酶标DNA探针与Y染色体DNA的探测

 用辣根过氧化物酶标记DNA的技术,制备了酶标基因探针。研究了酶标过程和产物的电泳行为;用斑点杂交和southern印迹杂交探测了单链、双链DNA,灵敏度可达pg水平,以此酶标的Y染色体特异的DNA片段作探针,进行了DNA杂交的性别分析,证明该探针能清楚地区别两性基因组DNA,这对基因的研究和诊断有一定实用价值。     

酶标探针在转基因植物检测中的应用

采用碱性磷酸酶标记DNA制备分子探针, 并首次在植物中应用。酶在苯醌作用下与单链DNA联结, 形成DNA和酶的共价复合物即酶标探针。此探针通过分子杂交与待测DNA结合, 再与酶的底物作用显色, 3～6h 内可观察结果。用此探针检测转基因植物中的UidA基因, 点杂交和Southern杂交结果表明, 所合成的酶标探针具有快速、准确、安全而经济的优点。点杂交证明外源UidA基因被成功转化到受体植物中, Southern 杂交对转基因的材料检测的结果证明, 该材料包含多个外源UidA基因拷贝, 初步确定其外源UidA基因拷贝数在5个以上。     

DNA探针的诊断应用

<正>使用重组DNA技术制备特异的分子(常为DNA)探针已为医学和兽医诊断实验室提高对传染病、遗传紊乱、恶性肿瘤的诊断提供了新的强有力的工具,同时也给完成如组织分型、亲缘关系试验这类工作提供了更灵敏感更特异的方法,该技术对法医学也是很有用的(如表1)。DNA探针作为诊断试剂的应用或使实验室减少周转时间,拓宽所检测、鉴别和/或定量的试样的范围,且由于简化     

直接酶标——化学发光分子杂交体系的建立

本文通过调整发光剂、氧化剂、发光增强剂的浓度,并加入一组辅助剂,改进了依赖过氧化物酶化学发光液的配方。并通过比较实验证明:该发光液的检测灵敏度高于其它原有的发光液,同时还发现合成后的酶标复合物可经CM-32离子交换柱阶段洗脱纯化,以提高杂交灵敏度。从而建立了一套直接酶标DNA探针——化学发光自显影分子杂交体系。其狭缝杂交灵敏度可达0.03pg。并利用该体系进行了单拷贝基因分析,Western印迹杂交及检测血清HBV DNA等的应用研究。     

HBV—DNA的生物素寡聚核苷酸探针快速检测

本文建立一种快速ＨＢＶ－ＤＮＡ检测方法,此法是基于生物素标记的寡聚核苷酸与硝酸纤维素滤膜上的靶ＤＮＡ杂交,然后通过Ｓｔｒｅｐｔｏａｖｉｄｉｎ－碱性磷酸酶偶联体及生色底物进行检测。本文研究了此法的实用性,表明对于实验室及临床的ＨＢＶ－ＤＮＡ检测,此法是一种快速（４小时）、灵敏（１－１０ｐｇ）、特异、方便的方法。     

用于HBV病毒快速诊断的分子信标探针设计

分子信标是一种高灵敏度、高特异性的新型荧光核酸探针.它在与互补DNA或RNA靶序列杂交时放出荧光.利用Genebank中调出已知HBV病毒ayr亚型基因组信息,通过BeaconDesigner4.0软件进行分子信标探针设计,共设计出6条分子信标探针,以便于为目前HBV病毒快速诊断提供参考.     

人胎盘碱性磷酸酯酶基因在毕赤酵母中的表达和活性检测

将人胎盘碱性磷酸酯酶 (hPLAP)基因克隆到质粒ppICZαA中并在巴斯德毕赤酵母Pichiapastoris蛋白酶缺陷菌株SMD1 1 6 8中诱导表达。结果表明 :2拷贝子的重组酵母诱导表达产物酶活性最高 ,拷贝Mut 和Muts 表型不同对hPLAP酶活性没有显著影响     

血清尿素氮、碱性磷酸酶与儿童营养不良的相关性分析

摘要 目的：探讨血清尿素氮（BUN）、碱性磷酸酶（AKP）与儿童营养不良的相关性。方法：选择2017年10月至2022年10月我院接诊的108例儿童,根据是否存在营养不良将受试儿分为营养不良组（38例）和营养良好组（70例）。检测血清BUN、AKP水平,分析BUN、AKP与受试儿身体组分的相关性。多因素Logistic回归分析影响儿童营养不良的因素,受试者工作特征曲线（ROC）分析BUN、AKP诊断儿童营养不良的价值。结果：营养不良组血清BUN、AKP水平,体脂百分含量（fat percentage,F%）、总体脂肪（TBF）、瘦体重（LBM）低于营养良好组（P＜0.05）。儿童血清BUN、AKP水平与F%、TBF、LBM呈正相关（P＜0.05）。消化不良是儿童营养不良的危险因素（P＜0.05）,出生体质量、BUN、AKP、LBM是儿童营养不良的保护因素（P＜0.05）。联合BUN和AKP诊断儿童营养不良的曲线下面积为0.879,高于单独BUN和AKP诊断（P＜0.05）。结论：营养不良儿童血清BUN、AKP水平降低,且与身体组分改变和营养不良风险增加有关,联合检测血清BUN和AKP水平有助于评估儿童营养不良风险。     

Effects of Magnesium Ions on Thermal Inactivation of Alkaline Phosphatase

The effect of Mg²⁺ on the thermal inactivation and unfolding of calf intestinal alkaline phosphatase has been studied at different temperatures and Mg²⁺ concentrations. Increasing the Mg²⁺ concentration in the denatured system significantly enhanced the inactivation and unfolding of the enzyme during thermal inactivation. The analysis of the kinetic course of substrate reaction during thermal inactivation showed that at 47°C the increased free Mg²⁺ concentration caused the inactivation rate to increase. Increasing the temperature strengthened the effect of Mg²⁺ on the thermal inactivation. Control experiment showed that this is not due to salt effect. The time course of fluorescence emission spectra showed that the emission maximum for Mg²⁺-containing system was always higher than that of Mg²⁺-free system, and the higher temperature enhanced this difference. In addition, Mg²⁺also enhanced the unfolding rate of the enzyme at 47°C. The potential biological significance of these results are discussed.     

南方鲇碱性磷酸酶的分离纯化及部分性质的研究

用正丁醇抽提,硫酸铵分级沉淀,ＤＥＡＥ－纤维素和ＳｅｐｈａｃｒｙｌＳ－２００柱层析,从南方鲇（Ｓｉｌｕｒｕｓ　ｍｅｒｉｄｉｏｎａｌｉｓ　Ｃｈｅｎ）肠粘膜中提取出碱性磷酸酶（ＡＫＰ）。提纯倍数为３９．５０倍,比活为６８．３５μ／ｍｇ蛋白,提取酶液经ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ只呈现一条区带。该酶的分子量为１３２１４０,Ｎ末端氨基酸为门冬氨酸,最适ｐＨ为１０．１０,７．５＞ｐＨ＞１１．５时不稳定,最适温度为４０℃左右,对热不很稳定,以磷酸苯二钠为底物其Ｋ_ｍ值为１．７２×１０~（－３）ｍｏｌ／Ｌ。Ｍｇ~（２＋）、Ｍｎ~（２＋）为该酶的激活剂,ＫＨ_２ＰＯ_４、Ｌ－ＣｙＳ、ＭＥ、ＤＦＰ、ＥＤＴＡ－Ｎａ_２为抑制剂。选用ＫＨ_２ＰＯ_４和ＤＦＰ作抑制类型的判断,结果表明,ＫＨ_２ＰＯ_４属竞争性掏剂,其抑制常数为２．３ｍｍｏｌ／Ｌ;ＤＦＰ为非竞争性抑制剂,抑制常数为１．０５ｍｍｏｌ／Ｌ。     

Oxidative Inactivation of Brain Alkaline Phosphatase Responsible for Hydrolysis of Phosphocholine

Abstract : Alkaline phosphatase, one of the enzymes responsible for the conversion of phosphocholine into choline, was purified from bovine brain membrane, where the phosphatase is bound as glycosylphosphatidylinositollinked protein, and subjected to oxidative inactivation. The phosphatase activity, based on the hydrolysis of p-nitrophenyl phosphate and phosphocholine, decreased slightly after the exposure to H₂O₂. Inclusion of Cu²⁺ in the incubation with 1 mM H₂O₂ led to a rapid decrease of activity in a time- and concentration-dependent manner. In comparison, the H₂O₂/Cu²⁺ system was much more effective than the H₂O₂/Fe²⁺ system in inactivating brain phosphatase. In a further study, it was observed that the hydroxy radical scavengers mannitol, ethanol, or benzoate failed to prevent against H₂O₂/Cu²⁺-induced inactivation of the phosphatase, excluding the involvement of extraneous hydroxy radicals in metalcatalyzed oxidation. In addition, it was found that both substrates, p-nitrophenyl phosphate and phosphocholine, and an inhibitor, phosphate ion, at their saturating concentrations exhibited a remarkable, although incomplete, protection against the inactivating action of H₂O₂/Cu²⁺. A similar protection was also expressed by divalent metal ions such as Mg²⁺ or Mn²⁺. Separately, it was found that H₂O₂/Fe²⁺-induced inactivation was prevented by p-nitrophenyl phosphate or Mg²⁺ but not phosphate ions. Thus, it is implied that phosphocholine-hydrolyzing alkaline phosphatase in brain membrane might be one of enzymes susceptible to metal-catalyzed oxidation.     

光标生物素HBV-DNA探针及其临床应用

本文报告了应用生物素交联光敏补骨酯素(Bp,Biotin-Psoralen)标记HBV DNA探针的动力学研究和临床应用结果。表明化学合成Bp经366nm光照与DNA发生加成反应。能标记dsDNA,也能标记ssDNA。加热可使Bp-dsDNA变性,强碱则不能使Bp-dsDNA变性。临床DNA检测结果表明,应用Bp-HBV探针杂交,DNA检出率与HBeAg符合率达99.08％,与~(32)P-HBV符合率达98.8％,提示光标记Bp-HBV探针灵敏度接近同位素探针。     

生物素标记HBV RNA探针的制备及应用

本文首次采用ＳＰ６５特殊质粒与人类的乙型肝炎病毒ＤＮＡ重组,制备了Ｂｉｏ－ＨＢＶ ＲＮＡ探针,能特异地与ＨＢＶ ＤＮＡ杂交,将该探针与缺口转移方法标记的Ｂｉｏ－ＨＢＶ ＤＮＡ探针进行了比较,结果显示出Ｂｉｏ－ＨＢＶ ＲＮＡ探针比Ｂｉｏ－ＨＢＶ ＤＮＡ探针的敏感性提高１０倍,并分别应用两种探针同时检测７０例乙肝病人血清中ＨＢＶ ＤＮＡ,阳性率各为３１．４２％、２８．５７％（Ｐ＞０．２５）。对Ｂｉｏ－     

Mammalian Brain Alkaline Phosphatase: Expression of Liver/Bone/Kidney Locus Comparison of Fetal and Adult Activities

The alkaline phosphatases (EC 3.1.3.1) are determined by at least three gene loci, which can be sharply distinguished one from another by their sensitivity to inhibition with various amino acids and peptides and by ther-mostability. Alkaline phosphatase is present in the brains of guinea pig, rat, mouse, hamster, squirrel, rabbit, cat, sheep, cow, tamarin, baboon, and man. The gene locus coding for alkaline phosphatase in all these brains is the liver/ bone/kidney locus, as indicated by thermostability studies and by inhibition studies with L-phenylalanine, L-homoarginine, and L-phenylalanylglycylglycine. The average brain alkaline phosphatase activity is about 35% of the average for the livers and only 7.2% and 4.4% of the average kidney and placental activities, respectively. During growth and development, brain alkaline phosphatase activity decreases in the mammals studied. The amount of change is tissue- and species-dependent.     

Kinetics of Inhibition of Green Crab (Scylla serrata) Alkaline Phosphatase by L-Cysteine

The inhibition of alkaline phosphatase from green crab (Scylla serrata) by L-cysteine has been studied. The results show that L-cysteine gives a mixed-type inhibition. The progress-of-substrate-reaction method previously described by Tsou [(1988), Adv. Enzymol. Related Areas Mol. Biol. 61, 391–436] was used to study the inactivation kinetics of the enzyme by L-cysteine. The microscopic rate constants were determined for reaction of the inhibitor with the free enzyme and the enzyme–substrate complex (ES) The results show that inactivation of the enzyme by L-cysteine is a slow, reversible reaction. Comparison of the inactivation rate constants of free enzyme and ES suggests that the presence of the substrate offers marked protection of this enzyme against inactivation by L-cysteine.