完全体外化牛细胞核移植的研究

本研究探讨了用体外成熟卵母细胞和体外受精胚胎进行核移植的可能性。本研究结果在我国首次获得牛核移植的成功。从屠宰场回收的卵巢中抽取卵球－卵母细胞复合体，采用以前报道的方法（石德顺等，广西农业大学学报’１９９４：１３：１－５）进行体外成熟（ＩＶＭ）和体外受精（ＩＶＦ），获得ＩＶＭ卵母细胞和ＩＶＦ胚胎，体外成熟２３－２４ｈ后，在含０．１％透明质酸酶的无钙镁ＰＢＳ（ＰＢＳ）内，用细管反复吹打去掉卵丘细胞。选择具有明显第一极体的卵母细胞通过显微操作移去第一极体及其附近约一半的细胞质进行去核，去核的卵细胞在成熟液内继续培养到ＩＶＭ３０ｈ．体外发育到８－３２细胞期的ＩＶＦ胚胎用作核供体．供体胚胎用含０．２５％Ｐｒｏｎａｓｅ（溶于ＰＢＳ￣－）溶解透明带，并用细管吹打将其分散成单个卵裂球，在ＩＶＭ３０ｈ将单个卵裂球显微注入去核卵母细胞的卵周隙内，并在ＩＶＭ３１ｈ用８０Ｖ／ｍｍ４０μｓ两次电脉冲（间隔１秒）来诱导卵裂球与去核卵母细胞的融合。融合卵在颗粒细胞单层培养滴内共培养，体外培养２４ｈ后观察卵裂结果，经体外培养５－８天后发育到桑椹和囊胚的核移植胚胎移植到同期发情的受体．２个月后直检妊娠情况．本实验中共操作１９４枚卵母细胞，     

Purification of platelet-derived endothelial cell growth inhibitor and its characterization as transforming growth factor-β type 1

In 1986, Brown and Clemmons (Proc. natl Acad. Sci. USA83 (1986) 3321) showed that platelets contain a substance, platelet-derived growth inhibitor (PDGI), that inhibits in vitro endothelial cell replication. Although platelets are rich in transforming grwoth factor (TGF-), PDGI was considered not to be related to TGF-, on the basis of its reported properties (extraction from platelets at neutral pH, binding to heparin-Sepharose). However, we purified PDGI to near homogeneity and showed that on the basis of HPLC retention behavior, in vitro growth inhibitory activities with several cell types, receptor binding, and immunoneutralization of growth inhibitory activity with specific anti-TGF- type 1 antibodies, PDGI is most probably identical with TGF- type 1.     

In vitro amiodarone protein binding and its interaction with warfarin

Summary The binding of amiodarone to human plasma protein and to bovine serum, albumin was studied by three different methods, ultracentrifugation, equilibrium dialysis and fluorescence spectroscopy. The fraction of amiodarone bound to plasma protein amounted to 96.3%. The changes in the binding properties of 1-anilino-naphthalene-8-sulfonate for bovine serum albumin using warfarin and amiodarone as independent inhibitors were analyzed in terms of binding site specificity. The findings indicated that amiodarone and warfarin have two different binding sites on bovine serum albumin, so a noncompetitive inhibition mechanism was indicated. On the basis of our data we cannot exclude other mechanisms of interaction besides direct displacement of one drug by another; nevertheless, metabolite interference between amiodarone and coagulation cofactors may better explain the enhancement of warfarin's pharmacological action in association with amiodarone.This work was partially funded by the CNR (National Research Council, Rome, Italy), Program on Clinical Pharmacology and Rare Diseases. The authors would like to thanks Drs E. Marzi and E. riva for their help.     

牛泡沫病毒BFV3026 gag基因的克隆及其在大肠杆菌中的表达

从牛泡沫病毒BFV3026感染的胎牛肺细胞中提取Hirt DNA,PCR扩增BFV3026　gag基因。序列分析确证后，将其克隆于原核表达载体pET32A，重组质粒转化大肠杆菌BL21（DE3），经IPTG诱导，使Gag蛋白得以高效表达。SDS－PAGE及Western blot证实：表达蛋白占菌体总蛋白的40%，本工作的完成为Gag蛋白功能的研究奠定基础。     

利用妊娠奶牛血液快速鉴定不同发育阶段的胚胎性别

采集妊娠奶牛静脉血液，从血浆中提取胎儿DNA，利用牛Y染色体上性别决定基因（sex-determining region Ylinked gene, SRY gene）的特异性序列设计引物，应用巢式PCR扩增SRY基因特异性片段，同时以牛肌动蛋白（β-Actin）特异性片段的扩增产物作为内参照，对32头妊娠奶牛早期胚胎性别进行鉴定。结果表明：从不同妊娠期奶牛血浆中提取胎儿游离DNA进行胚胎性别鉴定的总准确率为80％，其中1～2月龄、2～3月龄、4～8月龄胚胎性别鉴定的准确率分别为60％、85．7％、92．3％。     

IGF-Ⅰ及其受体在牛体外受精早期胚胎中的表达

采用成分明确的体外培养系统产生牛体外受精胚胎,结合荧光免疫定位的方法,利用激光共聚焦成像系统研究了在牛卵母细胞及不同发育阶段早期胚胎中IGF-Ⅰ及其受体的表达.结果表明,IGF-Ⅰ及其受体从未成熟的卵母细胞、成熟的卵母细胞到早期胚胎发育的各个阶段均有表达,在COC的颗粒细胞中也有表达.IGF-I在成熟卵母细胞中主要定位于细胞膜附近,在未成熟卵母细胞、2细胞期胚胎中分布于细胞膜及膜下区域,在8细胞期胚胎和桑椹胚中分布于整个细胞质中,在囊胚阶段主要位于滋胚层,内细胞团细胞有少量的表达.IGF-IR在未成熟卵母细胞和成熟卵母细胞以及2细胞、8细胞期胚胎中集中分布于卵裂球远离胚胎中心的细胞膜上,在桑椹胚期时遍布于整个胚胎细胞的细胞质中,在囊胚期时主要分布在滋胚层,内细胞团细胞有少量表达.     

有机磷农药与牛血清白蛋白的相互作用及分析应用

应用荧光光谱、同步荧光光谱和紫外吸收光谱研究了3种有机磷农药(敌百虫、乐果和甲基对硫磷)与牛血清白蛋白(BSA)的相互作用.实验表明敌百虫和甲基对硫磷与BSA的相互结合作用是单一静态猝灭过程,而乐果与BSA的相互结合作用是静态为主、动态为辅的猝灭过程.首次发现有机磷药物与BSA结合作用同药物疏水性直接相关:随着有机磷药物疏水性增大,猝灭常数KSV增大、BSA构象改变增大、药物与BSA的结合距离减小.有机磷药物与BSA的结合作用随着有机磷疏水性增强而增加.基于蛋白质与有机磷的结合作用研究,初步探讨了水产品中有机磷药物残留的提取方法,并利用气相色谱法检测了水产品中乐果和甲基对硫磷的含量,方法的回收率为85.71％～107.21％.该方法准确度高、简单、快速.     

变性剂对重组牛肠激酶活性的影响

肠激酶(EK,EC 3.4.21.9)是一种在基因工程产品中广泛应用的工具酶.以小分子荧光物质Gly-Asp-Asp-Asp-Asp-Lys-β-萘胺为底物,采用荧光跟踪法研究了变性剂盐酸胍、硫脲、尿素对重组牛肠激酶(rEK)活力的影响及抑制动力学.结果表明:盐酸胍、硫脲、尿素均对该酶的活性有较强的抑制作用,它们对该酶的半抑制浓度(IC50)分别为0.025,0.080和0.750 mol/L.研究的3种变性剂对该酶的抑制均显示可逆抑制机理;抑制类型也均为竞争性类型,测定它们对酶的抑制常数KI分别为0.015,0.060和0.553 mol/L.     

一种高灵敏度测定血红蛋白的化学发光分析方法

研究了在碱性介质和阳离子表面活性剂CTMAB存在下,牛血红蛋白催化鲁米诺-铁氰化钾的化学发光行为和化学发光反应的条件,提出一种流动注射-化学发光法测定牛血红蛋白的新方法.该方法测定牛血红蛋白的线性范围为5.18×10-10~1.29×10-6mol/L,r2=0.998 5;检出限(RSN=3)为1.2×10-10mol/L;回收率为(100±10)%;dRSD≤5%(n=6).