安氏隐孢子虫ABC2基因的克隆表达与营养物质转运

发布时间：2018-10-23 14:15

【摘要】：为探究安氏隐孢子虫(Cryptosporidium andersoni)ATP结合盒转运蛋白基因(CaABC2)对营养物质的转运功能,以安氏隐孢子虫基因组作为模板,设计合成CaABC2基因特异性引物,进行PCR扩增,获得CaABC2基因。将克隆的重组质粒CaABC2基因与真核表达质粒pEGFP-C1双酶切后再连接,构建重组真核质粒载体pEGFP-C1-CaABC2;再采用脂质体转染法将重组质粒pEGFP-C1-CaABC2转入小鼠肠上皮细胞(Intestinal epithelial cells,IEC),检测不同组IEC细胞对总胆固醇、还原型谷胱甘肽、葡萄糖和碱性磷酸酶的转运效果。结果表明:扩增出778bp的CaABC2基因,测序分析与预期片段大小一致;重组真核质粒载体pEGFP-C1-CaABC2转染小鼠IEC细胞后,观察到转染后的对照组和转染组IEC有绿色荧光;检测到在细胞内液中,转染组、对照组和空白组的总胆固醇浓度分别为15.99±0.12、11.80±0.35和12.43±0.16 mmol/L;还原型谷胱甘肽浓度分别为15.24±0.44、10.48±0.35和11.04±0.75mmol/L;在细胞外液中,转染组、对照组和空白组的总胆固醇浓度分别10.67±0.17、14.82±0.06和15.84±0.17 mmol/L;还原型谷胱甘肽浓度分别为10.20±0.79、14.60±0.45和15.60±2.50mmol/L。转染组总胆固醇、还原型谷胱甘肽浓度均显著大于空白组和对照组(P0.05),而在细胞外液中,转染组总胆固醇、还原型谷胱甘肽的浓度均显著小于空白组和对照组(P0.05)。在细胞内液和外液中,空白组与对照组总胆固醇、还原型谷胱甘肽的浓度之间均未见显著性差异(P0.05);转染组、对照组和空白组之间的葡萄糖和碱性磷酸酶浓度均没有统计学差异(P0.05)。CaABC2基因具有协助转运总胆固醇、还原型谷胱甘肽、葡萄糖和碱性磷酸酶的作用,其中转运总胆固醇效果最为明显。
[Abstract]:In order to investigate the transport function of (Cryptosporidium andersoni) ATP binding cassette transporter gene (CaABC2) from Cryptosporidium andersoni, the specific primers of CaABC2 gene were designed and synthesized using Cryptosporidium Angiosporidium genome as template, and the CaABC2 gene was obtained by PCR amplification. The cloned recombinant plasmid CaABC2 gene was digested with eukaryotic expression plasmid pEGFP-C1 and then ligated. The recombinant eukaryotic plasmid vector pEGFP-C1-CaABC2; was constructed and the recombinant plasmid pEGFP-C1-CaABC2 was transfected into mouse intestinal epithelial cells (Intestinal epithelial cells,IEC) by liposome transfection. The transport effects of different IEC cells on total cholesterol, reduced glutathione, glucose and alkaline phosphatase were detected. The results showed that the CaABC2 gene of 778bp was amplified and sequenced, and the size of the CaABC2 gene was the same as expected. After the recombinant eukaryotic plasmid pEGFP-C1-CaABC2 was transfected into mouse IEC cells, the green fluorescence of IEC was observed in the control group and the transfected group, and the green fluorescence was detected in the intracellular fluid. The total cholesterol concentrations in transfection group, control group and blank group were 15.99 卤0.121.80 卤0.35 and 12.43 卤0.16 mmol/L;, respectively, and the concentrations of reduced glutathione were 15.24 卤0.4410.48 卤0.35 and 11.04 卤0.75 mmol / L, respectively. The total cholesterol concentrations in the control group and the blank group were 10.67 卤0.17 卤14.82 卤0.06 and 15.84 卤0.17 mmol/L;, respectively, and the glutathione concentrations were 10.20 卤0.79 卤14.60 卤0.45 and 15.60 卤2.50 mmol / L, respectively. The concentrations of total cholesterol and reduced glutathione in transfection group were significantly higher than those in blank group and control group (P0.05), while in extracellular fluid, the concentrations of total cholesterol and reduced glutathione in transfection group were significantly lower than those in blank group and control group (P0.05). There was no significant difference in the concentration of total cholesterol and glutathione between the blank group and the control group (P0.05). There was no significant difference in glucose and alkaline phosphatase concentrations between the control group and the blank group (P0.05). The CaABC2 gene could facilitate the transport of total cholesterol, reduced glutathione, glucose and alkaline phosphatase. Among them, the effect of total cholesterol transport is the most obvious.
【作者单位】：安徽农业大学动物科技学院;安徽农业大学茶与食品学院;
【基金】：安徽省自然科学基金项目(1708085MC81) 国家自然科学基金项目(31001019)
【分类号】：S852.723

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