鉴定猪源性大肠杆菌K_(88)~ 株的三种简便方法

仔猪腹泻是一种由大肠杆菌引起的重要肠道传染病之一。其主要特点是发病急剧,发病率和死亡率高。目前,该病对工厂化养猪业的发展危害很大。自从φrskov等在猪源性大肠杆菌中发现K88抗原以来,许多学者陆续证明了这种抗原存在的普遍性。K88抗原是一种蛋白质性的仔猪小肠上皮细胞吸着素,它同肠道病原性大肠杆菌在小肠前端的定居有关,是一种重要的致病因子。这种抗原在低     

快速鉴定猪源性大肠杆菌K_(88)质粒的两种简便方法

本文叙述了快速鉴定猪源性大肠杆菌K_(88)质粒的两种简便方法——(1)60℃保温法;(2)煮沸法。这两种方法分别适用于从猪源性大肠杆菌中,人肠毒性大肠杆菌中以及转化子中快速筛检存在的细菌质粒。     

简便快速检测质粒DNA的方法 (一)可用于检测和筛选的快速法

细菌常常含有染色体外小的环状DNA遗传因子——质粒。它可以作为遗传工程中克隆的主要载体。它与某些细菌病原性有关,例如各种耐药性质粒,引起小儿腹泻和成年人旅游者腹泻的大肠杆菌肠毒素质粒,与金黄色葡萄球菌肠毒素B相关的质粒,引起猪、牛腹泻痢疾的K88、K99表面抗原质粒等等。因此无论在遗传工程技术,还是临床检验,都需要将质粒DNA检出,确定其大小,作为进一步的研究和分析。对于简便快速检测质粒DNA的方法已有一些报导,但有的并不简便。我们应用菌落溶解物法;直接从琼脂平板上挑取1—2个大菌落或少量菌苔裂解,进行琼脂糖凝胶电泳,检测的效果是满意的。     

痢疾杆菌毒力相关抗原分析

采用BA-immunoblot法,以肠道感染痢疾菌后的猴恢复期血清为抗体,对痢疾杆菌和侵袭性大肠杆菌共20株进行了抗原分析.发现有毒痢疾菌和侵袭性大肠菌均表达4种主要毒力相关抗原,其分子量分别为:78K,66K,44K和39K.而这4种抗原在无毒株是不存在的.采用痢疾病人恢复期血清分析也获得类似结果.还发现,豚鼠眼感染痢疾菌后,其恢复期血清与人及猴肠道感染痢疾菌后恢复期血清中主要抗体组分相似,而很少有与无毒株交叉的抗体.所分析的菌株均含120或140Mdal大质粒.文中对痢疾菌群、型间的交叉保护作用与4种共同抗原的关系进行了讨论.     

新疆北疆地区规模化牛场传染性犊牛腹泻分析

选择新疆北疆地区9个规模化牛场,开展传染性犊牛腹泻疫病调查与实验室检测.采集犊牛腹泻症状的病牛粪便或肛门拭子,经细菌培养、消化道ELISA抗原试剂盒检测轮状病毒、冠状病毒、大肠杆菌F5(K99)微小隐孢子虫,同时用漂浮法对样品进行球虫检测.对北疆3个地州的种畜场、规模场采集234份样品,细菌鉴别培养分离出15株致病性大肠杆菌,经药敏试验,对磺胺嘧啶、卡那霉素、头孢噻呋高敏感、替米考星和氟苯尼考中敏感,其他药物表现耐药;样品经消化道ELISA抗原试剂盒没有检测到冠状病毒抗原,检测出15份轮状病毒抗原,阳性率为6.41％,20份微小隐孢子虫抗原,阳性率为8.54％,7份大肠杆菌F5抗原,阳性率为2.99％;所有粪便样品均没有检测到球虫.本次调查新疆北疆规模化牛场牛传染性腹泻感染情况不容忽视,分别检出轮状病毒、微小隐孢子虫、大肠杆菌,通过药敏试验指导临床用药,为控制牛场犊牛腹泻提供临床治疗方案.     

CS3基因的克隆及结构分析

定居因子是产肠毒素大肠杆菌(ETEC)的主要毒力因子之一。它在ETEC对肠粘膜上皮细胞的粘附过程中起着重要的介导作用。在已知的定居因子中,CFA/Ⅱ为优势血清型定居因子抗原之一,它由CS1、CS2和CS3三种组分所构成。其中CS3组分为CFA/Ⅱ阳性菌的共有抗原。本工作运用DNA体外重组方法克隆了CS3基因,并对其结构进行了分析。     

分别含K88,K99和LT-B亚单位抗原基因的重组痘苗病毒的构建

本文构建了分别含K88,K99和LT-B亚单位抗原基因的重组痘苗病毒,并观察了这3种基因的表达情况。首先将K88亚单位基因克隆入痘苗转移或体PGJP-5中,得到嵌合质粒P_5-K88,将K99和LT-B亚单位基因分别克隆至痘苗转移载体PJ16中,得到重组质粒PJK-9和PLT-B。再经细胞体内同源重组技术,将以上3种基因分别插入到痘苗病毒天坛株的TK基因中,经5-BUdR的选择压力在人TK~--143细胞中挑选TK~-表型病毒,分别以相应的~(32)P标记的基因片段为探针进行杂交筛选重组病毒株。我们得到7株含K99亚单位基因的重组病毒V-K99(Ⅰ～Ⅷ),5株含K88亚单位基因的重组病毒V-K88(Ⅰ～Ⅴ);1株含LT-B亚单位基因的重组病毒V-LTB,ELISA检测结果表明,在以上3种重组病毒感染的人TK~--143细胞培养液和细胞裂解物中均未测出相应的基因表达产物。以上结果表明这3种基因可能不易在痘苗系统内有效表达。     

肠出血性大肠杆菌O157∶H7毒力蛋白NleB1的表达、纯化及多克隆抗体的制备与鉴定

目的：构建肠出血性大肠杆菌（ EHEC） O157∶H7毒力蛋白nleB1基因的原核表达载体,诱导表达重组蛋白,制备NleB1的抗血清。方法从EHEC O157∶H7 EDL933株的全基因组中PCR扩增出编码nleB1的990个碱基序列,构建原核表达载体pET24a-nleB1,将构建的重组表达载体的质粒pET24a-nleB1转化至大肠杆菌BL21（DE3）感受态细胞中,利用异丙硫半乳糖苷（IPTG）诱导融合蛋白His-NleB1表达,并利用镍柱进行亲和层析纯化和切胶纯化,以纯化后的融合蛋白His-NleB1为抗原免疫BALB/c小鼠,获得抗血清。 Western印迹和ELISA法鉴定获得的抗血清。结果成功构建了pET24a-nleB1原核表达载体,纯化得到融合蛋白His-NleB1,进而免疫BALB/c小鼠制备多克隆抗体,Western印迹和ELISA法证实多克隆抗体制备成功。结论成功获得了EHEC O157∶H7的毒力蛋白NleB1的多克隆抗体,为研究EHEC O157∶H7毒力蛋白NleB1的功能奠定了基础。     

黑色素瘤抗原MAGE-3基因的原核表达

肿瘤抗原基因的克隆、测序和表达是研究肿瘤免疫治疗性疫苗的基础.本研究采用Oligo V6.0基因分析软件,对人黑色素瘤抗原MAGE-3基因序列进行分析后设计引物,扩增和克隆MAGE-3基因片段(210 ～623位碱基),在大肠杆菌中诱导其表达,以期为制备MAGE-3抗体和肿瘤疫苗奠定基础.     

表达毒素源性大肠杆菌LT－B抗原的重组鼠伤寒沙门菌株免疫原性的研究

探讨了能稳定表达毒素源性大肠杆菌（ＥＴＥＣ）热敏肠毒素Ｂ亚基（ＬＴ－Ｂ）的重组鼠伤寒沙门菌株的安全性、免疫原性及其免疫保护力。用重组菌株活菌肌注免疫家兔，动物体内产生明显的抗鼠伤寒沙门菌抗体和抗ＬＴ－ＢｌｇＧ抗体。口服免疫小鼠，免疫后小鼠血清和肠液中均能测到抗ＬＴ－Ｂ抗体。兔肠结扎实验证明，重组菌株在达的ＬＴ－Ｂ没有ＬＴ的生物毒性。在小鼠安全性试验中，口服１６ｘ１０￣（１０）活菌３０ｄ后，小鼠存活率为１００％。免疫后的家兔肠结扎攻毒试验和小鼠攻毒试验结果表明，免疫后的动物对野生毒性人源ＥＴＥＣ、猪源ＥＴＥＣ、霍乱弧菌和鼠伤寒沙门菌的攻击均有一定的保护力．     

IgA肾病患者纤溶酶原激活物的变化及临床意义

为探讨IgA肾病 (IgAN)患者纤溶酶原激活物 (PA)的变化及临床意义 ,以纤维蛋白平板法检测 10 8例IgAN及 36例健康自愿者尿PA活性 ,以免疫组化方法观察了 2 7例IgAN及 6例正常人肾组织t PA、u PA抗原表达。结果显示 ,正常人肾组织t PA少量表达于肾小球毛细血管袢 ,u PA表达于所有节段的肾小管上皮细胞。IgAN肾组织t PA阳性率及肾小球t PA平均积分明显高于正常人 (P <0 0 1) ,与其增殖程度有关。IgAN患者u PA表达明显下调 ,尿PA活性下降 ,肾小管间质病变严重者尿PA活性下降更为明显。本研究表明 ,IgAN早期肾组织t PA表达增加 ,晚期下降 ;u PA表达减少 ;尿PA活性的检测有助于判断IgAN的病情。     

Generic method for imaging transgene expression.

We propose a generic method to report on gene expression based on the use of an antigen-antibody reporting system and visualization by MRI. This methodology was demonstrated using a truncated form of the H2K(k) antigen, tH2K(k), as the nonendogenous antigen to be imaged. HeLa cells, transfected to express tH2K(k), exposed to tH2K(k) antibodies conjugated to a superparamagnetic iron oxide particle, generated strong negative contrast compared to non-H2K(k) expressing cells by MRI. T(2) of the tH2K(k) expressing cells was 57.6 +/- 17.0 ms, compared to 424.0 +/- 38.7 and 445.4 +/- 47.2 ms for the mock transfected and nontransfected cells, respectively (P < 0.001). tH2K(k) expression in the former cells was confirmed by flow cytometry, fluorescence, and electron microscopy. The methodology can be adapted to image in vivo other nonendogenous antigens in cells/tissues.     

湿润烧伤膏治愈糖尿病下肢溃疡1例报告

糖尿病下肢溃疡是指糖尿病患者由于合并神经病变及各种不同程度末梢血管病变而导致下肢感染,溃疡形成和（或） 深部组织的破坏,是糖尿病慢性并发症之一,也是导致糖尿糖患者致残死亡的重要原因之一,主要临床表现为下肢溃疡和坏疽.国内糖尿病患者并发足坏疽的病例约占糖尿病足患者的0 9% ～1 7%,60岁以上的老年糖尿病患者并发糖尿病足坏疽的约占2 8% ～14 5%.通过全身综合治疗联合外用湿润烧伤膏（MEBO） 治疗1例糖尿病并发左胫前感染病例,取得了较好的疗效,现报道如下：     

The effect of circulating antigen on radioimmunodetection and monoclonal antibody localisation: studies in a normal rat model

The effect of circulating antigen (human kappa light chains (LC] on radioimmunoscintigraphy was examined in a normal rat model. Rats were implanted subcutaneously with kappa (test) and lambda (control) LC coupled to sepharose beads (500 micrograms LC/ml swollen gel). When 131I-labelled monoclonal antibody, K-1-21 was injected, clear images of specific antibody localisation to the kappa implant were seen 6 days later. The kappa implant: blood ratio (K:B) was 7.69 +/- 0.45, which represented an implant uptake of 2.52 +/- 0.20 percent injected dose per gram (%ID/g). When 131I-K-1-21 was preincubated with 125I-kappa LC at ratios of up to 1:10 antibody: antigen, (w/w) before injection; or if antigen was given daily by bolus injection or continuously infused via an osmotic pump, very clear specific images of antibody localisation to the kappa implant were still obtained. There was, however, a decline in the absolute uptake of label by the kappa implant. The greatest change was observed following continuous antigen infusion, when kappa implant uptake fell to 1.26 +/- 0.1 %ID/g. Nevertheless, the K:B ratio was still maintained at 6.8 +/- 0.2, due to increased clearance of antibody from the blood. Thus, in this case, where antigen and antibody might be expected to form small, soluble immune complexes, there was no major deleterious effect on radioimmunoscintigraphy.     

Results of an EC laboratory comparison on 40K, 90Sr and 137Cs in dried bilberry powder

The evaluation is presented of a laboratory comparison (LC) on ⁹⁰Sr, ⁴⁰K and ¹³⁷Cs in dried bilberries organised in 2011 by the IRMM. The activity concentrations reported by 88 participant laboratories are compared to the reference values of the new reference material IRMM-426 Wild Berries. Nine per cent and 17% of activity concentration results for ¹³⁷Cs and ⁴⁰K, respectively, deviate more than 20% from the reference values, a result worse than that obtained in previous LCs. For ⁹⁰Sr, about 88% of results lie within 30% of the reference value, better than observed in previous LCs. But only 58% of ⁹⁰Sr results are satisfactory in terms of the E_n criterion, indicating difficulties with a complete uncertainty estimation.     

抗人IgK和Igλ链单克隆抗体的制备及临床初步应用

用初乳和血清IgA免疫Balb/c小鼠的脾细胞与SP_2/O和NS-1瘤细胞融合,以ELISA双抗原夹心法检测获得5株杂交瘤细胞。其中3株(1D1、5C3、11A7)分泌抗人游离和结合型λ轻链;2株(4G12、14A6)分泌抗人游离和结合型K轻链。4G12与结合型K链反应比游离K链好。这些细胞株在长达1年的培养中仍能稳定分泌特异性单克隆抗体,经液氮冻存10个月后杂交瘤细胞仍保持原有的特性。用固相抗原竞争ELISA试验表明,两个抗K链McAb是针对不同的抗原位点,而3株抗λ链则针对相同的抗原位点。用于检测副蛋白和本周氏蛋白具有很好的特异性和敏感性。把McAb4G12-HRP和1D1-HRP混合,用于检测-EB病毒早期抗原(EA)和壳抗原(VCA)抗体被证明是满意的,同时试用于抗核抗体的检测也获得了初步的结果。     

The absolute counting of 166mHo, 58Co and 88Y.

In this study, absolute 4pi beta-gamma-coincidence counting was used to measure the mass activity of 166mHo, 58Co and 88Y. For 166mHo and 88Y, three gamma windows were set to study the systematic error caused by the different gamma window settings. To eliminate the effect arising from the large difference in counting efficiency of the 4pi beta counter between electron capture events and beta+ particles, the two-dimensional extrapolation method was used to measure 58Co. The measurement results for the three nuclides were linked with the BIPM SIR through two APMP regional comparisons, APMP.RI(II)-K2, Ho-166m and APMP.RI(II)K2.Co-58, Y-88. Results are presented.     

The effect of circulating antigen on radioimmunodetection and monoclonal antibody localisation: studies in a normal rat model

The effect of circulating antigen (human kappa light chains (LC)) on radioimmunoscintigraphy was examined in a normal rat model. Rats were implanted subcutaneously with kappa (test) and lambda (control) LC coupled to sepharose beads (500 g LC/ml swollen gel). When ¹³¹I-labelled monoclonal antibody, K-1-21 was injected, clear images of specific antibody localisation to the kappa implant were seen 6 days later. The kappa implant: blood ratio (K:B) was 7.69±0.45, which represented an implant uptake of 2.52±0.20 percent injected dose per gram (%ID/g). When ¹³¹I-K-1-21 was preincubated with ¹²⁵I-kappa LC at ratios of up to 1:10 antibody: antigen, (w/w) before injection; or if antigen was given daily by bolus injection or continuously infused via an osmotic pump, very clear specific images of antibody localisation to the kappa implant were still obtained. There was, however, a decline in the absolute uptake of label by the kappa implant. The greatest change was observed following continuous antigen infusion, when kappa implant uptake fell to 1.26±0.1 %ID/g. Nevertheless, the K:B ratio was still maintained at 6.8±0.2, due to increased clearance of antibody from the blood. Thus, in this case, where antigen and antibody might be expected to form small, soluble immune complexes, there was no major deleterious effect on radioimmunoscintigraphy.     

HLA与十二指肠溃疡病相关的研究

本文报告了31例十二指肠溃疡病与HLA-A、-B抗原相关研究。与50例北方汉族正常人对照,未能发现与十二指肠溃疡病有关的HLA抗原,但HLA-A30/31可能为十二指肠溃疡病的保护性抗原。     

皮层重建和皮层展开方法在临床应用中的探讨

目的：介绍皮层重建和皮层展开方法并且探讨该方法在临床中的应用。方法：选用3例视觉或矫正视觉正常的志愿者,进行功能像扫描和T1WI的轴位、三维结构像的扫描;采用FreeSurfer软件对图像重建并将皮层展开,应用枷软件进行视网膜定位功能像分析,最后绘制视网膜定位图。结果：经过皮层重建和皮层展开得到完整的展开的皮层,并用此方法划分视皮层功能区。结论：皮层重建展开的方法在临床可以有很大的应用价值。