Characterization and application of nucleocapsid protein of porcine reproductive and respiratory syndrome virus expressed in E.coli

从已构建的PRRSV ORF7重组质粒pUCm-T-ORF7中用PCR扩增ORF7基因亚克隆至表达载体pGEX-4T-3,构建重组表达质粒pGEX-4T-3-ORF7并转化大肠杆菌。经SDS-PAGE及Western blotting鉴定,成功表达了谷胱苷肽转移酶(GST)融合的核衣壳蛋白(N),重组N蛋白表达量约为菌体总蛋白的35%,主要以可溶的形式存在,且能形成同源二聚体。重组N蛋白经谷胱苷肽凝胶(glutathione sepharose 4B)亲和层析后得到高度纯化,并将该蛋白作为抗原建立了间接ELISA检测方法。利用该方法对某猪场76份猪血清进行检测并将结果与IDEXX公司ELISA试剂盒检测结果作比较,2种方法的总符合率达93.4%,检测结果之间差异不显著(P>0.05)。结果表明大肠杆菌表达的重组GST融合N蛋白具有良好的抗原性,因而有望利用该重组蛋白开发为试剂盒应用于临床PRRSV抗体的检测。Porcine reproductive and respiratory syndrome virus(PRRSV) FJ-1 was a newly identified virus isolate in Fujian province.The ORF7 gene of FJ-1 was amplified by RT-PCR and cloned into vector pUCm-T,then subcloned into expression vector of pGEX-4T-3.The recombinant GST-tagged nucleocapsid protein(rN) was expressed in E.coli and the molecular weight was approximately 38 000 as identified by SDS-PAGE and Western blotting.Expression level of the rN protein was approximately 35% of the total bacterial protein and mostly soluble.The rN protein was purified to homogeneity using GST affinity chromatography.Analysis of the rN protein under nonreducing conditions revealed that similar to native protein,the rN protein also possesses homo-dimerization property.An indirect enzyme-linked immunosorbent assay(ELISA) for detecting PRRSV antibody was developed using the purified rN protein as antigen.76 serum samples were detected by the method and the result was compared with that using IDEXX PRRS HerdChek ELISA kit.An identity of 93.4% was revealed between the two ELISA kits and no significant difference(P>0.05) was detected.The data indicate the rN protein has the potential usefulness for detection of the PRRSV antibodies.福建省科技攻关计划重点资助项目(2003N083);; 厦门市科技攻关计划重点资助项目(3502Z20031054

基于聚焦形貌恢复的刮板输送机链轮轮齿序列图像采集装置

由于现有刮板输送机链轮轮齿磨损测量手段不具备过程简捷、高精度、高效率和低成本等优势，基于聚焦形貌恢复，设计了一种链轮轮齿序列图像采集装置。通过控制该装置运动模块移动，实现工业相机与某个轮齿轴向与周向的对中，进而控制轮齿径向步进电机每步进一次，采集一帧照片，重复此循环过程100次，完成该轮齿序列图像的采集。实验测试结果表明，该序列图像采集装置能够快速地完成轮齿对中并采集到清晰的轮齿序列图像，可为刮板输送机链轮磨损测量提供硬件支撑和积累图像数据样本

Prokaryotic Expression and Diagnostic Application of ORF2 of Porcine Circovirus Type Ⅱ

II型猪圆环病毒(PCV2)是引起断奶仔猪多系统衰竭综合症(PMW S)的重要病原.本研究用PCR以构建的PCV2ORF2重组质粒pBS-T-ORF2为模板,扩增截短的ORF2(tORF2)基因亚克隆至表达载体pGEX-4T-3,构建重组表达质粒pGEX-tORF2并转化大肠杆菌BL21(DE3).经SDS-PAGE及W estern b lot鉴定,成功表达了谷胱甘肽S转移酶(GST)融合的tORF2,重组蛋白分子量约为45ku,表达量约为菌体总蛋白的20%,重组蛋白具有良好的免疫学活性.用tORF2重组蛋白对20份临床猪繁殖与呼吸障碍综合征(PRRS)阳性猪血清进行W estern b lot检测,有4份(20%)血清显示PCV2抗体阳性,说明PCV2与PRRSV存在共同感染现象.本研究初步证明表达的tORF2重组融合蛋白可以应用于临床检测.Porcine circovirus type Ⅱ(PCV2) plays a crucial role in the pathogenesis of post-weaning multisystemic wasting syndrome(PMWS) in swine.The ORF2 gene encoding the capsid protein of PCV2 was amplified by PCR and cloned into plasmid pBS-T to construct recombinant plasmid pBS-T-ORF2,which was used as template to amplify the truncated ORF2(tORF2).The tORF2 gene was consequently cloned into the expression vector of pGEX-4T-3 for prokaryotic expression in E.coli.Expression level of the recombinant GST-tagged tORF2 protein reached approximately 20% of the total bacterial proteins and its molecular weight was approximately 45 ku identified by SDS-PAGE and Western blot.The recombinant protein was used as antigen to establish a Western blot diagnostic assay.Twenty PRRS positive swine serum samples were detected by this Western blot assay.The positive rate of these sera was 20%(4/20),indicating that coinfection of PRRSV and PCV2 exist in swine.Taken together,the recombinant tORF2 protein reporteded in the present study may play a critical role in the detection of PCV2 antibodies immunologically.福建省科技攻关计划重点项目(2003N083);; 厦门市科技攻关计划重点项目(3502Z20031054)资

JUNO Sensitivity on Proton Decay p→νˉK+p\to \bar\nu K^+ Searches

The Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid scintillator detector designed to explore many topics in fundamental physics. In this paper, the potential on searching for proton decay in $p\to \bar\nu K^+$ mode with JUNO is investigated.The kaon and its decay particles feature a clear three-fold coincidence signature that results in a high efficiency for identification. Moreover, the excellent energy resolution of JUNO permits to suppress the sizable background caused by other delayed signals. Based on these advantages, the detection efficiency for the proton decay via $p\to \bar\nu K^+$ is 36.9% with a background level of 0.2 events after 10 years of data taking. The estimated sensitivity based on 200 kton-years exposure is $9.6 \times 10^{33}$ years, competitive with the current best limits on the proton lifetime in this channel

JUNO sensitivity on proton decay p → ν K + searches*

The Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid scintillator detector designed to explore many topics in fundamental physics. In this study, the potential of searching for proton decay in the $p\to \bar{\nu} K^+$ mode with JUNO is investigated. The kaon and its decay particles feature a clear three-fold coincidence signature that results in a high efficiency for identification. Moreover, the excellent energy resolution of JUNO permits suppression of the sizable background caused by other delayed signals. Based on these advantages, the detection efficiency for the proton decay via $p\to \bar{\nu} K^+$ is 36.9% ± 4.9% with a background level of $0.2\pm 0.05({\rm syst})\pm$$0.2({\rm stat})$ events after 10 years of data collection. The estimated sensitivity based on 200 kton-years of exposure is $9.6 \times 10^{33}$ years, which is competitive with the current best limits on the proton lifetime in this channel and complements the use of different detection technologies