ABSTRACT
To achieve efficient and stable expression of heterologous exogenetic protein or antigen in E. coli chromosome, the luciferase report gene was knocked in lacZ site of chromosome lac operon by using Red recombination system and selection-counterselection kan/sacB technology. The quantitative analysis of exogenous gene expression indicated that the target gene could be efficiently expressed at lacZ site of lac operon. The results confirmed the efficient screening and stable expression of heterologous protein or antigen on chromosome by using the recombinant engineering technique. This study demonstrated that the chromosome could be used as a vector for heterologous protein or antigen and the stable expression of exogenous gene on E. coli chromosome had no side effect on the bacterial growth and propagation.
Subject(s)
Bacteriophage lambda , Genetics , Chromosomes, Bacterial , Genetics , Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Gene Knock-In Techniques , Methods , Genes, Reporter , Genetics , Lac Operon , Genetics , Luciferases , Genetics , Recombination, Genetic , GeneticsABSTRACT
Objective To display of heterologous proteins on the surface of E. coli . Methods The 1653 bp luciferase report gene was knocked in Ipp and ompA genes of chromosome by Red recombine system and selection-counter selection kan/sacB. Results The quantitative analysis results of exogenous lu-ciferase expression displayed that it could be expressed as fusion with the outer membrane proteins on the cell surface. The fusion protein of foreign protein and outer membrane protein Lpp-OmpA-Luc could be high-effi-ciently displayed on cell surface. Conclusion The stable expression of exogenous gene on the surface of E. coli had no effect on the bacterial growth and propagation.
ABSTRACT
To identify the regulatory region that are responsible for the expression of mPC-1, we have isolated and characterized the mPC-1 gene promoter. Sequence analysis of the mPC-1 5' -flanking region and a series of truncated constructs were performed, which were transiently transfected into the prostate cancer cell lines and non-prostate cancer cell lines and analyzed through Dual-luciferase reporter assay system. The relative activity of mPC-1 gene promoter was by far higher than pGL3-control containing SV40 promoter and enhancer and p61-PSA containing hPSA 6 kb promoter in AR (androgen receptor, AR ) -positive prostate cancer cell lines. The region from 599 bp to 449 bp of mPC-1 promoter might contain a negative regulatory element. The expression of mPC-1 1.1 kb fragment is mainly restricted into prostate cancer cell lines. The relative activity of mPC-1 1.1 kb 5'-flanking region was regulated by androgen. The results demonstrated that the 1.1 kb fragment of mPC-1 5' -flanking region was relatively strong and prostate cancer cell specific promoter region.The 1.1 kb promoter of mPC-1 gene might be well suited to prostate cancer gene therapy if the promoter was properly modified.
ABSTRACT
Objective: To study the effects of rapid eye movement sleep (REMS) deprivation on active behavior and the expression of mRNA coding for inducible nitric oxide synthase (iNOS) of hypothalamus in rats. Methods: The flower pot technique was adopted to deprive the REMS of Sprague Dawley rats for 24 h, 48 h and 72 h respectively. The active behavior of rats were examined by the open field test (OFT). The expression of mRNA coding for iNOS of hypothalamus in rats was assayed by reverse transcription polymerase chain reaction (RT PCR). Results: The OFT scores were significantly increased in rats subjected to 24 h and 48 h REMS deprivation ( P 0.05). There was no difference in the expression of iNOS mRNA among 24 h, 48 h REMS deprivation and the control groups, but the expression was significantly increased in 72 h REMS deprivation group ( P
ABSTRACT
Objective: To investigate the changes of neuronal apoptosis induced by ischemia reperfusion in gerbil hippocampus CA 1 after hyperbaric oxygen(HBO) treatment, and to study the effects of HBO on the treatment of cerebral ischemia. Methods: Ischemia reperfusion model of gerbil was established. The effects of HBO treatment for 3 succesive days on neuronal apoptosis of hippocampus CA 1 were examined using H E and TUNEL staining methods. Results: The number of neuronal apoptosis in hippocampus CA 1 was significantly decreased in HBO treatment groups ( P