4株滑液囊支原体分离株P80脂蛋白的基因克隆及序列分析

为研究滑液囊支原体（Mycoplasma synoviae,MS）P80家族脂蛋白的功能及其异同,通过PCR方法从4株滑液囊支原体分离株中分别扩增P80-1,P80-2,P80-3蛋白的基因序列并进行测序,通过生物信息学方法对P80家族脂蛋白的蛋白序列进行分析,寻找保守结构域、抗原表位,并预测功能.结果表明,P80家族脂蛋白均为碱性亲水性稳定蛋白,在N端具有较强疏水性,只有P80-2蛋白具有跨膜螺旋区;P80家族脂蛋白信号肽的位置与疏水区结果相一致;3条P80蛋白具有12个相同的保守结构域及2～4个不同的结构域;有31～32个抗原表位;二级结构均以α-螺旋和无规则卷曲为主;三级结构预测显示,P80-1蛋白具有ABC转运蛋白的功能,P80-2蛋白为周质结合蛋白Ⅱ型,P80-3蛋白具有细胞黏附功能.研究结果可为MS P80家族脂蛋白功能的深入研究以及MS致病机制、病原诊断和疫苗研制提供参考.     

石家庄市猪链球菌分离鉴定及快速诊断方法研究

从石家庄市地区各县市猪场送检的疑似病例中分离得到72株猪源链球菌,选择其中具有一定区域、特定致病类型的代表性菌株18株,进行了病原分离和分群鉴定,并与标准株进行了对比试验.初步证实本地区流行菌株仍以兰氏分群C群及D群居多,以C群链球菌为主占50%,但D群有上升趋势占33.3%,此外还出现1株G群占5.6%,另有2株未定群的猪链球菌,其分布无明显的地域性.为检测石家庄地区SS分离株的毒力因子,选取了猪链球菌8种主要毒力因子基因,设计并合成8对引物,用PCR方法进行检测.结果显示:已知21株SS2菌株中90.5%表现为cps2/gdh+/gapdh+/ef+/mrp+/sly+/fbps+/orf2+;送检的疑似SS2的病例中,仅有10%左右表现为cps2/gdh+/gapdh+/ef+/mrp+/sly+/fbps+/orf2+.可见,石家庄地区的猪链球菌与国内其他地区SS2分离株的基因型不同,表明石家庄目前SS2的主要流行菌株是具有部分毒力因子的弱毒菌株.     

2型猪链球菌中国强毒株05ZYH33 ofs N-片段基因敲除株的构建

通过生物信息学分析2型猪链球菌（Streptococcus suis serotype 2,S．suis2）中国强毒株05ZYH33的基凶组,预测并发现猪链球菌血清浑浊因子（Opacityfactor of S．suis,OFS）的编码基因ofs.为了构建ofsN-末端片段ofs^37-683的基因敲除株,首先构建中间为壮观霉素抗性基因,两侧为ofs^37-683上、下游同源序列的基因敲除质粒,并对该质粒进行PCR和酶切鉴定．根据同源重组的原理,通过电转化方法筛选得到ofs^37-683基因敲除株．PCR、测序和RT—PCR分析结果均显示ofs^37-683已完全被壮观霉素抗性基因替代,证实ofs^37-683基因敲除株构建成功．该敲除株的获得为进一步研究ofs在猪链球菌2型致病机制中的作用奠定基础．     

棉花黄萎病菌T-DNA插入突变体功能基因及生物学性状分析

【目的】分析棉花黄萎病致病菌相关基因的功能揭示其中致病机制。【方法】通过ATMT法构建了大丽轮枝菌(Verticillium dahliae)650个T-DNA插入突变体。利用hiTAIL-PCR技术获得17株T-DNA插入体侧翼基因序列,通过blast比对分析表明,其中16个基因序列编码酶或蛋白。【结果】突变体的生物学性状与野生型菌株Vd991相比,出现黄色菌丝型2株,中间菌丝型6株;部分突变体在生长速率(7株)、产孢量(14株)和粗毒素产量(4株)上都发生了具有统计学意义的变化(p0.05);致病力测定表明,9株突变体的致病力较野生型有所降低,在p0.05水平具有统计学意义。【结论】该研究为大丽轮枝菌致病基因的筛选和鉴定提供了理论基础。     

肠道病毒71型外壳蛋白VP3在Pichia.pastoris酵母中的表达

利用逆转录聚合酶链式反应(RT-PCR)的方法克隆肠道病毒71型SHZH98株外壳蛋白VP3基因,连接T载体,经序列测定后,构建酵母分泌型表达质粒pPIC9K/VP3,经序列测定证实-factor信号肽和VP3的序列和阅读框正确后,用SacI酶切使之线性化,电转化法将目的基因整合到宿主菌GS115基因组上;经转化子表型筛选和PCR分析鉴定后,甲醇诱导,筛选到5株高效表达VP3蛋白的工程菌株.ELISA实验表明:重组蛋白VP3具有较好的免疫原性.     

角毛壳菌泛素结合酶基因的克隆和特性分析

利用BlastX将角毛壳菌(Chaetomium cupreum)几丁质诱导的菌丝体cDNA文库中的ESTs序列与NCBI的Nr(Non-redundant)数据库进行相似性搜索,获得泛素结合酶(ubiquitin conjugating enzyme,E2)基因的ESTs.以此结果设计引物进行RT-PCR,克隆该基因的cDNA序列(CcE2).该序列长600bp,开放阅读框444bp,编码147个氨基酸组成的多肽,蛋白分子量为16.6ku,是亲水性蛋白.BlastP分析表明该基因进化相当保守,与其他真菌的E2具有高度的相似性;三维结构分析显示CcE2仅包含一个功能结构域,在结构域的中央是决定CcE2活性的半胱氨酸残基活性位点.     

柳树痂囊腔菌的基因组测序和比较基因组分析

【目的】报道柳树痂囊腔菌(Elsinoë murrayae)的全基因组序列,与甜橙痂囊腔菌杨树致病型的基因组进行比较分析,为阐述柳树痂囊腔菌的致病和适应性机制提供参考。【方法】采用Illumina HiSeq 2500 测序仪对柳树痂囊腔菌的全基因组序列进行测序,预测蛋白编码基因,筛选与致病相关的碳水化合物活性酶基因、小分泌蛋白基因和次生代谢产物基因簇。根据痂囊腔属真菌基因的直系同源关系,筛选柳树痂囊腔菌和甜橙痂囊腔菌杨树致病型之间共有特异性的基因和二者之间差异基因,并进行GO富集分析。鉴定柳树痂囊腔菌的交配类型位点,使用特异性引物进行PCR,检测分离株的交配类型。【结果】组装获得了1个20.7 Mb基因组,完整度99%;预测出8 256个蛋白编码基因,其中包括486个碳水化合物活性酶基因,193个小分泌蛋白基因和16个次生代谢产物基因簇 (GenBank登录号:NKHZ00000000)。系统进化和共线性分析显示柳树痂囊腔菌和甜橙痂囊腔菌杨树致病型亲缘关系最近,两者之间具有12个在其他痂囊腔菌中没有的共有特异性基因。两个真菌的比较基因组分析,筛选出752和1 746个差异基因,主要参与碳水化合物代谢和毒素代谢的生物学过程。已有分离株的交配类型均为MAT1-2。【结论】获得柳树病原真菌-柳树痂囊腔菌的基因组,筛选出痂囊腔菌中负责寄主适应性的候选基因,分析了柳树痂囊腔菌交配系统,这可为柳树病害防治和柳树-病原真菌相互作用研究提供关键信息。     

抗原处理相关转运体( TAP) 的结构和功能研究进展

摘要: 抗原处理相关转运体( TAP) 属于 ABC( ATP-binding cassette) 转运体超家族的 B 亚家族,是由主要组织相容性 复合体( Major histocompatibility complex ,MHC) 上 2 个紧密连锁的基因 TAP1 和 TAP2 编码蛋白组成的异二聚体,位 于内质网和顺式高尔基膜上,功能是将抗原肽从细胞质转运进入内质网池,组装到 MHC I 类分子上构成复合体。 TAP 蛋白的缺乏和突变会影响抗原肽的提呈,最终损害免疫应答功能。     

胸膜肺炎放线杆菌Δlip40回复突变株的构建及鉴定

胸膜肺炎放线杆菌(APP)可引起猪胸膜肺炎,在一定程度上阻碍了养猪业的发展.目前,APP基因组中已得到有效鉴定的基因不足10%.为建立有效的APP功能基因研究体系,本文以前期构建的血清1型APP SLW01株外膜脂蛋白基因缺失突变株Δlip40为亲本,通过电转化法将携带有完整lip40基因的穿梭质粒pJFF-lip40导入Δlip40中,通过氯霉素抗性筛选获得回复突变株CΔlip40.对CΔlip40的生物学特性进行了初步分析发现,穿梭质粒可在APP中稳定遗传,并且APP生长能力未受穿梭质粒转入的影响.此外,SLW01和Δlip40对氯霉素高度敏感,但回复突变株CΔlip40较前两种菌株对氯霉素抗性明显增强,表明穿梭质粒pJFF224-XN抗性基因可在APP菌株中稳定表达.回复突变株CΔlip40的成功构建为分析脂蛋白Lip40致病机制提供了有效对照材料,本研究中建立的互补菌株构建系统以及APP抗性评价方法将有助于今后深入研究APP生物学特性及基因功能.     

棉花黄萎病菌 T-DNA 插入突变体功能基因及生物学性状分析

【目的】分析棉花黄萎病致病菌相关基因的功能揭示其中致病机制。【方法】通过 ATMT 法构建了大丽轮枝菌(Verticillium dahliae ) 650 个 T-DNA 插入突变体。利用 hiTAIL-PCR 技术获得 17 株 T-DNA 插入体侧翼基因序列,通过blast 比对分析表明,其中 16 个基因序列编码酶或蛋白。【结果】突变体的生物学性状与野生型菌株 Vd991 相比,出现黄色菌丝型 2 株,中间菌丝型 6 株;部分突变体在生长速率( 7 株)、产孢量( 14 株)和粗毒素产量( 4 株)上都发生了具有统计学意义的变化(p <0.05 );致病力测定表明, 9 株突变体的致病力较野生型有所降低,在 p <0.05 水平具有统计学意义。
     

Platelet secretory products inhibit lipoprotein metabolism in macrophages

Macrophages possess a receptor that binds low-density lipoproteins (LDL) containing lysine residues modified by acetylation (Ac LDL), acetoacetylation (AcAc LDL) or malondialdehyde treatment. This receptor (referred to as the Ac LDL receptor or scavenger receptor) internalizes the bound lipoprotein. As a consequence, massive amounts of cholesteryl esters accumulate so that macrophages in culture resemble foam cells found in atherosclerotic lesions. In an effort to identify an unmodified mammalian macromolecule that binds to the Ac LDL receptor, we investigated whether platelet secretory products affect the receptor-mediated endocytosis of chemically modified lipoproteins. Platelets are a potential source of such activity because they exist in close association with foam cells in developing atherosclerotic lesions. Our study demonstrates that human blood platelets secrete a product that inhibits the binding and uptake of AcAc LDL by mouse peritoneal macrophages and the subsequent accumulation of cholesteryl esters. This is the first indication that an endogenous macromolecule interacts with Ac LDL receptor on macrophages.     

Antibacterial properties and corrosion resistance of AISI 420 stainless steels implanted by silver and copper ions

Silver or copper ions are often chosen as antibacterial agents. But a few reports are concerned with these two antibacterial agents for preparation of antibacterial stainless steel (SS). The antibacterial properties and corrosion resistance of AISI 420 stainless steel implanted by silver and copper ions were investigated. Due to the cooperative antibacterial effect of silver and copper ions, the Ag/Cu implanted SS showed excellent antibacterial activities against both Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) at a total implantation dose of 2×1017 ions/cm2. Electrochemical polarization curves revealed that the corrosion resistance of Ag/Cu implanted SS was slightly enhanced as compared with that of un-implanted SS. The implanted layer was characterized by X-ray photoelectron spectroscopy (XPS). Core level XPS spectra indicate that the implanted silver and copper ions exist in metallic state in the implanted layer.     

Graphite-polypyrrole coated 316L stainless steel as bipolar plates for proton exchange membrane fuel cells

316L stainless steel (SS 316L) is quite attractive as bipolar plates in proton exchange membrane fuel cells (PEMFC). In this study, graphite-polypyrrole was coated on SS 316L by the method of cyclic voltammetry. The surface morphology and chemical composition of the graphite-polypyrrole composite coating were investigated by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). A simulated working environment of PEMFC was applied for testing the corrosion properties of graphite-polypyrrole coated SS 316L. The current densities in the simulated PEMFC anode and cathode conditions are around 3×10-9 and 9×10-5 A·cm-2, respectively. In addition, the interfacial contact resistance (ICR) was also investigated. The ICR value of graphite-polypyrrole coated SS 316L is much lower than that of bare SS 316L. Therefore, graphite-polypyrrole coated SS 316L indicates a great potential for the application in PEMFC.     

2型猪链球菌毒力因子

2型猪链球菌感染已成为影响全世界养猪业的重要问题之一.它主要引起猪脑膜炎、肺炎、心内膜炎、关节炎、败血症等,并可感染人.2型猪链球菌致病机理尚不太清楚,迄今为止,已知的毒力因子有荚膜多糖、溶菌酶释放蛋白、胞外蛋白因子、溶血素、粘附素等.     

The LDL receptor pathway delivers arachidonic acid for eicosanoid formation in cells stimulated by platelet-derived growth factor

Animal cells can convert 20-carbon polyunsaturated fatty acids into prostaglandins (PGs) and leukotrienes. These locally produced mediators of inflammatory and immunological reactions act in an autocrine or paracrine fashion. Arachidonic acid (AA), the precursor of most PGs and leukotrienes, is present in the form of lipid esters within plasma lipoproteins and cannot be synthesised de novo by animal cells. Therefore, AA or its plant-derived precursor, linoleic acid, must be provided to cells if PGs or leukotrienes are to be formed. Because several classes of lipoproteins, including low-density lipoproteins (LDL), very-low-density lipoproteins, and chylomicron remnants, are taken up by means of the LDL receptor, and because LDL and very-low-density lipoproteins, but not high-density lipoproteins, stimulate PG synthesis, we have suggested previously that PG formation is directly linked to the LDL pathway. Using fibroblasts with the receptor-negative phenotype of familial hypercholesterolaemia and anti-LDL receptor antibodies, we show here that LDL deliver AA for PG production and that an LDL receptor-dependent feedback mechanism inhibits the activity of PGH synthase, the rate-limiting enzyme of PG synthesis. These results indicate that the LDL pathway has a regulatory role in PG synthesis, in addition to its well-known role in the maintenance of cellular cholesterol homeostasis.     

全静电摄像管的结构设计

采用图案电极的静电聚集静电偏转摄像管，具有功耗低，扫描频率可变，分辨率高，体积小和重量轻等优点，该文分析了采用图案型聚集偏转电极的全静电摄像管的特点，介绍了整管的结构设计，设计并制作了２种不同结构，性能良好的２／３英寸全静电摄像管，中心分辨率为４５０电视行，畸行小于１．５％。     

稻粉虱Aleurooybotus indicus种群系统动态的模糊聚类分析

本文应用模糊聚类分析方法对稻粉虱Aleurooybotus indicus David of Subramanias种群系统动态进行模糊聚类分析,模糊聚类分析结果表明,在置信水平0.9640≤λi<0.9880的情况下,该种群系统分为6个亚系统:SS1、SS2、SS3、SS4、SS5和SS6。文中对各个亚系统的动态规律进行特征描述,以期为稻粉虱的综合防治提供理论依据。     

m序列扩频CDMA系统与混沌扩频CDMA系统在Rayleigh选择性衰落信道下的性能比较

对Logistic映射的混沌序列作为扩频序列构成的混沌扩频CDMA系统与传统的m序列扩频CDMA系统进行了对比研究，并且对其二者在Rayleigh选择性衰落信道下的性能在SPW上进行了仿真，分析，具有重要实际意义。     

RNA sequencing of pancreatic circulating tumour cells implicates WNT signalling in metastasis

Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases. Although these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device for efficient capture of CTCs from an endogenous mouse pancreatic cancer model and subjected CTCs to single-molecule RNA sequencing, identifying Wnt2 as a candidate gene enriched in CTCs. Expression of WNT2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensity in vivo. This effect is correlated with fibronectin upregulation and suppressed by inhibition of MAP3K7 (also known as TAK1) kinase. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple WNT genes, and pancreatic CTCs revealed enrichment for WNT signalling in 5 out of 11 cases. Thus, molecular analysis of CTCs may identify candidate therapeutic targets to prevent the distal spread of cancer.