Influencia del medio de cultivo sobre la cinética de crecimiento y expresión de la proteína recombinante Rv2626c de Mycobacterium tuberculosis H37Rv expresada en Streptomyces lividans TK24 / Influence of the culture medium on the kinetics of growth and expression of the recombinant protein Rv2626c of Mycobacterium tuberculosis H37Rv expressed in Streptomyces lividans TK24

Streptomyces lividans, ha ganado gran atención en los últimos tiempos, como vector de expresión y producción de proteínas de Mycobacterium tuberculosis de interés biomédico, como alternativa a su obtención tradicional en E. coli. La proteína Rv2626c, Proteína de Respuesta Hipóxica 1, es codificada por el gen Rv2626c (hrp1) de M. tuberculosis, perteneciente al regulón de la fase de latencia DosR. Esta proteína se sobre-expresa en la fase de latencia de la tuberculosis bajo condiciones de estrés como hipoxia y bajos niveles, de óxido nítrico. Se ha demostrado la inmunogenicidad y capacidad de esta proteína, de inducir citocinas características del patrón Th-1, tales como el interferón gamma. Por ello, nuestro grupo logró la obtención de Rv2626c por la tecnología del ADN recombinante usando como cepa hospedera Streptomyces lividans TK24. Con el objetivo de aumentar el nivel de expresión de la proteína recombinante, rRv2626c en este trabajo se ensayaron diferentes medios de cultivo, evaluando el crecimiento de la cepa transformada en condiciones de zaranda. Se determinó la cinética de crecimiento en el medio definido, formulado industrialmente en el Centro Nacional de Biopreparados: caldo Triptona Soya (TSB-BioCen) y en medio de cultivo equivalente preparado en el laboratorio. Para establecer la cinética de crecimiento, se utilizó el cálculo del peso seco y la determinación de la concentración de proteínas totales por la técnica de ácido bicinconinico (BCA) a diferentes tiempos del cultivo. Posteriormente, se identificó la proteína clonada mediante SDS-PAGE y Western Blotting, así como los niveles de expresión mediante análisis densitométrico. Los resultados indican que se alcanzaron los máximos niveles de densidad celular a las 36 h de cultivo y los más altos niveles de expresión proteica total y específica entre las 42 y 54 h. Con el medio químicamente definido TSB-Biocen preformulado en lugar del preparado en el laboratorio partiendo de los componentes, se logró reducir el tiempo óptimo para la expresión-secreción de la proteína rv2626c de 96 a 54 h. Los mejores resultados en la promoción de la expresión de la proteína recombinante se lograron con el medio definido TSB-BioCen, a las 48 h con 8,5% de rendimiento específico, superando en más de 10 veces los niveles de crecimiento celular obtenidos con el medio elaborado en el laboratorio y más de dos veces los niveles de secreción de la proteína recombinante(AU)

The use of Streptomyces as a bacterial cell factory for the secretory production of bio-active Mycobacterium tuberculosis proteins have gained a lot of attention in recent years, as a convenient alternative to the traditionally used Escherichia coli. The protein Rv2626c protein, also known as Hypoxic Response Protein 1 (HRP1), is encoded by the gene rv2626c (hrp1) of M. tuberculosis which belongs to the Dormancy Safety Regulator (DosR) regulon. This protein is over-expressed during the latency phase of tuberculosis under stress related conditions such as hypoxia and low, non-toxic, levels of nitric oxide and, the immunogenicity and ability to induce cytokines characteristic of the Th-1 pattern of this protein, such as gamma interferon, have been demonstrated. On this basis, our working group, succeeded in obtaining rRv2626c via recombinant DNA technology using Streptomyces lividans TK24 as the host cell. To improve the expression level of the protein, different culture media were used to evaluate the growth of the transformed strain in shaken flask conditions. Growth kinetic for the recombinant strain was evaluated in defined media of preformulated tryptic soya broth (TSB-Biocen), through the determination of dry weight as well as total protein concentration by Bicinconinic acid assay (BCA). Protein identification was done by SDS-PAGE, Western Blot and its expression level by densitometric analysis. Our results indicated a maximal cell density at 36 h of culture, with higher concentration of total and specific protein at 42-54 h in comparison with previous media used. With the chemically defined media a preformulated TSB-Biocen rather than individual components, culture time for expression/secretion of rRv2626c was reduced from 96 h to 54 h. The best results in expression-secretion levels of rv2626c protein were obtained with the TSB-Biocen defined media at 48 h of culture with 8.5% of specific yield. More than 10 fold increase in cellular growth and more than 2 fold increase in specific yield(AU)

Regulation of Hypoxic Response Elements on the Expression of Vascular Endothelial Growth Factor Gene Transfected to Rat Skeletal Myoblasts under Hypoxic Environment / 华中科技大学学报（医学）（英德文版）

Summary: The regulation of hypoxic response elements on the expression of vascular endothelial growth factor (VEGF) gene transfected to primary cultured rat skeletal myoblasts under hypoxic environment was investigated, pEGFP-C3-9HRE-CMV-VEGF vector was constructed with molecular biology technique and transfected to primary cultured rat skeletal myoblasts by lipofectamine in vitro. Gene expression of transfected myoblasts was detected by RT-PCR, Western blot and fluorescence microscope under different oxygen concentrations and different hypoxia time. The results showed that in hypoxia group, the VEGF gene bands were seen and with the decrease of oxygen concentrations and prolongation of hypoxia time, the expression of VEGF mRNA was obviously increased.Under hypoxic environment, the expression of VEGF protein in the transfected myoblasts was significantly increased. EGFP was expressed only under hypoxic environment but not under normoxic environment. It was concluded that hypoxia promoter could be constructed with HRE and regulate the expression of VEGF gene under hypoxic and normoxic environment, which could enhance the reliability of gene therapy.

Identification of Proteins Induced at Hypoxic and Low pH Conditions in Mycobacterium tuberculosis H37Rv

Mycobacterium tuberculosis likely reside within a granuloma as a dormant state. An area of necrosis forms at the center of lung granulomas. Within this area, the bacteria are deprived of nutrients and exposed to harsh conditions, including low pH and anoxia. The response of M. tuberculosis to low pH and low oxygen conditions was investigated in both cellular and extracellular proteins by two-dimensional polyacrylamide gel electrophoresis analysis and MALDITOF. Several proteins intensively expressed under low pH and/or hypoxic conditions were found. In the culture filtrate, PhoS1 (Rv0934) and ScoB (Rv2503c) were found in significant amounts under both the low oxygen and acidic stress conditions. These results indeed extend our understanding of acidic response as well as hypoxic in M. tuberculosis and provide an important insight into physiology of the latent bacilli.

In vitro killing effect of adenovirus-mediated herpes simplex virus thymidine kinase gene under regulation of hypoxic response element on hepatoma cell line HepG2 / 中国病理生理杂志

AIM:To investigate the in vitro killing effect of adenovirus-mediated herpes simplex virus thymidine kinase gene(HSV-TK) driven by hypoxic response element(HRE) on hepatoma cell line HepG2.METHODS:Recombinant adenoviral vector Ad-HRE-TK was constructed with HSV-TK under the control of HRE using AdEasy system.Then Ad-HRE-TK was transfected into hepatoma cell line HepG2 and the cells were cultured under normoxic or hypoxic conditions.After treated with GCV for 3 d,the sensitivity to GCV of HepG2 was measured by MTT method.RESULTS:Over 95% HepG2 cells infected with Ad-HRE-TK cultured under hypoxic condition were killed when the MOI was 100 and the concentration of GCV was 50 mg/L.On the contrary,no killing effect of GCV was observed in cells cultured under normoxic condition.CONCLUSION:HRE promotes the expression of HSV-TK specifically under hypoxic condition and induces the specific killing effect of GCV.

Is There Any Difference in Arterial Oxygenation between the Right and Left Thoracic Surgery under the Different One Lung Ventilation Mode? / 대한마취과학회지

BACKGROUND: Use of one lung anesthesia for thoracic surgery may compromize PaO2. The aim of this study was to compare the shunt and oxygenation effects of the application of CPAP and CPAP/PEEP between right and left thoracic surgery under one lung anesthesia. METHODS: 10 patients for right thoracic surgery were selected as group 1, and 10 patients for left thoracic surgery were selected as group 2. Measurements in each group, were made during each of the following stage. First 30 minutes, One lung anesthesia alone with 50% oxygen (control value), next 30 minutes, CPAP 10 cmH2O to upper lung with 50% oxygen (CPAP), and then CPAP 10 cmH2O to upper lung and PEEP 10 cmH2O to down lung with 50% oxygen for 30 minutes (CPAP/PEEP). RESULTS: PaO2 in CPAP and CPAP/PEEP were significantly increased as compare to control value at both group (P<0.05). Shunt percentage in CPAP and CPAP/PEEP were significantly decreased as compare to control value at both group (P<0.05). But, no statistically significant differences were observed between right and left thoracic surgery group in the PaO2 and shunt percentage. CONCLUSIONS: We confirmed that CPAP and CPAP/PEEP during one lung ventilation is thought to be effective method in preventing hypoxemia, but no differences were observed between right and left thoracic surgery group.