Novel biodegradable polymers as gene carriers

This study investigated two new biodegradable polymers as gene controlled-released coatings for gene transfer. Poly(ethylene glycol)-co-poly(D,L-lactic acid) (PELA) and poly(ethylene glycol)-co-poly(lactic acid)-co-poly(glycolic acid) random copolymer (PELGA) were synthesized and used as microspheres matrices with encapsulated plasmid pCH110. The plasmid loading efficiency, cytotoxicity, transfection efficiency and in vitro degradation and release profiles of microsphere complexes were evaluated in details. The biodegradable polymers showed high DNA loading efficiency and low cytotoxicity as gene controlled-released coatings, and the poly(ethylene glycol) (PEG) contents of polymer matrices influenced the diameter, loading efficiency and transfection efficiency of plasmid DNA within the microspheres. The average diameters of PELA and PELGA microspheres were between 0.5 and 1.5 microm, and the plasmid loading efficiency was 62 and 73% for PELA and PELGA microspheres with 10% PEG content, respectively. In vitro testing showed a gradual release profile of DNA from polymeric matrices. The polymers/DNA microspheres had high transfection efficiency and early gene expression and maintenance of gene expression level for up to 96 h, although transfection efficiency were slightly lower than that of liposome in the initial 24 h. The biodegradable polymeric materials possess potential superiority as gene carriers.     

Gemini surfactants: new synthetic vectors for gene transfection

The superior surfactant properties of cationic gemini surfactants are applied to the complex problem of introducing genes into cells. Of almost 250 new compounds tested, of some 20 different structural types, a majority showed very good transfection activity in vitro. The surfactant is shown to bind and compact DNA efficiently, and structural studies and calculations provide a working picture of the "lipoplex" formed. The lipoplex can penetrate the outer membranes of many cell types, to appear in the cytoplasm encapsulated within endosomes. Escape from the endosome--a key step for transfection--may be controlled by changes in the aggregation behavior of the lipoplex as the pH falls. The evidence suggests that DNA may be released from the lipoplex before entry into the nucleus, where the new gene can be expressed with high efficiency.     

Macrocyclic glycoclusters: from amphiphiles through nanoparticles to glycoviruses

Macrocyclic glycocluster amphiphiles are intended to be a covalent-bundle mimic of clustering glycolipid motifs on the cell membrane. They are irreversibly micellized to give glycocluster nanoparticles (GNPs); their masked hydrophobicity endows them with remarkable saccharide specificities in the interactions with biological saccharide receptors. The GNPs also exhibit unprecedented hydrogen-bond capacities; they are agglutinated with Na(2)HPO(4) and assembled on plasmid DNA in a number-, size-, and shape-controlled manner to give artificial glycoviral particles capable of transfection. Thus, the intrinsic function of viruses, that is, cell invasion followed by gene expression, is also intrinsic to size-regulated (approximately 50 nm) glycoviruses. The growth of glycocluster amphiphiles through nanoparticles to glycoviruses reveals a hierarchical adhesion control of the saccharide clusters.     

Cloning and expression of aClostridium thermocellum dna fragment that encodes a protein related to cellulosome component sL

Antibodies raised against the SL subunit of the Clostridium thermocellum cellulosome were used to screen a library of C. thermocellum chromosomal DNA fragments constructed in the vector lambda gt11. A DNA fragment that encoded a polypeptide that crossreacted with the anti-SL antibodies was isolated and its restriction map elucidated. No similarity with other previously cloned DNA fragments has been found. The anti-SL crossreacting polypeptide was isolated from recombinant Escherichia coli and found to have a mol mass of 37,000 Da and to possess low levels of CMCase and Avicelase activity. Using CMC as the substrate, a temperature optimum of 55 degrees C and a pH optimum of 6.6 were observed. These properties were compared to those of C. thermocellum SL isolated by electroelution from an SDS gel, which was also found to possess low levels of CMCase and Avicelase activities. In addition, the SL proteins produced in C. thermocellum and E. coli were able to interact positively against Avicel with an endoglucanase (Ss) purified from the C. thermocellum crude cellulase preparation, and with a recombinant protein that crossreacted with anti-Ss antibodies.     

Studies of poly-L-lysine-starch nanoparticle preparation and its application as gene carrier

The gene carrier system is the key factor in genetransfection and gene therapy. Suitable gene carriercan deliver the target gene into the receptor cells safely,highly efficiently, controllably, and then the gene isexpressed, thus accomplishing the gene tr…     

Total Synthesis and Expressions in E. coli of Three Redesigned Genes for the Mature Small Subunit (rbcS) of Rubisco Fron Tobacco, Wheat and Rice

Three structural genes, which code for the mature small subunits (rbcS) of ribulose-1,5-bisphosphate carboxylase/oxygenase from tobacco, wheat and rice, respectively,have been redesignedwith the aid of a computer,totally synthesized by combining chemical method and enzymatic ligation,and expressed in E. coli with high yields.     

叶酸和聚乙二醇接枝作基因载体用壳聚糖的合成与表征

本研究将叶酸和聚乙二醇接枝到四种不同分子量的壳聚糖氨基侧链上,以改善壳聚糖的靶向性和水溶性作基因载体。用FTIE、1HNMR、UV-Vis、DSC和TEM对产物进行了表征,结果表明,叶酸和聚乙二醇被成功地接枝到壳聚糖上,所制得的载体有望作为潜在的肿瘤细胞靶向基因载体。     

Chemical synthesis and cloning of human Β-endorphin gene inEscherichia coli

Total synthesis of human Β-endorphin gene has been designed for the expression in bacterial system. Eight individual oligonucleotides corresponding to the Β-endorphin gene were chemically synthesized and joined through the enzyme-catalyzed reaction. The final yield of the 111-nucleotide-long synthetic Β-endorphin gene construct was about 10% of the total oligonucleotide used. The synthetic human Β-endorphin gene was cloned into the bacteriumEscherichia coli, using pUC8 vector and shown to have the correct nucleotide sequences as designed.     

aprE基因表达的分子生物学和微量热法分析

利用分子生物学方法(SDS-PAGE和酶活检测法)未检测到所克隆的aprE基因在大肠杆菌中的表达产物(碱性蛋白酶), 而微量热法检测结果发现: 重组菌株的生长代谢产热曲线之间存在明显的差异. 根据这些差异, 分析了该基因的上、下游调控序列对该基因在大肠杆菌中表达的重要作用, 从而进一步对该基因进行了亚克隆, 得到了生物学方法可检测到的表达产物. 由此推测, 微量热技术有可能为检测外源基因表达及其调控, 以及为指导进一步基因工程操作提供一种新的快速灵敏的技术和方法.     

STRUCTURE OF THE MITOCHONDRIAL URFA6L GENE AND tRNA~(Lys) GENE FROM CARP

The carp mitochondrial URFA6L gene consists of 165 base pairs. The overall structural organization of the gene is very similar to that of the Xenopus URFA6L gene. Their nucleotide sequences exhibit 68% homology. The carp URFA6L gene encodes a protein of 54 amino acids. The amino acid composition of the protein is unusual because almost half of the residues consist of 5 hydrophobic amino acids(proline, tryptophan, leueine, isoleueine and tyrosine). A comparison between the amino acid sequences of 5 vertebrate URFA6L proteins and the yeast ATPase8 showed that they have weak but very important common structural features, suggesting that the vertebrate URFA6L proteins may function asATPase8. The nucleotide sequence of the lysine tRNA gene from carp has been determined and represented in cloverleaf secondary structure. Similar to amphibian and mammalian mitochondrial tRNA~(Lys) genes, the carp mitochondrial tRNA~(Tys) gene also has some unusual structural features as compared with its cytoplasmic counterpart