Cloning, expression and identification of the different fragments of polymorphic membrane protein I and its immunogenicity analysis of Chlamydia trachomatis serovar D

Abstract

Objective To obtain the full length (FL) and C-terminal fragment of polymorphic membrane protein I (PmpI) of Chlamydia trachomatis serovar D, and to study the immunogenicity of these proteins. Methods The target genes of PmpI-FL and PmpI-C were amplified by polymerase chain reaction (PCR) and inserted into the prokaryotic plasmid vector pGEX-6P-1. The recombinant plasmids pGEX-6P-1/PmpI-FL and pGEX-6P-1/PmpI-C were separately transformed into Escherichia.coli (E.coli) DH5α and were identified by enzyme digestion, sequencing and PCR. After the identification, the recombinant plasmids were separately transformed into E. coli BL21 and induced to express the proteins. The expected proteins were identified by Coomassie brilliant blue staining and Western blot, then purified by glutathione S-transferase (GST) MagBeads. The purified proteins were then injected into BALB/c mice to prepare the polyclonal antibodies against PmpI-FL or PmpI-C. Enzyme-linked immune sorbent assay (ELISA) was used for the quantitative detection of the specific antibody. Results The lengths of cloned target genes PmpI-FL and PmpI-C were 2 659 bp and 1 195 bp, respectively, and the sequences were consistent with those of Chlamydia trachomatis serovar D in GenBank. The molecular masses of target proteins were 122 000 and 69 000, respectively, which were confirmed by Coomassie brilliant blue staining and Western blot and then purified. The titers of the antibodies (anti-PmpI-FL and anti-PmpI-C) in sera of immunized mice detected by ELISA were 1∶12 800 and 1∶6 400, respectively. Conclusion The PmpI-FL-GST and PmpI-C-GST fusion proteins with high immunogenicity are successfully expressed and purified, which lays the foundation for further study. Key words: Chlamydia trachomatis; Polymorphic membrane protein; PmpI; Gene expression; Immunogenicity