安氏隐孢子虫ABC2基因的克隆表达与营养物质转运

摘要

To study the transportation function of Cryptosporidium andersoni ATP-binding cassette (CaABC) 2 gene,C.andersoni genome was used as PCR template,the primers for CaABC2 gene were designed according to information obtained from GenBank.The amplification of CaABC2 gene was conducted by PCR.The recombinant plasmid CaABC2 and linear pEGFP-C1 vector were prepared by double enzyme digestion and linked by T4 DNA ligase.The pEGFP-C1-CaABC2 recombinant plasmid was obtained,was then transfected and expressed in mouse IEC by liposome transfection.The concentration of nutrient (total cholesterol,reduced glutathione,glucose and alkaline phosphatase) in intracellular and extracellular fluids was analyzed.The results showed that:CaABC2 gene of 778 bp was obtained.The recombinant plasmid pEGFP-C1-CaABC2 was successfully transfected mouse IEC.Green fluorescence was observed in the control group and the transfect group.The concentrations of total cholesterol in the transfected group,the control group and the blank group were respectively 15.99 ± 0.12,11.80 ± 0.35 and 12.43 ± 0.16 mmol/L.The concentrations of reduced glutathione were respectively 15.24 ± 0.44,10.48 ± 0.35 and 11.04 ± 0.75 mmol/L in intracellular fluid.The concentrations of total cholesterol in the transfected group,the control group and the blank group were respectively 10.67 ± 0.17,14.82 ± 0.06 and 15.84 ± 0.17 mmol/L;The concentrations of reduced glutathione were respectively 10.20 ± 0.79,14.60 ± 0.45 and 15.60 ± 2.50 mmol/L in extracellular fluid.The concentrations of total cholesterol and reduced glutathione in the transfected group were significantly higher than those in the control group and the blank group in intracellular fluid (P＜0.05).However,the concentrations of total cholesterol and reduced glutathione in the transfected group were significantly less than those in the control group and the blank group in extracellular fluid (P＜0.05).No significant differences in the concentrations of total cholesterol and reduced glutathione were observed between the control group and the blank group (P＞0.05).The concentrations of glucose and alkaline phosphatase displayed no significant differences among the transfected group,control group and the blank group (P＞0.05).In conclusion,CaABC2 assisted the transportations of total cholesterol,reduced glutathione,glucose and alkaline phosphatase and its transporting effect on total cholesterol was most obvious.%为探究安氏隐孢子虫(Cryptosporidium andersoni)ATP结合盒转运蛋白基因(CaA BC2)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778 bp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±0.75 mmol/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±2.50 mmol/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(P＜0.05),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(P＜0.05).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(P＞0.05);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(P＞0.05).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.