Optimized Expression Of Chlamydiaphage PhiCPG1 Caspid Protein VP1 And Analysis Of Its Biological Effects

[Background] Chlamydia trachomatis(Ct)urogenital tract infections can cause persistent refractory sexually transmitted disease with minimal discernible clinical symptoms.With the increasingly serious phenomena of drug resistance and treatment failure,the application of bacteriophages in biotic therapy presents considerable potential.The lytic phage φCPG1 is specific for Chlamydia caviae,and VP1 is its major capsid protein.Purified wild-type VP1 inhibits the growth of Ct and the Chlamydia psittaci strain guinea pig inclusion conjunctivitis(GPIC).[Objective] To analyze the properties and structure of chlamydiaphage phiCPG1 capsid protein VP1 by various bioinformatics methods.In order to explore its best functional domains,the VP1 protein was truncated and prokaryotic expressed.The affinity between different types of truncated VP1 and Chlamydia trachomatis was detected by protein interaction assay.To clone the polymorphic membrane protein I(PmpI)gene,then express,purify,identify the immunogenicity of PmpI protein and analyze its biological characteristics.Research on the functional domains of VP1 protein,which play an important role in the adhesion mechanism of phage and chlamydia,provides an important basis for the structural analysis of VP1 protein and offer a new method for the treatment of Chlamydia trachomatis infection.[Methods] The secondary structure and B cell epitopes of the PmpI protein were predicted using bioinformatics software and biological databases.The PmpI gene from the D strain of Chlamydia trachomatis was cloned into prokaryotic expression vector to express the recombinant protein PmpI.A His?Bind Purification Kit was utilized to purify the recombinant protein.The localization of PmpI protein in Chlamydia trachomatis was observed by confocal microscopy.We make the evaluation of the structure and properties of the VP1 protein via bioinformatics analyses,codon optimization and sequence truncation and the biological effects of the truncated VP1 protein on Ct strains in vitro.The wild type and truncated form of VP1 were successfully expressed in E.coli.The cytotoxicity of truncated VP1 proteins to HeLa cells was determined using CCK-8 assay.The truncated VP1 proteins(50μg/mL)was incubated with Ct reference strain E serotype for 1 hour and added into in every step of Ct infection process.The number of Ct inclusion bodies was counted by iodine staining and the inhibition rate was calculated.The binding ability of different truncated VP1 protein to Chlamydia trachomatis PmpI protein was detected by Far-Western blot and GST pull-down.The functional domain of VP1 protein was explored according to the final experimental results.[Results] A comprehensive analysis of the secondary structure,flexible regions,hydrophilicity plot,antigenic index,and surface probability led to the prediction of 8 dominant B cell epitopes of PmpI protein and 12 B cell epitopes of VP1 protein.The all recombinant proteins were successfully constructed.Western blotting showed that the N-PmpI/FL-VP1 recombinant protein specifically reacted with an antiN-PmpI/FL-VP1 polyclonal antibody.The codon-optimized sequence of VP1 gene was obtained and its purification products were successfully obtained.After cytotoxicity and cell viability test,the concentration of the proteins in the intervention experiment was set as 50μg/mL.The inhibition rates of different truncated VP1 protein for Ct reference strain E serotype were from 83.2% to 6.8%.Among them,the inhibition rate of VP1-C protein which expressed 18~476 amino acid was the best,83.2%,and was higher than the FL-VP1.Meanwhile,the low-endotoxin bovine serum albumin(BSA)control group did not present inhibitory activity.Far-western blot and GST pull-down test were used to examine the different binding ability of every truncated VP1 protein with Chlamydia trachomatis PmpI protein.Based on the above results,it is presumed that the VP1 protein-specific region that binds to the Ct PmpI protein may be predominantly in the 158~330 amino acid region.The localization of Ct E-serotype PmpI protein in different culture periods was detected by confocal fluorescence microscopy.It was presumed that the PmpI protein,which is a autotransporter protein,is likely to have a conformational change from the intramembrane to the extramembrane.[Conclusion] Different truncated capsid protein VP1 had different inhibitory effects on the growth of Chlamydia trachomatis,and the possible interaction of VP1 protein was mainly in the region of 158~330 amino acid.Performing experiments on VP1 and its truncated form to predict the structure and determine the expression,characteristics and functions of the protein provide a foundation for future studies into the molecular mechanisms underlying the interactions between chlamydiaphages and chlamydia.However,more in-depth studies on VP1 are required.