Identification Of The Interaction Between Chlamydia Pneumoniae Inclusion Membrane Protein Cpn0308 And ACBD3

Chlamydia pneumoniae(Cpn)is an important atypical pathogen,which can cause respiratory diseases such as pneumonia and bronchitis,some studies have also shown that Cpn is closely related to extrapulmonary diseases like endocarditis and reactive arthritis.The survival of Chlamydia pneumoniae in host cells mainly depends on inclusion bodies which provide the living environment and required nutrition,and to evade host immunity.As a component of the inclusion body,inclusion membrane protein shoulders important biological functions,which has raised scholars' highly concern.Cpn0308 is a confirmed inclusion membrane protein,but its biological function remains unknown.In the previous experiment,using yeast two-hybrid assay we initially screened out the molecule interacting with Cpn0308 was Acyl-coenzyme A binding domain-containing 3(ACBD3).In this study,co-immunoprecipitation,GST pull-down assay and subcellular localization technique were adopted to further confirm the interaction between Cpn0308 and ACBD3.Firstly,primers were designed according to the sequence obtained from the yeast two-hybrid experiment.He La c DNA was used as a template,the ACBD3 gene fragment was amplified by PCR.To construct eukaryotic expression vector pc DNA3.1+/Flag-ACBD3,PCR products and pc DNA3.1+/Flag plasmid were double digested and ligated with T4 ligase.The recombinant plasmid was screened and identified by colony PCR,double digestion and plasmid sequencing.Secondly,the interaction between Cpn0308 and ACBD3 was verified with the co-immunoprecipitation assay.The recombinant plasmids pc DNA3.1/Myc-His-Cpn0308 and pc DNA3.1+/Flag-ACBD3 were co-transfected into He La cells by lipofectamine transfection method.After culturing for 48 hours,the cells were collected and lysed.The lysate was adsorbed to Myc beads overnight and detected with western blot.There were two bands at 33.5k D and 15 k D,corresponding to proteins ACBD3 and Cpn0308,respectively,which suggested Cpn0308 combining with ACBD3.Meanwhile,the GST pull-down assay was used to detecte the interaction.The bait protein GST-Cpn0308 and prey protein ACBD3 were prepared,respectively.Through the procedure of bait protein immobilization,prey protein capture,washing and detection with western blot,we found there were two bands at 39 k D and 33.5k D,corresponding to the protein GST-Cpn0308 and ACBD3,respectively,which further confirmed the interaction between Cpn0308 and ACBD3.Finally,subcellular localization assay was used to detect the interaction.Pc DNA3.1/Myc-His-Cpn0308 and pc DNA3.1+/ Flag-ACBD3 plasmids were co-transfected into He La cells.After culturing for 24 hours,the cells were fixed with acetone.Using rabbit anti-Myc and mouse anti-Flag antibodies as primary antibodies,goat anti-mouse Ig G-Cy3 and goat anti-rabbit Ig G-FITC antibodies as secondary antibodies,the expression of fusion proteins Myc-Cpn0308 and Flag-ACBD3 was detected by immunofluorescence staining.Cpn0308 was green fluorescence,ACBD3 was red fluorescence,the overlapped region showed yellow fluorescence,which confirmed the exogenous interaction between these two proteins.Based on the same method,pc DNA3.1/Myc-HisCpn0308 was transfected into He La cells.Using mouse anti-Cpn0308 and rabbit anti-ACBD3 antibodies as primary antibodies,goat anti-mouse Ig G-Cy3 and goat anti-rabbit Ig G-FITC antibodies as secondary antibodies,the proteins were identified by immunofluorescence staining.Cpn0308 was red fluorescence,ACBD3 was green fluorescence,the overlapped region showed yellow fluorescence,which confirmed the interaction between protein Cpn0308 and endogenous ACBD3.In summary,the results convincingly confirmed that Cpn0308 can interact with ACBD3,providing a basis for further studying its biological function,and which may be helpful to unveil the molecular mechanism of Cpn development.