关键词: 安氏隐孢子虫, ATP结合盒式转运蛋白1基因, 克隆, 离子转运

Abstract:

Objective  To investigate the transportation of intracellular and extracellular K+, Ca2+, Na+ and Mg2+ under the function of Cryptosporidium andersoni ATP-binding cassette（CaABC） 1 gene.  Methods  CaABC1 gene was amplified by PCR using specifically designed primers. The eukaryotic expression plasmid pEGFP-C1-CaABC1 was constructed, and transfected into mouse intestinal epithelial cells via liposome transfection. The blank（with no transfection） and control groups（transfected with empty plasmid pEGFP-C1） were also set. Changes in intracellular and extracellular K+, Ca2+, Na+ and Mg2+ concentrations were examined by the ion concentration assay kit. Results PCR amplification resulted in a 544 bp product. The recombinant plasmid pEGFP-C1-CaABC1 was successfully constructed. Green fluorescence was seen in the control and transfected groups, but not in the blank group. The concentrations of K+, Ca2+, Na+ and Mg2+ in intracellular fluid were （5.51 ± 0.51）, （1.98 ± 0.06）, （108.33 ± 1.33） and （0.93 ± 0.03） mmol/L in the blank group; （6.25 ± 0.70）, （1.90 ± 0.13）, （107.73 ± 1.79） and （0.87 ± 0.05） mmol/L in the control group; and （14.84 ± 0.90）, （3.40 ± 0.14）, （127.64 ± 1.49） and （1.72 ± 0.20） mmol/L in the transfected group. The concentrations of K+, Ca2+, Na+ and Mg2+ in extracellular fluid were （12.72 ± 0.83）, （3.72 ± 0.03）, （116.83 ± 1.04） and （2.02 ± 0.18） mmol/L in the blank group; （10.11 ± 0.90）, （3.58 ± 0.06）, （115.89 ± 1.86） and （1.71 ± 0.41） mmol/L in the control group; and （5.77 ± 0.21）, （1.29 ± 0.18）, （96.21 ± 1.19） and （0.64 ± 0.02） mmol/L in the transfected group. There were significant differences in K+, Ca2+ and Mg2+ concentrations between the transfected group and the control group.  Conclusion  CaABC1 participates in the transportation of K+, Ca2+ and Mg2+.

Key words: Cryptosporidium andersoni, ATP-binding cassette 1 gene, Cloning, Ion transportation