E2蛋白介导猪瘟病毒的细胞吸附与内化

【目的】已有研究显示,猪瘟病毒(CSFV)通过网格蛋白介导的内吞作用侵入宿主细胞,然而参与侵入过程的病毒蛋白尚待阐明。研究旨在明确E2蛋白在CSFV侵入过程中的作用。【方法】通过瞬时转染包装携带E2基因的慢病毒,将慢病毒转导悬浮293细胞构建稳定表达重组E2蛋白的悬浮293细胞系,利用亲和层析的方法纯化重组E2蛋白,同时优化表达时间以增加蛋白产量。利用SDS-PAGE和Western blotting分别鉴定了重组E2蛋白的表达水平及其反应原性。通过感染试验、吸附试验以及内化试验分别探究了E2蛋白对CSFV感染、吸附以及内化的影响。将重组E2蛋白免疫BALB/c小鼠制备多克隆抗体,通过阻断ELISA检测血清中多克隆抗体的阻断率。利用制备的多克隆抗体与CSFV感作后进行吸附和内化试验,进一步探究了E2蛋白在介导CSFV吸附和内化中的作用。【结果】通过倒置荧光显微镜观察转导慢病毒后的细胞系,与对照细胞相比可见明显的绿色荧光,表明慢病毒成功转导悬浮293细胞。SDS-PAGE结果显示,在还原和非还原条件下均出现与预期大小相符的蛋白条带,经Western blotting验证,上清中表达的重组E2蛋白能够被抗E2蛋白单克隆抗体WH303所识别,表明构建的悬浮293细胞系能够表达重组E2蛋白。通过优化表达条件,悬浮293细胞培养第8天时上清中重组E2蛋白浓度最高可达5.84μg·m L-1。可溶性蛋白阻断试验结果显示,重组E2蛋白具有阻断CSFV感染的活性。利用重组E2蛋白在病毒入侵过程中处理细胞对CSFV的感染同样具有显著抑制作用;阻断ELISA和中和试验结果显示,针对E2蛋白的多克隆抗体能够阻断CSFV感染,且具有良好的中和活性;吸附试验和内化试验证明,重组E2蛋白处理细胞能够显著抑制CSFV的内化,而利用多克隆抗体与CSFV感作后能够显著抑制其吸附。【结论】E2蛋白参与CSFV吸附和内化。     

SrfC of Pseudomonas cichorii JBC1 affects its attachment to the host surface and host tissue infection

Plant pathogenic Pseudomonas species produce effectors, toxins and cyclic lipopeptides to infect various host plants. Despite many studies aiming to understand the underlying mechanisms of virulence in Pseudomonas spp., the function of genes in the srf gene cluster including srfC, which was formerly known as HopL1, remains undetermined. To investigate the roles of srf genes in the virulence of the bacterial pathogen, each srfA, srfB, srfC and srfD gene from the srf cluster of Pseudomonas cichorii JBC1 was knocked out. When tomato seedlings were infected with the knockout mutants by flood inoculation, disease incidence was suppressed only in srfC-defective mutants (∆srfC) compared to wildtype. Interestingly, when the ∆srfC strain was inoculated directly into the apoplast of tomato leaves by vacuum infiltration, disease developed similar to that of the wildtype. In addition, the ∆srfC strain showed defective swarming motility and biofilm formation, and the attachment of ∆srfC cells on the leaf surface was significantly reduced compared to wildtype. Furthermore, droplets of the ∆srfC culture supernatant did not spread on a hydrophobic surface, unlike the wildtype. The results indicate that the SrfC protein plays important roles in swarming motility, biofilm formation and attachment/colonization on host surfaces; thus, it is beneficial for the pathogen's dispersion and entry into host tissues and consequently contributes to disease development. This study elucidates for the first time the functional role of srfC in P. cichorii virulence. The results will broaden understanding of plant and bacterial pathogen interactions.     

病毒灭毛剂等四种杀虫剂的室内药效比较试验

用病毒灭毛剂等四种杀虫剂进行室内灭杀草原毛虫药效试验。结果表明 ,病毒灭毛剂稀释 1 0 0倍的灭效为 1 0 0 %、5 0 0倍的灭效为 95 % ;杀灭灵稀释 1 0 0倍时灭效为 91 .5 % ,来福灵稀释 1 0 0倍的灭效为87.5 %。病毒灭毛剂灭效最好 ,敌杀死次之 ,杀灭灵和来福灵均较差。病毒灭毛剂是一种安全、经济、有效的生物药剂 ,充分利用和推广病毒灭毛剂对草地植被和环境保护具有重要意义     

几种物质使刺参脱离附着基效果的比较

水温4～10℃时于1L塑料瓶(容水800mL)中放入5头体质量0.5～7.4g的刺参,适应24h后加入不同体积(质量)分数的酒精(99.7%分析纯)(0%、0.5%、1.0%、2.0%、5.0%、10.0%)、氯化钾(99.7%分析纯)(0.00%、0.40%、0.60%、0.80%、0.10%、1.20%)、氯化钠(99.7%分析纯)(盐度45.0、50.0、55.0、60.0、65.0、80.0、100)和复方中草药(分苗灵)(1∶100、1∶200、1∶300、1∶400、1∶500、1∶600)溶液,观察刺参附着力的变化及化皮率。试验结果表明,酒精体积分数在2.0%以下时,刺参的自动脱落率达90%以上,高于2.0%时,化皮率明显增加。在盐度55.0以下浸泡20min后,刺参的自动脱落率随盐度的升高而上升,12℃时比8℃时增加了近30%。当分苗灵与海水的质量之比为1∶300以下时,刺参在其中浸泡30min后,脱落率最高,浸泡60min 90%以上的刺参脱落,未出现化皮、吐肠等异常现象,分苗后不用再进行消毒处理。     

酵母双杂交系统用于WSSV粘附蛋白VP37相关蛋白基因的研究

为研究WSSV的粘附蛋白VP37相关蛋白的基因，应用酵母双杂交系统Ⅲ筛选中国明对虾(Fenneropenaeus chinensis)鳃细胞全长cDNA文库。构建VP37基因的重组载体PGBKT7-VP37，把它转化酵母Y187后与含文库质粒的酵母AH109配合，在营养缺陷培养基上进行双杂交筛选。经过PCR鉴定确定了1个阳性克隆。测定该段cDNA序列并进行生物信息学分析，结果显示，此cDNA片段编码的蛋白与蛋白酶的同源性较高，该蛋白在WSSV识别并结合宿主的过程中所起的作用还有待于进一步研究。成功筛选了VP37相关蛋白的cDNA片段，为研究WSSV入侵以及感染致病的机制等方面提供了新线索。     

Influence of residents’ place attachment on heritage forest conservation awareness in a peri-urban area of Guangzhou,China

This study explored the role of community place attachment on heritage forest (feng shui woodland) conservation awareness in the peri-urban areas of Guangzhou. Questionnaire surveys were administered to glean the views of the local residents living near heritage forests. The results suggested that residents’ place attachment was significantly associated with positive perceptions, attitudes and ecologically responsible behaviours towards the heritage forests in their community. The findings revealed that traditional and cultural aspects can be an important focus of attention for local residents with higher levels of place attachment, as heritage forests offer not only ecosystem services but also feng shui value—a traditional vernacular belief in South China. This study clearly demonstrated that cultural bonding can be an important element in facilitating the environmental awareness of the local residents regarding heritage forest conservation.     

拖挂车辆挂接装置参数对车辆平顺性影响的理论研究

挂接装置是影响机组纵向行驶平顺性的重要因素,该文建立了预测挂接车辆行驶平顺性的计算模型,并从理论上研究了挂接装置刚度系数、阻尼系数、车速、路面不平度、载荷等因素对机组行驶平顺性的影响规律,为合理确定挂接装置参数提供了理论根据。     

全射流喷头射流元件附壁频率

基于全射流喷头射流元件的工作原理和内部流动状况,分析射流元件附壁频率的影响因素.利用元件两侧压差大小,建立附壁频率计算式,通过射流元件壁面脉动压力测量获得的附壁频率试验数据,频率计算值与试验值符合较好.计算与试验结果表明,附壁频率随元件腔室容积增大,信号水导管长度的变长而减小,随喷头工作压力增大,信号水流量的增大而增大,对于PXH30全射流喷头射流元件,获得信号水流量与附壁频率的线性关系式.在无因次数计算与分析中,喷嘴雷诺数对斯特劳哈数影响较小,斯特劳哈数随欧拉数的增大而减小.附壁频率的研究能指导射流元件的设计和全射流喷头工作状态的调节.     

甘蓝短散布元件对转基因表达效果的研究

为检验甘蓝短散布元件(SINE)对植物转基因表达的影响,采用PCR方法,从甘蓝基因组中克隆了一段短散布元件序列(Short Interspersed Nuclear Element,SINE),该SINE具有核基质结合区(matrix attachmentregion,MAR)的结构特征,将其构建到β-葡糖醛酸酶(β-glucuronidase,GUS)基因(uidA)的两侧翼,形成SINE调控的植物表达载体,采用农杆菌介导法,将含SINE序列和不含SINE序列的植物表达载体导入烟草中。对转基因植株进行GUS活性定量测定,结果表明,SINE表现出类似MAR的功能,可以提高外源uidA基因的表达水平,与不含SINE的转化植株相比,外源基因的平均表达水平提高了2倍,但转基因植株个体间表达水平存在较大的差异。