RESUMO
We used a simple and efficient method to construct the bicistronic eukaryotic expression vector pIRES2-NGF-VEGF165. The nerve growth factor (NGF) gene was obtained from the genomic DNA of human peripheral blood mononuclear cells by polymerase chain reaction. The NGF cDNA fragment was inserted into the multiple cloning sites of the pIRES2-EGFP vector to generate the bicistronic eukaryotic expression plasmid pIRES2-NGF-EGFP. The vascular endothelial growth factor 165 (VEGF165) gene was obtained from the pIRES2-VEGF165-EGFP plasmid by polymerase chain reaction. Next, the VEGF165 cDNA fragment was cloned into pIRES2-NGF-EGFP in place of enhanced green fluorescent protein creating the plasmid pIRES2-NGF-VEGF165. pIRES2-NGF-VEGF165 was transfected into HEK293 cells and reverse transcription-polymerase chain reaction and Western blot analysis were used to test the co-expression of double genes. The NGF and VEGF165 genes were cloned and the DNA was sequenced, which revealed that NGF and VEGF165 were consistent with the sequence recorded in GenBank. Restriction analysis showed that the NGF and VEGF165 genes were inserted into the expression vector pIRES2-EGFP. Transfection of pIRES2-NGF-VEGF165 into HEK293 cells resulted in expression of the double gene at the mRNA and protein levels. The NGF and VEGF165 coexpression plasmid provides a novel expression system, enabling further study of the functions of the NGF and VEGF165 genes.
Assuntos
Expressão Gênica , Vetores Genéticos/genética , Fator de Crescimento Neural/genética , Fator A de Crescimento do Endotélio Vascular/genética , Clonagem Molecular , Células HEK293 , Humanos , Fator de Crescimento Neural/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
We used a simple and efficient method to construct a bicistronic eukaryotic expression vector pIRES2-LIF-NT-3. The leukemia inhibitory factor (LIF) and neurotrophin-3 (NT-3) genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by polymerase chain reaction. The LIF cDNA fragment was inserted into the multiple cloning sites of a vector containing internal ribosome entry site and enhanced green fluorescent protein (EGFP) (pIRES2-EGFP) to generate the bicistronic eukaryotic expression plasmid pIRES2-LIF-EGFP. Next, the NT-3 cDNA fragment was cloned into pIRES2-LIF-EGFP in place of EGFP to create the plasmid pIRES2-LIF-NT-3. pIRES2-LIF-NT-3 was transfected into HEK293 cells and reverse transcription-polymerase chain reaction and Western blotting were used to test the co-expression of double genes. LIF and NT-3 genes were cloned and the DNA was sequenced. Sequencing analysis revealed that LIF and NT-3 were consistent with the sequence recorded in GenBank. Restriction analysis indicated that the LIF and NT-3 genes were inserted correctly into the expression vector pIRES2-EGFP. Following transfection of pIRES2-LIF-NT-3 into HEK293 cells, the double gene was expressed at the mRNA and protein levels. The LIF and NT-3 coexpression plasmid is a novel expression system that will enable further study of the functions of the LIF and NT-3 genes.
Assuntos
Clonagem Molecular/métodos , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Leucócitos Mononucleares/metabolismo , Neurotrofina 3 , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
We used a simple and efficient method to construct the bicistronic eukaryotic expression vector pIRES2-VEGF165-NT-3. The neurotrophin-3 (NT-3) gene was obtained from the genomic DNA of human peripheral blood mononuclear cells by polymerase chain reaction. The NT-3 cDNA fragment was cloned into the pIRES2-VEGF165-EGFP vector in place of enhanced green fluorescent protein (EGFP) to create the plasmid pIRES2-VEGF165-NT-3. Next, pIRES2-VEGF165-NT-3 was transfected into HEK293 cells, and reverse transcription-polymerase chain reaction and Western blotting were used to test co-expression of the double genes. The vascular endothelial growth factor 165 (VEGF165) and NT-3 genes were cloned; DNA sequencing analysis demonstrated that the VEGF165 and NT-3 sequences were the same as those recorded in GenBank. Restriction analysis indicated that the VEGF165 and NT-3 genes were correctly inserted into the expression vector pIRES2-EGFP. The double gene was expressed at both the mRNA and protein levels. The VEGF165 and NT-3 co-expression plasmid was successfully constructed, providing a novel expression system for further study of the functions of the VEGF165 and NT-3 genes.
Assuntos
Clonagem Molecular/métodos , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Leucócitos Mononucleares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
To investigate the variance of exogenous gene expression driven by different promoters by in vivo electroporation, 3 plasmid vectors carrying different promoters were selected, and their driving strength was compared in developing chicken embryos. The 3 promoters included: 1) the CAG promoter (containing the cytomegalovirus (CMV) immediate early enhancer and the chicken ß-actin promoter), 2) the CMV promoter (the human CMV immediate early region enhancer), and 3) the SV40 promoter (Simian virus 40). The intensity of GFP expression driven by the 3 promoters was detected by fluorescence microscopy. The results clearly showed that the expression intensity of the reporter gene differed significantly among the 3 promoters. Chicken ß-actin promoter induced the highest intensity of GFP expression, while SV40 promoter induced the lowest intensity. Our results indicate that plasmids with appropriate promoters should be carefully selected to obtain strong exogenous gene expression by in vivo electroporation.
Assuntos
Galinhas/metabolismo , DNA Viral/análise , Proteínas de Fluorescência Verde/metabolismo , Plasmídeos/genética , Animais , Embrião de Galinha , Galinhas/crescimento & desenvolvimento , Eletroporação , Expressão Gênica , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Microscopia de Fluorescência , Regiões Promotoras Genéticas , Medula Espinal/ultraestruturaRESUMO
Protocadherins constitute a large family belonging to the cadherin superfamily; they function in various tissues of a wide variety of multicellular organisms. However, their functions and expression modes are still unknown in many of these species and tissues. We developed a fast and low-cost method to produce polyclonal antibody against chicken protocadherin 1 (Pcdh1) that could be used in assays for immunological assessment of protein expression levels of chicken Pcdh1. Primers were designed with DNAStar, using the nuclear sequence of pcdh1 as a template; the pcdh101 fragment was amplified, identified by sequencing and cloned into expression vectors pGEX-2TK and pET-32a, separately, resulting in 2 recombinant plasmids, pGEX-2TK-pcdh101 and pET-32a-pcdh101. These were confirmed by double-enzyme digestion and sequencing. The recombinant expression vectors were transformed and expressed in Escherichia coli BL21. The recombinant oligopeptides glutathione-S-transferase (GST)-Pcdh101 and (His)6-Pcdh101 fused with the carrier protein GST and (His)6 separately, and were purified. Rats were immunized by injecting the emulsified GST-Pcdh101 antigen subcutaneously into their hind footpads, followed by a booster injection after 2 weeks. One week after the booster, the sera were collected and examined for antibody titer by indirect ELISA. The optimal dilution of this antiserum was 1:300. The specificity of the antiserum was confirmed by Western blotting. This antiserum had good specificity and could be used to detect chicken Pcdh1 in Western blot analysis. This method allows production of specific rat polyclonal antisera for Western blots in less than 1 month at a relatively low cost.
Assuntos
Anticorpos/genética , Anticorpos/imunologia , Proteínas Aviárias/imunologia , Caderinas/imunologia , Galinhas/genética , Animais , Anticorpos/análise , Formação de Anticorpos , Especificidade de Anticorpos , Embrião de Galinha , Galinhas/metabolismo , Primers do DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Imunização , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Invasive and noninvasive tests have been developed for the diagnosis of Helicobacter pylori infection. Because H pylori infection is acquired in childhood and adolescence, accurate diagnosis of the infection in the pediatric population is important. We conducted a study to compare invasive tests: culture, biopsy urease test, histology, and polymerase chain reaction on gastric biopsy specimens, with noninvasive tests: serology, (13)C-urea breath test, and a new diagnostic modality, stool antigen test to diagnose H pylori infection. A total of 53 children with symptoms were enrolled in this study, and all had completed the 7 diagnostic tests for H pylori. All the diagnostic tests except serology were excellent methods of diagnosing H pylori infection in children; the diagnostic accuracy was as follows: stool antigen test 96.2%, biopsy urease test 96.2%, histology 98.1%, polymerase chain reaction 94.3%, culture 98.1%, (13)C-urea breath test 100%, and serology 84.9%. The stool antigen test, being highly sensitive and specific, will be potentially very helpful in diagnosing H pylori infection in children.