To construct a recombinant adenovirus vector containing phosphatase and tensin homolog deleted on chromosome 10 (PTEN) using the pAdxsi system.PTEN cDNA from plasmid pcDNA3-PTEN was cloned into the shuttle plasmid pShuttle-GFP-CMV. The shuttle vector was transformed into competent DH5alpha strain with the vector pAdxsi to achieve the homologous recombination. The recombinant construct was subsequently linearized with PacI and transfected into HEK293 cells via Lipofectamine 2000. The recombinant adenovirus particles were collected, and after titration, the recombinant adenovirus was traced by monitoring GFP expression under fluorescence microscope. The expression of PTEN mRNA and protein in the recombinant adenovirus vector and airway smooth muscle cells were detected by PCR and Western blotting, respectively.GFP was expressed in HEK293 cells infected by recombinant adenovirus, and the expression intensity increased gradually with the passage of time, with obvious cytopathic effect (CPE) noted in the cells. After 3 cycles of amplification, the titer of adenovirus containing PTEN reached an appropriate level. The viral titer of pAdxsi-GFP-PTEN was 2x10(10) pfu/ml, and PTEN mRNA expression was detected by PCR. The homologous protein expressed in the infected human airway smooth muscle cells significantly increased in comparison with that in the control cells.The recombinant adenovirus containing PTEN is constructed successfully, which provides an experimental basis for studying the role of PTEN gene in asthma therapy.
The class of Omega glutathione transferases is newly identified with novel structural and functional characteristics. Human GSTO 1-1 (glutathione S-transferase Omega 1) is the first member of the GST Omega class. It was found to play a role in apoptosis and be in association with age-at-onset of AD and PD. In order to improve the understanding of the properties of other Omega class members, we screened a human fetal brain cDNA library and obtained the human GSTO2 (glutathione S-transferase Omega 2) cDNA. The full-length cDNA of human GSTO2 is 1179 bp long and encodes a protein of 243 amino acid residues. Expression pattern analysis revealed that GSTO2 was ubiquitously expressed at a low level, with a higher expression in pancreas and prostate. Enzyme assays showed that GSTO2 protein had activities similar to Omega class GSTs. It has detectable glutathione-dependent thiol transferase activity and glutathione-dependent dehydroascorbate reductase activity. But different from GSTO1-1, GSTO2 exhibits a high catalytic activity with CDNB. Subcellular localization analysis of GSTO2-EGFP fusion protein revealed that GSTO2 distributed to cytoplasm of COS-7 cells and both cytoplasm and nucleus of L-02, QGY-7703 and SMMC-7721 cells. Overexpression of GSTO2 induced apoptosis of L-02 cells detected by Annexin V-PE staining. The results suggest that GSTO2 may play an important role in cellular signaling.
Total RNA was isolated from the muscles of honeybee,and the cDNA sequence of triosephosphate isomerase gene was then cloned by RT-PCR and sequenced(GenBank accession No.EU760983).The gene was 744 bp in length,encoding 247 amino acids.The calculated molecular weight of the mature TPI protein was about 26.89 kD.The similarity between the AmTPI nucleotide sequence and those of Bombyx mori,Blattella germanica,Tenebrio molitor,Nasonia vitripennis and Oryza sativa TPI genes was over 69%,but the similarity between their amino acid sequences was over 59%.The target gene was cloned to the pGEX-4T-2 vector GST fusion expression system.The results indicated that the fusion protein was expressed successfully in the E.coli BL21(DE3)cells and the amount of the expressed protein accounted for 42.1% of the total proteins after 4 h induction.The recombinant fusion TPI protein was purified and concentrated to test its enzymatic properties.The brighter fluorescence recombinant EGFP vector is constructed for further study of expression in mammalian cells.
Currently, exogenous gene expression system based on retroviral vector has been widely used as efficient gene expression system in both gene therapeutic research and RNA interference. In this study, we evaluated the efficiency of exogenous gene expression mediated by the retroviral vector in mammalian cells. First, we constructed EGFP (enhanced green fluorescent protein) vector using pcDNA3.1(+) and retroviral vector pQCXIN as backbone vector respectively. Then, we transfected or infected HEK293 cells and CHO-K1 cells with above vector or corresponding retroviral virus, and measured the relative fluorescence intensity (RFI) of EGFP. The results showed that the RFI of the retroviral virus-infected cells was two times higher than that of the plasmid-transfected cells. Further experiments revealed repeated virus infection enhanced the expression of EGFP markedly, with RFI increasing twice after four rounds of virus infection. Furthermore, the EGFP expression in HEK293 cells mediated by the retroviral vector was more stable than transfected with plasmid pcDNA3.1(+). Finally, we further validated the efficiency of exogenous gene expression system based on the retroviral vector by expressing recombinant human activated protein C (rhAPC) in HEK293 cells. We obtained HEK293 cell lines with rhAPC expression between 10 and 15 microg/(10(6) cells d). In conclusion, the exogenous gene expression system based on the retroviral vector is an alternative method for the generation of stable and high-expressing mammalian cell lines.
A full-length cDNA with 3 479 bp was cloned from a fetal brain cDNA library. It contains an open reading frame encoding a 300-amino acid protein with a calculated molecular mass of 35 379 u and a pI of 5.35. The deduced protein, which was named as RNA-binding motif protein 13 (RBM13), shows 41% identity to MAK16 protein of S. cerevisiae. It contained the signals of a bipartite nuclear localization and an eukaryotic putative RNA-binding region RNP-1. Northern blot analysis showed that two sizes of human RBM13 messenger RNAs (3.6 kb and 2.2 kb) were widely appeared, with highest levels in heart, skeletal muscle, kidney and liver.
Abstract Background Drug–drug interactions (DDI) are prevalent in combination therapy, necessitating the importance of identifying and predicting potential DDI. While various artificial intelligence methods can predict and identify potential DDI, they often overlook the sequence information of drug molecules and fail to comprehensively consider the contribution of molecular substructures to DDI. Results In this paper, we proposed a novel model for DDI prediction based on sequence and substructure features (SSF-DDI) to address these issues. Our model integrates drug sequence features and structural features from the drug molecule graph, providing enhanced information for DDI prediction and enabling a more comprehensive and accurate representation of drug molecules. Conclusion The results of experiments and case studies have demonstrated that SSF-DDI significantly outperforms state-of-the-art DDI prediction models across multiple real datasets and settings. SSF-DDI performs better in predicting DDI involving unknown drugs, resulting in a 5.67% improvement in accuracy compared to state-of-the-art methods.
The enhancer-promoter looping model, in which enhancers activate their target genes via physical contact, has long dominated the field of gene regulation. However, the ubiquity of this model has been questioned due to evidence of alternative mechanisms and the lack of its systematic validation, primarily owing to the absence of suitable experimental techniques. In this study, we present a new MNase-based proximity ligation method called MChIP-C, allowing for the measurement of protein-mediated chromatin interactions at single-nucleosome resolution on a genome-wide scale. By applying MChIP-C to study H3K4me3 promoter-centered interactions in K562 cells, we found that it had greatly improved resolution and sensitivity compared to restriction endonuclease-based C-methods. This allowed us to identify EP300 histone acetyltransferase and the SWI/SNF remodeling complex as potential candidates for establishing and/or maintaining enhancer-promoter interactions. Finally, leveraging data from published CRISPRi screens, we found that most functionally-verified enhancers do physically interact with their cognate promoters, supporting the enhancer-promoter looping model.
A human tropomodlin gene (TMOD3) was cloned from a fetal brain cDNA library. The full-length cDNA was 1 042 bp with an open reading frame encoding a 352-amino acid protein. The deduced protein shows 82%, 41% and 35% identity to tropomodulin of rat, Drosophila and C. elegans, respectively. The TMOD3 gene was localized to human chromosome 15q21.2-q21.2 between markers D15S146 and D15S117. The tissue distribution of TMOD3 gene was determined by DNA microarray.
A triosephosphate isomerase(Tpi) gene of honey bee(Apis mellifera) was cloned by electronic cloning method based on the EST sequences from UniGene of NCBI,its sequence was identified by PCR and predicted by bio-informatics method.The results were as follows: the gene full length was 1768 bp;the gene had a complete open reading frame(ORF),which encoded the protein with 247 amino acids,whose nucleotide sequence had high conservation compared with the protein sequences of triosephosphate isomerase in fruit fly(Dorsophila melanogaster),silkworm(Bombyx mori),mealworm(Tenebrio molito),malaria mosquito(Anopheles gambiae) and tobacco budworm(Heliothis virescens).
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