Recombinant Expression And Indirect ELISA For COWP And HSP70 Proteins From Cryptosporidium andersoni

Abstract

Cryptosporidium spp. infect cattle at a high rates, and reduce milk production. Cryptosporidiosis has caused economic losses for the dairy industry. Studies in Western countries have shown that Cryptosporidium can also infect humans. Therefore, the development of methods for the early detection of Cryptosporidium is an important public health objective. Total RNA isolated from C. andersoni was used as template for generating cDNA encoding the COWP and HSP70 proteins. The recombinant plasmid, pET-32a(+)-COWP-HSP70, was constructed by double digestion and subcloning. The expression of the three recombinant proteins was induced in Escherichia coli BL21 using isopropyl-β-D-thiogalactopyranoside. The antigenicity of the recombinant proteins was examined using western blotting and indirect ELISA. The identities of the COWP fusion protein (CFP), HSP70 fusion protein (HFP), and COWP-HSP70 fusion protein (CHFP) were confirmed by BLAST searches of known sequences in GenBank respectively. The ELISA and western blot analyses indicated that all three of the proteins were highly immunogenic and antigenic. An indirect ELISA method was developed using the three recombinant proteins as coating antigens for the analysis of 40 clinical samples. The results showed that CHFP was the best candidate antigen for clinical testing, with a detection rate of 100%, compared with general parasitological screening. Above of all, the recombinant CHFP protein represents the best candidate antigen among three ones for detecting anti-Cryptosporidium antibodies in clinical samples. The development of the indirect ELISA lays the foundation for further research in immunodiagnosis and disease prevention of cryptosporidiosis.