绵羊棘球蚴病的病原学鉴定及组织病理学观察

从疑似棘球蚴病病羊的肝以及肺分离包囊,抽取囊液,离心取沉淀,分离基因组DNA,用PCR方法扩增NADH脱氢酶亚基1(ND1)基因并测序,对棘球蚴感染羊进行了病原学鉴定,并对患病羊的肝和肺病灶进行了病理学分析,以确定病原体的性质及所致的组织病理学变化。BLAST分析结果显示,所获ND1序列与细粒棘球绦虫(G1基因型)ND1序列的相似性达99.9%,判定该病例由G1型细粒棘球蚴引起。取包囊及其周围肝、肺组织进行石蜡切片、HE染色,观察结果显示,细粒棘球蚴包囊的形成对肝和肺造成的损伤是多方面的,除有压迫性组织萎缩,结缔组织及胆管大量增生造成的肝硬变外,尚见肝急性变性、过敏性炎症反应等。肺除压迫性萎陷外,还有间质增生炎症、囊液外渗引起的过敏反应等。本研究表明,采用PCR方法可以准确确定该病的病原性质,结合病理学观察分析,可揭示羊细粒棘球蚴病引起的组织病理学变化及器官损伤的机制。     

猪巨细胞病毒四川株gB基因的克隆表达及抗原性分析

为研究猪巨细胞病毒(PCMV)SC株gB蛋白的免疫学活性,采用PCR扩增PCMV SC株gB全基因和不含跨膜区及信号肽的gB基因片段,将后者克隆至pET-30a(+)载体,转化Rosetta(DE3)感受态细胞,经IPTG诱导以获得高效表达。将纯化蛋白免疫家兔制备多克隆抗体,分别用琼脂扩散试验和Western-blot对多克隆抗体和复性的gB蛋白进行检测。结果显示,扩增的PCMV gB基因全长2 580bp,编码860个氨基酸。与国内外参考株的核苷酸和氨基酸序列相似性分别为97.8%～99.5%和96.6%～99.0%;与其他9株β疱疹病毒核苷酸和氨基酸序列相似性分别为33.3%～41.4%和13.3%～49.4%;系统进化分析显示,PCMV与人疱疹病毒6型和7型属于一个分支。gB肽链N端1～23位氨基酸为信号肽,729～751位氨基酸间含有跨膜区。构建的表达载体pET30a-gB-B在Rosetta(DE3)感受态细胞中经IPTG诱导后以包涵体形式表达出大小约80ku的蛋白。所制备的血清琼扩抗体效价为1∶16,Western-blot显示重组gB蛋白可与PCMV阳性猪血清反应。结果表明,PCMV gB基因较为保守,与国内外不同地区的PCMV毒株具有较高的相似性,所表达的重组蛋白具有很好的抗原性。     

基于cytb基因对大熊猫等六种珍稀野生动物感染蛔虫的种系发育分析

为分析大熊猫、小熊猫、北极熊、黑熊四川亚种、浣熊和猕猴等6种野生动物蛔虫cytb基因的遗传多态性及其种系发育关系,对这6种野生动物寄生蛔虫cytb基因进行了扩增与测序分析,并分别构建NJ、MP和ML系统进化树。结果显示,六种野生动物寄生蛔虫的cytb基因经PCR扩增后共获得长度均为875bp的序列,其中大熊猫、小熊猫、北极熊、黑熊四川亚种和浣熊寄生的蛔虫cytb序列中包含了156个变异位点,而寄生于猕猴的蛔虫与寄生于猪的猪蛔虫在cytb序列上存在11个位点的差异。系统进化树显示,大熊猫等6种野生动物蛔虫聚类成贝蛔属分支。表明cytb基因适合于野生动物蛔虫的分类鉴定以及种系发育分析,可用于野生动物蛔虫的鉴别诊断。     

猪札幌病毒VP1基因的表达及ELISA抗体检测方法的建立

为了在大肠杆菌中表达猪札幌病毒VP1基因,并以纯化的重组蛋白为抗原建立猪札幌病毒血清抗体ELISA检测方法。采用RT-PCR技术扩增VP1基因,并将其克隆到pMD18-T载体中,再将所克隆的衣壳蛋白的编码序列亚克隆到表达载体pET-28a(+)中,将成功构建的原核表达质粒pETSAVCAP转化大肠杆菌Rosetta(DE3)进行诱导表达。用纯化的VP1蛋白作为抗原建立了猪札幌病毒血清抗体间接ELISA检测方法并初步用于临床样品的检测。结果显示,猪札幌病毒VP1基因可在大肠杆菌中稳定、高效地表达,Western-blot分析表明该重组蛋白具有良好的反应原性。经对反应条件进行优化,确定间接ELISA的抗原最佳包被浓度为0.5μg/mL,且不与其他常见猪病的阳性血清发生交叉反应,建立的ELISA方法具有较好的敏感性、特异性和重复性。通过对从多个省份收集的490份猪血清样品进行检测,阳性率为65.31%。表明,以大肠杆菌表达的猪札幌病毒VP1重组蛋白为抗原建立的间接ELISA方法可以用于猪札幌病毒抗体的检测。     

猪瘟病毒NASBA-ELISA检测方法的建立与应用

为建立猪瘟病毒NASBA-ELISA检测方法,以猪瘟病毒(CSFV)E2基因为研究对象,利用Prim-er 5.0和Blastn生物软件,设计并合成了特异的核酸序列依赖扩增(NASBA)引物和探针。经条件优化,确定的最佳扩增反应条件为42℃,反应1.5h。临床检测结果显示,用该方法可特异地扩增CSFV E2基因约155bp的目的片段,而对TGEV、PRRSV、PCV2、PPV、PEDV、APP和PK-15正常细胞等核酸的检测均为阴性,可最低检出1×100～1×101 copies/μL,其灵敏度比RT-PCR略高。应用该方法对采自重庆部分地区猪场的125份样品进行了检测;结果,猪瘟病毒阳性检出率为5.6%,NASBA-ELISA法与RT-PCR法的检出符合率为94.4%。结果表明,NASBA-ELISA方法可应用于CSFV的快速鉴别检测。     

鸭瘟病毒gD基因胞外区的原核表达及ELISA检测方法的建立与应用

为研究分离鉴定的鸭瘟病毒gD基因(GenBank登录号:EU195085)及其编码蛋白的应用,采用PCR方法从鸭瘟病毒CHv毒株DNA中获取编码gD胞外区基因729bp片段,构建重组表达载体pET32a-gD,转化受体菌E.coli BL21(DE3),IPTG诱导表达重组gD蛋白。结果显示,该重组蛋白分子质量约为31ku,为gD胞外区与载体6个组氨酸的融合表达产物,与gD胞外区预期大小一致(27.5ku);经离心收集菌体,超声裂解后,SDS-PAGE分析结果显示,gD蛋白主要以包涵体形式表达;将包涵体溶解后进行Ni-NTA亲和层析可较好地纯化重组蛋白。Western-blot检测显示,gD蛋白能与兔抗鸭瘟病毒血清特异结合,具有较好的免疫反应性。利用该蛋白建立了ELISA检测方法,优化了该方法的各个反应条件,并进行了初步应用,为该方法的临床应用奠定了基础。     

大鼠死后管家基因mRNA稳定性及其时序性降解与PMI相关性研究

目的研究脑组织及骨骼肌内β-actin、GAPDH、RPL32、PKG1、SDHA、RPL13、HPRT、TBP、YWHAZ死后稳定性及其相对表达量与死亡时间(PMI)的相关性。方法取健康成年SD大鼠33只,依据据死亡时间不同随机分为11组(0h、1h、3h、6h、12h、24h、2d、4d、8d、16d、24d),分别在各时间点取大鼠胫骨前肌及脑组织约50mg,提取各组织总RNA,利用RT-q PCR技术检测9种管家基因的m RNA相对表达量。运用ge Norm、Norm Finder和Best Keeper软件对基因稳定性进行评价,利用spss软件对内参标准化的m RNA指标进行回归分析。结果脑组织及骨骼肌中均为HPRT稳定性最高,适合用于本研究的内参基因。脑组织中SDHA、RPL32及TBP 3种指标的相对表达量变化与PMI有一定的线性关系。骨骼肌中m RNA指标相对表达量与PMI之间没有显著的相关性。结论脑组织及骨骼肌是较适宜推断晚期死亡时间的检材。脑组织中SDHA、RPL32及TBP 3种m RNA的相对表达量变化与PMI有一定的线性关系,有望成为推断PMI的辅助指标。     

浙江汉族人群21个非CODIS系统STR基因座的遗传多态性调查

目的调查21个非CODIS系统STR在浙江汉族人群的遗传多态性,为其法医学应用提供基础数据。方法应用AGCU21＋1荧光标记复合扩增系统,对浙江汉族481名无关个体进行21个STR基因座的复合扩增,用ABl3130XL型基因分析仪对扩增产物进行检测,并统计其STR基因座的群体遗传学参数。结果获得21个STR基因座的等位基因频率分布,分别检出8、11、9、10、8、9、6、5、13、9、6、15、8、7、8、9、8、9、9、16、7个等位基因,并分别获得21个STR基因座的H、He、DP、PM及EP等法医遗传学参数。结论21个STR基因座具有较强个体识别能力,可应用于法庭科学中的个体识别与亲权鉴定。     

Friendly neighbor or Trojan Horse? Assessing the interaction of soft law initiatives and the UN climate regime

Current global climate governance is characterized by increasing institutional proliferation. Within the last 5 years several non-legally binding initiatives have emerged, including (i) the Asia-Pacific Partnership on Clean Development and Climate and various other public–private partnerships working on the policy implementation level and (ii) the Group of Eight Gleneagles Dialogue on Climate Change, Clean Energy and Sustainable Development, and Major Economies Meeting on Energy Security and Climate Change as high-level political processes. As a first step toward analyzing the relationship between these parallel initiatives and the UN climate regime, this article looks at the negotiations of four UN-hosted climate meetings in 2007–2008, providing an examination of the interaction of ‘soft law’ climate initiatives and the ‘hard law’ UNFCCC/Kyoto Protocol process. The methodology of the study is based on participatory observations in the negotiations and document analysis of country and stakeholder positions. The analysis shows that the current multitude of processes in global climate governance entails potential institutional interaction. Deliberations of the key actors give some support to the claims of non-UN soft law being used to exert influence on the negotiations on a future climate regime within the UN context.

Comparing regulatory processes in genome editing and autonomous vehicles: How institutional environments shape sociotechnical imaginaries

This study compares the regulation of two emerging technologies, the CRISPR genome-editing system and Connected and Autonomous Vehicles (CAV) in the United States. The study draws on 33 in-depth interviews with innovation and governance experts to study the relationship between their regulatory environments and developing beliefs about these technologies. Using sociotechnical imaginaries as a framework, we explore how social actors envision technologically driven futures and the social order that enables them. These imaginaries are essential to emerging technologies, where experts build a framework of potentialities for innovation still underway. While scholarship has documented how sociotechnical imaginaries arise among policymakers, groups of scientists, state and local stakeholders, and public actors in different countries, less has been said about how regulatory organizations and their actors shape expectations around technologies that are in the early and middle stages of development. This article finds that regulatory institutions shape emerging imaginaries along three related axes: the distribution of authority, technological novelty, and risk. Interviewees negotiate these three contingencies differently based on relevant extant regulatory structures and ideologies, resulting in distinct imaginaries around each technology. CRISPR actors envision genome editing as largely diminishing biomedical harm and eventually suitable for health markets, while CAV actors diverge on whether self-driving cars alleviate or exacerbate risk and how they may enter roads. That organizational structures and practices of regulation inform broadly held sociotechnical imaginaries bears significance for studies of innovation trajectories, suggesting regulators can take an active role in shaping how risks and benefits of emerging technology are defined.