贝氏柯克斯体抑制宿主单核细胞的碳源代谢

贝氏柯克斯体是一种专性细胞内寄生的革兰阴性小球杆菌,为Q热的病原体。本文通过比较贝氏柯克斯体感染单核细胞和非感染单核细胞的蛋白表达谱,研究该病原体感染引发宿主细胞表达差异的蛋白。用贝氏柯克斯体I相毒株感染人单核细胞（THP一1）,采用等电聚焦和SDS-PAGE双向电泳对感染单核细胞和非感染单核细胞进行比较蛋白质组学研究,用电泳图谱分析软件发现它们之间的差异表达蛋白,用基质辅助激光解析电离飞行时间质谱仪鉴定差异表达蛋白。结果发现,电泳图谱分析发现137个差异蛋白,质谱分析鉴定到15种差异蛋白与细胞生理代谢有关。这些蛋白的表达变化说明贝氏柯克斯体感染单核细胞使宿主细胞碳源代谢受到抑制,由此可能影响单核细胞正常杀菌功能,而有利于贝氏柯克斯体在细胞内的生存。     

贝氏柯克斯体血清学反应蛋白芯片的研制

贝氏柯克斯体为Q热的病原菌.本研究对19个贝氏柯克斯体血清学反应阳性蛋白基因进行PCR扩增,将扩得的基因片段与表达载体连接,将重组质粒转化大肠杆菌并表达目的蛋白,采用Ni-NTA亲和层析柱纯化目的蛋白.将纯化的重组蛋白点在醛基化玻片上制备蛋白芯片.将贝氏柯克斯体新桥株实验感染小鼠血清分别对蛋白芯片进行血清学分析,结果显...     

基于CRISPRi的贝氏柯克斯体dotB基因沉默

目的 更简便快捷地研究贝氏柯克斯体基因功能。方法 基于CRISPRi原理构建携带化脓链球菌dCas9的重组质粒,将靶向贝氏柯克斯体dotB基因的sgRNA序列插入重组质粒中并将质粒经电转化导入贝氏柯克斯体,利用脱水四环素(aTC)诱导dCas9表达以抑制贝氏柯克斯体dotB基因的转录,从而建立基于CRISPRi系统的贝氏柯克斯体基因沉默技术。结果 在aTC诱导下,该CRISPRi系统中dCas9可正常表达,dotB在转录水平和蛋白水平均被抑制,dotB表达抑制后贝氏柯克斯体胞内生长繁殖水平降低。结论成功建立起基于CRISPRi的贝氏柯克斯体的基因沉默技术,为研究贝氏柯克斯体特定基因的生物学功能研究提供了技术支撑。     

云南玉龙鼠类携带贝氏柯克斯体的基因分型研究

为更好地了解云南省鼠类中贝氏柯克斯体Coxiella burnetii的流行情况,以便制定针对Q热的预防和控制措施,本研究采用巢式PCR方法检测云南省玉龙县捕获的鼠类携带贝氏柯克斯体的情况,并对阳性样本的贝氏柯克斯体进行多间隔区序列分型(multispacer sequence typing, MST)和多位点可变数目的串联重复分析(multilocus variable number tandem repeats analysis, MLVA)。结果显示,在玉龙共捕获到49只野生鼠类,其中24只携带贝氏柯克斯体(48.98%, 24/49)。基因分型结果显示阳性样本携带贝氏柯克斯体的MST型别可分为MST16和一种新的MST基因型别,但未能获得最终的MLVA基因型别。本研究表明云南玉龙的鼠类携带新的贝氏柯克斯体MST基因型别,可为我国Q热的溯源提供一定的理论依据和数据支撑。     

人类基因组DNA单核苷酸多态性的检测方法

单核苷酸多态性 (SNP)作为新的遗传标记对基因定位及相关疾病的研究意义重大。本文对近年来 9种SNP检测方法的原理、应用及优缺点 ,包括基于 FRET原理的 Taqman法和分子灯塔法 ;基于分子杂交技术的寡核苷酸连接分析、等位基因特异性寡核苷酸探针杂交法、动态等位基因特异性杂交法及 DNA芯片法 ;及质谱法、变性-高压液相色谱法和单个碱基延伸标记法进行综述     

利用基因芯片对少量细胞进行非整倍体检测的影响因素分析

目的 评估利用微阵列-比较基因组杂交技术检测少量细胞非整倍体的准确率及相关影响因素.方法 结合10K 2.0单核苷酸多态性(SNP)基因分型芯片平台与多重置换扩增技术(MDA),计算并比较扩增模板为1～10个细胞时各染色体拷贝数分析的准确率,评估影响芯片平台的拷贝数准确率的有关因素及其对染色体拷贝数异常的实际分辨率.结果 使用MDA-DNA作参照时,拷贝数分析的准确率[(79.3±2.9)%～(100.0±1.7)%]高于使用gDNA作参照时的准确率[(66.7±3.4)%～(89.5±3.3)%](P<0.001).随着模板增加至10细胞,芯片可在1 M平滑化处理的同时获得94%的分析准确率.对于单细胞MDA产物,缺失型非整倍体具有比获得型非整倍体更高的分析准确率[1C组(71.9±4.1)%～(95.5～2.0)%;1C-sDel-4组(81.4±3.7)%～(99.6±2.8)%],各组间差异均有统计学意义(P<0.01).结论 10K 2.0 SNP基因分型芯片平台结合多重置换扩增技术可有效对少量细胞进行非整倍体检测,选择MDA-DNA作为参照是提高分析准确率的最关键因素,而增加细胞模板与提升分析中的平滑化参数(即降低芯片的分辨率要求)也有助于改善拷贝数准确率,在同样的条件下,芯片更容易准确检出缺失型非整倍体.     

DNA倍体分析联合HPV-DNA23分型检测对ASC-US的诊断价值

目的 探讨DNA倍体分析联合HPV-DNA 23分型检测(HPV检测)对子宫颈液基细胞学涂片(liquid-based preparation,LBP)报告的"不能明确意义的非典型鳞状细胞(atypical squamous cells of undetermined significance,ASC-US)"的诊断价...     

乙肝病毒特异性核酶的构建及体外切割活性的初步研究

目的对我国伯氏考克斯体（Ｃｂ）分离株的２３ＳｒＲＮＡ基因插入片段（２３ＳＩＶＳ）作核酸序列分析。方法用聚合酶链反应（ＰＣＲ）扩增７株Ｃｂ中国分离株和２株外国参考株的２３ＳＩＶＳ,用双脱氧核苷酸法对扩增产物进行测序。结果重庆,四川,内蒙分离株以及Ｈｅｎｚｅｒｌｉｎｇ和Ｇｒｉｔａ等６株的ＩＶＳ序列均相同,ＹＳ－８,ＹＳ－９和ＹＨ－１１等云南分离株的ＩＶＳ与前６株的比较仅少一个７个碱基的重复序列。结论Ｃｂ２３ＳＩＶＳ为高度保守基因片段,与高度异质性的钩端螺旋体的同源性基因片段不同;云南株和其它６株的ＩＶＳ差别与Ｃｂ分离株的地理分布的不同有关,云南分离株可能为一亚种     

Survival of Coxiella burnetii within free-living amoeba Acanthamoeba castellanii

Objective   To determine whether Coxiella burnetti is able to survive within free-living ameoba.
Methods   When C. burnetii was co-cultivated with the free-living amoeba species Acanthamoeba castellanii .
Results   Viable bacteria were detected for 18 days. Microscopic studies confirmed the presence of bacteria within vacuoles, some of which appeared to be spore-like forms.
Conclusion   These results indicate that free-living amoebae could provide an intracellular niche for the spore formation and survival of C. burnetii in the environment.     

Q热立克次体单克隆抗体的产生及其特性的研究

Q热立克次体存在相变异的现象。随着相变异的出现,Q热立克次体的生物学特性、理化性质和抗原性等方面均有变化。这种变化的发生与Q热立克次体的表面结构和抗原组分的改变有密切关系。为了在分子水平上研究Q热立克次体,阐明相变异的机制或研究Q热立克次体的致病性、血清免疫学     

Application of fluorescent in situ hybridisation for demonstration of Coxiella burnetii in placentas from ruminant abortions

A fluorescent in situ hybridisation (FISH) assay targeting 16S ribosomal RNA was developed for detection of the zoonotic bacterium Coxiella burnetii in formalin-fixed, paraffin-embedded tissue, and applied on placentas from ruminant abortions. The applicability of the FISH assay was compared to immunohistochemistry (IHC) using human positive control serum in 12 cases of C. burnetii-associated placentitis as well as 7 negative control tissue samples. In all 12 cases the bacterium was detected within trophoblasts as well as free in the placental debris by both FISH and IHC. Extensive and significant infection by C. burnetii was revealed in 10 of the cases, whereas a slighter and focal distribution of the bacterium was observed in two cases. 90 aborted placentas from Danish ruminants were investigated by FISH. C. burnetii was detected in one bovine case only, representing the first confirmation of C. burnetii in Danish animals. The study shows that FISH targeting 16S ribosomal RNA is a feasible diagnostic tool for detection of C. burnetii in tissue samples and fully comparable to IHC.     

Vanin-1 controls granuloma formation and macrophage polarization in Coxiella burnetii infection

Q fever is caused by Coxiella burnetii, a bacterium that survives in MPhi. Vanin-1 is a membrane-anchored pantetheinase that controls tissue inflammation. Consequently, Vanin-1-deficient mice represent a unique mouse model in which stress-induced inflammation is limited by the reaction of resident tissue cells. To investigate the contribution of host tissues in the control of a bacterial infection, we infected Vanin-1-deficient mice with C. burnetii. Mortality and morbidity of mice were not affected. The lack of Vanin-1 had no effect on C. burnetii clearance but decreased the formation of granulomas in spleen and liver. Granuloma formation depends upon MPhi recruitment and activation in these tissues. Whereas the former was slightly impaired in mutant mice, the lack of Vanin-1 significantly affected the activation pattern of BM-derived MPhi stimulated by C. burnetii. While their microbicidal activity against C. burnetii was moderately impaired, they exhibited decreased inducible nitric oxide synthase (iNOS) and MCP-1 gene expression, and increased IL-10 and arginase expression. In liver from mutant mice, increased arginase expression and decreased expression of MCP-1 and iNOS were reminiscent of MPhi data. These results suggest a role of Vanin-1 in granuloma formation in response to C. burnetii by skewing MPhi activation toward an M2 program.     

青海省祁连县牦牛和山羊的莱姆病螺旋体和Q热柯克斯体血清抗体调查

为了解我国青海省祁连县家畜中莱姆病螺旋体及Q热柯克斯体的自然感染及复合感染状况,收集该地牦牛和山羊的血样标本,采用间接免疫荧光法分别检测血清莱姆病螺旋体及Q热柯克斯体抗体.共收集了200份血样,山羊及牦牛各100份,两种家畜均为当地的主要家畜.结果显示,牦牛血清中有11份为莱姆病螺旋体抗体阳性,23份为Q热柯克斯体抗体阳性 山羊血清中有17份为莱姆病螺旋体抗体阳性,27份为Q热柯克斯体抗体阳性 3只山羊和7头牦牛血清为双抗体阳性.结果提示该地区为莱姆病和Q热的自然疫源地,牦牛和山羊可能隐性感染了Q热柯克斯体和（或）莱姆病螺旋体.     

Seroprevalence of Coxiella burnetii and Brucella abortus among pregnant women

Coxiella burnetii and Brucella abortus are two intracellular bacteria implicated in zoonotic miscarriage. In the present study, C. burnetii and B. abortus seroprevalence was compared among women from London with and without miscarriage. Coxiella burnetii seroprevalence was high (4.6%, 95% CI 2.8–7.1) despite the rare apparent exposure of this urban population. Only two patients exhibited anti- B. abortus antibodies. As a result of the risk of chronic Q fever with endocarditis and/or hepatitis, the mode of Coxiella burnetii infection in this population merits further investigation.