Improved baculovirus system for transferring genes into insect and mammalian cells

A new set of eukaryotic expression vectors was constructed on the basis of baculoviruses. EcoRI fragments S, J, and P with the genes for late viral proteins p35 (polyhedrin), p39, and p10 were cloned from genomic DNA of the nuclear polyhedrosis virus. The promoter regions of these genes were used to construct double-and triple-promoter expression vectors. Baculovirus vectors containing an expression cassette with the cytomegalovirus promoter and the green fluorescent protein reporter gene were designed to express the cloned genes in cultured mammalian cells.     

Isolation and Characterization of the algD gene of Pseudomonas syringae pv. phaseolicola and its distribution among other Pseudomonads and related Organisms

The gene coding for GDP-mannose dehydrogenase ( algD ) was isolated from a Pseudomonas syringae pv. phaseolicola genomic library using a polymerase chain reaction-generated heterologous DNA-probe from Pseudomonas aeruginosa . A total of 2123 base pairs were sequenced (accession number AF001555) and analysed for homologies to the alginate gene cluster of P. aeruginosa . Downstream from algD an alg8 homologue was found suggesting a similar arrangement of the alginate gene cluster in P. syringae pv. phaseolicola to that in P. aeruginosa . Also, the deduced amino acid sequence of algD shows high similarity to that of P. aeruginosa (0.9) and Azotobacter vinelandii (0.88). Southern hybridization experiments revealed that algD is widely distributed among members of the Pseudomonas rRNA homology group I. Among others, sequences homologous to algD were detected in the P. syringae pathovars lachrymans , mori , morsprunorum, pisi , savastanoi, tabaci and tomato as well as in Pseudomonas amygdali . For most of the algD positive organisms synthesis of alginate has been reported by other studies. However, algD homologues were also detected for the species Pseudomonas corrugata , Pseudomonas marginalis and Pseudomonas avenae ( Acidovorax avenae ), for which alginate biosynthesis has not yet been reported.     

流感病毒M2蛋白胞外区与铜绿假单胞菌外毒素A融合蛋白的表达及其免疫原性研究

本研究构建了表达甲型流感病毒M2蛋白胞外区与铜绿假单胞菌外毒素A(PEA)融合蛋白的原核表达载体,根据铜绿假单胞菌外毒素A(PEA)核苷酸序列设计突变PCR引物并实施突变PCR,以获得PEA基因编码区第553位氨基酸密码子缺失的突变PEA(ntPE),从而产生无毒性的PEA突变基因,然后用合成的M2e编码区替换ntPE基因中的非必需区Ib,产生ntPE-M2e嵌合基因。将该嵌合基因导入pET表达载体以构建原核表达载体,将表达产物胶回收后与弗氏不完全佐剂联合皮下免疫BALB/c小鼠,终免两周后用5个LD50流感病毒A/PR/34/8株进行攻击。取动物血清作ELISA并取脾脏作ELISPOT试验结果表明,免疫组可以诱导小鼠产生抗M2e特异性抗体反应和细胞免疫反应并能够抑制病毒在肺内的复制。本研究为甲型流感病毒广谱疫苗的进一步研发打下了基础。     

Vector for regulated expression of cloned genes in a wide range of gram-negative bacteria.

A pKT231-based broad-host-range plasmid vector was constructed which enabled regulation of expression of cloned genes in a wide range of gram-negative bacteria. This vector, pNM185, contained upstream of its EcoRI, SstI, and SstII cloning sites the positively activated pm twin promoters of the TOL plasmid and xylS, the gene of the positive regulator of these promoters. Expression of cloned genes was induced with micromolar quantities of benzoate or m-toluate, the inexpensive coinducers of the pm promoters. Expression of a test gene, xylE, which specifies catechol 2,3-dioxygenase, cloned in this vector was tested in representative strains of a variety of gram-negative bacteria. Regulated expression of xylE was observed in most strains examined, and induced levels of enzyme representing up to 5% of total cellular protein and ratios of induced:noninduced levels of enzyme up to a factor of 600 were observed. The level of xylE gene expression in different bacteria tended to be correlated with their phylogenetic distance from Pseudomonas putida.     

Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors.

Two improved plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusions, have been constructed. These vectors allow fusion of any gene to the protein A moiety, giving fusion proteins which can be purified, in a one-step procedure by IgG affinity chromatography. One vector, pRIT2, is designed for temperature-inducible expression of intracellular fusion proteins in Escherichia coli and the other pRIT5, is a shuttle vector designed for secretion. The latter gives a periplasmatic fusion protein in E. coli and an extracellular protein in Gram-positive hosts such as Staphylococcus aureus. The usefulness of these vectors is exemplified by fusion of the protein A gene and the E. coli genes encoding the enzymes beta-galactosidase and alkaline phosphatase. High amounts of intact fusion protein are produced which can be immobilized on IgG-Sepharose in high yield (95-100%) without loss of enzymatic activity. Efficient secretion in both E. coli and S. aureus, was obtained for the alkaline phosphatase hybrid, in contrast to beta-galactosidase which was only expressed efficiently using the intracellular system. More than 80% of the protein A alkaline-phosphatase hybrid protein can be eluted from IgG affinity columns without loss of enzymatic activity.     

Detection and quantification of degradative genes in soils contaminated by toluene

Abstract: A method based on the polymerase chain reaction (PCR) was developed for a rapid and specific detection of toluene degradative genes in soil. The xylE gene coding for catechol 2,3-dioxygenase was chosen as a target gene. The detection threshold was evaluated in microcosms using a sterilized standard soil inoculated with various amounts of a degradative strain of Pseudomonas putida (mX). The extracted DNA was used as a template to amplify the xylE gene. PCR followed by hybridization with an internal probe allowed us to detect 10² bacteria per g of soil. In polluted soils, quantification of target DNA by competitive PCR was compared with enumeration of degradative microflora. This molecular method appeared to be rapid, sensitive and more suitable than the microbiological approach to estimate the biodegradative potential of a polluted soil.     

A new family of sugar-inducible expression vectors for Escherichia coli.

A set of 11 expression vectors was constructed, each of them harbouring a cloning cassette under the control of the araB promoter. Some of these vectors enable expression of foreign proteins in the cytoplasm, while others include a synthetic sequence coding for a very efficient secretion signal sequence. Other features are an f1 origin of replication (in plus or minus orientation) and a promoter(up) mutation that enhances the already very high level of expression from these vectors. With such a versatile vector family, cloning, sequencing and site-directed mutagenesis can be performed on the same vector, and the level of expression can be defined according to the specific constraints of a given protein.     

Cloning a synthetic gene for human stefin B and its expression in E. coli

A gene coding for human stefin B was synthesized by the solid-phase phosphite method and cloned in the pUC8 cloning vector. The insert with the verified DNA sequence was subcloned into two expression vectors and expressed in E. coli as a fusion protein with beta-galactosidase and as a native protein. The CNBr cleaved fusion protein and the native recombinant stefin B were inhibitory to papain and reacted with antibodies against human stefin B.     

Production of specific antibodies against protein A fusion proteins.

The gene for Staphylococcal protein A was fused to the coding sequence of bacterial beta-galactosidase, alkaline phosphatase and human insulin-like growth factor I (IGF-I). The fusion proteins, expressed in bacteria, were purified by affinity chromatography on IgG-Sepharose and antibodies were raised in rabbits. All three fusion proteins elicited specific antibodies against both the inserted protein sequences and the protein A moiety. In the case of IGF-I, the protein A moiety in the fusion protein may act as an adjuvant since native IGF-I alone is a poor immunogen. The results suggest that the protein A fusion system can be used for efficient antibody production against peptides or proteins expressed from cloned or synthetic genes. To facilitate such gene fusions a set of optimized vectors have been constructed.