猪脑心肌炎病毒VP2蛋白单克隆抗体的制备及其识别表位分析

为获得针对猪源EMCV结构蛋白VP2的特异性单克隆抗体,本试验以EMCV结构蛋白VP2为对象,将EMCVBJC3株VP2基因定向插入穿梭质粒中,构建重组腺病毒AdV—VP2,免疫BALB／c小鼠制备单克隆抗体,利用Pepscan技术对单抗识别的抗原位点进行分析。结果显示,间接ELISA及间接免疫荧光法筛选获得了2个阳性细胞克隆株,命名为C11和G8。经检测杂交瘤细胞培养上清抗体效价为1：1600,腹水效价分别为1：2．56×106和1：1.28×106,中和试验鉴定均无中和活性。Western—blot鉴定表明获得的2株单抗均能识别原核表达的重组VP2蛋白。亚类鉴定结果显示2株单抗均为IgG2b亚类,轻链均为κ型。Pepscan分析结果显示,这2株单抗可分别识别EMCVVP2蛋白上的2个B细胞线性表位。本研究获得的蛋白十分接近于其天然构象,很大程度上保证了病毒表面抗原位点的结构和功能。     

鸡包涵体肝炎过程中IL-2的mRNA原位杂交

FAV-Ⅷ型禽腺病毒口服接种感染2日龄SPF雏鸡,攻毒后取不同日龄的肝脏于福尔马林溶液中固定,并进行石蜡包埋,石蜡切片进行原位杂交染色。结果表明:着染部位主要出现在坏死灶或血管周围成团或散在的淋巴细胞胞浆内,在病情严重时5 dpi开始增多,12 dpi表达量达到高峰,以后逐渐减少,直到30 dpi基本消失。说明鸡包涵体肝炎过程中肝脏IL-2的mRNA大量增加,可能与T淋巴细胞的增多有关;而且IL-2作为免疫增强剂与抗病毒感染和病症恢复也有关。     

牛病毒性腹泻病毒E2蛋白纳米抗体筛选及反应原性检测

通过噬菌体展示技术筛选牛病毒性腹泻病毒(BVDV)重组E2蛋白特异性纳米抗体,验证纳米抗体反应原性.使用BVDV灭活疫苗免疫羊驼,分别在第0、21、49及70天采集全血,测得抗体效价后分离全血中淋巴细胞,提取总RNA,反转录后PCR扩增目的片段.目的片段和pCANTAB5E使用限制性内切酶酶切连接后转至TG1感受态细胞...     

猪繁殖与呼吸综合征病毒和猪圆环病毒2型基因重组质粒实时荧光定量PCR标准曲线的构建

根据猪繁殖与呼吸综合征病毒(PRRSV)ORF7和猪圆环病毒2型(PCV2)ORF1的核苷酸序列设计特异性引物,经RT-PCR/PCR扩增,分别扩增出大小为308 bp和417 bp的部分基因片段。用T4连接酶将目的片段与pGEM-Teasy载体系统连接,并转化到大肠杆菌DH-5α株感受态细胞。提取的质粒经PCR、酶切和测序鉴定,证实为PRRSV和PCV2的阳性重组质粒。将重组标准质粒10倍系列稀释后作为模板,通过实时荧光定量PCR方法(Real-time PCR),建立了PRRSV、PCV2的标准曲线及其直线回归方程,并确定其最佳读板温度。该方法具有线性关系好、特异性强、敏感性高、重复性好等特点,为分析PRRSV和PCV2共感染猪体内病毒的绝对含量提供了必要的技术平台。     

土壤CO2与喀斯特洞穴CO2季节变化响应分析

为了揭示土壤CO_2对洞穴内环境的意义,对大风洞和响水洞2016年洞内环境指标和上覆土壤的相关数据进行统计与回归分析。结果表明,洞内CO_2浓度、土壤CO_2浓度具有显著的季节性特征,土壤2#对应的洞内空气CO_2在夏冬两季差值达793~884mg/kg。土壤水PCO_2和滴水PCO_2在夏冬两季的差值最高可分别达1.12和0.41,因此洞内滴水PCO_2、土壤水PCO_2也表现出明显的季节性特征。土壤CO_2、洞内空气CO_2、洞内滴水PCO_2和土壤水PCO_2间均表现出一定的滞后现象,如4#土壤水PCO_2与滴水PCO_2的相关系数,由非滞后条件下的0.123上升至滞后条件下的0.596。同时在滞后条件下,土壤CO_2对洞内CO_2的贡献率的总和最高可达84.6%,高于非滞后条件下的值。     

Isolation and Identification of Orf Virus from Jiangsu Province and Phylogenetic Analysis of Their B2L Gene

The study was aimed to investigate prevalence of Orf virus (ORFV) in Jiangsu province in recent years and control Orf better. A total of 121 tissue samples were collected in some farms from 2013 to 2015 and subjected to PCR detection, viral isolation and phylogenetic analysis of B2L gene. Four samples were ORFV positive by PCR. The viruses were isolated by passaging in ovine fetal turbinate (OFTu) cells and MDBK cells, and were named as ORFV/Ovis/XZ/Jiangsu/2015/China, ORFV1/Ovis/DT/Jiangsu/2015/China, ORFV2/Ovis/DT/Jiangsu/2015/China and ORFV/Ovis/SL/Jiangsu/2015/China,respectively. The B2L gene was amplified and sequenced for the phylogenetic study. The nucleotide homology of these 4 strains was 98.5% to 100.0%. ORFV/Ovis/XZ/Jiangsu/2015/China, ORFV1/Ovis/DT/Jiangsu/2015/China and ORFV2/Ovis/DT/Jiangsu/2015/China, gathered into a cluster with SC-JY, GX-YB, JS-FX isolates and the nucleic acid homology of these strains was 97.8% to 100%. ORFV/Ovis/SL/Jiangsu/2015/China gathered into a cluster with LiaoNing, HuB and Gansu isolates, the nucleic acid homology was 98.8% to 98.9%. The nucleic acid homologies of 4 strains and ORFV strain China vaccine was 96.8% to 98.1%. The result showed that the ORFVs in Jiangsu province might be from different source. For controlling the spreading of this virus, it was necessary to carry out deep epidemiological survey in Jiangsu province.     

茶多酚的抗氧化作用及其机制

茶多酚(TP)是一种从绿茶和红茶中提取的纯天然复合物,具有清除自由基、防止DNA受损、调节细胞内抗氧化防御系统及抗癌等生物学功能。本文系统地对TP的组成成分、抗氧化作用、对丝裂原活化蛋白激酶-核因子酶2相关因子2-抗氧化反应原件(MAPK-Nrf2-ARE)信号转导通路的调控及其相关机制进行综述。     

超临界CO_2流体萃取兔肉腥味物质

【目的】探讨超临界CO_2流体(SFE-CO_2)萃取兔肉腥味物质的条件,确定腥味物质的种类、组成和含量,为兔肉脱腥以及加工提供理论基础。【方法】采用超临界CO_2流体对兔肉腥味物质进行萃取,以提取率为指标,在单因素试验基础上进行Box-Behnken响应面分析;定量加入内标物质2,4,6-三甲基吡啶(TMP),通过气相色谱质谱仪(GC-MS)对兔肉腥味物质进行分析鉴定。计算气味活度值(OAV),结合气相色谱-嗅闻(GC-O)和感官评价,确定兔肉腥味物质主体成分。【结果】单因素试验结果表明,当萃取温度为40℃时,提取率最高达到97.91%,峰面积为1.58×10~9;当萃取时间为4 h时,提取率最高,为97.83%,峰面积为2.42×10~9;萃取压力为25 MPa时,最高提取率为97.78%,峰面积3.78×108。其中,萃取温度和萃取压力不仅影响溶质扩散系数,还影响CO_2流体密度。温度增加时,尽管扩散系数增大,由于CO_2流体密度下降,提取率随之减小。虽然CO_2流体密度在高压下较大,但随着压力增加,可压缩性随之减小,由于扩散系数降低,溶质溶解度下降,因而提取率降低。当萃取时间过长时,一些非挥发性物质被溶出,使得萃取物质总量增加,降低了挥发性风味物质的比重。根据响应面分析,得出最优萃取条件为:萃取温度40.67℃、萃取压力25.67 MPa、萃取时间3.13 h,提取率为98.01%。而实际操作时,提取条件修正为萃取温度40℃、萃取压力25 MPa、萃取时间3 h,在此条件下提取率为98.11%。通过定量加入内标物质TMP,GC-MS定性定量分析得到兔肉挥发性风味物质5类,包括醛类、酸类、酯类、杂环类化合物和烃类,共计38种风味化合物。其中,酸类((1 394.25±3.45)μg·kg~(-1))酯类((569.26±1.23)μg·kg~(-1))烃类((471.82±1.11)μg·kg~(-1))醛类((168.46±0.97)μg·kg~(-1))杂环类((86.71±0.64)μg·kg~(-1))。通过计算得到兔肉挥发性风味物质中戊醛、己醛、己酸和2-戊基-呋喃的OAV值均大于1,且己酸己醛2-戊基-呋喃戊醛,表明这4种物质对兔肉腥味有重要贡献,而己酸对兔肉腥味的贡献最大。同时,气相色谱-嗅闻(GC-O)分析认为这4种物质均具有不同程度异味,包括肝脏腥味、草腥味、羊膻味和豆腥味等,感官分析表明萃取物有明显兔肉腥味。【结论】超临界CO_2流体萃取兔肉腥味物质可行,为研究兔肉腥味提供了新的提取方法。其最佳萃取条件为:时间3 h,温度40℃,压力25 MPa。戊醛、己醛、己酸和2-戊基-呋喃初步确定为兔肉腥味的主体成分。     

Polysaccharide Nucleic Acid of Bacillus Calmette Guerin Modulates Th1/Th2 Cytokine Gene Expression in Lipopolysaccharide-Induced Mastitis in Rats

The aim of this study was to evaluate, in rats, the changes in the T helper type 1 (Th 1)/Th2 radio in mammary glands after an intramammary infusion of lipopolysaccharide (LPS) and to characterize the moderating effects of the polysaecharide nucleic acid of Bacillus Calraette Guerin (BCG-PSN) on the mammary gland. In the control group, the levels of IL-2 and INF-γ, mRNA expression increased, whereas IL-4 mRNA expression decreased after LPS challenge. As a consequence, the INF-γ/IL-4 mRNA ratio was significantly higher at 3, 6, and 9 h post-infusion (PI) compared to the control value (O h; P<0.01).BCG-PSN increased mRNA expression of both INF-γ' and IL-4 before infusion of LPS. LPS challenge significantly the reduced Th1/Th2 eytokine ratio due to Th1 cytokine IFN-γ suppression and Th2 cytokine IL-4 upregulation compared with the control group. A significant reduction ofN-acety1-β-D-glucosaminidase (NAGase) was observed at 24 h PI in the BCG-PSN treatment group compared to the control group (P<0.05). Thus, it was demonstrated that level of BCG-PSN might change the Th1/Th2 ratio mainly by enhancing the Th2 immune response. This is the first report of a Th1/Th2 change induced by coliform mastitis and characterization of the effect of BCG-PSN on mammary gland inflammation. This study makes a better understanding of the mechanisms of coliform mastitis and provides a putative novel strategy for the prevention and/or treatment of mastitis.     

PS/2标准键盘与单片机的连接

分析了PS/2标准键盘的通信协议,阐述了其编码的扫描原理,提出了51单片机与PS/2键盘的接口设计,并通过LCD1602显示了键盘的输入情况。