RESUMEN
Protein drugs play an extremely important role in the prevention and treatment of diseases. But the properties of macromolecules hinder their effects on intracellular targets. Among the existing delivery strategies, penetrating peptides are more suitable for clinical research and treatment, and have gradually become the most important tool to deliver protein drugs. Therefore, the development of safe and effective penetrating peptide delivery vehicles is of great significance to the basic research and clinical application of biomedicine. In this paper, a self-releasing intracellular transporter LCA2 based on the enterotoxin A2 domain is designed. This carrier is composed of three parts: a linker, self-releasing enzyme sensitive sites (Cs), and the transmembrane domain LTA2. The fluorescent protein mCherry was used as the model protein to detect the properties of LCA2. The results of electrophoresis showed that the high-purity mCherryLCA2 fusion protein was obtained from the engineered bacteria containing pET24a(+)-ma2 recombinant plasmids, and mCherry could be effectively separated from LCA2 by low concentration trypsin. It was observed under a fluorescence microscope that LCA2 could transport mCherry into different types of cells. Flow cytometry has detected that the transport capacity of LCA2 has certain cellular differences. Confocal microscope fluorescence analysis and Western blotting results showed that the mCherry was transported to the endoplasmic reticulum by the LCA2 carrier, separated from LCA2 by cleavage of enzyme sensitive sites and released into the cell. The CCK-8 results showed that there was no significant change in cell viability within the dose range of 5-40 μg/ mL. These results demonstrate that LCA2 is a safe and effective self-releasing delivery vehicle, which can transport and release active proteins or protein drugs into cells.
RESUMEN
As a novel member of the IAP (Inhibitor of apoptosis protein) family, survivin was observed to be expressed in most human cancerous cells. Fusion protein TATm-survivin (T34A) has drawn considerable attention because it is a potential anti-tumor protein that can be transduced into cancer cell with the help of HIV-TAT domain. In this study, the cDNA encoding survivin was cloned by RT-PCR from human breast cancer cell lines B-Cap-37. An expression vector of pRSET-B-HIV-tatm-survivin (T34A) was constructed by PCR after survivin (T34A) was mutated by site-directed mutagenesis. Subsequently, the resultant plasmid was transformed into E. coli BL21 (DE3). Recombinant HIV-TATm-Survivin (T34A) protein was expressed efficiently with 0.5mM IPTG as inducer, reaching a yield of 650mg/liter (as inclusion body) in fermentation culture. The inclusion bodies were solubilized, refolded and purified to a purity of 96% by ion exchange chromatograghy and size-exclusion chromatography. Remarkable effects of the purified recombinant HIV-TATm-Survivin (T34A) on the morphology of cell line SW1990 and B-Cap-37 were observed after being administrated for 4h. MTT assay showed recombinant HIV-TATm-survivin (T34A) protein could inhibit significantly cell proliferation of SW1990 and B-Cap-37 and SSMC-7721 in vitro. Apoptosis rate and cell circle of SW1990 and B-Cap-37 that had been treated with target protein (final concentration 30 microg/mL) were detected with flow cytometry. Results revealed that more than 65% cancer cells were arrested at G1 phase. The study suggested that TATm-survivin (T34A) protein was a hopeful protein drug in the treatment of cancers by facilitating apoptosis of cancer cells. Key words recombinant HIV-TATm-Survivin (T34A), expression and purification, pro-apoptosis bioactivity, SW1990 and B-Cap-37 cancer cell lines
Asunto(s)
Humanos , Apoptosis , Proteínas Reguladoras de la Apoptosis , Genética , Farmacología , Secuencia de Bases , Línea Celular Tumoral , Escherichia coli , Genética , Metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión , Genética , Farmacología , Proteínas Recombinantes , Genética , Farmacología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Genética , FarmacologíaRESUMEN
<p><b>OBJECTIVE</b>To observe the protective effect of Acanthopanax senticosus saponins (ASS) on myocardial ischemia-reperfusion injury in rats.</p><p><b>METHOD</b>The myocardial ischemia-reperfusion model was induced by 30 min left anterior descending coronary occlusion and 120 min reperfusion in rats. The changes of myocardial infarct size (MIS), the serum creatine phosphokinase (CK) and lactate dehydrogenase (LDH) activity, the serum lipid peroxidation (LPO) content and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity and plasma endothelin (ET), angiotensin II (Ang II), prostacycline (PGI2) and thromboxane A2 (TXA2) levels and myocardial free fatty acid (FFA) content of infarct and noninfarct area were determined.</p><p><b>RESULT</b>In rats treated by ASS (in a dosage of 25, 50 and 100 mg x kg(-1) i.v. at 30 min after coronary occulusion), the MIS was significantly reduced, the serum CK and LDH activity, the plasma ET, Ang II and TXA2 level and myocardial FFA content declined, while plasma PGI2 level and PGI2/TXA2 was increased signficantly. In addition, serum LPO content declined, SOD and GSH-Px activity were increased markedly.</p><p><b>CONCLUSION</b>ASS has protective effect on myocardial ischemia-reperfusion injury, which may be due to its function of improving free radicals and myocardial metabolism, decreasing plasma ET, Ang II and TXA2 levels and increasing plasma PGI2 level and PGI2/TXA2 ratio etc.</p>
