Plasmid-encoded pgp3 protein of Chlamydia trachomatis serovar D: cloning, expression, identification, and evaluation of immunogenicity

Abstract

Objective To express and identify the plasmid-encoded pgp3 protein of Chlamydia trachomatis (Ct) serovar D,and to evaluate its immunogenicity.Methods The pgp3 gene of Ct serovar D was amplified by PCR and directly inserted into the prokaryotic expression plasmid vector pGEX-6P-1 through the pZeroBack vector.Then,the recombinant plasmid pGEX-6P-1/pgp3 was transformed into E.coli DH5α followed by enzymatic digestion,sequencing and PCR identification.After the identification,the recombinant plasmid was transformed into E.coli BL21 followed by isopropyl-β-D-thiogalactoside (IPTG) induction.The expressed protein was identified by Western blot,and purified by glutathione S-transferase (GST) MagBeads.Male New Zealand rabbits were immunized with the purified protein for three times.Blood samples were collected from these rabbits one week after the last immunization and subjected to the qualification and quantification of anti-pgp3 polyclonal antibodies by Western blot and enzymelinked immunosorbent assay (ELISA) respectively.Results The length of the cloned gene was 795 bp,which was consistent with the sequence of the pgp3 gene in GenBank.The recombinant fusion protein,whose relative molecular weight was 54 000,was successfully purified.The serum of immunized rabbits could react with the pgp3 protein,with the titer of anti-pgp3 polyclonal antibodies being 1 ∶ 25 600.Conclusions The pgp3-GST fusion protein with high immunogenicity is successfully expressed and purified,which may lay a foundation for further studies. Key words: Chlamydia trachomatis; Protein, pgp3; Recombinant fusion proteins; Immunization; Vaccines,synthetic