Subcellular Localization Analysis Of CT849 Protein In Chlamydia Trachomatis-Infected He La Cells And Screening Its Interacting Proteins

Objective:To investigate the subcellular localization of CT849 protein in Chlamydia trachomatis infected-HeLa cells and the possible interacting protein of CT849 protein in HeLa cells,and to provide more and reliable datas supportig for further understanding C.trachomatis pathogenicity.Methods:C.trachomatis serotype E was used to infect monolayer HeLa cells by centrifuge-assisted infection,then infected cells were fixed with 4%paraformaldehyde at 6 hours post infection(hpi),30hpi,and 42hpi,and blocked with high glucose culture containing 10%FBS after0.3%Triton-X100 treated.After primary antibody incubation and secondary antibody incubation,the slide was mounted and the nucleus of HeLa cells,C.trachomatis inclusions,CT849 protein were observed under a fluorescence microscope,then the subcellular localization of CT849 protein was analyzed.A proper amount of well-grown HeLa cells was extracted its total RNA with the Trizol method,then its mRNA was isolated and reversely transcripted into cDNA.Primary and secondary cDNA libraries of HeLa cells were constructed by Getway homologous recombination method using BP and LR enzymes,respectively.The primary and secondary library bacterial solutions were diluted by 1:1000,then were spreaded on LB plates,and 24 positive clones were selected for PCR amplification,to identify the capacity,recombination rate and the length of the libraries.The secondary library plasmids that was consistent with the standards were transformed into Y187 yeast to form a HeLa cell cDNA library.The sequence of ct849 gene was amplified by PCR,then pET28a-ct849 recombinant plasmid was constructed and its gene sequence was identified by sequencing.The pGBKT7-ct849 bait vector was constructed and was transformed into Y2Hgold yeast,and the self-activation and toxicity of this bait vector were tested.The bait recombinant plasmid-positive strains without toxicity and self-activation were combined with HeLa cell cDNA library for library screening and optimization,positive clones were sequenced and BLAST comparison analysis was performed to get proteins that may interact with the target protein CT849.Finally,The bioinformatics analysis was used to the screened suspected interacting proteins.Results:1.In the three points of 6hpi,30hpi,42hpi,the expression of CT849 protein was the most at 30h after infection with Ct observed by fluorescence microscopy,and the CT849 protein signal was seen in the cytoplasm of infected cells.2.211 positive clones grew on the LB plate coated with the primary library bacterial solution,and the CFU of the primary library capacity was 8.4×10~6cfu according to the calculation formula of the library capacity.24 clones were randomly selected on the LB plate for bacterial colonies PCR identification.The gel electrophoresis results showed that the fragment lengths were different,all were above 1Kbp,and the recombination rate was>90%.399 positive clones grew on the LB plate coated with the secondary library bacterial solution,and the total number of clones in this library capacity was 1.5×10~7cfu according to the formula of the library capacity calculation.24 clones were randomly selected on the LB plate for bacterial colonies PCR identification,gel electrophoresis showed that the fragment lengths were different,basically above 1Kbp,the recombination rate was>90%.These two libraries all meet the quality standards of library.3.The pET28a-ct849 recombinant vector was sequenced and BLAST comparison was used,the results showed that the vector was successfully constructed.The constructed recombinant bait plasmid pGBKT7-ct849 showed no toxicity to the host bacteria and could not activate the host bacteria reporter genes MEL1 and His3,it means that the bait plasmid can be used for screening library.4.Bait recombinant plasmid-positive strain and HeLa cells cDNA library were combinedly growth.Microscopic examination found combined growth yeast cells.Pressurized screening was used to the combined growers,and universal primer PCR reactions revealed that the size of the inserts in the library varied,with fragment sizes ranging from500-2000bp,indicating that the size of the genes carried by the prey plasmids contained in the combined growers were different.After sequencing analysis,50 suspected interacting proteins were identified,The protein classification analysis in the GO(Gene Ontology)analysis shows that it is mainly concentrated in three major categories of ribosomal proteins,adaptor proteins,and energy metabolism-related proteins.Conclusions:1.CT849 is localized in the cytoplasm of C.trachomatis infected-HeLa cells and may be a secreted protein.2.A total of 50 suspected interaction proteins were screened out,mainly including three classes of ribosomal proteins,adaptor proteins and energy metabolism-related proteins.