Development in synthesis of andrographolide derivatives / 中国中药杂志

Andrographolide is one of the major natural active constituents in Andrographis panniculata of Acanthaceae family. Andrographolide and its derivatives are proved to posses a wide range of biological activities, such as anti-inflammatory, antiviral, anti-tumor, anti-human immunodeficiency virus(HIV), hepatoprotective and α-glucosidase inhibition activity. In this paper, the development of synthesis of rographolide derivatives and their related pharmacological activities were reviewed.

Construction of Eukaryotic Expression Vector of siRNA Specific for BCR/ABL Fusion Gene and Its Effects on K562 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To construct eukaryotic expression vector of siRNA specific for BCR/ABL and to investigate the effect of recombinant plasmid on BCR/ABL and P210 protein expression in K562 cells.</p><p><b>METHODS</b>siRNA(small interfering RNA)was designed according to the Tuschl's principle of Ai-based medicine, and was converted into cDNA coding expression of shRNA(small hairpin RNAs)of siRNA for BCR/ABL fusion gene. The cDNA was synthesized and inserted into plasmid pTER. The pTER117 and pTER363 of recombinant plasmid being eukaryotic expression vector was controlled by the H1 promoter of RNA polymerase III, and identified by the restriction map and the sequence analysis. The recombinant plasmid did not only have the screening resisting antibiotics, its expression but also are induced by tetracycline (tet). After steadily transfection into K562 cells by Lipofectamine, their positive mono-cell clones being resistant to Zeocin were isolated. TaqMan real-time quantitative RT-PCR (RQ-PCR) and Western blot respectively detected expression of BCR/ABL mRNA and P210 protein. Trypaum blue dying was used to analyze the proliferation of K562 cells. Cell apoptosis was observed by flow cytometer.</p><p><b>RESULTS</b>the recombinant plasmid was steadily transfected into K562 cells by Lipofectamine 2000, Their positive mono-cell clones being resistant to Zeocin were isolated. The proliferation of K562 cells were remarkably inhibited by the recombinant plasmid induced gene expression by tetracycline. Tetracycline induced its expression for 48 h and 72 h. pTER117, pTER363 decreased the mRNA level of BCR/ABL 90%, 82% and 91.5%, 84%, respectively, P210 protein were almost measured in K562 cells. FCM analysis showed that the recombinant plasmid induced apoptosis in K562 cells, the apoptosis rate were respectively 34.4%, 58.1% in K562 cells treated by pTER117 for 48 h and 72 h, apoptosis rate were 31.8%, 54.6% by pTER363, but the control groups did not show these effects on K562 cells.</p><p><b>CONCLUSION</b>The siRNA eukaryotic expression vector against BCR/ABL mRNA has been successfully conctructed,and effectively inhibits the expression of BCR/ABL in K562 cells, inhibite cell growth and induce cell apoptosis.</p>

Comparison of three methods in construction fusion gene of influenza A virus Nucleoprotein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop a best method of constructing influenza NP fusion gene containing enhanced green fluorescent protein (EGFP).</p><p><b>METHODS</b>The full-length NP gene of influenza A was amplified by RT-PCR and was inserted into an eukaryotic expression vector pEGFP-N1 in order to construct a fusion gene of pEGFP-N1-NP using three different methods. Method one, NP gene containing restriction endonucleases and pEGFP-N1 were both digested using the same restrict enzymes and ligated, yielding the fusion gene of pEGFP-N1-NP. Method two, NP gene was cloned into pMD19-T Vector to construct a plasmid of pMD19-T-NP. The pMD19-T-NP cloned into pEGFP-N1 to construct the fusion gene of pEGFP-N1-NP; Method three, NP gene containing restriction endonucleases was cloned into pMD19-T Simple Vector to construct a plasmid of pMD19-T-NP. The pMD19-T-NP cloned into pEGFP-N1 to construct the fusion gene of pEGFP-N1-NP.</p><p><b>RESULTS</b>The fusion gene of recombinant eukaryotic expression vector pEGFP-N1-NP was successfully constructed by using method three.</p><p><b>CONCLUSIONS</b>The full-length NP gene is obtained and its fusion gene of recombinant eukaryotic expression plasmid is successfully constructed. This study provides foundation for further understanding the biological function of NP protein and the mechanism of diseases induced by influenza A virus.</p>

Atomic force microscopy investigation of the denudation of the envelope of influenza virus treated with Nonidet P-40 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Through observing the morphology and topography of the prepared influenza viruses (H1N1) treated with the different Nonidet P-40 solutions using atomic force microscopy (AFM), to explore the application of AFM on the research of the internal character of viral morphology and structural virology.</p><p><b>METHODS</b>The virus samples were treated with serial diluted Nonidet P-40 solutions from 0.05% to 0.20% and then investigated by AFM with the tapping mode in air at room temperature to obtain the morphology and topography changes including height data,amplitude data and phase data for both spherical and filamentous influenza virus A.</p><p><b>RESULTS</b>The serial AFM images show that the erosion degree of the virions is proportional with the improvement of NP-40 concentration,and partly denuded virion image appeared at 0.05% NP-40 treatment, which was revealed clearly on both amplitude images and phase images.</p><p><b>CONCLUSION</b>This work demonstrated for the first time that the internal topography of influenza virion could be revealed by AFM via suitable nonionic surfactants chemical dissection.</p>