Immobilization and Characterization of a Thermostable Lipase

Lipases have found a number of commercial applications. However, thermostable lipase immobilized on nanoparticle is not extensively characterized. In this study, a recombinant thermostable lipase (designated as TtL) from Thermus thermophilus WL was expressed in Escherichia coli and immobilized onto 3-APTES-modified Fe₃O₄@SiO₂ supermagnetic nanoparticles. Based on analyses with tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis, X-ray diffraction, transmission electron microscopy, and vibrating sample magnetometer observation, the diameter of immobilized lipase nanoparticle was 18.4 (±2.4)?nm, and its saturation magnetization value was 52.3 emu/g. The immobilized lipase could be separated from the reaction medium rapidly and easily in a magnetic field. The biochemical characterizations revealed that, comparing with the free one, the immobilized lipase exhibited better resistance to temperature, pH, metal ions, enzyme inhibitors, and detergents. The K _m value for the immobilized TtL (2.56 mg/mL) was found to be lower than that of the free one (3.74 mg/mL), showing that the immobilization improved the affinity of lipase for its substrate. In addition, the immobilized TtL exhibited good reusability. It retained more than 79.5 % of its initial activity after reusing for 10 cycles. Therefore, our study presented that the possibility of the efficient reuse of the thermostable lipase immobilized on supermagnetic nanoparticles made it attractive from the viewpoint of practical application.     

Microbiological Identification and Analysis of Swine Lungs Collected from Carcasses in Swine Farms, China

The primary objective of this 3 years study was to determine the prevalence of porcine pathogens of the lungs of swine in swine farms in southern China. A total of 5,420 samples were collected from 200 swine farms. The bacterium that was most commonly isolated was Streptococcus suis, with 10.24 % of the samples being positive, 114 lungs (2.1 %) were positive for pseudorabies virus and 263 (4.85 %) were positive for classical swine fever virus; much lower than positive for PRRSV (15.1 %, p = 0.023) and PCV2 (13.8 %, p = 0.038). lungs that were positive for PRRSV and/or PCV-2 have significantly increased odds of being positive for any of the S. suis (9.79 vs. 0.44 %, p = 0.003).     

猪胞内劳森菌感染的免疫应答与检测技术研究进展

胞内劳森菌(Lawsonia intracellularis, LI)常引起猪精神不振、食欲减退、血样下痢或突然死亡,严重损害养猪业。猪群感染该菌在肠道诱导免疫应答的研究较少。因该菌胞内寄生,基于细菌不同感染阶段动物免疫应答特点,检测方法会有所侧重。本文重点阐述了胞内劳森菌感染后机体免疫应答的特点,并对其检测技术进行综述,以期为新型检测技术研发及疫病防控提供参考。     

Magnesium promotes root growth and increases aluminum tolerance via modulation of nitric oxide production in Arabidopsis

Plant and Soil - Aluminum (Al) toxicity and magnesium (Mg) deficiency often coexist in acidic soils. Nitric oxide (NO) is involved in diverse physiological processes and stress responses. Here, we...     

PDCoV、SADS-CoV与SVA三重RT-PCR检测方法的建立与应用

【背景】猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)、新型猪急性腹泻综合征冠状病毒(swine acute diarrhea syndrome coronavirus,SADS-CoV)与塞内卡病毒A型(Seneca virus A,SVA)均为猪的新发病原,严重危害养猪业的发展。猪感染这3种新发病原难以根据临床症状诊断,因此亟须建立多重RT-PCR检测方法对疑似患病猪进行快速诊断,以降低经济损失。【目的】建立能同时检测PDCoV、SADS-CoV和SVA单一或混合感染的三重RT-PCR检测方法。【方法】参考GenBank中登录的PDCoV和SADS-CoV N基因、SVA L/P1基因保守区域,设计3对特异性引物,以温度梯度PCR法确定最适退火温度(T_m);采用方阵法优化其引物浓度;构建重组质粒PMD-PDCoV、PMD-SADS-CoV和PMD-SVA作为标准品确定最小检测量(limits of detection,LOD);以猪传染性胃肠炎病毒、猪流行性腹泻病毒、猪繁殖与呼吸综合征病毒等6种常见感染猪的病毒核酸样本为模板,确定三重RT-PCR法的特异性;以批间和批内实验验证其重复性;经检测疑似感染的临床样本并与已报道的检测方法进行比较,评估其临床应用效果。【结果】最佳退火温度为58.3℃;引物最佳浓度分别为0.5、0.25和0.25μmol/L;其敏感性高,PMD-PDCoV、PMD-SADS-CoV与PMD-SVA最低检测限分别为1、1和10copies/μL;其特异性强,仅对PDCoV、SADS-CoV和SVA有特异性条带,对其他病毒均无扩增条带;该方法重复性好,批间和批内实验检测结果均一致。经临床样本检测PDCoV、SADS-CoV和SVA的阳性率分别为65.85%、30.49%和57.32%,与已报道的检测方法一致。最后从13份PDCoV、SADS-CoV和SVA均为阳性的样品中随机选取5份样品进行测序,并做同源性及进化树分析,结果显示5份阳性病料的PCR扩增序列之间相似性较高,而且和参考序列之间也具有较高的相似性,均可达到96%以上。表明本研究建立的方法在应用于临床样品检测中具有较高的准确性和可靠性。【结论】建立了一种能够同时快速检测PDCoV、SADS-CoV和SVA的三重RT-PCR方法,为临床上检测上述3种病毒提供了技术支撑。     

牛巴氏杆菌病多重PCR检测技术的建立与应用

【背景】牛巴氏杆菌病是由血清型(A、B、E)多杀性巴氏杆菌(Pasteurella multocida,Pm)引起的一种严重危害养牛业的重要传染病,病原学聚合酶链反应(polymerase chain reaction,PCR)方法是诊断并防控该病的有效手段。【目的】建立检测血清型(A、B、E)多杀性巴氏杆菌的多重PCR方法,为临床诊断牛巴氏杆菌病和病原分型提供技术支撑。【方法】参考多杀性巴氏杆菌hyaD-hyaC基因、bcbD基因和ecbJ基因特异区域,设计3对特异性引物,以温度梯度PCR法确定适宜退火温度(T_m);采用棋盘试验优化引物浓度并初步建立多重PCR方法;采用重组质粒标准品及阳性菌株菌液确定其敏感性(最小检测量);以8种常见牛感染病原体[溶血性曼氏杆菌(Mannheimia hemolytica)C1655、大肠埃希氏菌(Escherichia coli)C237、产单核细胞李氏杆菌(Listeria monocytogenes)C1597、金黄色葡萄球菌(Staphylococcus aureus)C3053、都柏林沙门氏菌(Salmonella dublin)C79351、副结核分枝杆菌(Mycobacterium paratuberculosis)C1625、牛传染性鼻气管炎病毒(bovine infectious rhinotracheitis virus)CAV1546和牛支原体分离株(Mycoplasma bovis)C65-1]核酸样本确定其特异性;制备3批诊断试剂,对敏感性和特异性样品进行批间和批内试验,确定其重复性;运用建立的方法使用3种不同型号的PCR仪检测敏感性和特异性样品,确定其适用性;通过检测临床样本及人工模拟感染样本评价临床应用效果。【结果】在T_m为55℃时,3对引物浓度分别为0.25、0.30和0.20μmol/L条件下建立多重PCR方法较优,可以同时检测多杀性巴氏杆菌血清A型(821bp)、血清B型(203bp)和血清E型(363bp);该方法敏感性高,对重组质粒标准品pMD-A、pMD-B和pMD-E检测限分别为43.080、3.710和4.350copies/μL,对阳性菌液最低检出限均为10²CFU细菌;其特异性强,仅对血清型(A、B、E)多杀性巴氏杆菌有特异性扩增条带,同时对其他病原体均无扩增条带;该方法重复性良好,批间与批内试验均一致;临床样本及人工模拟感染样本检测结果显示与病原分离鉴定符合率为100%。【结论】成功建立了一种可鉴定不同血清型的牛多杀性巴氏杆菌多重PCR检测方法。     

牛种布鲁氏菌2308不同途径小鼠感染模型的建立与评估

[背景] 布鲁氏菌可经口、皮肤、黏膜和呼吸道感染人和动物。小鼠是布鲁氏菌研究中最常用的模型动物。[目的] 建立牛种布鲁氏菌2308不同途径和剂量感染BALB/c小鼠的模型,为布鲁氏菌小鼠感染试验提供参考。[方法] 用10¹-10⁵ CFU这5个不同感染剂量,分别经注射、口服和点眼方式感染BALB/c小鼠。在感染后不同时间点采集小鼠血清,检测IgG、IgM、IgA抗体含量、脾脏重量及脾脏含菌量,评价布鲁氏菌经不同途径感染BALB/c小鼠的效果。[结果] 10 CFU是注射感染BALB/c小鼠的最小感染剂量;10⁵ CFU是口服感染BALB/c小鼠的最小感染剂量。10¹-10⁵ CFU这5个不同感染剂量经点眼途径均未能成功感染BALB/c小鼠。在10⁵ CFU感染剂量下,口服与注射感染组小鼠每克脾脏平均含菌量分别为10^5.673 CFU/g和10^5.009 CFU/g,无显著差异（P>0.05）,但口服感染组小鼠脾脏平均重量为0.310 g,显著高于注射感染组0.165 g （P<0.01）。在试验期内,注射感染组和口服感染组小鼠体内IgG抗体的滴度均随感染时间延长而持续升高;整体上,口服感染组IgG抗体峰值显著高于注射感染组;2组IgM抗体变化趋势一致;口服感染组有2只小鼠在感染28 d后产生IgA抗体,注射感染组均未检测到IgA抗体。[结论] 建立了牛种布鲁氏菌2308通过不同途径感染BALB/c小鼠的模型。     

HBV相关肝癌组织中微生物宏蛋白质组特征分析

越来越多的证据表明微生物菌群和肝脏疾病有着密切联系,但是HBV相关肝细胞癌(hepatitis B virus(HBV)-related hepatocellular carcinoma,HCC)人群肝脏组织内的微生物菌群种类和数量仍不清楚.本研究对来自165个HCC人群的肝癌组织和对应癌旁组织的质谱数据进行宏蛋白质组...