Preliminary Identification Of The Molecules In HeLa Cells Interacting With CT224 From Chlamydia Trachomatis

Chlamydia trachomatis(Ct)is a strict living intracellular parasitic pathogen with a unique developmental cycle.It replicates in the inclusion formed after infecting host cells.Inclusion membrane proteins(Incs)are a class of specific proteins mainly encoded by Chlamydia genes.And as an important part of the inclusion,they can maintain the growth and development of Chlamydia by regulating many biological processes of host cells.CT224 is an Incs that has been confirmed by experiment and three molecules with high possibility of interaction with it have been analyzed and predicted by affinity purification-mass spectrometry combined with bioinformatics: vitellogenic carboxypeptidase-like protein(CPVL),TNF receptor-associated factor 7(TRAF7)and peripheral-type benzodiazepine receptor-associated protein 1(BZRAP1).However,whether these molecules really interact with CT224 needs to be verified by experiments.Therefore,on the basis of the preparation of anti-CT224 polyclonal antibody,the above molecules which may interact with CT224 in He La cells will further confirmed by GST pull-down technique and co-immunoprecipitation technique,which lay the foundation for further study of the biological function of CT224.Firstly,preparation of the polyclonal antibody against CT224.The prokaryotic expression vector p GEX-6P-2-Ct224 was constructed via molecular cloning technique,then induced and expressed as the GSTCT224 fusion protein.After purification by affinity chromatography,GST-CT224 fusion protein was immunized into BALB/c mice.One week after the last immunization,the blood of mice was collected and the serum was isolated.The constructed pc DNA3.1/myc-His(-)A-Ct224 was transfected into He La cells and cultured for 36 h.After fixed,permeated and blocked,the cells were incubated with 1:300 mouse anti-GST-CT224 serum(experimental group)and 1:800 mouse anti-Myc antibody(control group 1),respectively.The secondary antibody of both groups was goat anti-mouse Ig G antibody labeled with Cy3 and were observed under laser confocal microscope after the cell nucleus were stained with DAPI.At the same time,untransfected He La cells were set as the control group 2,and the primary antibody and secondary antibody were the same as the experimental group.The result showed that there were red fluorescence signals in the experimental group and control group 1,and no fluorescence signal in control group 2.The He La cells transfected with pc DNA3.1/myc-His(-)A-Ct224 for48 h were collected,and the supernatant of the cells lysate was divided into two groups.1:2 000 mouse anti-GST-CT224 serum was served as the primary antibody of the experimental group,and 1:3 000 mouse anti-Myc antibody was served as that of the control group.The secondary antibody was HRP-labeled goat anti-mouse Ig G antibody in the both groups.The Western Blot result showed that there was only one band in both groups and the position was the same.The above experiments indicated that the polyclonal antibody against CT224 was successfully prepared.Secondly,verification of the interacting molecules of CT224 with GST pull-down technique.The GST-CT224 fusion protein(experimental group)and GST protein(control group 1)were used as bait proteins respectively after purified by GST beads,then co-incubated with the He La cells lysate supernatant.The precipitates after centrifugation were analyzed by Western Blot.At the same time,the supernatant of He La cell lysate and unincubated GST-CT224 were used as control(control group 2 and control group 3,respectively).After electrophoresis,membrane transferred and blocked,the antibodies of CT224,CPVL,TRAF7 and BZRAP1 were incubated as primary antibody in each group,and then the corresponding HRP-labeled goat anti-mouse Ig G antibody or HRP-labeled goat anti-rabbit Ig G antibody was added and developed by ECL.The result showed that the bands corresponding to CT224,TRAF7 and BZRAP1 appeared in the experimental group,while no bands in the control group 1.There were corresponding bands of CPVL,TRAF7 and BZRAP1 in the control group 2,and only the band of CT224 appeared in the control group 3.The above result suggested that CT224 could interact with TRAF7 and BZRAP1 in vitro,but not with CPVL.Finally,confirmation of the interacting molecules of CT224 using co-immunoprecipitation technique.The supernatant of He La cells lysate was collected after transfected with pc DNA3.1/myc-His(-)A-Ct224 and added to Myc beads,then adsorbed and centrifuged.The obtained precipitate was set as the experimental group.The He La cells transfected with pc DNA3.1/myc-His(-)A(treated with the same way as the experimental group)was set as control group 1,and the supernatant of untransfected He La cells lysate was set as control group 2.After electrophoresis,membrane transferred and blocked,the antibodies of CT224,CPVL,TRAF7 and BZRAP1 were incubated as primary antibody in each group,then the corresponding secondary antibody(HRP-labeled goat anti-mouse Ig G antibody or HRP-labeled goat anti-rabbit Ig G antibody)were added and developed by ECL.The result showed that the bands corresponding to CT224,TRAF7 and BZRAP1 appeared in the experimental group,while no bands in the control group 1.There were corresponding bands of CPVL,TRAF7 and BZRAP1 in the control group 2.The above result demonstrated that CT224 could interact with TRAF7 and BZRAP1 in He La cells,but not with CPVL.In conclusion,this study further proved that the CT224 interacts with TRAF7 and BZRAP1,but not with CPVL.It is speculated that CT224 may be involved in interfering with the proapoptotic pathway of host cells and inhibiting the host immune response,which will provide an experimental basis for further exploring the biological function of CT224.