产肠毒素大肠杆菌K88ab/K88ad菌毛操纵子fae全基因的克隆、表达及生物学活性的初步研究

为研究K88ab/K88ad菌毛对细胞的黏附作用,本研究分别以产肠毒素E.coli (ETEC) K88ab C83901株和K88ad C83903株基因组DNA为模板,采用PCR技术扩增这两种K88菌毛操纵子fae基因(均约7.9 kb).将其分别克隆于表达质粒pBR322中构建pBR-K88ab和pBR-K88ad重组质粒,并将其分别转化至不含任何菌毛的E.coli SE5000株中.该重组菌能够分别与鼠抗K88菌毛阳性血清和抗K88菌毛单克隆抗体(MAb)产生凝集反应;在电镜下观察到重组菌表面大量表达K88菌毛.采用热抽提法提取其体外表达的K88ab和K88ad菌毛,SDS-PAGE电泳检测结果显示,菌毛蛋白的分子量约为26 ku.玻板凝集试验和western blot结果表明:重组表达的K88ab及K88ad菌毛与K88+参考株菌毛均能够被抗K88菌毛阳性血清和MAb识别.以猪小肠上皮细胞系IPEC-J2为模型进行黏附和黏附抑制试验,结果表明表达K88菌毛的重组菌及K88+参考株均能够黏附于IPEC-J2上皮细胞表面;而且阳性血清和MAb能够有效抑制重组菌或K88+参考株对猪小肠上皮细胞系的黏附结合.     

西南“旱三熟”区不同作物和秸秆覆盖对土壤团聚体及固碳潜力的影响

为了探讨西南"旱三熟"(小麦/玉米/大豆)套作种植模式下农田土壤团聚体的分布特征及有机碳含量变化情况,进而估算该模式下的土壤固碳潜力,在重庆北碚西南大学试验农场对传统耕作(traditional tillage,T)和传统耕作+秸秆覆盖(traditional tillage+straw mulching,TS)2种处理下的土壤团聚体进行筛分和测定。结果表明,3种作物种植下的2mm粒径与2~0.25mm粒径团聚体含量此消彼长,存在显著的负相关关系(r=-0.985,P0.05)。土壤团聚体结构对不同作物的响应不同,水稳性大团聚体(0.25mm粒径)含量在小麦和大豆种植后高达90%左右,玉米种植后约为80%,说明种植玉米有利于土壤水稳性微团聚体的形成。2~0.25mm粒径团聚体的有机碳含量最高,而水稳性微团聚体的两个粒径团聚体有机碳含量相差不大,有机碳含量在团聚体中的分布规律不受种植作物和耕作方式的影响。秸秆覆盖显著提高了0~5cm和5~10cm土层的本土及各个粒径中的有机碳含量,且5~10cm土层团聚体有机碳受秸秆覆盖的影响较大。通过估算固碳潜力发现,玉米条带的土壤固碳潜力显著大于小麦-大豆条带,在耕作处理保持一致的情况下,土壤团聚体有机碳含量对农作物的响应不同。因此,在西南"旱三熟"地区,农田土壤团聚体分布特征和不同粒径有机碳含量受到耕作措施和种植作物的双重影响,土壤固碳潜力主要由水稳性大团聚体的固碳能力决定,水稳性大团聚体更易受到耕作措施和种植作物的影响,在实践中通过秸秆还田提高土壤固碳外,合理安排农作物也有助于提高土壤的固碳能力。     

鸡传染性支气管炎病毒(IBV)ZZ2004株核衣壳蛋白基因的克隆及原核表达

本研究旨在克隆并表达鸡传染性支气管炎病毒（IBV）的核衣壳蛋白（N）。根据IBV ZZ2004株的N基因设计1对引物,提取IBV ZZ2004株的RNA,利用RT-PCR技术扩增大小约1.23 kb的片段,将其插入克隆载体pGEM-T构建pGEM-T-N。将pGEM-T-N用EcoRⅠ和XhoⅠ进行双酶切,将酶切后的N基因片段克隆到表达载体pGEX-6P-1,转入大肠杆菌BL21（DE3）菌中,经IPTG诱导表达,进行SDS-PAGE电泳,结果显示外源基因获得了表达且为可溶性蛋白;Western blotting检测结果显示N蛋白可与相应的IBV ZZ2004阳性血清发生特异性反应,结果表明N蛋白有一定的生物活性。本研究为IBV诊断试剂盒及新型IBV重组亚单位疫苗研制奠定基础。     

猪蛋白质二硫化物异构酶A4基因克隆及密码子偏好性分析

试验旨在研究猪蛋白质二硫化物异构酶(protein disulfide isomerase A4,PDIA4)基因的密码子使用特性及其与其他物种密码子使用的差异。试验以香猪子宫组织为试验材料,采用RT-PCR方法扩增获得猪PDIA4基因,参考从NCBI数据库中下载的18个不同物种PDIA4基因序列,对猪PDIA4基因的密码子使用特性及其与其他物种密码子的使用差异和进化关系进行分析,同时对猪PDIA4基因与4个模式生物密码子使用偏性进行比较。结果表明,猪PDIA4基因编码区长1 941 bp,共编码646个氨基酸。与NCBI数据库公布的野猪PDIA4基因(GenBank登录号:NM_001267834.1)的相似性达99%。猪PDIA4基因偏好使用G/C结尾的密码子,27种密码子为猪PDIA4基因的偏好密码子(RSCU>1),其中使用频率最高的前3个密码子分别为CTG(RSCU=3.69)、GCC(RSCU=2.31)和CGC(RSCU=2.29)。比较19个物种PDIA4基因发现,不同物种间PDIA4基因的有效密码子数(ENc)、密码子适应指数(CAI)、GC含量以及T、C、A、G、G+C在密码子第3位含量(T3s、C3s、A3s、G3s、GC3s)均存在差异,且密码子偏好以G/C结尾;聚类分析发现,基于CDS序列的系统进化树更符合19个物种的真实分类系统。在密码子使用频率上,猪PDIA4基因与小鼠基因组间密码子使用偏好程度更为接近,故小鼠更适合作为猪PDIA4基因的遗传转化和异源表达的受体系统。     

气候变化对藏药独一味适生区分布格局的影响

本研究通过实地调查,运用最大熵(MaxEnt)模型和环境气候数据模拟未来气候变化对青藏高原地区藏药独一味(Lamiophlomis rotata(Benth.)Kudo)适生区空间分布格局的影响,探索其适生区变迁规律.通过ArcGIS和SPSS对模拟结果分析显示,当前气候条件下独一味最适宜生长地区主要分布于青藏高原地区...     

Cell-based cancer gene therapy: breaking tolerance or inducing autoimmunity?

This review examines the mechanisms involved in anti-tumor immunity and how peptides present in many tumor types (tumor-associated antigens) are recognized by T cells from tumor-bearing cancer patients. Tumor-associated antigens are derived from proteins that are also expressed in normal cells. It is predicted that immune responses to such peptides will be compromised by self-tolerance or that stimulation of effective immune responses will be accompanied by autoimmunity. We also consider that the immunity induced against two autoantigens, which are highly conserved in vertebrates, involve qualitatively different mechanisms, such as the production of antibodies and cell-mediated immune responses. However, both pathways lead to tumor immunity and identical phenotypic manifestations of autoimmunity. Appropriate selection of the optimal tumor antigen is critical for the induction of an anti-tumor immune response. Thus, we stress that the methods for antigen presentation using dendritic cells play a critical role in the development of tumor vaccines, to break immune tolerance and induce a strong immune response against them. The viability and feasibility of expansion of canine dendritic cells from bone marrow and peripheral blood ex vivo for the treatment of spontaneous cancers in dogs is also discussed.     

Identification of RSVP14 and RSVP20 components by two-dimensional electrophoresis and Western-blotting.

We have already shown that RSVP14 and RSVP20, two ram seminal plasma (SP) proteins postulated to be involved in sperm capacitation and gamete interaction can protect spermatozoa against cold-shock. In this study, we use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the analysis of SP proteins of Rasa Aragonesa rams, using enhanced protein solubilization in the presence of tributyl phosphine (TBP) and a polyacrylamide linear gradient gel with a narrow pH range (4-7). The image analysis of the 2D map detected 195 protein spots, with isoelectric points (pIs) ranging from 4.5 to 6.6, and molecular weight (M(r)) from 11.7 to 90.4. Staining of 2D gels with Pro-Q Emerald 300 Glycoprotein Stain revealed that most significant proteins in ram SP are glycosylated. The removing of protein N-linked oligosaccharides improved the gel resolution. 2D-PAGE analysis of the whole fraction 6 (F6) separated from ram SP by exclusion chromatography showed six main protein spots, four (a, b, c, d) in the 14 kDa and two (e, f) in the 20 kDa region. Western-blot analyses indicated that the anti-P14 antibody recognized four spots on the SP map, 4, 5, 6 and 7, that matched with spots a, b, c, d of F6 map. The anti-P20 antibody recognized spots 13 and 14 of SP map that corresponded to spots e, f of F6 map. The deduced sequences by de novo sequencing evidenced that protein spots 7 and 13 have significant similarities to BSP family, while protein spots 4 and 14 did not appear to be homologous with any reported protein in the current mammalian Proteinbank databases.     

夏季日粮中主要营养水平对肉种母鸡生产性能的影响

为研究夏季日粮中粗脂肪、粗蛋白、赖氨酸和蛋氨酸水平对肉种鸡生产性能的影响,27周龄良凤肉用种母鸡随机分为4个处理,每个处理3个重复,每个重复为490只,处理一饲喂对照日粮(CF2.7%,CP16.5%,Lys0.83%,Met0.40%),处理二饲喂高脂肪日粮(CF5.43%),处理三饲喂高蛋白日粮(CP18.2%),处理四饲喂高赖氨酸和蛋氨酸日粮(Lys0.91%,Met0.44%),试验期共112天。结果表明,对照日粮的营养水平是适合的,在此基础上分别增加日粮的粗脂肪水平、粗蛋白水平、蛋氨酸和赖氨酸水平,都不能提高肉种母鸡的生产性能,也不能减轻夏季炎热带来的影响。