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.