RESUMO
In order to clone and express alcohol dehydrogenase II (ADH2) gene from Saccharomyces cerevisiae in E. coli BL21 (DE3) efficiently, we extracted the total RNA as template and obtained ADH2 gene by RT-PCR and connected ADH2 gene to pTAT plasmids to gain recombinant expression plasmid pTAT-ADH2, then transformed this recombinant expression plasmid pTAT-ADH2 into E. coli BL21 (DE3). The recombinant was induced by IPTG to express ADH2. After purification, ADH2 activity was tested in vitro and toxicologic test was done in mouse. Sequence test showed that the acquired fragments exhibited 90% homology to ADH2 gene sequence from GenBank report. The target gene expressed efficiently and took up to approximant 50% of total protein by SDS-PAGE and band scanning analysis. The purified protein exhibited the identified activity through biochemical test and mouse toxicological test. As a result, the acquired ADH2 gene was highly homology to the published sequence and expressed at a high level in E. coli BL21 (DE3), more importantly, ADH2 proved to have ethanol dehydrogenase activity.
Assuntos
Animais , Camundongos , Álcool Desidrogenase , Genética , Clonagem Molecular , Escherichia coli , Genética , Metabolismo , Vetores Genéticos , Distribuição Aleatória , Proteínas Recombinantes , Genética , Saccharomyces cerevisiae , Genética , Proteínas de Saccharomyces cerevisiae , GenéticaRESUMO
One pair of primers were designed and synthesized based on the cDNA sequence encoding Homo sapiens poly (ADP-ribose) polymerase family, member 10 (PARP10) reported on the GenBank. The cDNA sequence encoding PARP10 was cloned from 293FT cell by RT-PCR. Then the RT-PCR product was cloned into pCMV-Myc and pEGFP-C1 plasmids. The interaction between PARP10 and beta-actin was identified through immuno-precipitation and laser confocal microscopy. Extensive expression of PARP10 in mouse tissues was confirmed by RT-PCR. Besides, Western blotting analysis indicated that cell injury caused by UV treatment could promote the expression of PARP10. The results in this paper would benefit further study of PARP10.
Assuntos
Animais , Humanos , Camundongos , Actinas , Metabolismo , Poli(ADP-Ribose) Polimerases , Metabolismo , Efeitos da Radiação , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas , Metabolismo , Efeitos da Radiação , Estresse Fisiológico , Efeitos da Radiação , Distribuição Tecidual , Raios UltravioletaRESUMO
NS1 is a non-structural protein of the influenza A virus, which could only be expressed when cells are infected. The effect of NS1 protein on host cell is still not clear. To understand the role of NS1 protein in cell infection, recombinant plasmid pCMV-myc-NS1 was constructed, and then transfected into A549 cells. Two-dimensional electrophoresis was employed to analyze proteins regulated by NS1 that could reflect the interaction between influenza virus and host cells at the protein level. The influence of NS1 on cell proliferation and cell cycle was also studied. The result showed that not only could NS1 remarkably affect metabolism, but it could also slow down cell proliferation through blocking cell cycle.