广西家畜布鲁氏菌病的监测分析

目的 对广西家畜布鲁氏菌病进行监测。方法 采用虎红平板凝集和试管凝集试验对2009-2011年广西14个市的16 143份家畜血清进行布鲁氏菌抗体监测;同时对1 070份家畜脾、胎衣、流产胎儿等样本以及10份布鲁氏菌试管凝集试验抗体阳性家畜的子宫或睾丸进行布鲁氏菌分离和鉴定。结果 2009年有1头种公猪,2010年有1头牛和8头山羊被检出为布鲁氏菌抗体阳性;经生化特性和PCR鉴定有1株从布鲁氏菌试管凝集试验抗体阳性羊内脏分离到的细菌被鉴定为羊种布鲁氏菌。对分离的羊种布鲁氏菌毒力基因VirB8的克隆测序结果显示,VirB8基因在布鲁氏菌种型间高度保守。结论 虽然广西家畜布鲁氏菌防治仍然达到稳定控制标准,但是需加强家畜引种和动物流通检疫工作,防止因引种和动物流通而将布鲁氏菌引入广西。     

布鲁氏菌外膜蛋白 VirB4在感染胚胎滋养层细胞中的作用分析

目的 筛选布鲁氏菌侵染牛胚胎滋养层细胞过程中与Ⅳ型分泌系统VirB4蛋白结合的潜在靶蛋白。方法 设计引物并PCR扩增布鲁氏菌的virB4基因,构建表达载体pGBKT7-VirB4,酶切鉴定,测序分析正确后,转化酿酒酵母菌感受态细胞Y187,进行自激活和毒性检测;建立布鲁氏菌侵染牛胚胎滋养层细胞模型,构建布鲁氏菌侵染牛胚胎滋养层细胞cDNA文库;采用酵母双杂交技术筛选与VirB4相互作用的滋养层细胞蛋白,实时定量PCR检测靶蛋白表达量的变化。结果 成功构建了pGBKT7-virB4诱饵质粒,转入Y187后无毒性,不能自激活;获得了布鲁氏菌侵染牛胚胎滋养层细胞cDNA文库;筛选到了13个阳性质粒,其中蛋白辅酶Q₁₀和SLC3A2在布鲁氏菌侵染后mRNA表达量均增加。结论 本试验对VirB4蛋白与宿主细胞的互作研究为进一步阐明布鲁氏菌感染宿主细胞的发病机制奠定了基础。     

MALDI-TOF-MS鉴定布鲁氏菌方法建立和评价

目的 为了快速、准确、便捷的检测和鉴定布鲁氏菌,本研究拟建立鉴定布鲁氏菌属的MALDI-TOF-MS方法,利用现场分离菌株对该方法进行特异性和敏感性评价。方法 收集布鲁氏菌标准菌株和地方流行株,应用MALDI-TOF-MS采集图谱,获取独特的蛋白质指纹图谱,汇总成标准图谱,建立布鲁氏菌鉴定数据库。并用39株布鲁氏菌菌株,对建立的数据库进行验证。结果 经数据库鉴定,39株布鲁氏菌菌株与数据库中布鲁氏菌的匹配分数全部大于2.300,均报告为布鲁氏菌,表明鉴定结果的可信度很高。 聚类分型结果表明,在蛋白质水平,MALDI-TOF-MS将测试的布鲁氏菌分成3大类。结论 MALDI-TOF-MS对布鲁氏菌进行鉴定,具有快速、准确、灵敏等优点,可以实现对布鲁氏菌属的准确鉴定,对布鲁氏菌病的临床诊断具有较高的使用价值。     

免疫-PCR检测肾综合征出血热患者血清中抗核衣壳蛋白抗体方法的建立

目的　建立高灵敏性和高特异性的免疫 -PCR方法检测肾综合征出血热 (HFRS)患者血清中抗核衣壳蛋白抗体。方法　用汉坦病毒 ( 76 - 118株 )重组核衣壳蛋白为抗原包被聚苯乙烯微滴度板 ,与经ELISA法和临床均已确诊为HFRS患者的血清进行抗原抗体特异性反应 ,以链霉亲和素为桥梁将生物素标记的抗体与生物素标记的DNA指示分子相连 ,PCR扩增DNA指示分子 ,通过检测指示分子DNA来反映肾综合征出血热患者血清中抗核衣壳蛋白抗体的水平 ,并与传统的ELISA法进行比较。结果　免疫 -PCR检测肾综合征出血热患者血清中抗核衣壳蛋白抗体的敏感性是ELISA法的 2 5 0 0 0倍 ;30例HFRS阳性患者血清免疫 -PCR检测均为阳性 ,2 0例甲型肝炎阳性血清和 2 0例正常人血清免疫 -PCR检测均为阴性。结论　免疫 -PCR方法敏感性高 ,特异性强 ,有可能作为一种高敏感性的检测方法用于HFRS的临床辅助诊断     

牛种布鲁氏菌A19-△VirB12标记疫苗双重实时荧光定量PCR方-法的建立

目的 建立一种区分牛种布鲁氏菌A19-△VirB12标记疫苗株与布鲁氏菌野毒感染株的双重荧光定量PCR方-法。方法 分别以布鲁氏菌4型分泌系统中VirB8基因、VirB12基因序列设计2对引物及探针,优化实时荧光PCR反应体系及条件。以牛种布鲁氏菌A19-△VirB12标记疫苗株、牛种布鲁氏菌A19疫苗株、羊种布鲁氏菌M5疫苗株以及猪种布鲁氏菌S2疫苗株、大肠杆菌、沙门氏菌基因组DNA进行 Realtime-PCR扩增,评价该方-法特异性。分别构建布鲁氏菌VirB12基因和VirB8基因片段阳性质粒,10倍系列稀释后进行Realtime-PCR扩增,测定该方-法的敏感性。结果 本方-法具有良好的特异性,牛种布鲁氏菌A19疫苗株、羊种布鲁氏菌M5疫苗株以及猪种布鲁氏菌S2疫苗株基因组DNA同时出现VirB8基因与VirB12基因阳性扩增,牛种布鲁氏菌A19-△VirB12标记疫苗株仅出现VirB8基因阳性扩增,大肠杆菌、沙门氏菌均未扩增出目的条带,对VirB8基因及VirB12基因片段阳性质粒的检测限分别为约10² copies/μL和10³ copies/μL。该方-法仅用于鉴别区分牛种布鲁氏菌A19-△VirB12标记疫苗株与布鲁氏菌野毒株。结论 本研究建立的布鲁氏菌双重Realtime-PCR方-法,具有良好的特异性和敏感性,为今后鉴别牛种布鲁氏菌A19-△VirB12分子标记疫苗免疫牛与自然感染牛提供技术支撑。     

布鲁氏菌AP-PCR最优反应体系的建立

目的建立布鲁氏菌的随机扩增DNA多态性的优化反应体系，用于布鲁氏菌分子流行病学研究。方法利用几种随机引物及其组合(Pl、P2、P3、P4、P5、OPLO4、P3／5、P4／5)进行随机引物PCR的反应(AP—PCR，又称RAPD)，及重复片断引物ERICIR／ERIC2的随机扩增多态性；另外针对优选引物P5和OPLO4，分别对Mg^2 浓度、dNTPs浓度、模板DNA浓度、引物的浓度及扩增过程中的退火温度等影响反应结果的因素，进行了一系列优化组合方案的试验及系统分析。结果引物Ps、OPLO4和引物组合P4，5可以得到布鲁氏菌的高分辨率的分型带谱，并建立相应的优化反应体系。结论在严格的优化反应条件下。建立简单、快捷、灵敏度高的RAPD方法，用于布鲁氏菌的DNA多态性研究，为布鲁氏菌分子流行病学的深入研究提供资料。     

牛羊猪种布鲁氏菌快速鉴别PCR方法的建立及评价

目的 建立一种可在一个反应体系中同时鉴别布鲁氏菌及牛羊猪种布鲁氏菌的快速PCR鉴别方法。方法 将布鲁氏菌及牛羊猪种布鲁氏菌的扩增引物进行优化组合,建立全新的BAMS-PCR方法;随后对该方法的特异性和灵敏度进行评价,并对现场分离的219株布鲁氏菌进行鉴别,评价其在布鲁氏菌鉴别中的应用价值。最后,用该方法对临床标本中布鲁氏菌的DNA进行扩增,评价其在临床诊断中的实用性。结果 BAMS-PCR方法可在同一反应中同时鉴别布鲁氏菌及牛种(1,2,4型),羊种(1,2和3型)和猪种(1型)布鲁氏菌,并有较好的特异性和敏感性。布鲁氏菌属,牛种,猪种和羊种布鲁氏菌引物的检测灵敏度分别为10 pg/μL,100 pg/μL, 10 pg/μL和100 pg/μL。BAMS-PCR对219株临床分离菌株的鉴定结果和常规生物分型方法的鉴定符合率为100%。经BAMS-PCR检测,97份临床血清样本仅6份为阳性,全血和组织(羊脾)样本全部为阴性。结论 BAMS-PCR是一种简便、快速、高效、准确的布鲁氏菌鉴别方法,可作为临床分离菌株的首选鉴别方法。     

布鲁氏菌微滴式数字PCR方法的建立与应用

目的 建立可对布鲁氏菌准确定量的微滴式数字PCR(Droplet digital PCR,ddPCR)方法.方法 使用以布鲁氏菌bscp 31基因为靶标的引物和探针,摸索最佳的引物和探针工作浓度,确定反应体系和扩增条件,评价所建立方法的灵敏性、特异性及对疫情相关血液标本的检测效果.结果 ddPCR反应体系中最佳引物和探...     

布鲁氏菌多重荧光定量PCR方法的建立与应用北大核心CSCD

目的探讨应用多重荧光定量PCR方法检测待检者血液、血清以及环境样本中布鲁氏菌的价值。方法根据149名待测者的就诊情况进行分类,其中疑似组75人、治疗组74人。对收集的环境和待检者的样本应用多重荧光定量PCR方法检测,结果进行统计分析。将3对引物、探针的目的基因克隆到PUC57载体上制作阳性标准品,进行灵敏度检测和标准曲线的绘制。结果通过对149名待检者的样本进行荧光定量PCR检测,单重、双重和三重方法的阳性率分别为79.2%、34.2%和27.5%。单重与双重,单重与三重荧光定量方法的阳性率差异均有统计学意义,χ^(2)值分别为55.42和73.42,P<0.05。布鲁氏菌属的单重荧光定量PCR结果生成的标准曲线为y=-3.7732x+43.188,R^(2)=0.9953,线性关系良好,最低检测范围为101 copies/μL。经过多重荧光定量方法检测的环境样本结果全部为阳性。结论单重荧光定量PCR方法灵敏度较好,建议与血清学方法联合使用对高危人群进行定期筛查,三重荧光定量方法可以在一次实验中既能完成属水平的鉴定又能区分牛种和羊种布鲁氏菌,对于环境样品具有良好的扩增效果,2种方法对于布鲁氏菌病的预防控制具有重要作用。     

基于样品检测的布鲁氏菌荧光定量PCR方法的建立

目的 本文建立一种基于样品检测的特异性好、灵敏性高的布鲁氏菌荧光定量PCR检测方法。方法 以哺乳动物beta-actin基因和外源重组质粒为内参,针对布鲁氏菌属特异性基因IS711,建立用于样品检测的布鲁氏菌荧光定量PCR方法,并验证其敏感性、特异性及稳定性,绘制标准曲线。将该方法用于哺乳动物样品检测,并与细菌分离培养检测结果比较。结果 荧光定量PCR方法对布鲁氏菌检测有良好的特异性,3对引物对阴性对照菌均无非特异性扩增;该方法用于样品检测的最低检测限为17拷贝;内外源内参同时存在条件下,布鲁氏菌荧光定量PCR的标准曲线相关系数为R²=0.997(Y=-3.12X+40.9)。将该方法直接用于181份动物样本的检测,并与分离培养结果相比,荧光定量PCR检测阳性率为11.4%,细菌分离培养检测阳性率为9.9%,且细菌分离培养阳性样品与荧光定量PCR阳性样品中编号基本一致。结论 本文建立的荧光定量PCR检测方法灵敏性高、特异性及稳定性好,可直接应用于样品的检测,检测结果受内参基因质控。     

弓形虫毒力决定因子Rop18的研究进展

Rop18是一种通过数量性状遗传位点等分析手段新近发现的弓形虫毒力决定因子,其基因的高表达与强毒I型RH株对实验小鼠模型的急性毒力直接相关。Rop18可通过其丝氨酸／苏氨酸蛋白激酶活性,干扰宿主细胞南IFN-（诱导表达的免疫相关GTP酶IRGs蛋白以及内质网相关转录因子ATF6（的抗弓形体机制,使弓形虫具备强毒力。目前对Rop18的研究已成为棒状体蛋白激酶家族的研究热点。本文综述了Rop18的发现过程、序列和结构特征、作用机制和应用前景等方面的研究进展。     

rDNA-ITS2-PCR assay for grouping the cryptic species of Anopheles culicifacies complex (Diptera: Culicidae)

Anopheles culicifacies, a predominant vector of malaria in India exists as a complex of five sibling species A, B, C, D and E, of which, except species B, all the rest are vectors with varying vectorial capacities. With a combination of PCR assays, it is possible to identify all the five members of this species complex. These assays include amplification of the rDNA-ITS2 region followed by digestion of the ITS2 amplicon using restriction enzyme, Rsa I which groups the five members of the An. culicifacies complex into two categories: species A and D forming one category and species B, C and E forming another. The samples grouped thus are then subjected to two allele-specific PCR assays (AD-PCR and BCE-PCR), which has been designed using sequence differences in the mitochondrial cytochrome oxidase II (CO II) subunit. The AD-PCR assay distinguishes species A and D, whereas the BCE-PCR assay distinguishes species B, C and E. In the present study, the differences in the ITS2 region of the five species was used to design a PCR assay which groups the five members into the same two categories as obtained after digestion of the ITS2-PCR product. This assay uses a common forward primer based on the 5.8S region and two reverse primers, which is specific for the two categories. Amplification of a PCR product of size 253bp indicates the presence of species A/D, while a product of size 409bp indicates the presence of species B/C/E. By using this ITS2 PCR assay, the three-step procedure is reduced to two cutting down the time and cost involved. The ITS2 PCR assay has been validated on specimens collected from different regions of India and the results confirm to the earlier reports on the distribution of the members of the An. culicifacies complex.     

梅毒螺旋体阿奇霉素耐药的分子生物学检测方法的建立

目的建立梅毒螺旋体阿奇霉素耐药的分子生物学检测方法。方法为了提高检测的敏感性和特异性,应用套式聚合酶链反应（Nested-PCR）扩增样本DNA;限制性酶切反应检测阿奇霉素耐药突变;并对PCR产物和酶切产物进行测序以验证实验结果。结果套式PCR能很好地扩增样本DNA,耐阿奇霉素的菌株经酶切后形成188bp和440bp两个片段,而不耐药者不发生酶切反应。测序结果显示,耐药者的2058位DNA从A突变为G,而野生型者无此突变。结论利用套式PCR和限制性酶切分析法,成功建立了梅毒螺旋体阿奇霉素耐药的分子生物学检测方法 ,为梅毒螺旋体的耐药监测以及进一步的分子流行病学研究,提供了实验室基础。     

Evaluation of a tissue factor dependent factor V assay to detect factor V Leiden: demonstration of high sensitivity and specificity for a generally applicable assay for activated protein C resistance

Resistance to the anticoagulant effects of activated protein C (APC) is now considered the most prevalent cause of inherited thrombophilia. The great majority of patients with activated protein C resistance (APCR) have a missense mutation in the factor V molecule (factor V Leiden, FVR506Q) resulting in defective inactivation of factor Va due to a loss of an APC cleavage site. The diagnosis of APCR has been based upon the inability of APC to prolong the activated partial thromboplastin (aPTT) clotting time in subjects with APCR. However, this assay has a number of deficiencies which limit its general use. We have evaluated a newly described one-stage tissue factor dependent factor V coagulation assay for APCR in 117 patients and controls and compared the results of this assay in a blinded manner to a polymerase chain reaction (PCR) based assay for the molecular defect of factor V Leiden. 43% (50 /117) of the patients studied were receiving coumadin or heparin, or had a lupus anticoagulant. The tissue factor dependent factor V assay had 100% specificity and sensitivity for factor V Leiden and successfully predicted a homozygous state in the three documented homozygotes. The PCR-based assay for factor V Leiden resulted in a single false positive assay due to a silent A to C transition at nucleotide 1692 resulting in the loss of the Mnl restriction endonuclease cleavage site. The single-stage tissue factor dependent factor V assay is a highly sensitive and generally applicable assay for APCR.     

Establishment of competitive binding assay to detect and differentiate hepatitis E virus infection

Introduction and ObjectivesThis study was undertaken to demonstrate a promising approach for detection and differentiation the serum immunoglobulin G (IgG) against hepatitis E virus (anti-HEV IgG) using a competitive binding assay established with known genotype-specific monoclonal antibodies (mAbs) 2B1 and 4C5.Materials and methodsThe mAb 2B1 derived from genotype 1 hepatitis E virus (HEV) antigen and specifically reacted with genotype 1, 2 antigens; 4C5 induced by genotype 4 HEV antigen was specific to genotypes 3, 4 antigens. The 2B1 and 4C5 were labeled with Horseradish peroxidase (HRP), respectively. Subsequently, the titers of coated antigens and HRP-conjugated mAbs for establishment of competitive binding assay were determined by enzyme linked immunosorbent assay (ELISA). And then, the competitive binding assay was performed to assess the inhibition percentage of mAbs binding to antigens inhibited by different genotypes anti-HEV IgG.ResultsThe results of competitive binding assay revealed that genotype 1 anti-HEV IgG could inhibit the binding of mAb 2B1 to genotype 1 antigen more strongly than that of mAb 4C5 to genotype 4 antigen. Whereas, the genotype 3 or 4 anti-HEV IgG could inhibit the binding of mAb 4C5 to genotype 4 antigen more remarkably than that of mAb 2B1 to genotype 1 antigen.ConclusionsThese findings provided us a valuable approach for detection and differentiation the HEV infection derived from genotypes 1, 2 (human) or genotypes 3, 4 (zoonosis).     

Activation of Rab8 guanine nucleotide exchange factor Rabin8 by ERK1/2 in response to EGF signaling

Exocytosis is tightly regulated in many cellular processes, from neurite expansion to tumor proliferation. Rab8, a member of the Rab family of small GTPases, plays an important role in membrane trafficking from the trans-Golgi network and recycling endosomes to the plasma membrane. Rabin8 is a guanine nucleotide exchange factor (GEF) and major activator of Rab8. Investigating how Rabin8 is activated in cells is thus pivotal to the understanding of the regulation of exocytosis. Here we show that phosphorylation serves as an important mechanism for Rabin8 activation. We identified Rabin8 as a direct phospho-substrate of ERK1/2 in response to EGF signaling. At the molecular level, ERK phosphorylation relieves the autoinhibition of Rabin8, thus promoting its GEF activity. We further demonstrate that blocking ERK1/2-mediated phosphorylation of Rabin8 inhibits transferrin recycling to the plasma membrane. Together, our results suggest that ERK1/2 activate Rabin8 to regulate vesicular trafficking to the plasma membrane in response to extracellular signaling.Rab GTPases constitute the largest family of small GTPases and are important regulators of membrane trafficking in eukaryotic cells (1). Rab proteins cycle between the GDP- and GTP-bound states. Guanine nucleotide exchange factors (GEFs) stimulate the dissociation of GDP from Rab proteins to allow subsequent GTP loading, thus switching the Rab proteins to their active conformation (2–6). Although a number of GEFs have been identified as critical regulators of specific Rab GTPases, how these GEFs are regulated in cells remains unclear.Rab8a and its paralogue, Rab8b (henceforth referred together as “Rab8”) are members of the Rab family of proteins that mediate membrane trafficking from the trans-Golgi network (TGN) and recycling endosomes to the plasma membrane (7–12). Rabin8 is a GEF and major activator of Rab8 in cells (8, 13). To understand the molecular mechanism of Rab8 activation, it is pivotal to understand how Rabin8 itself is regulated in cells. Pioneering studies in yeast demonstrated that Sec2p, the yeast homolog of Rabin8, is a direct downstream effector of Ypt32p (the yeast homolog of Rab11), which mediates the budding of vesicles from TGN (14). Ypt32p, together with phosphatidylinositol 4-phosphate [PI(4)P], mediates the membrane recruitment of Sec2p (15). Similar to its yeast counterparts, Rabin8 is a direct downstream effector of Rab11, which mediates the generation of secretory vesicles from TGN and recycling endosomes (16, 17). Our previous studies have shown that Rab11 kinetically stimulates the GEF activity of Rabin8 toward Rab8 (16, 18). Although studies in yeast and mammalian cells have revealed different types of regulation of Sec2p/Rabin8 by Ypt32p/Rab11 (i.e., membrane recruitment vs. kinetic activation), the Rab cascade serves to coordinate the generation of vesicles from the donor compartments to its subsequent transport and fusion (3, 4, 19, 20). The Rab11-Rabin8-Rab8 signaling cascade is implicated in a number of cell biological processes, such as primary ciliogenesis and cystogenesis (16, 17, 20, 21). Despite the recent progress in this field, the molecular nature of Rabin8 activation remains unknown.In this study, using biochemical and biophysical approaches, we demonstrate that Rabin8 has an autoinhibitory conformation. We identify Rabin8 as a direct phospho-substrate of ERK1/2, the principle kinases in the Ras-MEK-ERK cascade in response to EGF signaling. ERK1/2 phosphorylation relieves the autoinhibition and kinetically stimulates the GEF activity of Rabin8 toward Rab8, thus regulating vesicular trafficking to the plasma membrane.     

嗜水气单胞菌毒力因子特性及作用机理研究进展

嗜水气单胞菌是一类广泛存在于水环境的常见菌,已成为人-兽-鱼共患病原菌。近年来,对其研究越来越多,对其毒力因子的研究,有利于深入了解其致病机理,探索药物和免疫防治的有效方法。本文综述了嗜水气单胞菌的毒力因子及其特性、致病机理等内容。     

抗ALT抗体间接ELISA检测方法的建立

目的建立免疫小鼠体内抗人丙氨酸氨基转移酶(ALT)抗体的间接ELISA检测方法,以期作为融合后杂交瘤细胞株的筛选体系。方法利用人ALT为包被抗原,以1∶40 000稀释的辣根过氧化物酶标记的羊抗鼠IgG为二抗,采用棋盘滴定法确定抗原和对照血清(阴性、阳性)最佳工作浓度及ELISA封闭液的浓度建立ELISA体系。结果包被抗原和血清的适宜浓度或稀释度分别为1∶5 000和1∶1 000;用4%BSA封闭18 h~24 h效果最好。结论建立的ELISA方法稳定,简便易行,能准确检测免疫小鼠血清抗体的效价,并可用于抗ALT单克隆抗体杂交瘤细胞株的筛选。