Cloning and expression of CP23 gene encoding a surface antigen on sporozoite of Cryptosporidium parvum

Objective To construct a recombinant plasmid containing CP23 gene of Cryptosporidium parvum and obtain recombinant protein. Methods Genomic DNA of C. parvum was extracted from oocysts of C. parvum. CP23 gene fragment was amplified from genomic DNA of C. parvum by PCR. The purified PCR fragment was ligated into the pMD18-T Simple Vector, then subcloned into the pET-30a(+). The recombinant plasmid was transformed into E. coli DH5α. The positive clones were screened and identified by PCR from bacteria directly and digested via double enzymes following with a sequencing of recombinant plasmid. The recombinant plasmid was transformed into E. coli BL21 (DE3); the expression of protein was induced by IPTG. The expression product was identified by SDS-PAGE and Western blot, which was preceded with rabbit anti-C. parvum serum produced by our laboratory. Results The CP23 gene fragment was amplified correctly as the size of gene was about 342 bp; including an open reading fragment encoding 113 amino acids residues. The molecular weight of the whole expression protein was 27 ku on SDS-PAGE in accordance with the result of prestained protein marker. The expressed protein was further identified by Western blot performed with rabbit anti-C. parvum serum. Conclusion The recombinant plasmid is successfully constructed. The protein of CP23 gene of C. parvum is over-expressed and recognized by anti-serum. Our work would lay solid foundations for the diagnosis of cryptosporidiosis as well as study of vaccines.