从狗蜱(Hemaphysails campanulatahoeppliana)分离出嗜神经性病毒

1953年在北京从狗蜱Hemutphslis cantpamdata hoepplicta 分离出二株嗜神经性病毒,经免疫学证明此二株为同一种病毒。此种病毒经接种于白鼬鼠可引骚脊髓灰白質炎。此种病毒与脑炎日本乙型、型路易型、春夏型、马脑脊髓炎西型的标准免疫虹清无交互中和反应,与鼠脑脊髓炎FA株及淋巴球性脉络霞脑膜炎亦舞交互免疫。根据多种试验进行分析研究,我们认为这种病毒是从狗蜱Hentpltysalis campanu-lata hoeppliana 分离出来的可使白鼬鼠发生脊髓灰白質炎的病毒。经我们用实验室保存的人的和鼠的标准病毒鉴定,这种病毒可能是一种新的嗜神经性病毒。     

黑曲霉An-76木聚糖酶系的酶学研究

用分子筛和离子交换等色谱分离技术,由黑曲霉An-76的木聚糖酶系中分离纯化到一种β-木糖苷酶(βx)和三种内切-β-木聚糖酶组分(EX1,EX 2,EX 3)。这几种酶均达到凝胶电泳纯和聚焦电泳纯。用凝胶过滤方法测得3X的分子量为147000,用凝胶电泳法测得EX1、EX2和EX3的分子量分别为2 3000、22000和41 000;βX、EX1、EX2和EX 3的等电点分别为4．6、5．9、4．1和3．9。本文还研究了各种酶的最适反应温度和PH、酶的热稳定性和pH稳定性;研究了金属离子和巯基试剂对酶活力的影响、动力学参数、氨基酸组成、底物持异性和反应产物等。各酶组份不具有分解纤维素的交叉活力。巯基试剂能完全抑制卢βX活力,其活力丧失可被半胱氨酸恢复。     

应用分值计算法优选SS琼脂配方的研究

用分值计算法对四批ＳＳ琼脂质量检测,分值均小于质控标准分值５６．１２５。主要问题是抑制大肠杆菌（Ｅ.ｃｏｌｉ）生长和促鼠伤寒沙门氏菌（Ｓｔｙｐｈｉｍｕｒｉｕｍ）,痢疾志贺氏菌（Ｓ．Ｄｙｓｅｎｉｃｒｉａｅ）生长的能力不够。调整ＳＳ琼脂配方中各试剂的用量进行筛选,结果表明：０．５％胆盐抑制大肠杆菌（Ｅ/ｃｏｌｉ）能力达到分值质控要求,但对鼠伤寒沙门氏菌（Ｓｔｙｐｈｉｍｕｒｉｕｍ）和痢疾志贺氏菌（Ｓ．Ｄｙｓｅｎｉｃｒｉａｅ）的生     

农杆菌介导的苏云金杆菌抗虫基因cryIA(b)和cryIA(c)在水稻中的遗传转化及蛋白表达

通过农杆菌介导法用含有抗潮霉素和GUS基因的双元载体将杀虫结晶蛋白基因cryIA(b)和cryIA(c)导入到籼、粳稻幼穗愈伤组织中,然后经过在含有不同浓度潮霉素的培养基上进行数次筛选,获得一批Bt转基因株。经PCR、Southern杂交及Western印迹分析证实此二基因已整合进水稻中,饲虫试验结果表明,转基因株具有100%杀虫率。     

Smad3基因剔除致骨关节炎小鼠血清蛋白质组双向凝胶电泳分析

Smad3基因剔除导致小鼠骨关节炎。为了进一步深入研究Smad3基因缺失导致骨关节炎形成的分子机制,寻找骨关节炎发生早期的分子变化。用双向电泳技术结合肽质量指纹谱技术对Smad3基因剔除小鼠和野生型小鼠血清蛋白质组进行了初步分析。对其中7个表达差异蛋白质进行了鉴定,并探讨了所鉴定蛋白质与骨关节炎发生的关系。为揭示SMAD3介导的TGF-β信号在骨骼发育中的重要作用提供了线索。      

杜氏盐藻psaB基因cDNA的克隆与序列分析

根据真核生物莱茵衣藻(Chlamydomonas reinhardtii)、Chlamydomonas moewusii、Chlorella vulgaris以及Mesostigma viride的psaB基因的氨基酸高度保守序列,设计一对简并引物,利用TRIzol试剂提取杜氏盐藻（Dunaliella salina）细胞的总RNA,通过RT-PCR,得到的一段长为1.8kb左右的cDNA片段。PCR产物经T-A克隆并测序分析以及测序结果推导成氨基酸序列进行同源性比较,表明所克隆的1815bp序列为杜氏盐藻psaB cDNA片段,GenBank收录号为AY820754。根据已经得到的psaB序列推导成氨基酸序列与一些已知物种的psaB基因相比较,同源性分别为Chlamydomonas reinhardtii 92%,Chlamydomonas moewusii 91%,Chlorella vulgaris 86% , Mesostigma viride 85%,Physcomitrella patens subsp.Patens 85%, Nephroselmis olivacea 84%。据此可推断本实验中所克隆的序列为杜氏盐藻psaBcDNA序列。     

地衣芽孢杆菌产生碱性蛋白酶的动力学研究

应用自动控制发酵设备,首先进行分批发酵试验摸索了地衣芽孢杆菌2709生长与代谢的基本规律。然后采用补料分批发酵方法限制生长基质浓度,测定了一系列(S_I,μ_I)、(μ_j,q_pj)数据,获得K_S、μ_max、α、β等参数的值,并且推导出了细胞生长与产物合成的动力学公式,从而证明了用Monod方程描述地衣芽孢杆菌2709生长速率与基质浓度关系的合理性和合成碱性蛋白酶的发酵属于生长部分关联型。     

含庚型肝炎病毒E2基因片段质粒DNA能诱发相当强烈的体液免疫应答

研究了庚型肝炎病毒E2(HGVE2 )基因片段作为DNA疫苗的可行性。将来自于质粒pThioHis-E2编码HGVE2的基因片段 (559bp)亚克隆到质粒pCMV-S中 ,使之和HBsAg基因位于同一阅读框 ,形成重组质粒pCMV-S-E2。用纯化的质粒pCMV-S-E2DNA注射到昆明小鼠后腿四头肌中来免疫小鼠 ,同时用pCMV-S作为对照。间隔 14天再加强一次免疫。在加强免疫后的第 8天眼眶取血。用E2-GST融合蛋白作为固定化抗原 ,通过ELISA检测受试小鼠的体液免疫应答。结果表明 ,用质粒pCMV-S-EDNA免疫的小鼠可以产生很强的体液免疫应答。     

克鲁维酵母Y-85合成菊粉酶最适条件的研究

采用响应面方法（ResponseSurfaceMethod,RSM）对克鲁维酵母（Kluyveromycessp．）Y－85产菊粉酶培养基成份进行了优选,和正交试验相比,该法选出的最适培养基的酶发酵水平提高28％。用15L自控发酵罐进行产酶条件控制试验,并在1000L罐上进行5批次酶发酵中试,平均菊粉酶活性达68.9u／ml。     

苏芸金杆菌的质粒检测及载晶体蛋白合成基因的质粒在不同菌株间的传递

分析了苏芸金杆菌(Bacillus thuringiensis)7个变种19个野生菌株及无晶体(sp+cr-)和无芽孢(sp-cr+)突变株的质粒图型,证实了苏芸金杆菌的质粒组成既有变种的特异性,亦有菌株的特异性。载有晶体蛋白质合成基因的质粒,B. thuringiensis var．Kurstaki HD-191 菌株可能为42和50Mdal,B．Thuringiensis var. aizawai HD-282菌株为47Mdal, B．Thuringiensis var．Israelensis IPS-82菌株为57Mdal的质粒,而B．Thuringiensis var. wuhanensis 140 菌株的此种基因则可能不在质粒上而在染色体中。HD-191载晶体蛋白合成基因的质粒以很高频率被传递给其无晶体突变株HD-1并在后者细胞中表达。B. thuringiensis var. israelensisIPS-82菌株和 B．Thuringiensis var. kurstaki 无晶体突变株HD—1之间载晶体蛋白合成基因质粒的传递未能成功,提示可能存在着不相容性的障碍。     

The effect of yoghurt on some components of the gut microflora and on the metabolism of lactose in the rat

Feeding yoghurt or base milk (from which the yoghurt was prepared by fermentation) to rats increased the counts of coliforms in the gut whereas the counts of lactobacilli were reduced by yoghurt but not by the base milk. Lactobacillus bulgaricus survived in the guts of gnotobiotic and conventional rats when yoghurt was fed continuously. Streptococcus thermophilus also survived in gnotobiotic rats but its ability to survive in conventional rats could not be examined. Both organisms failed to colonise the gut when a small inoculum of yoghurt was administered orally to germfree rats maintained on the stock diet. Streptococcus thermophilus but not Lact. bulgaricus grew in the rat diet when tested in vitro. Two enzyme systems (beta-galactosidase and lactase) were studied using, respectively, o-nitrophenyl-beta-D-galactopyranoside (ONPG) and lactose as the test substrates. Enzyme levels estimated with both substrates increased in the gut contents when rats were fed yoghurt but an increase was only found with ONPG in the intestinal mucosa fraction. The bacterial origin of all this increased activity is discussed. The other lactose-containing diets did not affect enzyme activity to the same degree. Feeding yoghurt changed the lactobacillus flora from one which was predominantly heterofermentative (Lact. reuteri ) to one which was predominantly homofermentative (Lact. salivarius).     

Beta-galactosidase for distinguishing between Citrobacter and Salmonella

Detection of beta-galactosidase with the aid of o-nitrophenyl-beta-d-galactopyranoside (ONPG) was examined as a means for distinguishing between Citrobacter and Salmonella. Several factors which influence sensitivity and reliability of the test were studied. A bacteriostat, sodium azide, was included to permit prolonged incubation of weak and negative strains of enteric bacilli. By the procedure described, salmonellae gave negative ONPG tests; all of 171 strains of Citrobacter gave positive tests.     

A sensitive method for the detection of beta-galactosidase in transfected mammalian cells.

A sensitive method has been developed for the detection of E. coli beta-galactosidase in transfected HeLa cells. The chromogenic substrate, CPRG (chlorophenol red-beta-D-galactopyranoside), was compared with ONPG (o-nitrophenyl-beta-D-galactopyranoside) by kinetic analysis with purified beta-galactosidase. The Km for CPRG was 1.35 mM and the Vmax was 21.4, whereas the Km for ONPG was 2.42 and the Vmax was 41.1. CPRG at 8.0 mM (6-fold Km) gave 86% of the Vmax and was used as the standard concentration for quantitation of enzyme levels. The Vmax for CPRG was half that for ONPG, and chlorophenol red has an extinction coefficient that is 21-fold higher than o-nitrophenol; these factors make CPRG about 10-fold greater in sensitivity for the quantitation of enzyme levels. The use of Nonidet P-40 to lyse the cells and the use of CPRG as substrate permitted the rapid detection of low levels of enzyme production from transfected human cells that could not be detected using ONPG.     

Use of the contour approach for visualizing the dynamic behavior of intermediates during O-nitrophenyl-β-d-galactoside hydrolysis by β-galactosidase

ONPG disappearance and ONP appearance were synchronously measured during ONPG hydrolysis by β-galactosidase using in situ on-line UV–vis spectroscopy. Intermediate formation was determined by the formula d[ONPG]/dt − d[ONP]/dt. The combined effects of temperature and time on ν_inst and ν_inc during the conversion of ONPG to ONP were expressed by the isogram method in which contour plots were used. Based on this approach, new insights were obtained into the irreversible-continuous conversion of ONPG to ONP during hydrolysis. The intermediate was a moving mass that flowed in three-dimensional space from the substrate to the product. The results of this study support the use of the isogram method for understanding the mechanisms of enzyme-catalyzed reactions via the dynamic resolution approach.     

Induction and general properties of beta-galactosidase and beta-galactoside permease in Pseudomonas BAL-31.

The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside (ONPG) by BAL-31, a marine Pseudomonas that acts as a host for bacteriophage PM2, was studied with intact cells and with cell-free extracts. A transport system for ONPG in whole cells and a beta-galactosidase activity in extracts were evident for cells grown on lactose minimal medium. It was found that the addition of isopropylthio-beta-D-galactopyranoside (IPTG) to cells growing in rich medium induced an ONPG hydrolytic activity detectable in cell extracts but cryptic in whole cells. The existence of a transport system for IPTG, which remained cryptic for ONPG, became apparent from studies of the rates of induction of beta-galactosidase as a function of cell mass at different concentrations of IPTG. The main properties of beta-galactosidase and the lactose transport system of BAL-31 were studied in terms of how they were affected by pH, temperature, or by the presence of several sugars. IPTG competitively inhibits the hydrolysis of ONPG by cell extracts. In cells pregrown on lactose, IPTG slightly inhibits the transport of ONPG. Glucose, and with less efficiency lactose, also inhibits the hydrolysis of ONPG in cell extracts. The growth of cells on lactose minimal medium was inhibited by the addition of IPTG. A mechanism for this inhibition and for the inhibition of ONPG transport by IPTG is discussed.     

Scanning assay of β-galactosidase activity

β-galactosidase, encoded by the lacZ gene in E. coli, can cleave lactose and structurally related compounds to galactose and glucose or structurally related products. Its activity can be measured using an artificial substrate, o-nitrophenyl-β-D-galactopyranoside (ONPG). Miller firstly described the standard quantitative assay of β-galactosidase activity in the cells of bacterial cultures by disrupting the cell membrane with the permeabilization solution instead of preparing cell extracts. Therefore, β-galactosidase became one of the most widely used reporters of gene expression in molecular biology to reflect intracellular gene expression difference. But the Miller assay procedure could not monitor the β-galactosidase reaction in real time and its results were greatly influenced by some operations in the Miller procedure, such as permeabilization time, reaction time and concentration of the cell suspension. A scanning method based on the Miller method to determine the intracellular β-galactosidase activity in E. coli Tuner (DE3) expressing β-galactosidase in real time was developed and the permeabilization time of cells was optimized for that. The comparison of 3 assays of β-galactosidase activity (Miller, colorimetric and scanning) was made. The results proved that scanning method for the determination of enzyme activity with using ONPG as substrate is simple, fast and reproducible.     

Galactosidase activity of lactose-positive Neisseria

The chromogenic substrate o-nitrophenyl-beta-d-galactopyranoside (ONPG) was hydrolyzed by lactose-positive Neisseria. Eight strains of pharyngeal origin were examined. In culture reactions, seven strains resembled Neisseria meningitidis with the exception that they produced acid from 1% (w/v) lactose. An eighth strain (V8) differed in that it did not form acid from maltose or from 1% lactose. However, acid formation was observed in 10% lactose cultures of strain V8, suggesting that entry of lactose occurred by passive diffusion, rather than as a result of permease activity. The enzymes which hydrolyzed ONPG were produced constitutively by the cells of all eight strains. Thus, specific activity in these strains was not increased by prior exposure to lactose, or to two other possible inducers, isopropyl-beta-d-thiogalactoside or methyl-beta-d-thiogalactoside. Study of cell-free extracts of one strain showed that the enzyme was heat-labile, having a half-life of 10 min at 45 C. The enzyme was unstable at low protein concentrations, but it was protected completely or partially when albumin or manganous ions were added. The enzyme appeared to be a typical beta-galactosidase: alpha-galactosides (melibiose and p-nitrophenyl-alpha-d-galactopyranoside) were not hydrolyzed, activity against ONPG was not dependent upon inorganic phosphate, and galactose was released by cleavage of ONPG. ONPG hydrolysis provided a simple and rapid method for detecting lactose-positive Neisseria.     

Membrane malfunctions in freeze-dried Escherichia coli

Freeze drying and exposure to oxygen of E. coli causes damage to the bacterial cytoplasmic membrane. Freeze-drying itself produces an injury to the transport system for ONPG and potassium, so as to make the membrane leaky to these compounds. This damage is partially repaired upon incubation of the reconstituted bacteria in nutrient medium. When, however, freeze dried bacteria are not held in vacuo before reconstitution, but exposed to oxygen, this damage to the bacterial membrane becomes more extensive and irreversible.     

静电纺丝纳米纤维体外释放与蛋白活性的研究

目的:通过选择不同的模型蛋白,探讨准确的研究静电纺丝纳米纤维支架的体外释放和快速的测定蛋白活性的方法.方法:通过O/W乳液法静电纺丝制备纳米纤维,并用扫描电镜对纳米纤维表面进行了表征.以GM-CSF为模型蛋白,采用ELISA双抗体夹心法考察纤维的体外释放行为;以BSA为模型蛋白,用SEC-H-PLC比较纤维制备前后蛋白的聚集情况;以β-半乳糖苷酶为模型蛋白,用ONPG法比较纤维制备前后酶的催化活性.结果:纤维表面平滑,直径均一,呈现互相连通的三维网状结构.纤维在5天内释放90％以上;纤维中回收的BSA单体比例为66.53％;β-半乳糖苷酶在纤维中的催化活性保持原活性的3.37％.结论:通过选择不同的模型蛋白,能够准确的测定静电纺丝纤维的体外释放,快速的考察纤维中的蛋白活性,对于更好的研究蛋白药物纳米纤维支架具有重要的参考价值.     

Rapid confirmation of Clostridium perfringens by using chromogenic and fluorogenic substrates

The use of 4-methylumbelliferyl phosphate (MUP) and ortho-nitrophenyl-beta-D-galactopyranoside (ONPG) for the identification of Clostridium perfringens was investigated. A liquid assay containing both MUP and ONPG was a highly specific alternative method for C. perfringens confirmation, reducing incubation time from 48 to only 4 h. The assay solution is easy to prepare, does not require anaerobic conditions for use, and has an extended shelf life.