Identification Of The Interaction Between Of Chlamydia Trachomatis CT813 And CREB3

Chlamydia trachomatis(CT)is obligate intracellular parasites of eukaryotic cell with a unique biphasic life cycle.It can cause urogenital tract infection disease,female pelvic inflammatory disease,ectopic pregnancy and infertility,which cause scholars to pay attention.CT infects host cells through the original body(ER)into the host cells,then ER differentiates into quickly the reticular body(RB)in the cytoplasmic vacuoles.After RB replication,RB becomes new EB,the EB infects adjacent cells.CT in the inclusion body completes its entire biosynthesis process.In order to maintain their own growth and development,CT exchange materials and transfer information for host cells by its inclusion body membrane.In this process,chlamydia inclusion body membrane plays a very important role in interaction between CT and host cells.Inc is an important component of inclusion bodies,encoded by chlamydia genes.It is specific in the chlamydia.Inc has two main features: an N-terminal region can encode a type III secretion signal and a C-terminal hydrophilic region containing at least twotransmembrane helices,Incs N-terminal and C-terminal regions are exposed to the host cytoplasm.When RB is formed,Incs can be secreted by type III secretion system(T3SS),which plays an important role in the growth and development of chlamydia.Therefore,it is very important for Incs research.It has been speculated that there are more than 50 putative inclusion body membrane proteins encoded by genome of Chlamydia trachomatis.CT813 is one of the predicted and confirmed inclusion body membrane proteins,and preliminary research indicates CT813 in CT infection of urinary tract diseases in women has immunogenicity but the detailed biological function of CT813 is not clear.Therefore,we did some researches about this protein.Previous experiments showed that the molecules interacting with CT813 protein were screened by yeast two-hybrid technique.These molecules were cyclic adenylate-response element-binding protein 3(CREB3),galactin lectin-1(LGALS1),ribosomal protein L10a(RPL10a)and tubulin 37E-16(RP1-37E16).Now,In this study,cell co-localization technique and co-immunoprecipitation technique were used to validate the interaction between the two proteins.Firstly,the primers of CT813 and CREB3 were designed according to Gen Bank.The target fragment CT813 and CREB3 wereamplified by PCR.The recombinant expression vector pc DNA3.1+/ Myc-His A-CT813 pc DNA3.1+/Flag-CREB3 were constructed res pe-ctively.The recombinant plas-mid was successfully constructed by double digestion,colony PCR and sequencing.Secondly,subcellular co-localization technique was used to verify the interaction between CT813 protein and exogenous CREB3 protein.The recombinant plasmid pc DNA3.1+/Myc-His A-CT813 and pc DNA3.1+/Flag-CREB3 were co-transfected into He La cells(experimental group).The plasmid pc DNA3.1+/Myc-His and the recombinant plasmid pc DNA3.1+/ Flag-CREB3 were co-transfected into He La cells(control group).After 24 hours,they were fixed with acetone,blocked with goat serum,incubated with primary antibody(mouse anti-Myc monoclonal antibody,rabbit anti-CREB3 mono clonalantibody),and then washed.They were incubated with second antibody(anti-mouse anti-mouse Ig G-Cy3 antibody,goat anti-rabbit Ig G-FITC antibody),and were washed.At last,cell nucleus were stained with DAPI and washed again,then observed with the laser confocal microscope.The results showed that there was the CT813 protein in red fluorescence and the CREB3 protein in green fluorescence,the overlapped region showed yellow fluorescence in different levels.In the control group,the CREB3 protein showed green fluorescence,and it did not overlap with the label Myc.So,theCT813 protein interact with the exogenous CREB3 protein.Thirdly,the co-immunoprecipitation technique was used to verify the interaction between the CT813 protein and the exogenous CREB3 protein again.The recombinant plasmid pc DNA3.1+/MycHis A-CT813 and pc DNA3.1+/Flag-CREB3 were co-transfected into He La cells(experimental group).The plasmid pc DNA3.1+/ MycHis and recombinant pc DNA3.1+/Flag-CREB3 were co-transfected into He La cells.The recombinant pc DNA3.1+/Myc-His A-CT813 was transfected into He La cells(control group).After 48 hours,the cells were lysed(on ice for 30 min)and centrifuged,the supernatant was adsorbed with anti-Myc agarose(beads).The adsorbed beads were washed and tested by Western blot.?-actin from He La cells without transfection was used as control(?-actin control group).The results showed that there were bands at 33 k D and 46 k D respectly in the experiment group,which consistent with CT813 and CREB3 protein respectively.In the control group,there were no band in group co-transfected with empty plasmid and pc DNA3.1+/ FlagCREB3;In the pc DNA3.1+/Myc-His A-CT813 group,there had a band at 33 k D,the size was consistent with CT813;In?-actin group,there is a band at 43 k D.Those results indicated that CT813 indeed interacted with exogenous CREB3.Finally,subcellular co-localization technique was used toconfirm the interaction between the CT813 and the endogenous CREB3.CREB3 is a transcription factor of the leucine zipper c AMP element-response binding protein family,it locates in the endop-lasmic reticulum,so endoplasmic reticulum calcium binding protein(calnexin)was selected as a reference.He La cells were fixed with acetone,blocked with goat serum and incubated with primary antibody(mouse anti-calnexin monoclonal antibody,rabbit anti–CR EB3 monoclonal antibody).After being washed,they were incubated with second antibody(anti-mouse anti-mouse Ig G-Cy3 antibody,goat anti-rabbit Ig G-FITC antibody)and then washed.At last,cell nucleus were stained with DAPI,washed again,and then observed with confocal laser scanning microscopy.The results showed calnexin protein in red fluorescence and CREB3 protein in green fluorescence.The overlapped part had yellow fluorescence.So,CREB3 was confirmed to be located in the endoplasmic reticulum again.The recombinant plasmid pc DNA3.1+/Myc-His A-CT813 was co-transfe-cted into He La cells(experiment group).After 24 hours,they were fixed with acetone,blocked with goat serum,incubated with primary antibody(mouse anti-Myc monoclonal antibody,rabbit anti-CREB3 monoclonal antibody),and then washed.They were incubated with second antibody(anti-mouse anti-mouse Ig G-Cy3 antibody,goat anti-rabbit Ig G-FITC antibody).Cell nucleus were stained withDAPI and washed again,then observed with the laser confocal microscope.The results showed that there was the CT813 in red fluorescence and the CREB3 in green fluorescence,the overl-apped region showed yellow fluorescence in different levels,which proved the CT813 protein interacted with the endogenous CREB3 protein.In conclusion,CT813 could interact with CREB3 protein.It might lay a foundation for further exploring the biological function of CT813 and clarifying the pathogenic mechanism of Chlamydia trachomatis.