低分子量神经丝蛋白(NF-L)的体外组装

利用超速离心和离子交换层析技术,从牛脊髓中分离纯化了神经丝蛋白三组分:NF-L,NF-M和NF-H。应用电镜负染色和金属投影方法研究神经丝的形态结构与NF-L的体外组装,结果表明:神经丝由10nm的核心纤维与外周的丝状突起组成;在近似生理条件下,NF-L可在60min内组装成10nm纤维,纤维由4根亚丝缠绕而成;在碱性缓冲液中,NaCl能促进NF-L装配成短纤维,这种10nm的短纤维无法连接成长纤维。     

Coil-1 of rod domain of NF-L is essential for its assembly in vivo

Neurofilaments take highly ordered structures composed of parallel arrays of 10 nm filaments linked to each other with frequent cross-bridge. It is composed of three components named NF-L, NF-M and NF-H. NF-L is able to form filamentous network alone in Sf9 cells, while M could not. To identify which domain is essential for the assembly of NF-L, two chimera proteins named ML and MML were constructed: ML was composed of the head domain of NF-M and other domains of NF-L; MML was composed of the head and Coil-1 domains of NF-M and Coil-2 and tail domains of NFL. ML was not only able to form filaments in Sf9 cells, but also co-assemble with NF-M into parallel filamentous bundies. MML could not assemble into filaments. Thus the Coil-1 domain of NF-L was essential for its assembly.     

Nuclear reconstitution of demembranatedOrychophragmus violaceus sperm inXenopus laevis egg extracts

The cell-free extracts from animal Xenopus laevis egg could induce chromatin decon- densation and pronuclear formation from demembranated plant (Orychophragmus violaceus)sperm. The demembranated Orychophragmus violaceus sperm began to swell in 30 min incubation, and then were gradually decondensed. The reassembly of nuclear envelope in the reconstituted nuclei had been visualized by means of electron microscopy and fluorescent microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nucleus, with a double membrane, was similar to those nuclei after fertilization. Transmission electron microscope micrograph of the whole mount prepared nuclear matrix-lamina showed the reconstituted nucleus to be filled with a dense network.     

衣藻淀粉核中存在类角蛋白中间纤维网架

采用选择性抽提结合 ＤＧＤ（ｄｉｅｔｈｙｌｅｎｅ ｇｌｙｃｏｌ ｄｉｓｔｅａｒａｔｅ） 包埋电镜技术在衣藻（Ｃｈｌａｍｙｄｏｍ ｏｎｓａ ｓｐ．）淀粉核中观察到直径１０ ｎｍ 的纤维网架,以哺乳动物角蛋白抗体进行免疫胶体金标记,在淀粉核区域有很强的特异性结合,间接免疫荧光染色得到相似结果,表明在衣藻淀粉核中存在类角蛋白中间纤维网络骨架     

镉诱导蚯蚓金属硫蛋白的分离纯化及其溶液构象的研究

镉诱导蚯蚓匀浆液经热沉淀,乙醇沉淀后,经凝胶过滤ＳｅｐｈａｃｒｙＳ－２００柱层析,得三个镉结合蛋白峰,分子量依次为４０,１７及９ｋＤ。这三个组份再分别经ＤＥＡＥＳｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ柱层析,分别得三、三、二个镉结合蛋白峰。根据光谱学特征、巯基含量及氨基酸组成等分析,认为凝胶过滤第二峰为金属硫蛋白（ＭＴ）,其经ＤＥＡＥ柱层析所得三个峰为ＭＴ三个亚型。对蚯蚓ＭＴ三个亚型在不同ＰＨ条件下     

非细胞体系核重建过程中两类膜泡在环形片层及核被膜形成中的作用

非洲爪蟾卵经钙离子载体A 23187激活后,在10,000g下离心得到爪蟾卵提取物。Lambda DNA加入上述提取物可构建出染色质结构,并在染色质表面重建核被膜,同时在染色质外的区域形成环形片层。核被膜与环形片层有相似的发生途径,它们都是由两类在形态、大小、膜结构上有明显差别的膜泡融合而来。首先是直径200nm的圆形小膜泡相互融合成双层膜片层,同时核孔复合体在双层膜上大量装配,以这些双层膜片层为基础,光滑的大膜泡与之融合导致环形片层的扩张与核被膜的成熟。     

猪瘟病毒E2(gp55)基因的克隆表达及其DNA疫苗的初步研究

用RTPCR方法从中国标准强毒株石门毒的细胞培养物中 扩增获得了其结构蛋白E2基因cDNA,将之克隆到pGEM5Z T载体,用双脱氧链终止法测定其核苷酸序列,并推导出其对应氨基酸序列,与几个代表毒株Alfort株、Brescia株和C株相应序列进行比较,所测核苷酸序列与各株的同源性分别为84.7%、92.6%和95.2%,氨基酸序列的同源性分别为89.4%,92.6%和94.6%;将此E2片段亚克隆至真核表达载体pcDNA3.1,构建表达CSFV E2蛋白的重组质粒pcE2,用脂质体转染法将pcE2导入cos7细胞进行瞬时表达,用针对E2蛋白的特异性单抗以间接免疫荧光法检测,结果E2蛋白在cos7细胞中获得了正确表达,随之将pcE2质粒DNA进行小鼠肌内接种免疫,ELISA法检测证实在免疫后2周和4 周的小鼠体内可诱导出较为明显的阳性血清,并高于E2单抗的阳性对照,病毒中和试验也表明DNA免疫后小鼠体内可诱导产生CSFV中和抗体;同时构建了能在昆虫细胞Sf9中表达GSTE2和GSTGFPE2融合蛋白的重组杆状病毒;上述研究结果为研制针对CSFV的DNA疫苗,亚单位疫苗及其诊断试剂打下了基础。     

Sequence analysis of keratin-like proteins and cloning of intermediate filament-like cDNA from higher plant cells

Two keratin-like proteins of 64 and 55 ku were purified from suspension cells of Caucus carota L, and their partial amino acid sequences were determined. The homological analysis showed that the sequence from the 64 ku protein was highly homological to p-glucosidase, and that from the 55 ku protein had no significant homologue in GenBank. Using conservative sequence of animal IF proteins as primer, we cloned a cDNA fragment from Daucus carota L. Southern blot and Northern blot results indicated that this cDNA fragment was a single copy gene and expressed both in suspension cells and leaves. Homological analysis revealed that it had moderate homology to a variety of a-helical proteins. Our results might shed more light on molecular characterization of IF existence in higher plant.     

核体合成自身DNA与病毒DNA的能力

近年来,用松胞素B(简称CB)诱导细胞去核的技术已被许多学者应用于细胞内大分子代谢及其调控过程的核质关系研究中(Colonne et al.,1980;Premveer et al.,1980;White et al., 1982)。我们在对CB诱导的去核产物的研究基础上(何大澄等1982),对核体内RNA的合成进行了研究,表明核体合成RNA的能力与核体贴壁和所带质膜的完整性以及胞质量多少有关(戎宪辉等1984)。此外,我们还研究了两种小RNA病毒和痘病毒的核酸在胞质体内的复制,证明它们的复制可以不依赖细胞核     

哺乳动物细胞去核产物(胞质体及核体)的结构与功能研究

1967年Carter发现细胞松弛素可以诱发组织培养细胞的自发排核之后,Prescott(1972)借助细胞松弛素存在下的离心处理,使这一排核现象普遍化,从而确立了体外细胞去核的标准方法。经过不断改进(croce et al., 1974;Veomett et al., 1976;Lucas etal., 1976;Wigler et al., 1975),现在,这一技术已广泛应用于细胞学研究的各个重要领域(Mc Burney et al., 1979;Goldman et al., 1974;du Bols et al., 1980)。细胞去核技术及其应用的研究在我国已有初步开展(陈瑞铭等,1979;沈鼎武等,1980)。本实验对二种上皮型传代细胞系进行了去核手术,用扫描电镜和透射电镜对所获得的胞质体