Thymidine Kinase 1(TK1)重组蛋白的表达、纯化及鉴定

为获得具有免疫原性的TK1重组蛋白。通过构建能够表达TK1蛋白的重组菌BL21-pET32a-TK1,采用大肠杆菌pET32a表达系统,优化IPTG浓度、诱导温度、诱导时间使BL21-pET32a-TK1重组菌表达目的蛋白的作用条件最佳。表达产物用镍离子亲和层析纯化获得TK1蛋白,并用SDS-PAGE和Western blot进行检测。用TK1重组蛋白免疫BALB/c小鼠制备单克隆抗体,检测蛋白质免疫原性。实验结果表明,成功构建能够表达TK1蛋白的重组菌BL21-pET32aTK1,在37℃条件下,IPTG浓度为0.2mmol/L、诱导6h时重组蛋白TK1表达量最高。镍离子亲和层析梯度洗脱在80mmol/L咪唑条件下TK1蛋白纯度最大,灰度分析为87.3%,浓缩后蛋白质浓度为5.96mg/ml。用该蛋白质制备杂交瘤共获得10株稳定分泌TK1抗体的阳性单克隆细胞株,表明TK1重组蛋白具有较好的免疫原性。成功获得可溶性、抗原活性高、免疫原性强的TK1重组蛋白,为肿瘤科学及临床应用研究提供物质支撑。     

Effect of growing media and their constituents on fungus gnat, Bradysia sp, nr, coprophila （Lintner） adults

This study was conducted to determine the attractiveness of two growing media, commonly utilized in greenhouses, to fungus gnat, Bradysia sp. nr. coprophila adults. The constituents of the most attractive growing medium tested were determined by gas chromatography analysis using a steam-distillation procedure. We found that fungus gnat adults were more attracted to the growing medium, SB300 Universal Professional Growing Mix, which contains composted bark, than to another growing medium （Sunshine LC1 Mix） and their components when tested in a series of laboratory experiments using multiple-choice experimental arenas. A higher percentage of fungus gnat adults were attracted to moist SB300 （92%） than SB300 growing medium that had been oven dried （8%）. In addition, fungus gnat adults preferred SB300 although they had been reared on Sunshine LC1 Mix. When comparing the SB300 fresh from the bag to growing medium that had been pasteurized and moistened with water, gas chromatographic-mass spectroscopic data showed there were declines in several terpenoid constituents as well as an increase in fatty acids and cyclosulfur. The results of this study indicate that B. sp. nr. coprophila adults prefer certain growing media, which may assist greenhouse producers in managing fungus gnats in crop production systems.