Biological Characteristics Of Plamsid-encoded Pgp5 Protein Of Chlamydia Muridarum And Its Mechanism Of Regulating Gene Expression

[Background] Chlamydia trachomatis(C.t)is an obligate intracellular pathogen that infects genital tract epithelial cells.There are an estimated 313 million new cases of Chlamydia trachomatis infections worldwide each year.Although most cases are spontaneously resolved,the persistence of infection may cause more severe severer inflammation.As the infection rate of chlamydia increases and the drug resistance increases,it is urgent to explore new treatment method.Our previous study found that Chlamydiamuridarum plasmid-encoded protein pgp5 plays an important role in the pathogenesis of chlamydial fallopian tubes.This finding provides a new research direction for the study of chlamydia treatment.[Objective]The pgp5 gene encoding the Chlamydia muridarum plasmid and its purified protein were obtained in vitro,and rabbit-derived polyclonal antibody was prepared.The biological characteristics of the protein were analyzed from the aspects of protein expression level,mRNA level and solubility.The molecular mechanism of protein regulation gene expression was explored by experiments.[Methods] Indirect immunofluorescence was used to detect the infection rate of Chlamydia muridarum.The total DNA of HeLa cells infected with Chlamydia muridarum was extracted,and the target gene was amplified by polymerase chain reaction(PCR).Two sets of primers containing restriction sites were designed to make recombinant plasmids that the His tag on pET28 a was located upstream and downstream of the pgp5 gene protein.Then the recombinant plasmid pET28a/pgp5 was transformed into Escherichia coli DH5α,and the plasmid was extracted for digestion,sequencing and identification by PCR.The recombinant plasmid pET28a/pgp5 was transformed into Escherichia coli Rosetta(DE3),and induced by IPTG which expressed protein was identified by SDS-PAGE and purified by nickel column.The rabbits were immunized with the purified renatured protein to prepare polyclonal antibodies,and the antibody titer was determined by indirect ELISA.The indirect immunofluorescence were used to detect the biological activity of the antibody and antibody specificity was detected by Western blotting.The total RNA of cells infected with CM strain was extracted by Trizol method,and reverse transcribedinto cDNA.Real-time quantitative gene amplification fluorescence detection system(qPCR)was used to detect the expression of pgp5 protein RNA at different time points.The total protein of HeLa cells infected with Chlamydia muridarum was extracted,and the expression of pgp5 at different time points was detected by Western blotting.The gene fragment of the promoter region of the target genes TC0181,TC0319,TC0357,and TC0419 was amplified by polymerase chain reaction(PCR).After purification by a gel recovery kit,EMSA assay was performed to detect the binding of each gene to pgp5 protein.[Results] The infection rate of Chlamydia muridarum was up to 90%,which guaranteed the accuracy of subsequent experiments.The length of the pgp5 gene fragment obtained by the two recombinant plasmids was 795 bp,and the search sequence was consistent with Genebank.the search sequenceof which was consistent with Genebank.SDS-PAGE electrophoresis showed that the recombinant plasmid protein pgp5 was a precipitated protein.Polyclonal antibodies were successfully prepared by immunizing rabbits.The polyclonal antibody titer of ELISA was as high as1:100,000.While Western blotting identified polyclonal antibodies with good specificity and also demonstrated that the pgp5 protein in Chlamydia muridarum was a soluble protein.The results of cellular immunofluorescence showed that the antibody specifically binds bound to Chlamydia muridarum cultured in vitro.qPCR results showed that the RNA content of pgp5 protein in Chlamydia growth and development cycle was high at early at an early stage and then decreased with time.Western blot showed that pgp5 protein in Chlamydia growth and development cycle was not detected at an early stage,and it was expressed at 12 h and then increased over time.EMSA results showed that pgp5 protein could not bind tobe combined with partial gene fragments in the upstream promoter regions of TC0181,TC0319,TC0357,and TC0419.The EMSA results showed that the pgp5 protein could not bind to a upstream promoter regionspecific DNA fragment of the TC0181,TC0319,TC0357,TC0419.[Conclusion] The recombinant plasmid was successfully constructed,and the pgp5 protein with strong immunogenicity was successfully expressed and purified.The pgp5 was successfully carried out and polyclonal antibody with high titer,highspecificity and strong bological activity was successfully prepared.The solubility analysis of the protein was detected successfully.It find that the transcriptional level and protein level of the pgp5 protein at different time points,and explored possible binding sites between the pgp5 protein and the Chlamydia gene.It provides an important basis for the study of the mechanism of pgp5 protein leading to fallopian tube disease,which can provide assistance for the diagnosis of Chlamydia trachomatis,and also provide new ideas for the study of Chlamydia vaccine.