iss基因与鸡大肠杆菌致病力的关系

为探讨iss基因的存在及其核苷酸序列的差异与鸡大肠杆菌致病力的关系,分别采用普通PCR及套式PCR方法检测iss基因在21株鸡大肠杆菌菌株中的分布,并进行序列测定,分析菌株携带iss基因的情况及不同毒力的菌株间iss基因序列的差异。结果显示,经套式PCR检测21株鸡大肠杆菌均携带iss基因,而普通PCR检测显示仅有13株携带iss基因;16株菌株iss基因与国外禽大肠杆菌iss基因参考序列同源性为100%;其余5株中,E1与参考序列同源性为93.5%,E33、O78与参考序列同源性为99.4%,E4、E21与参考序列同源性为99.7%。推测,iss基因可能在不同来源、不同毒力、不同血清型的鸡大肠杆菌中都普遍存在,仅是拷贝数高低不同,而不是目前普遍认为的"有或无"的关系,并且iss基因序列是高度保守的。iss基因与鸡大肠杆菌致病力的相关性可能与它所定位的质粒拷贝数有关,与iss基因的序列差异无关。     

牛病毒性腹泻病毒RT-PCR检测方法的建立及应用

根据GenBank中登录的牛病毒性腹泻病毒(BVDV)5′UTR基因序列,设计合成了1对特异性引物,建立了检测BVDV218bp片段的RT-PCR方法。通过对该方法的特异性、敏感性和重复性进行试验,结果显示,该方法从BVDV标准毒株OregonC24V中扩增出了218bp的特异性片段,而对猪瘟病毒、蓝舌病病毒、牛传染性鼻气管炎病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、MDBK正常细胞的扩增结果均为阴性。经对标准毒株的细胞毒进行检测,其敏感度达10-1TCID50。不同人员用该方法进行检测,结果均一致,表明其重复性好。应用该方法对70份临床疑似发病猪样品进行检测,结果检出11份阳性,阳性检出率约为15.7%。在对5份细胞培养用犊牛血清的检测中,亦检出2份阳性。表明,建立的RT-PCR方法具有特异、灵敏、高效、快速的特点,可用于BVDV的临床检测及流行病学监测。     

传染性羊痒病抗性基因分型方法的建立

根据GenBank中登录的绵羊朊蛋白编码基因PRNP序列,设计并合成了可用于单核苷酸多态性(SNP)快速分析的3对引物和7条分别标记了FAM和VIC荧光染料的MGB探针,建立了一种利用荧光定量PCR扩增反应对羊痒病抗性基因进行筛选的方法。结果表明,设计的引物及探针具有特异性和高效扩增性,能够用于PRNP羊痒病抗性基因的快速分型。借助该方法可建立一整套完善的预警监测体系来预防该病的发生,也可用于羊痒病的常规实验室诊断。     

大熊猫轮状病毒CH-1株NSP4基因的克隆与生物信息学分析

用MA-104细胞增殖大熊猫轮状病毒(RV),并提取总RNA,经RT-PCR扩增,获得NSP4基因,将其克隆到载体进行测序,应用生物信息学方法初步分析NSP4基因的结构及功能,并与不同动物的RV构建系统进化树。结果显示,获得了长750bp的大熊猫RVNSP4基因,此序列包含1个528bp的完整开放阅读框,编码175个氨基酸;预测大熊猫RVNSP4基因的理论分子质量为20223.7u,等电点为6.84,半衰期为30h,不稳定系数为30.40,总平均亲水性为-0.240,疏水性为-2.400～2.622;无信号肽序列;跨膜结构分析表明,NSP4有1个跨膜区,其N端位于病毒膜外区,C端位于病毒膜内区;抗原表位预测显示,NSP4有6个抗原决定簇;结构预测显示,其可能包含2个N-糖基化作用位点、6个蛋白激酶C磷酸化作用位点、3个酪蛋白激酶Ⅱ磷酸化作用位点和1个酪氨酸激酶磷酸化作用位点;系统进化树分析显示,大熊猫RV NSP4基因与猪RV NSP4基因的进化距离最近。     

乙型脑炎病毒E蛋白基因的原核表达及ELISA抗体检测方法的建立

通过PCR方法扩增流行性乙型脑炎病毒(JEV)E基因,全长1 500 bp,将其连入经双酶切的pET-28a(+)载体,构建了重组原核表达质粒pET28a-E.将pET28a-E转化大肠杆菌BL21(DE3)后,经IPTG诱导,进行SDS-PAGE分析.结果显示,E基因在大肠杆菌BL21中获得高效表达,表达的蛋白分子质量约53 ku.Western-blot分析表明,该表达产物具有良好的抗原性.在此基础上,利用该蛋白初步建立了检测猪JEV抗体的间接ELISA方法,并用武汉科前生物制品公司生产的猪JEV ELISA抗体检测试剂盒同时对250份临床采集的猪血清样品进行了检测,结果这两种方法的符合率达到92%,表明建立的ELISA方法具有较高的灵敏性和特异性.     

Procedurally Robust Risk Assessment Framework for Novel Genetically Engineered Organisms and Gene Drives

In this article, a new framework for improving risk assessments of novel genetically engineered organisms (GEOs) is developed and applied. The Procedurally Robust Risk Assessment Framework (PRRAF) provides a set of principles and criteria for assessing and enhancing risk assessment protocols for GEOs under conditions of high uncertainty. The application of PRRAF is demonstrated using the case of a genetically engineered mosquito designed to kill its wild population and therefore decrease disease transmission. Assessments for regulatory approval of this genetically engineered insect fall short of several PPRAF criteria under the principles of humility, procedural validity, inclusion, anticipation, and reflexivity. With the emergence of GEOs designed to spread in ecosystems, such as those with gene drives, it will become increasingly important for regulatory agencies and technology developers to bolster their risk analysis methods and processes prior to field testing. PRRAF can be used as a flexible guide for doing so within a variety of institutional, regulatory, and governance contexts.     

Lutheran血型系统的分子遗传学基础

Lutheran血型系统合有18个抗原,LU1／LU2、LU18／LU19抗原位于LU／B—CAM糖蛋白上。LU／B—CAM糖蛋白的蛋白质部分由LU基因表迭。LU基因位于19q13．2—13．3,合15个外显子、14个内含子。LU基因在转录时发生变位剪接,产生2．5、4．0kb前体mRNA。LU基因在镰状红细胞、卵巢癌和结肠上皮细胞癌呈高表达。LU／B—CAM糖蛋白是层粘素特异性受体。     

A rare mutation in the primer binding region of the Amelogenin X homologue gene

Use of Amelogenin locus typing as a gender marker incorporated in STR multiplexes is common practice in forensic genetics analysis. Among 5534 Polish male individuals tested using the SGMPlus kit, one was found to lack the amelogenin X-specific homologue (0.02%). The same result was obtained with other commercial kits which also amplify the amelogenin locus, namely ProfilerPlus and PowerPlex16. When alternative amelogenin primers external to but encompassing the initial amplicon were applied, an X homologue product was seen. Sequencing of the X homologue amelogenin allele revealed C to G mutation located at the most 3′ base of the commonly used amelogenin reverse primer. To our knowledge, this mutation and failure to amplify the X homologue of the amelogenin gene has not been reported for the European population.     

线粒体16SrRNA和Cytb基因复合扩增进行种属鉴定

目的 建立一种用于种属鉴定的线粒体DNA16SrRNA基因和细胞色素b基因荧光标记复合扩增检测体系。方法 利用引物设计软件Primer 5．0对mtDNA序列的16SrRNA基因和细胞色素b基因各设计一对引物,建立复合扩增体系,分别扩增人和牛、猪、狗、鸡、草鱼5种常见动物,用310遗传分析仪对产物进行分析。结果 人和5种动物DNA扩增产物均出现两个峰。Cytb通用引物的扩增产物为人与动物的共有峰,为358bp;16SrRNA基因的扩增产物为人与动物间存在位置差异的特异峰,位于231～256bp之间。结论 该复合扩增体系可以明确区分人和5种动物样本,可用于种属鉴定。     

Spatial and temporal influences on bacterial profiling of forensic soil samples

Bacterial content may be helpful in differentiating forensic soil samples; however, the effectiveness of bacterial profiling depends on several factors, including uniqueness among different habitat types, the level of heterogeneity within a habitat, and changes in bacterial communities over time. To examine these, soils from five diverse habitats were tested over a 1 year period using terminal restriction fragment length polymorphism (TRFLP) analysis. Soil samples were collected at central locations monthly, and 10 feet in cardinal directions quarterly. Similarity indices were found to be least related among habitats, while the greatest bacterial similarities existed among collection locations within a habitat. Temporally, however, bacterial content varied considerably, and there was substantial overlap in similarity indices among habitats during different parts of the year. Taken together, the results indicate that while bacterial DNA profiling may be useful for forensic soil analysis, certain variables, particularly time, must be considered.