Molecular Cloning, Expression and Characterization of Plasmid Encoding Rhomboid 4 (ROM4) of Tachyzoite of RH Strain.

BACKGROUND

The objective of this study was to clone, express and characterize the gene encoding rhomboid 4 () proteins, a vital gene in surface adhesion and host cell invasion process of tachyzoite of in an appropriate expression vector and eukaryotic cell for production of recombinant protein.

METHODS

RNA was isolated from tachyzoites (RH strain) and complementary DNA was synthesized. Oligonucleotide primer pair was designed based on gene sequence with I and RI restriction sites at 5' end of forward and reverse primers, respectively. gene was amplified by PCR, cloned into pTG19-T vector and the recombinant plasmid was sequenced. The gene was subcloned into pcDNA3 plasmid and expressed in CHO cells as eukaryotic cell. SDS-PAGE and western blotting were performed for protein determination and verification.

RESULTS

Cloning of gene in pTG19-T vector was confirmed by colony-PCR and enzymatic digestion. The results of enzymatic digestion and gene sequencing confirmed successful cloning and subcloning procedures. The nucleotide sequence of the cloned gene showed 99% homology compared to the corresponding sequences of original gene. SDS-PAGE and western blotting analyses of the purified protein revealed a single band having expected size of 65 kDa.

CONCLUSION

This eukaryotic expression system is an appropriate system for high-level recombinant protein production of gene from tachyzoites used as antigenic component for serological assay and vaccine development.