抗微囊藻毒素单克隆抗体的研制

为研制抗微囊藻毒素(Microcystins,简称MC)的特异性单克隆抗体,用MC-KLH偶联物免疫6～8周龄雌性BalB/c小鼠,取脾细胞与小鼠骨髓瘤细胞Sp2/0融合,经过3～4次亚克隆建立稳定分泌抗MC的杂交瘤细胞株。将杂交瘤细胞注射入BalB/c小鼠腹腔,生产出抗MC的单克隆抗体。得到了抗微囊藻毒素单克隆抗体细胞株,命名为G5,抗体亚类为IgG2a,亲和力常数2.9×10-11mol/L,分子量150KD,加标回收率在81.5%～125.0%之间,与其他结构类似物的交叉反应率<1%。筛选出的抗微囊藻毒素单克隆抗体具有较好的特异性。     

抗神经性贝毒素PbTx-2单克隆抗体的制备及鉴定

研究采用混合酸酐法将神经性贝毒素PbTx-2与牛血清白蛋白(BSA)、卵清白蛋白(OVA)偶联制备完全抗原。用免疫原PbTx-BSA免疫8周龄BALB/c小鼠,取脾细胞与小鼠骨髓瘤细胞SP2/0融合,经过4次亚克隆获得一株可稳定分泌抗PbTx-2的杂交瘤细胞株4B5,间接ELISA法测得腹水效价为1:5.12×104,抗体亚类为IgM,亲和常数为2.32×107mol/L。     

ThermoFisher Scientific检测食品中五种黄曲霉毒素的完整解决方案

黄曲霉毒素因具有致癌性及强急性毒性,所以在食品和农产品贸易中为必检项目,尤其经过蒙牛乳制品中黄曲霉毒素M1超标事件后,人们对黄曲霉毒素的关注愈来愈热.黄曲霉毒素是霉菌的二级代谢产物,目前主要关注的黄曲霉毒素有黄曲霉毒素B1、B2、G1、G2、M1,其中黄曲霉毒素B1的毒性和致癌性最强,而黄曲霉毒素M1是黄曲霉毒素B1的代谢物.我国规定,乳品及乳制品中黄曲霉毒素M1限量为0.5μg/kg,粮食中黄曲霉毒素B1为10μg/kg.     

高特异性黄曲霉毒素B_1单克隆抗体的制备及特性研究

本研究将黄曲霉毒素B1转化为半缩醛B2a,在硼氢化钠（NaBH4）还原作用下与载体蛋白偶联制备完全抗原。将制备的完全抗原免疫Balb/c小鼠,经4次免疫后取其脾脏与小鼠骨髓瘤细胞Sp2/0细胞融合,采用半固体培养基筛选后鉴定,获得杂交瘤细胞株3A12,抗体的灵敏度可达6.1±0.025ng/mL,抗体与其它黄曲霉毒素B2、G1及G2的交叉反应率依次为7.8%、20.2%及0.6%,与黄曲霉毒素M1交叉反应率小于0.1%。本研究为研发花生等农产品黄曲霉毒素B1特异性免疫分析技术及产品奠定了重要基础。     

定性测定羊奶中黄曲霉毒素M1残留高灵敏度检测卡的研制

研制定性测定羊奶中黄曲霉毒素M1残留的高灵敏度检测卡.在硝酸纤维素膜上预包被黄曲霉毒素M1全抗原作为检测线,以及预包被羊抗鼠二抗作为质控线.将抗黄曲霉毒素M1单克隆抗体用胶体金标记作为金标垫.将样品垫、金标垫、预包被黄曲霉毒素M1的硝酸纤维素膜以及吸水垫依次粘贴在PVC板上,切割成试纸条并组装成检测卡.结果表明:该检测卡的检测限为0.5μg/L,对黄曲霉毒素M1的交叉反应率为100％,而与同族的其他物质的交叉反应率小于5％.假阳性率小于5％,假阴性率为0％,检测卡常温储存,保质期18个月.本研究的检测卡,可快速准确地应用于羊奶中黄曲霉毒素M1残留的定性检测,为快速准确地测定羊奶中黄曲霉毒素M1残留提供了依据.     

牛乳铁蛋白LF单克隆抗体的制备

制备牛乳铁蛋白单克隆抗体,研究利用标准LF蛋白作为免疫原,免疫六周龄Balb/c雌性小鼠,运用淋巴细胞杂交瘤技术,通过间接ELISA筛选,采用有限稀释法,经过3次克隆筛选,获得两株稳定分泌抗LF蛋白的杂交瘤细胞株,命名为:L1,并进行了亚类鉴定。结果表明:该杂交瘤细胞培养上清抗体效价L1为1:4.56×104;腹水L1为1:5.21×106;L1分泌的单克隆抗体为IgG1型,轻链为λ链抗体。     

牛奶及奶粉中黄曲霉毒素M1的快速测定

采用免疫亲和柱－荧光光度法快速测定了牛乳及乳制品中黄曲霉毒素M1。试样经过离心、脱脂、过滤后滤液经过键合有黄曲霉毒素M1特殊抗体的免疫亲和柱净化，此抗体对黄曲霉毒素M1具有专一的识别能力，黄曲霉毒素M1键合在分离柱中的抗体上，用甲醇与水之比为10：90的混合液将免疫亲和柱上杂质除去；以甲醇与水之比为80：20的混合液通过分离柱洗脱；加入溴溶液衍生，以提高测定灵敏度，衍生化后的洗脱液于荧光光度计中测定黄曲霉毒素M1，检测低限为0．1μg/kg；在0．1－1．0μg/kg范围内，回收率为89．6％－96．1％，变异系数为0．52％－5．8％，分析一个样品的时间小于30min，分析过程中不使用黄曲霉毒素M1标准物质，结果表明，本方法具有准确、简单、快速、安全等优点，可以满足少量和批量样品的检测需要。     

亲水亲脂平衡柱萃取高效液相色谱法快速测定干酪中的黄曲霉毒素M1

研究了利用RP-HPLC对干酪中黄曲霉毒素M1进行测定,分别采用乙腈、甲醇、丙酮作为提取溶剂对干酪样品中的黄曲霉毒素M1进行提取,利用一种亲水亲脂平衡柱萃取黄曲霉毒素M1,然后上机检测,采用质量分数为25％的乙腈水溶液为流动相,流速1 mL/min,利用荧光检测器进行检测.结果表明,采用乙腈作为干酪中黄曲霉毒素M1的提取试剂比其他2种溶剂更为适宜;OASIS HLB固相萃取净化柱对于黄曲霉毒素M1的分离纯化效果很好;干酪中的黄曲霉毒素在8mm内被快速检出,平均同收率较高,相对标准偏差(RSD)为1.4％～5.7％,最低检出限为0.037μg/kg.该方法是一种针对干酪中黄曲霉毒素M1的快速高效的检测方法.     

粮油作物中伏马菌素B1免疫测定方法的建立

旨在制备伏马菌素B_1单克隆抗体,并建立伏马菌素B_1免疫学检测方法。以制备的免疫原FB_1-BSA免疫小鼠,利用细胞融合技术建立能分泌抗FB1抗体的杂交瘤细胞株,体内诱生腹水的方法制备FB_1单抗。基于制备的FB_1单抗,建立间接竞争ELISA检测分析方法,并对检测方法性能进行了初步鉴定。结果表明,通过动物免疫、细胞融合筛选到1株分泌抗FB_1抗体的杂交瘤细胞株3F6。建立的间接竞争ELISA检测分析方法检测范围40~600 ng/mL之间,检测下限达到40 ng/mL,半数抑制浓度IC_(50)为136.81 ng/mL,除与FB1特异性反应外,与同系物FB2及FB3交叉反应率分别为11.65%和7.85%,与黄曲霉毒素B_1、黄曲霉毒素M_1、β-玉米赤霉烯醇、呕吐毒素、T-2毒素、玉米赤酶烯酮、赭曲霉毒素A、玉米赤霉酮、α-玉米赤霉醇交叉反应率均低于0.2%,样品回收率在88.89%到115.45%之间,平均101.91%,变异系数为7.43%。本研究建立的ELISA方法与LC-MS-MS检测相同的样品时,二者的检测结果没有显著差异(P0.05)。本研究初步研发出灵敏度符合检测要求、特异较强、准确度较高、简便快捷的FB1免疫学检测方法 。     

高分子微球免疫吸附剂的制备及其对黄曲霉毒素M1的吸附性能

制备用于牛乳中吸附黄曲霉毒M1的免疫吸附剂。以乙醇和水溶液为反应介质,聚乙烯吡咯烷酮为分散 剂,偶氮二异丁腈为引发剂,采用分散聚合法制备苯乙烯-甲基丙烯酸缩水甘油酯共聚物微球。利用微球表面的环 氧基团将抗黄曲霉毒M1的单克隆抗体固定在微球表面,获得免疫吸附剂。制备的微球粒径约为1.7 μm,微球中环氧 基量在67.2%～71.6%之间,制备的免疫吸附剂对乳中黄曲霉毒素M1的吸附量达1.2 μg/g以上。该免疫吸附剂对乳中 黄曲霉毒素M1的选择性吸附较好,是去除乳中黄曲霉毒素M1较理想的免疫吸附材料。     

黑色素生物合成抑制剂产生菌株的筛选

采集了中国 1 1个省的土样和草样共 2 3 1份 ,分离出了 1 70 0株链霉菌 .筛选结果是 :3 9 2 4号菌株在比基尼链霉菌筛选模型中抑制圈的直径为 2 0mm ,属于直径较大者 ;其黑色素生物合成抑制剂初提物对CV 1细胞的急性细胞毒性 (致毒质量浓度 )为 1 0mg/mL ,属于毒性较小者 .根据以上试验结果 ,作者确定 3 9 2 4号菌株为黑色素生物合成抑制剂研究的试验菌株 .     

Increase in desulfurization activity of Rhodococcus erythropolis KA2-5-1 using ethanol feeding

Rhodococcus erythropolis KA2-5-1 is one of the best strains for the desulfurization of dibenzothiophene (DBT) via a sulfur-specific pathway in which DBT is converted to the end product, 2-hydroxybiphenyl, by releasing sulfite via DBT-sulfone and 2-(2'-hydroxyphenyl) benzene sulphinate. The objective of this research is to develop a culture method in order to attain a high cell density with a high level of specific desulfurization activity. Compared with glucose or glycerol, ethanol was found to be a preferable carbon source for obtaining a high specific activity (SA) of desulfurization. When the amount of DBT fed was restricted by feeding 2.9 mg-DBT/g-ethanol solution, the maximum SA and final cell concentration were 135.5 (mmol-2HBP/kg-dry cell weight-h) and 37 (g-dry cell weight/l), respectively. On the other hand, when glucose or glycerol was used as a carbon source, the SA was lower than 50 (mM-2HBP/kg-dry cell weight-h) and the final cell concentration was also lower than 27 (g-dry cell weight/l). The activities of the desulfurization enzymes in R. erythropolis KA2-5-1 grown on ethanol were remarkably higher than when the strain was grown on glucose or glycerol. It was also suggested that NADH, which is produced by the biochemical reaction of NAD with ethanol catalyzed by alcohol dehydrogenase, might contribute to the conversion of FMN to FMNH2, which is a coenzyme for the activities of desulfurization enzymes.     

In vitro toxicity tests of diethyleneglycol with cell cultures and an automated bacterial test system

Diethyleneglycol was tested for its general cytotoxic effects in three cell culture test systems and in a novel, automated bacterial test system. The first cytotoxic effects were detected in the cell culture test systems at DEG-concentrations of 1 g/1 and 3g/1. The bacterial test system showed a minimal inhibitory concentration of 5 g/1 DEG (20% growth reduction). Morphological observations showed evidence of membrane damage to the cultured cells by DEG. With the bacterial test system one could determine the amount of DEG semiquantitatively in wines to which this material had been added.     

双亲灭活米曲霉原生质体融合中原生质体制备的研究

对双亲灭活米曲霉原生质体融合育种中原生质体制备的条件进行了研究,将菌丝的预处理与细胞壁的酶解合为一步,并讨论了DTT的加入量。结果表明,原生质体制备的最佳破壁酶为纤维素酶、溶壁酶、蜗牛酶3种酶混合浓度比为5:3:1;菌丝培养15h;酶解时加入3mol/L DTT;酶解2.5h;0.8mol/L NaCl作为渗压稳定剂。所得双亲菌株原生质体的融合率为3.31%。     

Translational regulator RpL10p/Grc5p interacts physically and functionally with Sed1p,a dynamic component of the yeast cell surface

Biogenesis of an active ribosome complement and a dynamic cell surface complement are two major determinants of cellular growth. In yeast, the 60S ribosomal subunit protein RpL10p/Grc5p functions during successive stages in ribosome biogenesis, specifically rRNA processing, nucle(ol)ar preribosomal subunit assembly, nucleo-cytoplasmic transport and cytoplasmic maturation of ribosomes. Here, we report that a two-hybrid screen identified yeast genes SED1, ACS2 and PLB3 as encoding proteins physically interacting with both ribosomal RpL10p/Grc5p and its human homologue hRpL10p/QMp. SED1 encodes a differentially expressed cell wall protein which is proposed to be first transiently secreted to the plasma membrane as a GPI (glycosylated derivative of phosphoinositol)-anchored form and to be then transferred to the glucan layer of the cell wall. Ectopic expression of SED1 rescues both the aberrant growth phenotype and the translation defect of grc5-1(ts) temperature-sensitive cells. Furthermore, we report that Sed1p associates with translating ribosomes suggesting a novel, cytoplasmic role for Sed1p. ACS2 encodes one of the two yeast acetyl-CoA synthases and represents a key enzyme in one of several metabolic routes to produce acetyl-CoA, which in turn is indispensable for lipid biosynthesis. PLB3 encodes a phospholipase, which is active in the breakdown of membrane lipids. Our results support the view that Grc5p/RpL10p links ribosome function to membrane turnover and cell surface biogenesis.     

酒酒球菌高密度培养条件的优化

酒酒球菌（Oenococcus oeni）主导的苹果酸-乳酸（MLF）发酵是优质葡萄酒生产的重要工艺环节,制备高活菌数的酒酒球菌发酵剂是保障该环节顺利进行的重要前提之一。研究发现,一定量的L-苹果酸可以有效促进酒酒球菌的生长,并通过高密度培养条件优化提高酒酒球菌菌体密度。结果表明,通过正交试验得出的最佳高密度培养条件为初始pH值5.1、接种量3%、L-苹果酸添加量1 g/L。在此优化条件下,酒酒球菌ES-1的菌体密度较高,为（7.33±0.40）×109 CFU/mL;进一步结合化学中和法和半连续培养法,可使最终菌体密度达（1.67±0.11）×1010 CFU/mL,是对照组的11倍。该研究获得的高密度培养优化方案可为制备优质本土酒酒球菌发酵剂奠定基础。     

RGD1 genetically interacts with MID2 and SLG1, encoding two putative sensors for cell integrity signalling in Saccharomyces cerevisiae

The RGD1 gene was identified during systematic genome sequencing of Saccharomyces cerevisiae. To further understand Rgd1p function, we set up a synthetic lethal screen for genes interacting with RGD1. Study of one lethal mutant made it possible to identify the SLG1 and MID2 genes. The gene SLG1/HCS77/WSC1 was mutated in the original synthetic lethal strain, whereas MID2/SMS1 acted as a monocopy suppressor. The SLG1 gene has been described to be an upstream component in the yeast PKC pathway and encodes a putative cell surface sensor for the activation of cell integrity signalling. First identified by viability loss of shmoos after pheromone exposure, and since found in different genetic screens, MID2 was recently reported as also encoding an upstream activator of the PKC pathway. The RGD1 gene showed genetic interactions with both sensors of cell integrity pathway. The rgd1 slg1 synthetic lethality was rescued by osmotic stabilization, as expected for mutants altered in cell wall integrity. The slight viability defect of rgd1 in minimal medium, which was exacerbated by mid2, was not osmoremediated. As for mutants altered in PKC pathway, the accumulation of small-budded dead cells in slg1, rgd1 and mid2 after heat shock was prevented by 1 M sorbitol. In addition, the rgd1 strain also displayed dead shmoos after pheromone treatment, like mid2. Taken together, the present results indicate close functional links between RGD1, MID2 and SLG1 and suggest that RGD1 and MID2 interact in a cell integrity signalling functionally linked to the PKC pathway.     

Production of Beta-D-Galactosidase from Kluyveromyces fragilis Grown in Cheese Whey

Two strains of Kluyveromyces fragilis (145 and 276) and one of Kluyveromyces lactis were tested for their abilities to produce beta-D-galactosidase in cheese whey. Kluyveromyces fragilis 145 was selected for its higher beta-D-galactosidase activity per cell at the end of the exponential growth phase.Addition of ammonium sulfate (.3%) and yeast extract (.1%) to the deproteinized cheese whey increased cell mass and enzyme yield.Addition of 3% lactose did not affect beta-D-galactosidase activity per cell, which responded positively to a reduction in aeration from 1 to .25 air volume/medium volume/min.The harvested yeast cells were ethanol- and acetone-permeabilized to enhance lactose hydrolysis by beta-D-galactosidase.     

Possibility of insulin-producing cells derived from mouse embryonic stem cells for diabetes treatment

Insulin injection therapy is the principal current treatment of type 1 diabetes. Patients, however, suffer from various complications generated by insufficient control of blood glucose levels over a long period. Therefore, a method which can infuse insulin in response to changes of blood glucose levels is eagerly desired. Transplantation of insulin releasing cells derived from embryonic stem (ES) cells has been expected to be one of promising approaches to realize this requirement. In this study, ES cell progeny which were derived in culture media with/without fetal calf serum contained two distinct kinds of cells immunostained by anti-insulin and anti-C-peptide antibodies. The cytoplasm and nuclei of one type of cell were immunoreactive against antibodies for insulin, while the other kind of cell only had the cytoplasm stained by the anti-insulin antibody. The first cell type was the major population of insulin-positive cells in serum-free medium, while the latter kind of cells was the major population in medium containing serum. Interestingly, the latter insulin-positive cells could be also immunostained by anti-C-peptide antibodies, and was observed even after nine subcultures in medium containing serum. Although there still remain many issues to be addressed in order to definitely demonstrate that insulin-positive cells derived from ES cells to be truly beta cells in the islets, these properties of the obtained cells are believed to promising cells for treatment of type 1 diabetes.     

阳电极面积对微生物燃料电池产电性能的影响

微生物燃料电池（MFC）最具应用前景之一是处理废水的同时能够产生电能。以糖蜜废水作为阳极基质,以金属离子的电镀废水做阴极溶液,研究了双室微生物燃料电池不同电极面积对产电性能和COD的影响。结果发现,当外电阻为300Q时,大反应器微生物燃料电池A．（阳极面积为78．15cm^2）及小反应器微生物燃料电池～（阳极面积为76．8cm^2）最大功率密度分别为0．28mW／cm^2和0．22mW／cm^2。在前200个小时内,A：电池在第60个小时时产生最大电压71．1mV和最大电流189．5μA,A,在第190个小时时产生最大电压81．1mV和最大电流228．1μA。同时,当Zn^2＋作阴极溶液时,小反应器微生物燃料电池阳极溶液的COD去除率在1．5％到7．02％之间,大反应器微生物燃料电池阳极溶液的COD去除率在0到14．96％之间。阴极中Zn^2＋去除率A1中为28．6％,A2为21．2％。