鹅细小病毒vp1基因片段的原核表达及抗血清的制备

利用PCR技术扩增了鹅细小病毒(Goose parvovirus , GPV)HG5/82株vp1基因5'端长662 bp的基因片段，将其克隆到pMD18-T Simple载体后，转化入大肠杆菌(Escherichia coli) DH5α细胞。筛选阳性质粒，并通过BamHⅠ和Hind Ⅲ将外源基因定向克隆到原核表达载体pPROEXTMHTb，所得的阳性重组质粒经序列测定，证明外源片段正确插入到pPROEXTMHTb的预期位置。将转入外源基因的工程菌经0.6 mmol/L IPTG诱导，SDS-PAGE电泳表明， 外源基因表达，融合蛋白分子量约为32 kD。通过薄层扫描分析显示，融合蛋白表达量占工程菌菌体总蛋白的22.8%。经诱导后的工程菌用6 mol/L盐酸胍裂解，再经超声处理后离心，用镍离子亲和树脂对裂解产物的上清进行纯化。纯化所得的融合蛋白免疫白兔，制备了兔抗鹅细小病毒结构蛋白的抗血清。Western blot结果表明，抗血清与表达的融合蛋白及亲本病毒的VP1和VP2都具有反应性。     

Soil properties associated with organic matter-mediated suppression of bean root rot in field soil amended with fresh and composted paper mill residuals

The ability of an organic amendment to suppress soil-borne disease is mediated by the complex interactions between biotic and abiotic soil factors. Various microbiological and physicochemical soil properties were measured in field soils with histories of receiving 4 or 5 years of spring additions of paper mill residuals (PMR), PMR composted alone (PMRC), PMR composted with bark (PMRB), or no amendment under a conventionally managed vegetable crop rotation. The objectives of this study were to (i) determine the residual and re-amendment effects of the organic materials on root rot disease severity; (ii) determine the influence of amendment type on the structure of bacterial communities associated with snap bean roots grown in these soils; and (iii) quantify the relative contributions of microbiological and physicochemical properties to root rot suppression in the field and greenhouse. While all amendment types significantly suppressed root rot disease compared to non-amended soils in both environments, only soils amended with PMR or PMRB sustained suppressive conditions 1 year after the most recent amendment event. Disease severity was inversely related to microbial activity (fluorescein diacetate assay) in recently amended soils only. Terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16s rRNA gene was performed to obtain bacterial profiles. Principal component analysis (PCA) of terminal restriction fragments (TRFs) revealed general differences in bacterial community composition (PC1) among amendment types, and specific TRFs contributed to these differences. Correlation and multiple regression analyses of the measured soil variables revealed that the composition of root-associated bacterial communities and the amount of particulate organic matter—carbon in bulk soils imparted independent and relatively equal contributions to the variation in disease severity documented in the field and greenhouse. Together, our findings provide evidence that disease suppression induced by annual PMR inputs was mediated by their differential effects on bacterial communities and the amount and quality of organic matter in these soils.     

Arthroscopically‐assisted removal of an osteochondral fragment from the equine elbow using a caudo‐medial approach

An 8‐year‐old Polo pony mare was admitted for investigation of lameness related to swelling of the elbow. An intra‐articular fragment from the proximo‐medial radius was identified on radiographic examination. The fragment was removed arthroscopically under general anaesthesia via a caudo‐medial approach to the elbow joint.     

砾石覆盖度测量方法研究

地表砾石是各种水文和侵蚀等过程综合作用的产物,是土壤退化和生态系统恶化的一个标志;反过来这些砾石也影响到侵蚀的各个过程。分别利用视点框架法和照相法对不同粒径样本下(2~6mm,6~20mm,20~60mm)的砾石覆盖度进行了测量,对比分析了不同砾石覆盖度测量方法对其精度的影响。结果表明,采用两种测量方法观测砾石覆盖度均能达到一定精度,视点框架法测量的砾石覆盖度相对误差变化范围为-7.84%~80.86%,平均相对误差为15.69%,照相法测量的砾石覆盖度相对误差变化范围为-48.35%~9.89%,平均相对误差为-11.69%。随着砾石覆盖度的增加,视点框架法测量的相对误差绝对值表现为减小。视点框架法测量的最大相对误差均值的绝对值随着抽样次数的增加而减少,当抽样次数达到约30,相对误差减小的趋势减缓。     

小鼠ret中间片段真核表达载体的构建

利用RT-PCR法从小鼠睾丸组织中获得ret的中间片段,首先将其连接到pMD18-T载体上,转化DH5α后通过阳性克隆子的鉴定以及序列测定,将序列正确的ret中间片段双酶切下来后连接到真核表达载体pcDNA3.1上.结果表明成功构建了ret中间片段的真核表达载体pcDNA3.1-ret.     

梅与其近缘种亲缘关系的AFLP分析

以包括野梅在内的15个梅品种、1个野生种和其近缘种杏、山杏、桃、山桃、李和‘垂枝’毛樱桃、‘紫叶’李为试材,运用AFLP标记,采用7对引物组合(E-ACTM-CAT、E-GAM-CTC、E-ACTM-CCA、E-ACTM-CAC、E-ACTCTC、E-ACTCAG、E-ACCM-CAG)选择性扩增得到287个多态性条带的6888条带数据,使用NTSYSpc2.1t软件,采用Nei’s(72)遗传距离,SAHNClustering进行不加权成对算术平均法聚类分析,得到了梅品种与近缘种的亲缘关系与形态学、育种记录等相关记录相符的结论,并支持梅花二元分类系统的相关分类。     

扇脉杓兰AFLP反应体系的建立

通过SDS法、高盐低pH法、常规CTAB法、高盐CTAB法和改良CTAB法提取扇脉杓兰(Cypripedium japonicum Thunb.)幼叶基因组DNA,并对其AFLP反应体系进行了优化。结果表明:改良CTAB法所提取的DNA电泳条带清晰无污染,A260和A280比值在1.8～2.0,用限制性内切酶酶切后条带均一,酶切时间以4 h为宜。最终确定50μL PCR反应体系的最佳条件为:预扩增产物稀释20倍4μL,dNTP(2.5 mmol/L)3μL,Mg2(+25 mmol/L)4μL,MseⅠ(10μmol/L)和EcoRⅠ(10μmol/L)各1.5μL,Taq DNA聚合酶(5 U/μL)0.3μL。     

猪肺炎支原体P216基因片段的表达及黏附活性研究

本研究旨在研究猪肺炎支原体P216蛋白的黏附活性并初步建立猪肺炎支原体蛋白黏附模型。根据软件分析和文献报道从P216全基因中选取亲水性、抗原性、黏附性较好的目的片段,利用PCR从Mhp NJ株扩增P216基因片段,克隆到表达载体pET-32a(+)中,获得重组质粒pET-32a(+)/P216,经IPTG诱导获得重组蛋白,通过Western blot和间接免疫荧光试验检测重组蛋白的免疫原性及黏附活性。结果表明,PCR扩增的目的基因大小为l 636bp;SDS-PAGE检测重组蛋白相对分子质量为80.1ku;Western blot检测表明重组蛋白能与Mhp阳性血清发生特异性反应;间接免疫荧光试验表明该蛋白对猪肺炎支原体黏附SJPL细胞产生占位抑制作用。结果提示,P216蛋白具有良好的黏附活性,能黏附SJPL细胞,从而为猪肺炎支原体其他黏附因子的研究奠定基础。     

Genomic lineage of Salmonella enterica serovar Dublin

Thirty five strains of the host adapted Salmonella serotype Dublin (S. Dublin) have been characterized by IS200 patterns, ribotyping, pulsed-field gel electrophoresis (PFGE), restriction fragment polymorphism after hybridization with five randomly cloned DNA-framents of S. enteridis (RFLP), and plasmid profiling in order to divide the strains into ‘genomic lines’. For comparison, 20 other strains of 9 different group-D serotypes were included. The IS200 patterns were identical in all strains of S. Dublin examined. These patterns were different from those observed in other group-D Salmonella with the exception of one strain S. Enteritidis phage type 11 and a strain of S. Rostock. The insertion element IS200 was not detected in strains of S. Dar-es-Salam, S. (II) 9,12:z -, and S. Panama. RFLP, based on probing with five random cloned chromosomal fragments gave the same pattern in all strains except for one isolate from the UK. This strain was also found to have an unique PFGE pattern and ribotype. Among the remaining strains, three different PFGE patterns and 7 different ribotypes were observed. Based on all four typing methods, 8 different ‘genomic lines’ of S. Dublin were identified. The same grouping could be obtained from the use of ribotyping alone, but PFGE and RFLP were found to provide valuable information on possible relationships between ribotypes. Seven different plasmid profiles and a group of strains without plasmids were observed. In several cases, the same plasmid profile was shown to be present in more than one ‘genomic line’.