In vitro construction of a recombinant human embryonic brain-derived neurotrophin-4 gene and pEGFP-N1 vector

BACKGROUND: Neurotrophin-4 (NT-4) can promote neuronal growth, development, differentiation, maturation, and survival. NT-4 can also improve recovery and regeneration of injured neurons, but cannot pass through the blood-brain barrier, which limits its ac

Biochemical properties of C78SC96S rhFGF-2: A double point-mutated rhFGF-2 increases obviously its activity

Fibroblast growth factor-2 (FGF-2) is a multifunctional polypeptide that affects many cellular functions and phenomena. The wild-type recombinant human fibroblast growth factor rhFGF-2(W) and the mutant C78SC96S rhFGF-2(M) were expressed in Escherichia coli and their products were purified. The results by the means of fluorescence spectroscopy and CD spectrums, suggested that due to its decreased hydrophobicity rhFGF-2 is not deposited as an inclusion body. The mitogenic activity of the expressed rhFGF-2(M) on 3T3 fibroblasts was shown to be 10-fold more than the expressed rhFGF-2(W) of which the biological activity was a little less than that of the standard rhbFGF(W), indicating that the increased biological activity was due to the change of its secondary structure, dimerization and affinity binding to FGF receptor (FGFR).

重组人胰岛素样生长因子1对db/db小鼠血糖的影响

下载PDF阅读器目的:研究重组人胰岛素样生长因子1(rhIGF-1)对近似于人类2型糖尿病的db/db小鼠的降血糖作用.方法:正常饲养db/db及同品系的正常小鼠,定期监测血糖和血胰岛素水平.待db/db小鼠表现出明显的高血糖及高胰岛素血症后随机分为4组,每日皮下注射溶媒、胰岛素或不同剂量的rhlGF-1,共2周.首次给药后每30min测定血糖1次,观测不同干预方式的即时降糖效果.再于每次给药前测定血糖,观察2周内的变化趋势.结果:db/db小鼠饲养至8周龄时表现出明显的血糖升高及高胰岛素血症.所用剂量的胰岛素对db/db小鼠未表现出明显的降血糖作用;rhIGF-1特别是高剂量rhIGF-1即时及短期降糖效果均较明显,可有效保护db/db小鼠.结论:rhIGF-1对db/db小鼠有较好的降血糖作用,对伴有明显胰岛素抵抗的糖尿病患者具有临床应用前景.

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.

Rapid molecular weight determination of recombinant hirudin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The molecular weight of recombinant hirudin ( rHV-2) was determined rapidly by matrix-assisted laser desorption/ionization time of fight mass spectrometry (MALDI-TOF-MS). The effects of the three types of matrixes were compared and discussed, alpha-cynao-4-hydroxycinnamic acid was proved to be the best matrix. It showed that MALDI-TOF-MS was superior to the traditional method of molecular weight determination of the biological macromolecules. The mass spectrum data proved that the primary structure of rHV-2 was correct and there was no amino acid deletion, mutation and modification in its expression, refolding and purification.

Recombinant expression of rt-PA gene (encoding Reteplase) in gametophytes of the seaweed Laminaria japonica (Laminariales, Phaeophyta)

The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the recombinant expression in gametophyte was investigated by particle bombardment with the rt-PA gene encoding the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction (AMI). Transgenic gametophytes were selected by their resistance to herbicide phosphiothricin (PPT), and proliferated in an established bubble column photo-bioreactor. According to the results from quantitative ELISA, Southern blotting, and fibrin agarose plate assay (FAPA) for bioactivity, it was showed that the rt-PA gene had been integrated into the genome of gametophytes of L. japonica, and the expression product showed the expected bioactivity, implying the proper post-transcript modification in haploid gametophyte.

云南基诺族及汉族特发性全面强直-阵挛性癫(癎)与CLCN2基因的相关性

目的 研究氯离子通道CLC-2基因是否与中国云南地区基诺族及汉族特发性全面强直-阵挛性癫(癎)(IGTCS)相关.方法 以14例云南西双版纳傣族自治州景洪市基诺乡基诺族IGTCS患者及其16名未发病亲属、67例云南籍汉族IGTCS患者及57名云南籍汉族健康体检者为对照,对常染色体3q26上CLCN2基因的内含子2及外显子5、19(内含子18)进行研究,采用PCR及直接基因测序技术,应用病例-对照研究法对CLCN2基因与云南基诺族及汉族IGTCS进行相关性分析.结果 CLCN2基因的内含子2及外显子5、19在病例组和对照组中均没有发现已报道的易患突变,但我们在对外显子19的序列测定过程中发现了其上游内含子18的146位上存在1个单核苷酸多态性位点:146T→C.该位点的3种基因型(TT、TC、CC)在汉族病例组(9、3、29例)和汉族对照组(22、9、26例)之间的分布差异有统计学意义(x2=16.079,P＜0.05);在基诺族组(基诺族病例组+基诺族亲属组,6、12、12例)与汉族对照组(22、9、26例)之间分布差异亦有统计学意义(x2=7.027,P＜0.05).汉族病例组与汉族对照组间TT型与非TT型基因型(分别为9、32例和22、35例)、TC型与非TC型基因型(分别为3、38例和9、48例)比较差异有统计学意义(x2=10.694,OR=4.121,P＜0.05;x2=11.592,OR=0.238,P＜0.05).结论 CLCN2基因内含子18的多态性位点146T→C可能是中国云南地区基诺族与汉族IGTCS患者的1个相关性位点,且在本组有限的样本数量研究中,此SNP位点在两个民族IGTCS患者之间的分布无民族差异.基因型TT为IGTCS的1个保护性因素,基因型TC则增加了患者的易患性.

Mapping chromosomal homologies between humans and two langurs (Semnopithecus francoisi and S-Phayrei) by chromosome painting

Chromosomal homologies were established between human and two Chinese langurs (Semnopithecus francoisi, 2n=44, and S. phayrei, 2n=44) by chromosome painting with chromosome-specific DNA probes of all human chromosomes except the Y. Both langur species showed identical hybridization patterns in addition to similar G-banding patterns. In total, 23 human chromosome-specific probes detected 30 homologous chromosome segments in a haploid langur genome. Except for human chromosomes 1, 2, 6, 16 and 19 probes, which each gave signals on two non-homologous langur chromosomes respectively, all other probes each hybridized to a single chromosome. The results indicate a high degree of conservation of chromosomal synteny between human and these two Chinese langurs. The human chromosome 2 probe painted the entire euchromatic regions of langur chromosomes 14 and 19. Human chromosome 1 probe hybridized to three regions on langur autosomes, one region on langur chromosome 4 and two regions on langur chromosome 5. Human 19 probe hybridized on the same pattern to one region on chromosome 4 and to two regions on langur chromosome 5, where it alternated with the human chromosome 1 probe. Human 6 and 16 probes both hybridized to one region on each of the two langur autosomes 15 and 18. Only two langur chromosomes (12 and 21) were each labelled by probes specific for two whole human chromosomes (14 and 15 and 21 and 22 respectively). Comparison of the hybridization patterns of human painting probes on these two langurs with the data on other Old World primates suggests that reciprocal and Robertsonian translocations as will as inversions could have occurred since the divergance of human and the langurs from a common ancestor. This comparison also indicates that Asian colobines are karyotypically more closely related to each other that to African colobines.

Effects of rhFSH regimen and time interval on ovarian responses to repeated stimulation cycles in rhesus monkeys during a physiologic breeding season

We studied the effects of repeated stimulation by recombinant human FSH (rhFSH) at various time intervals during a physiologic breeding season in rhesus monkeys. Ovarian recovery and responses were assessed by ultrasonography, serum steroid concentrations

Dynamic changes in ovarian follicles measured by ultrasonography during gonadotropin stimulation in rhesus monkeys

The objective was to study dynamic changes of ovaries in rhesus macaques stimulated by gonadotropins to identify an indicator for predicting ovarian response to stimulation. Twenty-one cycling monkeys were given 36 IU/d recombinant human follicle-stimulat

Growth hormone gene transfer in common carp

The first successful case of transgenic fish was achieved in 1984. It is in a model system that the integration and expression of recombinant human growth hormone (hGH) in host red common carp (Cyprinus carpio, red var.) have been thoroughly studied. Recently, the integration sites have been recovered and characterized. Compared with non-transgenic peers, hGH-transgenic fish are prior in dietary utilization and growth performance. In view of bio-safety and bio-ethics, an "all-fish" construct CAgcGH, grass carp growth hormone fused with common carp P-actin promoter, has been generated and transferred into Yellow River carp (C carpio, local strain in Yellow River) fertilized eggs. Under middle-scale trial, CAgcGH-transgenics show higher growth rate and food conversion efficiency than the controls, which is consistent to laboratory findings. To avoid the potential impact of transgenic fish on the environment, a sterile strain of transgenic triploid fish has been successfully produced. The "all-fish" transgenic common carp is also approved safe enough as daily food, according to a test based on the pathological principles of new medicines issued by the Ministry of Health of China. The "all-fish" transgenic common carp with growth enhancement is now ready for market, but looking for governmental authorization. (C) 2003 Editions scientifiques et medicales Elsevier SAS and Ifremer/IRD/Inra/Cemagref. All rights reserved.

重组人内皮抑素的基因表达与表征

内皮抑素具有抑制内皮细胞增殖和抗肿瘤新生血管生成活性，在小鼠实验中对多种肿瘤取得了有效的结果，有望成为一种新型抗肿瘤药物。本论文从基因的获取到在大肠杆菌中的高效表达，系统地研究了重组内皮抑素的全过程，并对其体内、体外的生物学活性及多种光谱学性质进行了深入的研究。采用Trizol试剂改进法从人胚肝组织中制备细胞mR NA，通过RT-PCR方法获得人内皮抑素基因。应用基因工程技术构建了亚克隆载体和表达载体，在大肠杆菌BL21（DE3）中以包涵体方式表达出N-末端带有6XHisTag标签的融合蛋白，表达量最多达到菌体总蛋白的42％。建立了包涵体蛋白的复性和纯化方法，得到具有较高纯度的重组内皮抑素蛋白。首次用质谱法证实从大肠杆菌的包涵体中获得的重组蛋白，因宿主菌中水解甲硫氨酸的酶不能对所有包涵体蛋白起作用，而在目的蛋白中会棍有N-末端具有甲硫氨酸的产物。对内皮抑素基因做定点突变，首次将内皮抑素蛋白的N-末端锌离子结合位点的的His2、His4突变成Leu2、Va14，构建了点突变的亚克隆载体，为以后研究其作用机理提供了条件。重组内皮抑素能够抑制鸡胚尿囊膜血管生成和人脐静脉内皮细胞ECV-304的增殖，在小鼠体内实验中对黑色素瘤产生良好的抑瘤效果（P＜0.01）。通过细胞周期分析，首次发现重组内皮抑素在G2期抑制 ECV-304细胞的增殖。电镜观察发现受内皮抑素作用的ECV-304细胞，产生了凋亡小体并出现多种细胞凋亡特征。同时，首次发现内皮抑素能使Balb／c 3T3细胞产生凋亡。计算机结构模拟结果表明重组内皮抑素蛋白与天然蛋白具有相似的空间结构。研究了内皮抑素与锌离子、肝素、稀土离子作用后蛋白质的光谱学性质和二级结构的变化，首次发现偏酸性pH对肝素与内皮抑素的结合作用有特殊影响，为研究内皮抑素特异抑制肿瘤组织新生血管的生成提供了实验依据。从人胚肝组织出发初步完成了人硒蛋白P亚克隆载体的构建。

改性纳米羟基磷灰石/PLGA复合材料的制备及生物活性

以低聚乳酸接枝改性的羟基磷灰石纳米粒子(op-HA)和聚丙交酯-乙交酯(PLGA)制备的生物可降解纳米复合材料(op-HA/PLGA)为研究对象,采用FTIR,TGA,ESEM和EDX分析其接枝反应、接枝率、表面形貌和钙磷沉积情况,通过在材料膜表面接种兔成骨细胞进行体外培养,采用荧光染色、NIH ImageJ图像分析和Real-time PCR综合评价细胞在材料表面的形态、黏附面积比、增殖能力和基因表达水平,以此评价新型骨修复纳米复合材料op-HA/PLGA的表面性质和生物活性.研究结果表明,op-HA的表面接枝率为8.3%,掺入至PLGA后可形成富含钙磷的粗糙表面,促进成骨细胞的黏附、扩展和增殖,提高Ⅰ型胶原蛋白(Collagen-Ⅰ)、骨形态蛋白-2(BMP-2)和骨连接蛋白(Osteonectin)的基因表达水平,提高材料的钙磷沉积能力.op-HA/PLGA具有良好的细胞相容性和...

环境变迁在中国文明形成中的作用

The formation of civilization, one of great marks in the history of human's society development, has been remained one of the hottest topics in the world. Many theories have been put ford to explain its causes and mechanisms. Although more attentions have been paid to its development, the role of environmental change should not be ignored. In this paper, the level of ancient farming productivity was analyzed, the mechanisms and the process of Chinese ancient civilization formation was explored, and some causes why Chinese ancient civilization shows many different features from other 5 ancient civilizations of the world was analyzed. The main results and conclusions are presented as followed. 1. Compared with the productivity level of other five ancient civilizations, the productivity of ancient China characterized by a feature of extensive not intensive cultivation was lower than that of other five ancient civilizations whose agriculture were based on irrigation. 2. The 5 5000 a B.P. cold event may have facilitated the formation of Egypt and Mesopotamian ancient civilizations and also have had an influence on the development of Neolithic culture in China. 3. The 4 000 a B.P. cold event, which may be the coldest period since the Younger Dryas cold event and signifies the changes from the early Holocene Climate Optimum to late Holocene in many regions of the world, resulted in the great migration of the Indo-European peoples from north Europe to other part of the World and the collapses of ancient civilizations in Egypt, Indus and the Mesopotamian and the collapse of five Neolithic cultures around central China. More important than that is the emergence of Chinese civilization during the same period. Many theories have been put ford to explain why it was in Zhongyuan area not other places whose Neolithic cultures seem more advanced that gave rise to civilization. For now no theory could explain it satisfiedly. Archaeological evidence clearly demonstrate that war was prevailed the whole China especially during the late Longshan culture period, so it seemed war has played a very important role in the emergence of China ancient civilization. Carneiro sees two conditions as essential to the formation of complex societies in concert with warfare, i.e. population growth and environmental circumscription. It was generally through that China couldn't evolved into the environmental circumscription and population pressure because China has extensive areas to live, but that depends on situations. The environmental circumscription area was formed due to the 4000a B.P. cold event and companied flooding disasters, while the population pressure is formed due to three factors; 1) population grow rapidly because of the suitable environment provided by the Holocene Optimum and thus laid its foundations for the ancient human population; 2) population pressure is also related to the primitive agricultural level characterized by extensive not intensive cultivation; 3) population pressure was mainly related to the great migrations of people to the same areas; 4) population pressure was also related to productivity decrease due to the 4 000a B.P. cold event. 4. When population pressure is formed, war is the most possible way to solve the intensions between population and the limited cultivated land and then resulted in the formation of civilization. In this way the climate change during the 4 000a B.P. cold event may have facilitated the emergence of Chinese ancient civilization. Their detailed relations could also be further understood in this way: The first birth places of China ancient civilization could be in Changjiang areas or (and) Daihai area, Shandong province rather than in central China and the emergence time of ancient civilization formed in central China should be delayed if the 4 000a B.P. cold event and companied flooding disasters didn't occurred.