Five novel antimicrobial peptides from skin secretions of the frog, Amolops loloensis

While investigating antimicrobial peptide diversity of Amolops loloensis, five novel antimicrobial peptides belonging to two families were identified from skin secretions of this frog. The first family including two members is esculentin-2-AL (esculentin-2-ALa and -ALb): the second family including three members is temporin-AL (temporin-ALd to -ALf). The family of esculentin-2-AL is composed of 37 amino acid residues (aa); the family of temporin-AL is composed of 16, 13 and 10 aa, respectively. All of these antimicrobial peptides showed antimicrobial activities against tested microorganisms. cDNAs encoding precursors of esculentin-2-ALs and temporin-ALs were cloned from the skin cDNA library of A. loloensis. All the precursors share similar overall structures. There is a typical prohormone processing signal (Lys-Arg) located between the acidic propiece and the mature peptide. The antimicrobial peptide family of esculentin-2 is firstly reported in the genus of Amolops. Combined with previous reports, a total of four antimicrobial peptide families have been identified from the genus of Amolops; three of them are also found in the genus of Rana. These results suggest the possible evolutionary connection between the genera Amolops and Rana. (C) 2009 Elsevier Inc. All rights reserved.

Genetic diversity and divergence in Chinese yak (Bos grunniens) populations inferred from blood protein electrophoresis

In 6 Chinese yak (Bos. grunniens) populations including 177 yaks, 34 blood protein loci were studied by horizontal starch gel electrophoresis, four of these loci (AKP: ALB, LDH-1, TF) were found to be polymorphic. The percentage of polymorphic loci(P) is 0.118, the mean individual heterozygosity(H) is 0.015, which means a low level of genetic diversity in the whole Chinese yak population. The coefficient of gene differentiation (G(ST)) is 0.0625, which indicated an almost-indistinguishable divergence among different populations at the level of blood protein electrophoresis.

Genetic diversity of cattle in South China as revealed by blood protein electrophoresis

Genetic variation of 31 blood protein loci in 236 cattle from eight South China populations (including mithan, Bos frontalis) and a Holstein population was investigated by means of horizontal starch gel electrophoresis. Thirteen loci (ALB, CAR, Hb-b, Np, PGM, Amy-I, PEP-B, AKP, 6PGD, Cp, Pa, EsD, and TF) were found to be polymorphic. The comparison of average heterozygosities (H) shows that all the native cattle embrace a rich genetic diversity Our results on protein polymorphism suggest that cattle in China originated mainly from Bos indicus and Bos taurus; Xuwen, Hainan, Wenshan, and Dehong cattle and the Dehong zebu are close to zebu-type cattle, and Diqing and Zhaotong cattle are close to the taurine. The mithan was very different from other native cattle, and we suggest that its origin was complicated and may be influenced by other cattle species.

体外分步诱导猕猴胚胎干细胞为肝细胞

采用分阶段诱导方法模拟肝细胞体内发育,建立体外诱导猕猴胚胎干细胞(rhesus monkey embryonic stem cells,rESCs)分化为成熟肝细胞的体系,对研究以ES细胞为基础的临床替代治疗人类晚期肝脏疾病具有重要的意义.将rESCs团块在含有10%FBS的DMEM培养基中悬浮培养11d,形成含有早期内胚层细胞的拟胚体(embryonic bodies,EB)并开始表达早期肝细胞的部分基因或蛋白,将11日龄EB接种至包被有ECM的组织培养皿,分阶段加入aFGF、BMP-4及OSM.经aFGF和BMP-4诱导7～10d后,分化细胞形态变为具有双核的多角形细胞,表达早期和中期肝细胞特异性的蛋白(AFP、ALB及CK18)和基因(AFP、ALB、APOH,G-6-P及TAT),并具有储存糖原的功能.撤除aFGF和BMP-4,添加OSM继续诱导7～10 d,分化的细胞表达成熟肝细胞所特有基因CYP1B1和ADH1C,并具有摄取靛青绿的能力.

藏獒血液蛋白遗传多样性的研究

采用不连续垂直板聚丙烯酰胺凝胶电泳技术,对河曲藏獒、青海藏獒、青海藏狮犬和青海土种犬共4个群体103只犬的9个血液蛋白基因座(Tf、Po、Es-1、Es-2、Sα_2:、Hb、Alb、Pr、Amy)的多态性进行了检测,分析了两个藏獒群体的群体内和群体间的遗传变异,并以Nei氏标准遗传距离(D)为基础用UPGMA法探讨了不同犬群之间的遗传关系.结果表明,在4个被测犬群中,Tf、Po、Es-1、Es-2和Sα_2 5个基因座上存在多态性,其中Tf、Es-1和Po分别由3个等住基因所控制,Es-2和Sα_2,分别由2个等位基因所控制,而Hb、Alb、Pr和Amy基因座均呈现单态;河曲藏獒群体内遗传变异较青海藏獒丰富,而两个藏獒群体间的遗传分化程度很低(G_(ST)=0.0187);以Nei氏标准遗传距离(D)为基础的UPGMA法聚类结果表明.青海藏獒与青海藏狮犬和青海土种犬的遗传关系近于河曲藏獒.

藏獒群体遗传结构及遗传分化研究

采用不连续垂直板聚丙烯酰胺凝胶电泳技术,对河曲藏獒和青海藏獒2个群体86只犬的9个结构基因座(Tf、Po、Es-1、Es-2、Sα2、Hb、Alb、Pr、Amy)的遗传变异情况进行了检测,探讨了2个藏獒群体的遗传结构和遗传分化。结果表明,河曲藏獒群体内遗传变异较青海藏獒丰富,而2个藏獒群体间的遗传分化程度很低(GST=0.018 7);较大的基因流(Nm=14.543 7)是2个藏獒不同地理群体间遗传分化水平低的主要原因。

XDistribution and Length2weight Relationships of Glyptosternoid Fishes in the Drung River Basin , Yunnan

鱼类主要分布于青藏高原周边, 是一群适合于急流生活的　科鱼类。在独龙江流域7 个点共采 集271 号　　鱼类标本(隶属　属、　属和异齿　属) , 结合各采集点数据绘制大鳍异齿　、藏　和扁头　的分布 直方图, 结果显示, 藏　( E. labiatum) 在流速较快的独龙江下游各采集点的种群数量较上游高, 更适应急流生 境。同时研究了这3 种　　鱼类的体长与体重的关系, 参数b (W = aLb) 的范围在218201 和310131 之间, 均为异 速生长; 藏　的生长类型最接近等速生长。

人工养殖条件下暗纹东方鲀的生长发育特性

对连续3年人工养殖的暗纹东方鲀的体重、体长、肝重、肝体系数、性腺系数和肥满度等指标进行分析,并用体长、体重相关式W=aLb进行拟合,研究其生长、发育情况.结果表明:池养暗纹东方鲀体长与体重的相关式为W=0.0557L2.9029,性成熟前肝脏与体长的相关式为W=0.0033L3.1875,肥满度为0.05±0.01,明显高于野生暗纹东方鲀.通过对性腺系数和肝体系数的分析得出,池养暗纹东方鲀如果培育良好,性腺能较好地发育.

Study on the hydrolysis/precipitation behavior of Keggin Al-13 and Al-30 polymers in polyaluminum solutions

The hydrolysis/precipitation behaviors of Al3+, Al-13 and Al-30 under conditions typical for flocculation in water treatment were investigated by studying the particulates' size development, charge characteristics, chemical species and speciation transformation of coagulant hydrolysis precipitates. The optimal pH conditions for hydrolysis precipitates formation for AlCl3, PAC(A113) and PAC(A130) were 6.5-7.5, 8.5-9.5, and 7.5-9.5, respectively. The precipitates' formation rate increased with the increase in dosage, and the relative rates were AlCl3 >> PAC(A130) > PACA113. The precipitates' size increased when the dosage increased from 50 mu M to 200 mu M, but it decreased when the dosage increased to 800 AM. The Zeta potential of coagulant hydrolysis precipitates decreased with the increase in pH for the three coagulants. The isoelectric points of the freshly formed precipitates for AlCl3, PAC(A113) and PAC(A130) were 7.3, 9.6 and 9.2, respectively. The Zeta potentials of AlCl3 hydrolysis precipitates were lower than those of PAC(A113) and PAC(A130) when pH > 5.0. The Zeta potential of PAC(A130) hydrolysis precipitates was higher than that of PACA113 at the acidic side, but lower at the alkaline side. The dosage had no obvious effect on the Zeta potential of hydrolysis precipitates under fixed pH conditions. The increase in Zeta potential with the increase in dosage under uncontrolled pH conditions was due to the pH depression caused by coagulant addition. Al-Ferron research indicated that the hydrolysis precipitates of AlCl3 were composed of amorphous AI(OH)3 precipitates, but those of PACA113 and PACA130 were composed of aggregates of Al-13 and Al-30, respectively. Al3+ was the most un-stable species in coagulants, and its hydrolysis was remarkably influenced by solution pH. Al-13 and Al-30 species were very stable, and solution pH and aging had little effect on the chemical species of their hydrolysis products. The research method involving coagulant hydrolysis precipitates based on Al-Ferron reaction kinetics was studied in detail. The Al species classification based on complex reaction kinetic of hydrolysis precipitates and Ferron reagent was different from that measured in a conventional coagulant assay using the Al--Ferron method. The chemical composition of Al-a, Al-b and Al-c depended on coagulant and solution pH. The Al-b measured in the current case was different from Keggin Al-13, and the high Alb content in the AlCl3 hydrolysis precipitates could not used as testimony that most of the Al3+ Was converted to highly charged Al-13 species during AlCl3 coagulation.

铝及其形态的分析方法

我在研究生期间所做的论文为铝及其形态的分析方法。为什么要做这项工作呢？许多研究表明：①铝是天然水和土壤中十分重要的pH缓冲剂，②铝能影响土壤中象磷和有机碳这些重要元素的环比，③铝对动物、植物及人体都有害，这些过程都与铝的形成有关，因此弄清铝的形态及测定各种形态的浓度十分有意义。由于铝在水中含量较低，且水中含有其它共存离子，因此围绕铝形态的分析，首先建立了一种抗干扰，高灵敏的方法来测定总铝量及各种形态铝的浓度。利用铬天青s试剂测定铝的方法很多，铝与铬天青s能形成红色的二元配合物，但其灵敏度低，稳定性差。近年来人们大多采用铝－铬天青S－表面活性剂三元体系，提高了灵敏度和选择性。本文研究了铝－铬天青S－溴代＋元烷基吡啶三元显色体系，试验确定了三元显色体系的最佳显色条件。其条件如下：实验结果及选定的条件 影响因素 实验结果 选定条件 显色酸度 pH 5.5～7.5 6.0 1 * 10~(-4)M CAS溶剂用量 0.3～0.5ml 0.4ml 5 * ~(-3)M CPB的用量 0.5～1.5ml 0.9ml此三元体系十分稳定，在常温下，显色液放置40分钟才能基本发色完全，其吸光度在24小时内无变化。实验方法为于50ml容量瓶中，加入铝标准液，然后加0.4ml 1 * 10~(-4)M CAS溶液，0.9ml CPB 5 * 10~(-3)M溶液，加入用盐酸调好的pH≈6的六次甲基四胺缓冲液5ml，用水定容，放置40分钟，于644nm处，1cm比色池，以试剂空白作参电测定吸光度。当铝量为0～5ug/50ml时，有色配合物遵从电尔定律，其线性相关系数为0.9999，表观摩尔吸光系数ε_(644nm) = 1.43 * 10~5 l·mol~(-1)·cm~(-1)。本法做了三十六种共存离子的影响实验，多数常见离子不干扰铝的测定，此法主要用于水中铝的测定，家Gr~(3+), Ln~(3+), Ti~(4+), Sn~(4+), Be~(2+)这些离子对铝离子测定的干扰尽管很大，但它们在水中的含量很低，因此可以不考察其干扰作用，F~-和PO_4~(3-)都易与铝离子形成配合物，但水中F~-，PO_4~(≡)含量很小，一般情况下不干扰铝的测定。本法主要考虑了Cu~(2+), Fe~(3+)对铝测定的干扰，用硫脲掩蔽Cu~(2+), 用抗坏血酸掩蔽Fe~(3+), 得到了满意的结果。应用本法测定实际水样，五次测定结果的相对标准偏差为5％，用标准加入法测得其回收率在92～104％之间。由以上工作可以看出，由于CPB的加入，使得Al-CAS-CPB三元体系的摩尔吸光系数较Al-CAS二元配合物的大3.5 倍，最大吸收波长也产生了较大的红移，因此，有必要进一步探讨一下此三元体系的反应机理。目前，阳离子表面活性剂对显色反应作用机理的研究较多，但尚没取得一致的见解，这些研究结果可以归纳为四个方面，1．拟均相萃取模型，2．电荷胶米模型，3．双区作用模型，4．配位体－配位体相互作用的理论和协同微扰机理。本文以Al-CAS-CPB体系作为代表，通过Al-CAS-CPB三元配合物的结构、吸收光谱的变化和表面能力测定的结果探讨了CPB作用机理。利用电泳和离子交换实验说明了Al-CAS-CPB的配阴离子，利用平衡移动法和直线法测得Al：CAS的配位电为1：2，用等摩尔连续变换法测定Al：CPB的配位电为1：4，因而配合物的组成比Al：CAS：CPB＝1：2：4。最后推出此配合物的可能结构。从CAS、Al-CAS、Al-CAS-CPB的结构出发，研究了配位体之间相互作用对CAS中大共轭π键中π－电子流动难易的影响，成功地解释了Al-CAS-CPB最大吸收波长产生红移的原因。通过对CAS溶液浓度与三元配合物最大吸收波长、吸光度影响的研究，说明CPB有三个作用，一是与Al、CAS反应形成三元配合物，同时提高了Al与CAS的配位电；二能与CASi试剂产生缔合作用；三是能够形成胶米；增大了三元配合物在水中的溶解充，这三种作用相互制约，并与Al、CAS及CPB之间的摩尔比有关。通过对配合物吸光度，表面张力和表面活性剂浓度关系的研究及CMC值前后此三元体系吸光度、表面张力变化的比较，结果表明，单分子和形成胶米的表面活性剂同样具有增敏作用，这与郑用熙提出的双区作用机理相一致。在CMC值前，单分子表面活性剂与Al、CAS形成三元配合物而起增敏作用，在CMC值以后，CPB与Al、CAS形成胶米配合物而产生增敏作用。最后，讨论了三元体系的最大吸收波长与表面活性剂浓度的关系，结果表明，随CPB浓度的增大，三元体系的最大吸收波长发生蓝移。产生蓝移的原因可以从结构化学的角度得到解释，导致最大吸收波长不同的原因是在CMC前后，Al-CAS-CPB三元配合物所处的微环境不同。对稀酸性水体中铝形态的分离分析及低浓度铝的测定已有人进行研究，普遍认为无机单核铝是致毒因子，因此无机单核铝的浓度较其实际浓度对生命物质的意义更为重要。本文基本上采用Dirscoll形态分离法进行测定土壤酸性浸出液中铝的形态，用Al-CAS-CPB三元显色反应测定各部分铝形态和浓度。其各部分铝形态的分离如下：总活性铝（Al_r）：将样品用0.2um微孔滤膜过滤，用1N HCl将样品酸化为pH等于1，持续24小时，用Al-CAS-CPB测得其中铝含量。总单核态铝（Ala）：将样品用0.2um微孔膜过滤，直接用Al-CAS-CPB法测定铝量。稳定单核铝（Alo）：稳定的单核铝和不稳定的单核铝通过阳离子交换树脂分离。稳定单核铝通过阳离子树脂后，不能被树脂交换，用Al-CAS-CPB法可以直接流出液中铝量为Alo。不稳定单核铝(Alb)为Ala-Alo。在铝形态的分离过程中，我们使用了过滤和树脂交换二种分离方法。对此做了较详细的条件实验。研究了pH值对滤液中铝的影响，随pH值升高，滤液中铝量减小，这就说明，pH值升高，可能形成某些不能通过滤膜的形态，另外也可能是由膜吸附引起的。本文还比较了用静态平衡法和动态法阳离子交换分离稳定单核铝和不稳定单核铝，指出静态平衡法受到溶液pH值的制约，平衡所需时间过长，容易引起溶液中铝形态的变化和沾污，而动态法则能克服这些缺点，因此在实验中采用动态法。把形态分离的方法应用到土壤酸性浸提液中，结果表明，不稳定的单核铝形态(Alb)远远大于稳定的单核铝形态，而不稳定的单核铝形态中含有对植物的致毒因子，因而酸雨能导致森林死亡，农作物发育不良。

猕猴多潜能成体肝前体细胞及猕猴ES 定向分化为胆管上皮细胞的研究

为解决供体器官的不足，以细胞移植为基础的替代疗法已成为治疗不可逆肝 脏疾病新的希望。 肝前体（干）细胞（Hepatic progenitor cellS，HPCs）和 胚胎干细胞（embryoic stem cells, ES）由于其特殊的细胞特性已成为细胞替 代治疗理想的种源细胞。 然而一方面包括人在内的灵长类动物的正常成体肝来 源的HPCs 的分离依然是很困难的；另一方面，ES 细胞来源的肝细胞和胆管细胞 的生成效率依旧很低。因此有必要建立稳定的高效的灵长类动物HPCs 细胞分离 培养体系及ES 细胞的肝细胞或胆管细胞分化体系以满足供体细胞的不足；这种 体系的建立还有利于研究肝细胞生物学如分化机制、自我更新机制等方面的重要 基础问题。 本研究以猕猴为实验模型，研究了正常成体肝来源的猕猴HPCs 分离、纯化 的条件，系统地鉴定了猕猴HPCs 的细胞特性和体内、外分化潜能，并评价了体 内移植效果。 同时以rES 为材料，建立了rES 高效分化为限定性内胚层 （definitive endoderm cells, DE）和胆管上皮细胞的分化体系。主要实验结 果包括：1）： FBS、EGF、HGF 及rat tail collagen (鼠尾胶原)是分离培养正 常成体猕猴来源的肝上皮前体细胞(rhesus monkey liver epithelial progenitor cells, mLEPCs)所必需的，mLEPCs 在此培养体系中至少可以扩增20 代或5 个月以上，并仍然保持原有的细胞特性；mLEPCs 呈现典型的上皮细胞形 态，并表达HPCs 细胞特有的表达模式即同时表达肝细胞和胆管细胞相关基因 （ALB，APOH，CX43，IB4）或蛋白（CK7，CK8，CK18）；在适宜的分化体系下， mLEPCs 可分化为功能性的肝细胞，形成具有胆管上皮细胞的胆管样结构，并能 转分化形成肌肉样细胞、肌样成纤维细胞及少突样细胞；移植入肝损伤的免疫抑 制的小鼠体内后，mLEPCs 能参与受体肝组织的再生，并能分化成ALB 阳性的肝 细胞；体内定位发现mLEPCs 与胆管区的细胞有相似的免疫原性，提示mLEPCs 可能来源于胆管区。2）：rES 在高浓度的acitvin A（100ng/ml）和低浓度的血 清(1%)单层诱导体系下可定向分化得到高比率的限定性内胚层细胞（definitive endoderm cells，DE 细胞）(约80%)； 高比率的DE 细胞的得到还与rES 细胞的接种密度相关；BMP4 和FGF1 可诱导DE 细胞高效向胆管上皮细胞分化（约90%）， 但并不能得到肝细胞；而Notch 信号通路可维持DE 细胞的存活，并决定着DE 细胞向胆管细胞分化，在Notch 信号通路失活的情形下，即使存在BMP4 和FGF1 都不能促使DE 细胞向胆管细胞分化。 本实验首次成功建立了正常猕猴成体肝HPCs 分离培养体系，证实了分离得 到的猕猴肝上皮前体细胞不但具有正常HPCs 的增殖活力和参与受体肝组织的再 生能力，而且还具有三个胚层的分化潜能，这一结果将为以HPCs 为基础的细胞 替代治疗人类肝脏疾病的实现提供了可能，并首次证明了HPCs 也可以像某些少 数成体干细胞一样具有三个胚层得分化潜能。 此外，本研究建立了rES 高效定 向分化为DE 细胞和胆管细胞的分化体系，这一方法的建立将促进灵长类动物的 DE 细胞的发育机制研究，同时也可为高比率的内胚层功能细胞（如胰岛细胞、 肝细胞、肺细胞）的获得提供丰富的种源细胞和平台。

人载脂蛋白APOA5的克隆表达纯化及相互作用蛋白研究

人类的载脂蛋白A5（apolipoprotein A5，APOA5）是一个新近发现的载脂蛋白家族成员。它在血浆中的含量比其他载脂蛋白低1-2个数量级，但能显著影响血浆三酰甘油水平，对血脂代谢具有重要意义，可以作为降血脂药物治疗中一个强有力的潜在靶标。 由于APOA5在血浆中含量低，直接从血浆中分离纯化很困难，国内一直没有报道简易可靠的纯化方法。为进一步研究APOA5的生物学特性，探讨其与TG代谢中的其它关键成分之间的相互关系，揭示其在脂类代谢相关疾病中的重要地位，必须有大量的蛋白和抗体用于基础研究。因此本研究首先利用基因工程技术，诱导表达纯化APOA5蛋白，免疫动物制备多克隆抗体，为进一步研究人肝脏细胞中APOA5的相互作用蛋白，研究APOA5蛋白在肝脏细胞中的功能奠定基础。 为了深入研究APOA5在肝脏中如何行使功能，我们采用细菌双杂交技术寻找与APOA5相互作用的蛋白因子。并采用Pull-down技术，免疫荧光及免疫共沉淀技术进一步确证其在体外和体内的相互作用关系，为进一步阐明APOA5在体内的生理功能提供了新的线索。 第一部分 APOA5基因的克隆、原核表达、纯化及其多克隆抗体的制备 本研究首先应用基因克隆技术，从人肝癌细胞系SMMC-7721的cDNA中扩增出1.1 kb的ApoA5基因全长序列。然后将其克隆至表达载体pThioHisD，构建原核表达载体pTH-APOA5。该重组质粒转化至大肠杆菌 BL21（DE3），成功实现人APOA5融合蛋白在大肠杆菌中的表达。经发酵得到高效表达的融合蛋白。 融合蛋白在 IPGT 诱导下以包涵体的形式大量表达。利用融合蛋白上的一段组氨酸序列，用镍离子亲和柱进行纯化和复性后，获得较高纯度的人APOA5融合蛋白。利用该融合蛋白免疫新西兰大耳白兔，获得了高效价的兔抗人APOA5多克隆抗体，Western Blot结果显示此多克隆抗体与APOA5特异性结合。 第二部分 细菌双杂交筛选与APOA5相互作用的蛋白 本实验首先构建了pBT-APOA5重组质粒，经双酶切、PCR和测序鉴定证明重组诱饵质粒构建成功，并进行了表达、自激活鉴定。Western Blot鉴定证实报告菌株中表达了分子量为 68 kD左右的重组融合蛋白，与预测的分子量APOA5（41 kD）/lamda cI （27 kD）一致。自激活实验证明诱饵蛋白不能单独激活报告基因，可用于筛选人肝脏cDNA文库。经过双重抗性筛选和回复筛选，分离出10个阳性克隆。对结果进行生物信息学分析，得到7个与APOA5相互作用的蛋白，其中BI1为细胞凋亡调节因子；ATP6、CYTB、ND2、COX-1为线粒体表达蛋白； ALB、TTR为血清蛋白。 第三部分 APOA5与BI1相互作用的确证 首先构建了BI1的原核表达载体pGEX-5X-3-BI1，利用Pull-down实验检测了APOA5与BI1在体外具有相互作用。然后构建了BI1的真核表达载体pCDNA3.1-HA-BI1和APOA5的真核表达载体pCDNA3.1-APOA5，并验证其表达。通过免疫荧光细胞内共定位研究发现，靶蛋白APOA5主要分布于胞浆，与BI1在HEK293细胞有共定位，即APOA5与BI1存在相互作用的可能。最后利用免疫共沉淀手段，在HEK293细胞中确证了靶蛋白APOA5与BI1在体内的相互作用。 上述研究结果，为深入研究APOA5在体内的生物学功能提供了新的思路。 Apolipoprotein A5 (APOA5) is a newly discovered protein belongs to apolipoprotein family. APOA5’s concentration is 1-2 orders of magnitude lower than other apolipoproteins in the circulation. APOA5 significantly affected plasma triglyceride levels, which is important on lipid metabolism. APOA5 has strong potential to be used as a hypolipidemic drug target. Large amount of APOA5 protein and antibodies are needed in basic research, such as biological characteristics study of the APOA5, its relationship with other key components in TG metabolism, its role played in Lipid metabolism-related diseases. Due to its low concentration in plasma, separation and purification of APOA5 from the plasma is very difficult. Until now no report on simple and reliable method for purification has been published in China. In this study, we firstly got APOA5 recombinant protein using genetic engineering technology. The purified recombinant protein was used to immunize rabbits to get antiserum. It is important for further study of the APOA5 protein-interacting protein. And it lays the foundation for studing APOA5 function in liver. In order to study APOA5 function in liver, we used bacterial two-hybrid technology to find the APOA5 protein interactor. Pull-down, immunofluorescence and immunoprecipitation techniques were used to further confirm the interaction between APOA5 with its interactor in vitro and in vivo. All of these stdudies provided new clues on its physiological functions in vivo. Part I: Cloning, prokaryotic expression, purification and polyclonal antibody preparation of APOA5 First of all, we amplified APOA5 CDS sequence from the human hepatoma cell line SMMC-7721, and subcloned into Expression vector pThioHisD, and got the recombinants named pTH-APOA5. The plasmid was transformed to BL21 (DE3). E. coli BL21(DE3) cells bearing the pTH-APOA5 plasmid were cultured and APOA5 protein synthesis was induced by the addition of IPTG. Recombinant protein was expression in the form of inclusion. Inclusion bodies were dissolved in phosphate-buffered saline containing 8 M urea and 40 mM imidazole, then applied to a Ni2+ affinity column, and were eluted in a buffer containing 4 M urea and 200 mM imidazole. Fractions containing the APOA5 protein were pooled and dialyzed against buffer containing phosphate-buffered saline. Antiserum to recombinant human APOA5 was generated by immuning rabbit. Western Blot showed that this antiserum specific binding with APOA5. Part II Two-hybrid system screening protein interactions with the APOA5 The coding sequence of human APOA5 was amplified using synthetic oligonucleotide primers from pTH-APOA5 vector and was subcloned into the pBT plasmidc to yield pBT-APOA5 vector. DNA sequencing was performed to verify that no unwanted mutations occurred during the process of plasmid vector construction. We verified recombinant protein expression and tested self-activation by pBT-APOA5 prior to screening. Western Blot verified inducing a 68 kD band, consistent with the predicted molecular weight (APOA5 41 kD, lamda cI 27 kD). pBT-APOA5 can be used for screening human liver cDNA library because it can not self-activation. Totally 10 positive clones were isolated. The nucleotide sequence of the positive clones were determined and compared to NCBI nucleotide sequence databases. We got 7 protein which interact with APOA5, included BI1(Apoptosis regulator); ATP6, CYTB, ND2, COX-1(Mitochondrial protein) and ALB, TTR(Serum protein). Part III Confirming of interaction between APOA5 with BI1 pGEX-5X-3-BI1 vector was subcloned at first. Pull-down experiments were used to detect the interaction between APOA5 with BI1 in vitro. Later, pCDNA3.1-HA-BI1 and pCDNA3.1-APOA5 were subcloned. Through immunofluorescence co-localization study, we found APOA5 mainly distributed in the cytoplasm. APOA5 is co-localization with BI1 in HEK293 cells. Finally, we verified interaction between APOA5 with BI1 in vivo through immunoprecipitation.

Biological effect of rare earth (I) - Content and distribution of rare earth in normal human plasma

The content and distribution of rare earth(RE) in normal human plasma have been investigated by ultrafiltration, FPLC and ICP-MS methods, The results showed that there are trace RE in normal human plasma, and their contents are in accordance with their abundance, The RE can bond with immunoglobulin G(IgG), transferrin(Tf) and albumin(Alb) species, but mostly bond with Tf.

稀土生物效应研究(Ⅰ)──正常人血浆中稀土含量及其物种分布

采用ICP-MS及FPLC等技术研究了正常人血浆中稀土含量及其物种分布．结果表明，正常人血浆中含有痕量的稀土(总量为1.413.3μｇ/L)，每种稀土含量与其天然丰度一致；稀土物种主要集中于大分子蛋白组分中，与免疫球蛋白G(IgG)、运铁蛋白(Tf)、血清白蛋白(Alb)等均有作用，其中Tf结合稀土量较多

丁二烯聚合过程中补加某一组分对顺丁橡胶结构和性能的影响

在接近顺丁橡胶(BR)工业生产配方的基础上,在聚合过程中分别补加适当的B/Bd,Al/Bd,Al—B陈化液和H_2O含量等组分,试图调节BR的微观结构和某些物理性能,如支化度、门尼粘度、△ML值和浓溶液粘度等。