抗冻蛋白基因转化植物的研究展望

重新评估了用美洲拟鲽抗冻蛋白基因转化烟草的研究，认为植物耐寒性状的提高与转基因植物中冻抗蛋白含量呈正相关是转基因植物成功的关键性指标。美洲拟鲽抗冻蛋白的空间结构与冷冻诱导蛋白相似而且都保护细胞膜免受冻害，因而提出了用冷诱导蛋白调节元的顺式作用元件和反式作用因子的调控序列来控制抗冻蛋白基因在植物中冷诱导表达的思路。     

蛋白质二级结构中同义密码子与氨基酸上下文关联的偏好型

通过对人(Homo sapiens)的117个蛋白质和大肠杆菌(E．coli)的87个蛋白质的统计分析，发现mRNA序列中的同义密码子与氨基酸的上下文关联是和蛋白质二级结构有关的，并给出了各二级结构中有意义的上下文关联型．讨论了该结果对蛋白质结构预测的改进意义及其在基因工程领域可能的应用。     

rhBMP-6的表达和纯化

骨形态发生蛋白(bone　morphogenetic protein，BMP)—6属于转化生长因子(transforming growth fac-tor)—β超基因家族，在骨的形成中具有重要作用，并受雌激素选择性上调．本研究利用RT—PCR从人胎盘组织中扩增BMP—6成熟肽的DNA片段，克隆到pGEx—5x—1中，构建GST—BMP6融合蛋白表达载体pGEx—BMP—6，转化E.coli DH5a．克隆质粒转化的工程菌，经IPTG诱导4h后，高效表达GST—BMP6融合蛋白，在SDS—PAGE上出现一新蛋白带，分子量约为42kD，约占总蛋白的25％，表达产物以包涵体形式存在．菌体超声破碎，经0．3％N—十二烷基肌氨酸钠(SKL)溶解包涵体，离心后的上清液加入0．6％　Triton　x—100整合SKL，然后利用谷胱甘肽Sepharose—4B亲和层析纯化．结合GST—BMP6的介质经洗涤、xa因子酶切得到纯度达95％的rhBMP—6蛋白。     

两种不同示踪剂用于蛋白质与抗原分子微阵列信号检测的对比研究

以氧化型琼脂糖凝胶膜为基片，制作蛋白质与抗原分子微阵列，建立了检测固相人IgG,游离IgG和自身抗体分子的免疫学测定模式，分别以辣根过氧化物酶－二氨基联苯胺一双氧水（HRPDAB－H2O2）和胶体金－硝酸银－对苯二酚2种示踪系统作为蛋白质与抗原分子微阵列信号显示剂，并对其作用的原理与特点进行讨论，检测结果发现基于胶体金的免疫金银染色法（IGSS）比固相酶免疫测定法（EIA）敏感，2种示踪系统对固相人IgG的最低检出量分别为0．05ng和0．5ng；而对血清游离IgG的最低检出量IGSS法比EIA法至少高出10倍，两法用于自身抗体的测定亦表现出较好的重复性，在14例HCV感染者中，自身抗体的总体检出率分别为38．6％和31．4％，结果具有显著的同步性，故同时辅以2种示踪剂并用于阵列芯片的联合测试，有助于提高自身抗体的总体检出水平。     

Inhibitory effect of the adenovirus type 5 E1A protein expressed in the yeast system

The human adenovirus type 5 E1A, a tumor- suppressor gene[1], codes for two major related proteins of 243 amino acids (12S) and 289 amino acids (13S) by al-ternative splicing in two exons[2]. Studies have been shown that E1A can regulate expression of many genes and cell cycle[3]. Both in vitro and in vivo experiments indicated that E1A could induce tumor cells differentia-tion, convert tumor cells into an epithelial phenotype, in-hibit tumor cell growth and metastasis and strongly en-ha…     

Roles of protein kinase C in oocyte meiotic maturation and fertilization

Protein kinase C (PKC) is a superfamily of Ser/Thr protein kinases that is distributed widely in eukaryotes. It plays key regulatory roles at multiple steps of oocyte meiotic maturation and fertilization. During the process of meiotic maturation, the activation of PKC in cumulus cells stimulates meiotic maturation, whereas the activation of PKC in oocytes results in the inhibition of germinal vesicle breakdown. PKC activity increases following the meiotic maturation, and decreases at the transition of metaphase/anaphase in meiosis I, so as to facilitate the release of the first polar body and the entry of meiosis II. In fertilization of mammalian oocytes, PKC may act as one of the downstream targets of Ca2+ to stimulate the cortical granule exocytosis, release the oocytes from MII arrest and to induce pronucleus formation. PKC is also involved in the regulation of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Several PKC isoforms have been identified in mammalian oocytes, and there is evidence showing that classical PKCs may be the principal mediator of oocyte cortical reaction.     

Dependence of mutual information of big protein sequence

The mutual information function is used to describe the auto-correlation of amino acids in protein. We find two interesting phenomenon: (1) for any given big protein, the mutual information function l(k) is almost a const, where k is the length of gap. (2) for any two sequence similar proteins, the mutual information are nearly the same. As a consequent, we may use mutual information of protein as a character for sequences comparison. Foundation item: Supported by the National Natural Science Foundation of China (30170214) Biography: Shi Feng ( 1966-), male, Ph. D, Associate professor, research direction: bioinformatics.     

N-terminal of L protein of vesicular stomatitis virus contains a new signal sequence

The L protein (241kD) of vesicular stomatitis virus (VSV) is the most important snbnnit of the replication complex. The existence of specific localization signal in the L protein was investigated by making recombinant constructs expressing truncated mutants of the L protein fused to green fluorescent protein (GFP) in transient transfection assays. The chimeric genes encoding varied N-terminal of L and GFP gene were put under the control of T7 promoter or CMV promoter. The fusion proteins were transiently expressed in BHK-21, COS-7, CHO or Hep G2 cells. When more than 120 residues were deleted or only 96 residues were kept on the N-terminal, the fusion proteins were shown to be distributed throughout the cells, cytoplasm and nucleus under the confocal microscope. However, other chimeric proteins with 120 or more amino acids were dotted and distributed in the perinuclear regions. And the fusion protein with 96—120 aa has the similar distribution. A thirteen-residue peptide QGYSFLHEVDKEA (108—120) was identified as localization signal, whose function would be absolutely distributed with the deficiency of D or V. Our results show that there is an independent localizing signal in N-terminal domain of L protein of VSV and this functional signal is conserved in different cell lines.     

Analysis of cDNA sequence, protein structure and expression of parotid secretory protein in pig

Parotid secretory protein (PSP) secreted abundantly in saliva, whose function is related with the anti-bacterial effect. The PSP cDNA has been isolated from pig parotid glands by 3′ and 5′ rapid amplification of cDNA end (RACE), based on the conserved signal peptide region among the known mammalian PSP. The result of homologous comparison shows that pig PSP and human PSP shares the high identity at the level of the primary, secondary and tertiary protein structure. A search for functionally significant protein motifs revealed a unique amino acid sequence pattern consisting of the residues Leu-X(6)-Leu-X(6)-Leu-X(7)-Leu-X(6)-Leu-X(6)-Leu near the amino-terminal portion of the protein, which is important to its function. RT-PCR, Dot blot and Northern blot analysis demonstrated that PSP was strongly expressed in parotid glands, but not in other tissues.