减少水体污染的作用.结果显示,该模式使蔬菜产量提高了30%,能显著改善土壤质量,基本没有废水排放到示范区以外,不构成水体污染.该生产模式绿色环保,具有良好的经济效益和社会效益,值得推广.

Objective] To determine the effects of fermentation conditions on the pig manure fermentation.[Method] We researched the effects of fermentation time, fermentation season and compound bacteria additive dosage on the fermentation effects of pig manure.Crop planting test was carried out.It was compared with the fertilizer and biogas slurry so as to verify the fertilizer efficiency of pig farm.[ Result] The best manure fer-mentation time was around 7 days, and the fertilizer elements reached the maximum value;fermentation effect in summer was better, ammonium nitrogen and nitrate content in manure increased sharply;adding bacteria additive into the manure could contribute to fermentation.manure in pig farm and fertilizer had equal fertilizer efficiency.[Conclusion] Fermentation time, season and compound bacteria additive can affect the fertilizer effect of pig manure fermentation, so we should attach importance to them in production.

In this study,the performance of three kinds of solid-liquid separation equipments such as screw extrusion solidliquid separator,roller extrusion filter solid-liquid separator and horizontal scroll decanter centrifuge for piggery wastes were analyzed.The evaluation index included the content of suspended particles,COD (chemical oxygen demand),total nitrogen and total phosphorus in the liquid after treatment and the operation costs.The results showed that the horizontal scroll decanter centrifuge had the lowest content of suspended particles,COD,total nitrogen and total phosphorus values;screw extrusion solid-liquid separator and roller extrusion filter solid-liquid separator had less energy consumption;horizontal scroll decanter centrifuge had lower additional investment.It suggested that the horizontal scroll decanter centrifuge is more suitable for preliminary treatment of piggery wastes.

Despite the powerful impact gene expression markers like the green fluorescent protein (GFP) or enhanced GFP (EGFP) exert on linking the expression of recombinant protein for selection of high producers in recent years, there is still a strong incentive to develop more economical and efficient methods for isolating mammalian cell clones secreting high levels of recombinant proteins. Here we present a new method based on the co-expression of EGFP that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the related protein are linked by an internal ribosome entry site and thus are transcribed into the same mRNA in an independent translation process. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein in each clone. By expressing recombinant porcine β-defensin 1 in Marc 145 cells, we demonstrate the robustness and performance of this technique. The method can be served as an alternative to identify high-producer clones with various cell sorting methods.

To study the eukaryotic expression of duck defense, duck defensins genes AvBD2, AvBD10 and AvBD12 were ar-tificia11y synthesized and inserted into the eukaryotic expression p1asmid pDoub1eEx-EGFP-f1ag-N to construct recombinant ex-pression p1asmids. After identification by enzyme digestion, recombinant expression p1asmids were transfected into HEK293 ce11s respective1y. And the expression of target genes were ana1yzed by using RT-PCR and Westem-b1ot. The resu1ts showed that target genes were inserted into the eukaryotic expression p1asmid successfu1; 24 hours after the transfection of recombi-nant expression p1asmids into HEK293 ce11s, the expression of green f1uorescent protein were visib1e under the f1uorescence microscope; RT-PCR cou1d detected the target genes' mRNA, but western b1ot did not detect the expression of target pro-teins. This study constructed eukaryotic expression vectors of three duck defenses, and try to express the defensins in HEK293 ce11s. It provided reference for further research of eukaryotic expression of duck defensins.

TAT穿膜肽是近年来发现的一类富含碱性氨基酸的短链多肽,具有较强的穿透细胞膜的能力,与核酸分子、药物蛋白分子甚至病毒体表面分子链接后,能将这些物质顺利带入细胞内发挥作用,被称为"生物导弹".笔者从4个方面对TAT穿膜肽进行介绍,不仅为深入阐述TAT蛋白转导肽跨膜转导作用的功能意义提供了依据,而且为TAT蛋白转导肽在微生物工程、蛋白质工程及药物靶向治疗等领域的潜在应用价值提供了重要参考.

Previous studies revealed that thymus is a targeted immune organ in malnutrition, and high-boron stress is harmful for immune organs. African ostrich is the living fossil of ancient birds and the food animals in modern life. There is no report about the effect of boron intake on thymus of ostrich. The purpose of present study was to evaluate the effect of excessive boron stress on ostrich thymus and the potential role of TLR3/4 signals in this process. Histological analysis demonstrated that long-term boron stress (640 mg/L for 90 days) did not disrupt ostrich thymic structure during postnatal development. However, the numbers of apoptotic cells showed an increased tendency, and the expression of autophagy and proliferation markers increased significantly in ostrich thymus after boron treatment. Next, we examined the expression of TLR3 and TLR4 with their downstream molecular in thymus under boron stress. Since ostrich genome was not available when we started the research, we first cloned ostrich TLR3 TLR4 cDNA from thymus. Ostrich TLR4 was close to white-throated Tinamou. Whole avian TLR4 codons were under purify selection during evolution, whereas 80 codons were under positive selection. TLR3 and TLR4 were expressed in ostrich thymus and bursa of fabricius as was revealed by quantitative real-time PCR (qRT-PCR). TLR4 expression increased with age but significantly decreased after boron treatment, whereas TLR3 expression showed the similar tendency. Their downstream molecular factors (IRF1, JNK, ERK, p38, IL-6 and IFN) did not change significantly in thymus, except that p100 was significantly increased under boron stress when analyzed by qRT-PCR or western blot. Taken together, these results suggest that ostrich thymus developed resistance against long-term excessive boron stress, possibly by accelerating intrathymic cell death and proliferation, which may bypass the TLR3/4 pathway. In addition, attenuated TLRs activity may explain the reduced inflammatory response to pathogens under boron stress.

The degree of brain development can be expressed by the levels of brain brain-derived neurotrophic factor (BDNF). BDNF plays an irreplaceable role in the process of neuronal development, protection, and restoration. The aim of the present study was to evaluate the effects of boric acid supplementation in water on the ostrich chick neuronal development. One-day-old healthy animals were supplemented with boron in drinking water at various concentrations, and the potential effects of boric acid on brain development were tested by a series of experiments. The histological changes in brain were observed by hematoxylin and eosin (HE) staining and Nissl staining. Expression of BDNF was analyzed by immunohistochemistry, quantitative real-time PCR (QRT-PCR), and enzyme linked immunosorbent assay (ELISA). Apoptosis was evaluated with Dutp-biotin nick end labeling (TUNEL) reaction, and caspase-3 was detected with QRT-PCR. The results were as follows: (1) under the light microscope, the neuron structure was well developed with abundance of neurites and intact cell morphology when animals were fed with less than 160 mg/L of boric acid (groups II, III, IV). Adversely, when boric acid doses were higher than 320 mg/L(groups V, VI), the high-dose boric acid neuron structure was damaged with less neurites, particularly at 640 mg/L; (2) the quantity of BDNF expression in groups II, III, and IV was increased while it was decreased in groups V and VI when compared with that in group I; (3) TUNEL reaction and the caspase-3 mRNA level showed that the amount of cell apoptosis in group II, group III, and group IV were decreased, but increased in group V and group VI significantly. These results indicated that appropriate supplementation of boric acid, especially at 160 mg/L, could promote ostrich chicks’ brain development by promoting the BDNF expression and reducing cell apoptosis. Conversely, high dose of boric acid particularly in 640 mg/L would damage the neuron structure of ostrich chick brain by inhibiting the BDNF expression and increasing cell apoptosis. Taken together, the 160 mg/L boric acid supplementation may be the optimal dose for the brain development of ostrich chicks.

采用RT-PCR方法从15日龄的仔鸡肾组织的总RNA中扩增出鸡防御素基因(GAL-2)序列,然后将其亚克隆到载体pIRES2-EGFP构建成真核表达载体pIRES2-EGFP-GAL-2.经PCR鉴定、限制性内切酶酶切分析和克隆片段序列测定、比较,证实了重组质粒的正确性.将构建好的真核表达质粒转染到Marc145细胞中进行瞬时表达,荧光检测证实细胞转染成功.pIRES2-EGFP-GAL-2真核表达载体的成功构建为下一步在细胞水平研究GAL蛋白功能以及进一步将其开发为兽用生物制品奠定了基础.

为分析鸭胚肾细胞用于鸭肝炎病毒增殖的可能性,从鸭胚肾细胞的制备、体外培养、鸭肝炎弱毒在鸭胚肾细胞中的增殖等方面入手,初步分析了鸭胚肾细胞体外培养和用于鸭肝炎病毒增殖的可行性.结果表明,鸭胚肾细胞能够体外培养并形成良好的细胞单层,形态多为多边形或梭形;原代鸭胚肾细胞可以用于增殖鸭肝炎弱毒A66株,毒价(ELD50)达10-4.2/0.2 mL.说明鸭胚肾细胞适合用于鸭肝炎病毒的增殖,有望用于鸭肝炎病毒疫苗的生产.

为了研究猪β-防御素-1 (PBD-1)在肺、肝、睾丸和大脑中的表达情况,笔者采集健康公猪的肺、肝、睾丸和大脑组织,利用RT-PCR和免疫组织化学染色法分析PBD-1在不同组织中的表达情况.结果显示,PBD-1在公猪的肺、肝、睾丸和大脑4种组织中均有不同程度的表达,其中肺泡上皮、生精小管内部和大脑中的免疫阳性信号较多.PBD-1在公猪的4种器官组织中广泛分布,提示PBD-1不仅在抵御外界微生物感染中发挥作用,可能在其他生理活动中也发挥着重要的作用.

B cell activating factor (BAFF), which belongs to the tumor necrosis factor (TNF) family, is testified to play a critical role in B cell survival, proliferation, maturation and immunoglobulin secretion. In the present study, the cDNA of open reading frame (ORF) in African ostrich (Struthio camelus) BAFF (designated OsBAFF) was cloned by reverse transcription-PCR (RT-PCR). The OsBAFF gene encodes a 288-amino acid protein containing a predicted transmembrane domain and a putative furin protease cleavage site like BAFFs from chicken (cBAFF), quail (qBAFF), duck (dBAFF), goose (gBAFF) and dove (doBAFF). RT-PCR analysis showed that the OsBAFF gene is strongly expressed in the bursa of Fabricius, thymus, spleen, and bone marrow. The soluble OsBAFF had been cloned into pET28a. SDS-PAGE and Western blotting analysis confirmed that the soluble fusion protein His-OsBAFF was efficiently expressed in Escherichia coli Rosset (DE3). In vitro, purified OsBAFF was not only able to promote the survival of African ostrich bursal lymphocytes, but also able to co-stimulate proliferation of mouse splenic B cells. The expression of OsBAFF in lymphocyte cells was higher than the control after LPS stimulation. These findings indicated that OsBAFF plays an important role in survival and proliferation of African ostrich bursal lymphocytes, which may provide valuable information for research into the immune system of African ostrich and OsBAFF may serve as a potential immunologic factor for enhancing immunological efficacy in African ostrich and any other birds.

笔者使用人工合成TAT-CD46基因,通过T4连接酶将TAT-CD46基因与原核表达载体PET-23a进行连接,经PCR鉴定、限制性内切酶酶切分析和克隆片段序列测定,得到重组质粒PET-23a-TAT-CD46.转化大肠杆菌,诱导TAT-CD46融合蛋白表达,Ni-NTA Superflow柱纯化后,将融合蛋白与猪瘟病毒液一起加入培养的ST细胞中,观察荧光进入细胞的情况.经免疫荧光证实,TAT-CD46融合蛋白能够提高猪瘟病毒对ST细胞的感染能力,可以有效促进CSFV在ST细胞中的增殖速率,这为进一步提高猪瘟病毒效价奠定了基础.

β-lactamase was found in raw milk in a pasture of Wuhan,the laboratory testing results indicated that it was generated by some cows common pathogenic bacteria. The drug sensitive test and enzyme production rate of cows pathogenic bacteria was conduct in this study, aiming to provide a theoretical basis for clinical prevention and treatment.

硼是广泛分布于自然界的微量元素,具有多种生物学作用.研究表明,硼与某些动物的生长发育和新陈代谢过程密切相关,可能是动物不可或缺的元素.适量的硼可促进动物的生长发育,维持动物的正常生理功能;高剂量的硼则对动物产生毒害作用.目前主要以试验动物为模型,研究硼对动物的营养与毒性作用,从分子水平探讨硼的作用机理.论文结合了硼在动物营养和毒性作用方面的最新研究进展,对硼在提高动物的生长速度、促进动物胚胎发育、改善动物骨代谢等方面的营养作用及发育毒性、生殖毒性和免疫毒性进行综述,旨在为硼的生物学和营养学研究提供参考.

Cell Mycoplasma contamination in the process of producing animal biological products(especially the passage cell line) is a worldwide problem. Cell Mycoplasma contamination can exert a negative influence on culture and display of biological function of cells for production, ultimately affect producing biological products, bring huge losses to the biological products enterprises. Therefore it is necessary to establish an efficient, rapid, sensitive method for detecting cell isolation and culture method. A nested PCR method of detecting Mycoplasma is established, which is efficient, rapid, sensitive and can minimize the appearance of the false negative and false positive results. The establishment of the method have certain significance reference for standardized management of biological products enterprises engaging in cell culture and animal vaccines.

动物防御素是一类内源性阳离子短肽,广泛分布于包括人类在内的各种哺乳动物体内,具有广谱抗菌活性,又被称为抗菌肽(AMPS).早在1963年,美国科学家Zeya等人在研究豚鼠白细胞裂解物时就发现该类具有抗菌活性的物质[1].随后,人们在昆虫、鸟类、鱼类、两栖类、爬行类和哺乳类等动物体内均发现有防御素的存在.到目前为止,已从多种动物和人体内分离出百余种防御素.哺乳动物防御素的抗微生物机理独特,不易产生耐药性,是机体抵抗外来致病性微生物侵袭的重要武器.