[Cloning and Ion Transportation of Cryptosporidium andersoni ATP-binding Cassette 1 Gene].

Objective: To investigate the transportation of intracellular and extracellular K(+), Ca(2+), Na(+) and Mg(2+) 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(+), Ca(2+), Na(+) and Mg(2+) 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(+), Ca(2+), Na(+) and Mg(2+) 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+.

Cloning and Iron Transportation of Nucleotide Binding Domain of Cryptosporidium andersoni ATP-Binding Cassette (CaABC) Gene.

Cryptosporidium andersoni ATP-binding cassette (CaABC) is an important membrane protein involved in substrate transport across the membrane. In this research, the nucleotide binding domain (NBD) of CaABC gene was amplified by PCR, and the eukaryotic expression vector of pEGFP-C1-CaNBD was reconstructed. Then, the recombinant plasmid of pEGFP-C1-CaNBD was transformed into the mouse intestinal epithelial cells (IECs) to study the iron transportation function of CaABC. The results indicated that NBD region of CaABC gene can significantly elevate the transport efficiency of Ca(2+), Mg(2+), K(+), and HCO3 (-) in IECs (P<0.05). The significance of this study is to find the ATPase inhibitors for NBD region of CaABC gene and to inhibit ATP binding and nutrient transport of CaABC transporter. Thus, C. andersoni will be killed by inhibition of nutrient uptake. This will open up a new way for treatment of cryptosporidiosis.

[Isolation and identification of cow-origin Cryptosporidium isolates in Hefei].

OBJECTIVE: To isolate cow-origin Cryptosporidium in Hefei, and identify its species. METHODS: 285 dairy cattle fecal samples collected from a farm in Hefei were examined by using floating saturated solution of sucrose and modified acid-fast staining. Cryptosporidium oocysts were isolated and purified from positive fecal samples. Genetic DNA was extracted to be the template. According to the sequence of 18S rRNA gene and HSP70 gene from Cryptosporidium sp., the primers were designed and synthesized. The PCR products were amplified by PCR and nested-PCR. The nested PCR products were cloned and sequenced. Homology searches and phylogenic tree construction were done by DNAStar software. RESULTS: Five fecal samples were positive by morphological methods with an infection rate of 1.8% (5/285). Oocysts from the 5 positive fecal samples were elliptical or ovoid detected by using floating saturated solution of sucrose and modified acid-fast staining with the size of 7.37 microm x 6.13 microm and 7.58 microm x 6.20 microm, and a shape index of 1.20 and 1.22, respectively. Nested-PCR resulted in a 18S rRNA and HSP70 gene fragments with approximately 250 bp and 325 bp, respectively. The five isolates showed a high level of nucleic acid identity with sequence data of the 18S rRNA gene of Cryptosporidium andersoni (DQ989573), and they were clustered in the same clade. The highest HSP70 gene sequence identity was found among the five isolates and other reported C. andersoni isolates (AY954892 and DQ989576), and they were placed into the same clade. CONCLUSION: The cow-origin Cryptosporidium isolates derived from Hefei is Cryptosporidium andersoni.

[Research progress on nutrient transport and metabolism of Cryptosporidium].

Cryptosporidiosis is a worldwide zoonotic disease, and Cryptosporidium is coccidia-like parasite that develops in epithelial cells in digestive and respiratory tracts of human and animals. This review summarizes the specific function structure of Cryptosporidium, nutrient uptake, transport, metabolism, and the impact of Cryptosporidium on host nutrient absorption.