贝类产品中软骨藻酸的HPLC检测方法

建立了高效液相色谱测定贝类产品中软骨藻酸的检测方法,具体过程为:样品先经甲醇浸提后,再用甲醇-水(V（甲醇）∶V（水）=1∶1）提取2次,合并提取液后用Zorbax SB300-C18柱(2.1 mm×250 mm,5 μm)进行分离,以乙腈-0.2%磷酸水溶液（含0.1%三乙胺）（V（乙腈）∶V（磷酸水溶液）=12∶88）为流动相于室温下等度洗脱,流速为0.25 mL/min,进样量20 μL,检测波长为242 nm.结果表明,在建立的提取方法和色谱条件下,软骨藻酸从复杂的样品基体中被分离出来,在0.02~1.0 mg/L质量浓度范围内具有良好的线性关系（r＞0.999）,样品的平均加标回收率为108%,测量方法的RSD为3.5%（n=6）,检出限为23.5 ng/g.所建立的检测方法可适用于对贝类产品中软骨藻酸的快速检测.     

ELISA法快速测定McAb培养上清中鼠源IgG质量浓度

用小鼠IgG纯品免疫新西兰大白兔,获得兔抗小鼠IgG多克隆抗体.以兔抗小鼠lgG多抗包被聚苯乙烯微孔板.配以辣根过氧化物酶标记的山羊抗小鼠IgG抗体和以3,3'5,5-四甲基联苯胺(TMB)为底物,建立了快速测定McAb培养上清中鼠源IgG的双抗体夹心ELISA方法.该方法的检测线性范围是7.02ng/mL至449.38ng/mL之间,回收率在94.83%-115.15%之间,变异系数在1.45%-9.94%之间.显然用该方法测定MeAb培养上清中鼠源IgG质量浓度是可行的.     

Immunochemical identification of gelsolin in maize pollen

Gelsolin is a representative of a type of actin-binding proteins (ABPs) universally found in eukaryotes. It plays role in nucleation, capping and severing of actin filamentsin vitro. In our experiment, gelsolin was purified from pig plasma and the polyclonal antibodies against it were prepared. The crude extracts of maize pollen were immunodetected by Western-blotting with polyclonal antibody and monoclonal antibody respectively. The immunodetection results show that gelsolin exists in maize pollen and its molecular weight is about 91 ku, similar to that of gelsolin found in animal tissues.     

HPLC-MS/MS法快速测定鲜鱼肉中孔雀石绿及其代谢物含量

建立了鲜鱼肉中孔雀石绿及其代谢物隐色孔雀石绿残留量的高效液相色谱-串联质谱测定方法.采用电喷雾正离子模式离子化,选择反应扫描模式定量,孔雀石绿的定量监测离子对为m/z:329313,隐色孔雀石绿的定量监测离子对为m/z:331239,孔雀石绿的检出限为0.002 2ng/m L,定量限为0.007 3 ng/m L;隐色孔雀石绿的检出限为0.043 3 ng/m L,定量限为0.14 ng/m L,线性范围均为1~10 ng/m L,相对标准偏差均小于4%(n=6),方法的回收率均大于90%.     

心肌肌钙蛋白I-C复合物酶联免疫检测方法的建立

为了制备抗人心肌肌钙蛋白I(cTnI)单克隆抗体、建立检测人cTnI链酶亲和素-生物素双抗体夹心酶联免疫吸附测定(ELISA)方法,用基因工程表达的人cTnI免疫雌性BALB/c小鼠,以杂交瘤技术制备得到三株特异性抗人cTnI的单克隆抗体记为6G3,6A6,3G9.三株杂交瘤细胞中6G3和6A6为IgG1,3G9为IgG2b,腹水效价分别为4×10-5,4×10-5和10-5.亲和常数为5.0×109,6.4×109和3.8×109mol-1.Westernblotting结果表明6G3和6A6能够识别cTnI-C复合物.把6G3抗体生物素化并制备抗cTnI、cTnC和cTnI-C的大鼠多克隆抗体.以多抗作为固相捕捉抗体,生物素化6G3抗体作为检测抗体,建立检测cTnI的双抗体夹心ELISA方法并初步应用于临床病人标本检测.以cTnI-C复合物多抗捕捉检测方法具有良好的灵敏度、特异性和准确性,灵敏度为0.9ng/mL,较包被其他抗体捕捉方法提高1~2倍.批内变异系数为5.03%,回收率为94.56%.测定17例AMI病人和18例血清样本临床诊断结果符合率为100%.     

Effects of ubiquitin-proteasome pathway on mouse sperm capacitation, acrosome reaction and in vitro fertilization

Chlortetracycline (CTC) fluorescence patterns were used to study changes in the patterns B and AR of mouse sperm after incubation with reagents that would block the UPP. They were the monoclonal antibody against ubiquitinated proteins—UCP1; the polyclonal antibody against ubiquitin—anti-Ub, and a special inhibitor against proteasome—ALLN. Furthermore, we treated the capacitated sperm or the eggs with these reagents separately and tested whether the normal in vitro fertilization was blocked or not. Results illustrate that UCP1, anti-Ub, and ALLN have little effects on sperm capacitation and acrosome reaction, but they do inhibit fusion of mouse sperm with eggs, which suggests that UPP play an important role in mouse in vitro fertilization.     

Preparation of a recombinant adeno-associated viral vector with a mutation of human factor Ⅸ in large scale and its expression in vitro and in vivo

A series of adeno-associated viral vectors containing a mutation of human factor Ⅸ (hFⅨR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFⅨR338A were obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFⅨR338A was prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFⅨR338A viral stocks on a large scale and higher titer was established, which can be used for industrial purpose. The titer of rAAV-hFⅨR338A was more than 1.25×10¹² particle/mL, and then, a mammalian cell line, C2C12 and the factor Ⅸ knock-out mice were transfected with the rAAV-hFⅨR338A in vitro and in vivo. The results show that the high-level expression of rAAV-hFⅨR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng·(10⁶ cells)^-1·(24 h)^-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFⅨR338A, which was as high as the expression of rAAV-hFⅨ-wt (2565.76±64.36) ng·(10⁶ cells)^-1·(24 h)^-1 in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFⅨ-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFⅨR338A is about 2.46 times higher than that of hFⅨ-wt. It was first reported that a mutation of human factor Ⅸ was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFⅨR338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFⅨ.     

水产品中麻痹性贝类毒素的微流体免疫检测技术研究

鉴于我国在贝类毒素检测方面相对薄弱的现状,将微流体免疫芯片技术和化学发光分析法相结合来检测贝类毒素,旨在探索一种快速、高效、低耗以及灵敏的检测手段以弥补方法缺陷。选择研究最为广泛的麻痹性贝类毒素-石房蛤毒素（STX）作为研究对象,利用基于酶联免疫反应（ELISA）为原理的石房蛤毒素检测试剂盒在微流体免疫芯片-化学发光检测平台进行实验,绘制出STX标准曲线并计算出检测限。首先在羧基聚苯乙烯微球表面包被抗体并进行定性分析,然后对底物流速和底物溶液的稀释倍数等进行优化,最后对市场上的贝类进行了实际样品的检测。通过绘制标准曲线得到STX的检测限为0.005675ng/mL,而传统方法的检测限为5ng/mL,表明应用微流体芯片技术可以大幅度提高食品安全检测的灵敏度。     

石墨炉原子吸收法测定重酒石酸去甲肾上腺素中痕量钯

采用石墨炉原子吸收法测定重酒石酸去甲肾上腺素中的痕量钯,对石墨炉测钯的条件进行了优化,选定的石墨炉升温条件为:干燥100℃,灰化1 000℃,原子化2 300℃,除残2 600℃。用于实际样品测定,方法的精密度为3.45%,检出限为1.55 ng/mL,样品测定结果令人满意。     

Effects of 2,3,7,8-TCDD and benzo[a]pyrene on modulating vitellogenin expression in primary culture of crucian carp （Carassius auratus） hepatocytes

Vitellogenin (Vtg) is the precursor of yolk protein. Its expression and secretion are estrogen-regulated and are crucial for oocyte maturation. An in ritro xenoestrogen screening model was established by measuring Vtg induction in cultured primary hepatocytes from crucian carp. Vtg production was detected by biotin-avidin sandwich ELISA method while Vtg and cytochrome P4501A1 (CYP1A1) mRNA induction were measured by semi- quantitative PCR-primer dropping technique. Vtg and Vtg mRNA were dose-dependently induced by diethylstilbestrol (DES, 0.2-200 ng/mL) in hepatocytes of crucian carp. Co-treatment of the DES-induced hepatocytes with either 2,3,7,8-TCDD (TCDD, 0.1-4 pg/mL) or benzo[a]pyrene (B[a]P, 5-1000 ng/mL) resulted in a reduction of Vtg production and an increment of CYP1A1 mRNA expression both in a dose dependent manner, indicating the anti-estrogenic effects of the compounds. However, at lower tested concentrations, TCDD (0.1, 0.2 pg/mL), B[a]P (5 ng/mL)seemed to have a potentiating effect on Vtg expression and secretion, although by their own these compounds had no observable estrogenic effect on Vtg induction. Tamoxifen (a selective estrogen receptor modulators, 1 nmol/L-1μmol/L),and β-naphtho-flavone (β-NF, an aryl hydrocarbon receptor inducing compounds, 2.5-1000 ng/mL) also were employed to study the possible interactions in DES-induced Vtg expression. In co-treatment of the DES-induced hepatocytes with β-NF or tamoxifen, the decrease in Vtg production did parallel induction of CYP1A1 for β-NF, but tamoxifen inhibited Vtg induction did not parallel induced CYP1A1 expression in all test concentrations. On the contrary, it was found that in co-treatment of the TCDD-induced hepatocytes with DES, TCDD induced CYP1A1 mRNA production was inhibited by DES also. These results implicated a possible cross talk between estrogen receptor- and aryl hydrocarbon receptor-mediated pathways in the hepatocytes.     

Biological aspects on the cultures of the entomophthoralean fungus<Emphasis Type="Italic">Pandora delphacis</Emphasis> grown on broomcorn millets

Among entomopathogenic fungi the Entomophthor- ales plays an important role in natural control of insect pests through induction of epizootics in insect popula-tions[1—3]. Since most species of the fungal group are 4 - characteristic with their special capability of actively dis-charging conidia to favor infection and dissemination in host populations, they are ideal targets for epizootiologi-cal study, being considered as important microbial control agents. Currently, 66 species …     

轮状病毒间接免疫荧光的检测方法

以兔抗SA11多克隆抗体为一抗、 Alexa 488标记的山羊抗兔抗体为二抗, 通过对反应条件的优化建立轮状病毒（RV）检测的间接免疫荧光法（IFA）. 实验结果表明, IFA最佳工作条件为: MA104细胞密度为每孔2×10⁴个, 胰酶活化轮状病毒质量浓度为1 μg/mL, 培养时间为18 h, 一抗最适稀释度为1∶800, 二抗最适稀释度为1∶500.     

Ovicidal activity of two fungal pathogens （Hyphomycetes）against Tetranychus cinnabarinus （Acarina：Tetranichidae）

The carmine spider mite, Tetranychus cinna-barinus, is an economically important pest that devastates varieties of crops worldwide and develops significant resistance to common chemical pesticides, most of which lack ovicidal activity. In the present study, two isolates of entomopathogenic fungi, Beuaveria bassiana SG8702 and Pae-cilomyces fumosoroseus Pfrl53, were bioassayed against T. cinnabarinus eggs at 25 ℃ under a photophase of 12 : 12 (L:D). Infected eggs on Vicia faba var. minor leaves failed to hatch due to distortion and shrinkage and had fungal outgrowths when maintained under moist conditions. Sprays of B. bassiana conidia to T. cinnabarinus eggs (on leaves) at the concentrations of 58, 298 and 1306 conidia/mm2 (3 replicates per concentration and 35-65 fresh mite eggs per replicate) resulted in corrected egg mortalities of 20.4±4.2%, 36.0±7.6% and 64.6±12.5% (F=43.14, P <0.01), respectively; sprays of P. fumosoroseus at 129, 402 and 2328 conidia/mm2 caused egg mortalities of 16.1±11.1%,     

流动注射-冷蒸汽发生AAS法测定烟草样品中的痕量汞

 研究了以KBH₄为还原剂的流动注射-冷蒸汽发生原子吸收光谱法,测定烟草中痕量汞.就其分析条件对灵敏度影响进行了优化研究.本方法共存离子干扰小,精密度较高(平行测定10ng/mL的标准样品7次的相对标准偏差为2.80%)、检测限好(0.4ng/mL),线性范围在0.4～60ng/mL之间.利用HNO₃-H₂O₂体系微波消解进行了样品前处理,有较高的样品回收率(98.15%).测定结果令人满意.     

Cloning and Expression of BTB/POZ-Domain from Zebrafish gcl （Germ Cell-Less）, and Appraisement of Its Polyclonal-Antibody

In this study, a recombinant pET28c-gBTB/POZ was constructed by cloning the sequence of the BTB/POZ domain of the zebrafish gcl （germ cell-less） into the expression vector pET28c, and pET28c-gBTB/POZ was transformed into BL21（DE3） pLysS strain to express the fusion protein for the preparation of antibody. Polyclonal-antibody against the GCL-BTB/POZ domain was prepared by immunizing rabbit with the fusion protein, and the Western Blot and immuno-histochemical analysis were performed to detect the quantity of the polyclonal-antibody. The result indicates that the polyclonal-antibodies were of good quantity and specification. Further studies will be performed to demonstrate the function and expression pattern of the GCL protein during the development process of zebrafish with the polyclonal-antibody.     

P-5m八肽多克隆抗体的制备及应用

目的制备P-5m八肽的多克隆抗体并进行初步鉴定和应用,为研究P-5m八肽的功能及作用机制获得重要的实验工具.方法将9-氟甲氧羰基(Fmoc-)固相合成法合成的P-5m八肽纯化后与载体蛋白-血蓝素联接;取偶联后的多肽皮下注射免疫新西兰白兔,并加强免疫得到抗血清,以C18反相色谱分离柱分离纯化;通过间接ELISA方法测定血清效价;利用Transwell实验和Western blot方法初步鉴定多克隆抗体拮抗P-5m八肽抑制肿瘤细胞转移的效果.结果纯化后的抗体经间接ELISA测定效价均大于1∶160 000,表明获得高效价的多抗;通过Transwell实验证明该抗体能够封闭P-5m对肿瘤细胞侵袭的抑制能力;通过Western blot方法证实此抗体效价较高,特异性较强;该多克隆抗体能够封闭P-5m在SW620细胞中上调E-cadherin表达的作用,并阻遏P-5m对MMP-2和MMP-9表达的下调作用,与对照组比较差异具有统计学意义(P0.05).结论 P-5m多克隆抗体可用于Transwell和Western blot等实验,为研究P-5m八肽的功能及抗肿瘤转移作用机制提供实验依据.     

Effects of temperature on the expression of STN7 and phosphorylation of LHCII proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In Arabidopsis thaliana, STN7 kinase is required for phosphorylation of LHCII and for state transitions. In this paper, a hydrophilic polypeptide, derived from the amino acid sequence of STN7, was conjugated to a carrier protein, bovine serum albumin （BSA）, to obtain the polyclonal antibody. Immunogenicity and specificity of the polyclonal antibody were evaluated by agar gel immunodiffusion （AGID） test and Western blot analysis. The results show that besides the phosphorylation of LHCII proteins, also the expression of STN7 was regulated by temperature conditions. In addition, the change tendency of LHCII proteins phosphorylation was not only coherent with expression of STN7 with respect to increasing temperature, but also closely related to state transitions. These results would provide useful information for studying regulatory mechanism of LHCII proteins phosphorylation and expression of STN7.     

HPLC法测定重楼块根中4种重楼皂苷含量研究

建立HPLC测定重楼块根中4种重楼皂苷含量的方法。结果表明:重楼皂苷Ⅰ质量浓度在0.054~0.540 mg·mL^-1范围内线性关系良好,r=0.999 6 (n=5),回收率为99.12%,RSD为1.95%;重楼皂苷Ⅱ质量浓度在0.043 4~0.434 mg·mL^-1范围内线性关系良好,r=0.990 8 (n=5),回收率为99.10%,RSD为1.89%;重楼皂苷Ⅵ质量浓度在0.012 6~0.126 mg·mL^-1范围内线性关系良好,r=0.993 3 (n=5),回收率为99.46%,RSD为2.01%;重楼皂苷Ⅶ质量浓度在0.010 3~0.103 mg·mL^-1范围内线性关系良好,r=0.999 1 (n=5),回收率为96.52%,RSD为2.25%。HPLC法能够准确检测出重楼块根的主要皂苷成分及其含量,且操作简便,可为重楼中4种主要皂苷含量的检测提供科学方法。     

Investigation of Ru （ phen ）3^2＋-Hydrazine-Ce（Ⅳ） Chemiluminescence System and Its Analytical Application

0 IntroductionThbiep ycroidmipnlee)xru ctahteinoinu mof(Ⅱ r)u t(he Rniuu(mbip,y m)3ai2n +ly) atrnids- t(r2is -,(21’ -,10-phenanthroline)ruthenium(Ⅱ) (Ru(phen)32 +) ,is a kindof sensitive analytical reagent for electrogenerated chemilumi-nescence(ECL) and chemiluminescence(CL) ,on which a par-ticular review has been presented[1]. The earliest publicationon the synthesis of Ru(bipy)32 +appeared in 1936[2], andduringthefollowing30 years ,theinvestigations relatedtothissubstance were only in…