High-level expression of whiG——A key gene for Streptomyces differentiation in Escherichia coli

Six nucleotides located in the region of translation start site of whiG were changed. whiG was amplified by PCR technique. Reformed sequences were determined. This gene was directly subcloned into expression vector pET11c containing strong T7 promoter, and the recombinant plasmid was introduced into E. coli BL21(DE3), which could be induced by IPTG to produce T7 RNA polymerase. The SDS-PAGE result showed that whiG highly expressed in E. coli BL21(DE3), and the yield of whiG product was about 20% of insoluble proteins in cell. whiG product (σwhiG) was further identified by Western blot hybridization after making its antibody. whiG gene was subcloned into Streptomyces plasmid pIJ6021, and then it was introduced into sporulation deficient mutant C71 from Streptomyces coelicolor. The result showed that C71 could restore sporulation and σwhiG has biological functions.     

Flexible manipulation of Omb levels in the endogenous expression region of Drosophila wing by combinational overexpression and suppression strategy

Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments.The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms.Driven by a given tissue-specific Gal4,expressing UAS-transgene or UAS-RNAi(RNA interference)could be used to up-or down-regulate target gene expression,respectively.However,the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock.Here,we describe a simple way to modulate optomotor blind(omb)expression levels in its endogenous expression region of the wing disc.We co-expressed UAS-omb and UAS-omb-RNAi together under the control of dpp-Gal4 driver which is expressed in the omb expression region of the wing pouch.The repression effect is more sensitive to temperature than that of overexpression.At low temperature,overexpression plays a dominant role but the efficiency is attenuated by UAS-omb-RNAi.In contrast,at high temperature RNAi predominates in gene expression regulation.By this strategy,we could manipulate omb expression levels at a moderate level.It allows us to manipulate omb expression levels in the same tissue between overexpression and repression at different stages by temperature control.     

Expression of HSV-1 ICP0 Antigen Peptide in Prokaryotic Cells and Preparation of Specific Antibody

As an immediate-early protein of herpes simplex virus, infected-cell polypeptide 0 (ICP0) exhibits complicated interactions with host cells, and its regulatory function on gene expression is of great importance. Since the ICP0 encoding sequence contains many rare codons which are absent in E.coli, and ICP0 is highly unstable in prokaryotic cells, expression of entire ICP0 in prokaryotic cells has never been reported. In order to further investigate the function of ICP0, a recombinant plasmid was constructed by subcloning a cDNA fragment encoding an amino-terminal of 105 residues of the ICP0 protein into pGEX-5x-1 vector. The resulting GST-105 fusion antigen peptide was expressed with high efficiency in E.coli. Antibodies prepared after the immunization of mice with purified fusion protein can recognize not only the denatured ICP0 protein, but also the native ICP0 protein with normal biological conformation.     

含荧光素酶基因的黑胸大蠊浓核病毒重组质粒的构建与表达

A luciferase gene has been inserted into the recombinant plasmid PfDNV-pUC119 which contained partly deletion of genome of Periplanete fuliginosa densovirus(PfDNV.)The recombinant plasmid with luciferase gene was co-transfrected with PfDNV-pUC 119 into Periplanele fuliginosa larvae and had a high luciferase gene expression in enteron of the transfected larvae.     

中国株HIV-1外膜蛋白真核表达载体的构建与表达

To construct the eukaryotic expression vector of HIV-1 gp120 gene and observe its expression in vitro, the recombinant expression vector pVAX1GP120 was constructed by inserting the gp120 gene into the eukaryotic expression vector pVAX1. The pVAX1GP120 was transfected into Vero cells by lipofectamine and the expressed product was detected by indirect immunofluore- scence.Restriction enzymes digestion analysis and sequencing results revealed that the recombinant expression vector pVAX1GP120 has been constructed successfully. The indirect immunofluorescence result showed green fluorescence on the membrane of transfected cells. The constructed eukaryotic expression vector of HIV-1 gp120 can be expressed in vitro, which lay the foundation for the further study of HIV-1 DNA vaccine.     

Expression of HSV-1 ICPO Antigen Peptide in Prokaryotic Cells and Preparation of Specific Antibody

As an immediate-early protein of herpes simplex virus, infected-cell polypeptide 0 （ICP0） exhibits complicated interactions with host cells, and its regulatory function on gene expression is of great importance. Since the ICP0 encoding sequence contains many rare codons which are absent in E.coli, and ICP0 is highly unstable in prokaryotic cells, expression of entire ICP0 in prokaryotic cells has never been reported. In order to further investigate the function of ICP0, a recombinant plasmid was constructed by subcloning a cDNA fragment encoding an amino-terminal of 105 residues of the ICP0 protein into pGEX-5x-1 vector. The resulting GST-105 fusion antigen peptide was expressed with high efficiency in E.coli. Antibodies prepared after the immunization of mice with purified fusion protein can recognize not only the denatured ICPO protein, but also the native ICPO protein with normal biological conformation.     

A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus (rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1 (rHSV-1) designated HSV1-rc/AUL2, which expressed adeno-associated virus type2 (AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein (GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/AUL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit (TU) or 4.28×104 particles per cell. Therefore, compared     

An Arabidopsis embryonic lethal mutant with reduced expression of alanyl—t RNA synthetase gene

In present paper,one of the T-DNA insertional embryonic lethal mutant of Arabidopsis is identified and designated as acd mutant.The embryo developmant of this mutant is arrested in globular stage,The cell division pattern is abnormal during early embryogenesis and results in distubed cellular differentiation.Most of mutant embryos are finally degenerated and aborted in globular stage,However,a few of them still can germinate in agar palte and produce seedlings with shoter hypoctyl and distorted shoot meristem.To understand the molecular basis of the phenotype of this mutant,the joint fragment of T-DNA/plant DNA is isolated by plasmid rescue and Dig-labeled as probe for cDNA library screening.According to the sequence analysis and similarity searching,a 936 bp cDNA sequence(EMBL accession #:Y12555)from selectoed positive clone shows a 99.8%(923/925bp) sequence homolgy with Alanyl-tRNA Synthetase(AlaRS) gene of Arabidopsis thaliana.Furthermore,the data of in situ hybridization experiment indicate that the expression of Ala RS gene is weak in early embryogenesis and declines along with globular embryodevelopment in this mutant Accordingly,the reduced expression of Ala RS gene may be closely related to the morphological changes in early embryogenesis of this lethal mutant.