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目的研究安氏隐孢子虫(Cryptosporidium andersoni)ATP结合盒式转运(ATP-binding cassette,ABC)蛋白1基因对细胞内液和细胞外液中K~+、Ca~(2+)、Na~+和Mg~(2+)的转运作用。方法设计特异性引物,从安氏隐孢子虫基因组中PCR扩增CaABC1。构建真核表达质粒pEGFP-C1-CaABC1,采用脂质体转染法将重组质粒转染至小鼠肠上皮细胞(intestinal epithelial cells,IEC)中表达,设空白组(未转染质粒组)、对照组(转染空质粒pEGFP-C1组)和转染组(转染重组质粒pEGFP-C1-CaABC1组)。用溶液离子浓度检测试剂盒检测IEC细胞内液和细胞外液中的K~+、Ca~(2+)、Na~+和Mg~(2+)的浓度。结果扩增出544 bp的CaABC1基因,构建了真核表达质粒载体pEGFP-C1-CaABC1。转染小鼠IEC后,空白组未观察到绿色荧光,对照组和转染组均观察到绿色荧光。在细胞内液中,空白组K~+、Ca~(2+)、Na~+和Mg~(2+)的浓度分别为(5.51±0.51)、(1.98±0.06)、(108.33±1.33)和(0.93±0.03)mmol/L,对照组分别为(6.25±0.70)、(1.90±0.13)、(107.73±1.79)和(0.87±0.05)mmol/L,转染组分别为(14.84±0.90)、(3.40±0.14)、(127.64±1.49)和(1.72±0.20)mmol/L。在细胞外液中,空白组K~+、Ca~(2+)、Na~+和Mg~(2+)的浓度分别为(12.72±0.83)、(3.72±0.03)、(116.83±1.04)和(2.02±0.18)mmol/L,对照组分别为(10.11±0.90)、(3.58±0.06)、(115.89±1.86)和(1.71±0.41)mmol/L,转染组分别为(5.77±0.21)、(1.29±0.18)、(96.21±1.19)和(0.64±0.02)mmol/L。转染组K~+、Ca~(2+)和Mg~(2+)与对照组相比,差异均有统计学意义(P<0.05)。结论CaABC1蛋白具有协助转运K~+、Ca~(2+)和Mg~(2+)离子的作用。
Objective To study the effect of ATP-binding cassette (ABC) 1 gene of Cryptosporidium andersoni on cytokines such as K ~ +, Ca ~ (2 +), Na ~ + And Mg ~ (2+) transport. Methods Specific primers were designed to amplify CaABC1 from Cryptosporidium parvum genome. The eukaryotic expression plasmid pEGFP-C1-CaABC1 was constructed and transfected into mouse intestinal epithelial cells (IEC) by lipofection. The blank plasmid (untransfected plasmid) Control group (transfected with empty plasmid pEGFP-C1 group) and transfected group (transfected with recombinant plasmid pEGFP-C1-CaABC1 group). The concentrations of K ~ +, Ca ~ (2 +), Na ~ + and Mg ~ (2+) in IEC cells and extracellular fluids were detected by using the kit. Results The 544 bp CaABC1 gene was amplified and the eukaryotic expression vector pEGFP-C1-CaABC1 was constructed. After transfection of mouse IEC, no green fluorescence was observed in the blank group, and green fluorescence was observed in both the control group and the transfected group. The concentrations of K ~ +, Ca ~ (2 +), Na ~ + and Mg ~ (2+) in the blank group were (5.51 ± 0.51), (1.98 ± 0.06) and (108.33 ± 1.33) And (0.93 ± 0.03) mmol / L in the control group were (6.25 ± 0.70), (1.90 ± 0.13), (107.73 ± 1.79) and ), (3.40 ± 0.14), (127.64 ± 1.49) and (1.72 ± 0.20) mmol / L, respectively. The concentrations of K ~ +, Ca ~ (2 +), Na ~ + and Mg ~ (2+) in the extracellular fluid were (12.72 ± 0.83), (3.72 ± 0.03) and (116.83 ± 1.04) And (2.02 ± 0.18) mmol / L in the control group were (10.11 ± 0.90), (3.58 ± 0.06), (115.89 ± 1.86) and (1.71 ± 0.41) mmol / L respectively ), (1.29 ± 0.18), (96.21 ± 1.19) and (0.64 ± 0.02) mmol / L, respectively. Compared with the control group, the levels of K ~ +, Ca ~ (2+) and Mg ~ (2+) in the transfected group were significantly different (P <0.05). Conclusion CaABC1 has the function of assisting the transport of K ~ +, Ca ~ (2+) and Mg ~ (2+) ions.