Screening And Preliminary Identification Of Related Molecules In Hela Cell Interacting With Cpn0146

Chlamydia pneumonia（Cpn）is a kind of pathogenic microorganism with strict intracellular parasitic and unique two-phase development cycle,which easily causes respiratory tract infection and is closely related to the occurrence and development of cardiovascular and cerebrovascular diseases.Studies have found that some secreted proteins and inclusion membrane proteins（Incs）encoded by chlamydia gene play an important role in chlamydia development.Cpn0146 was one of the inclusion membrane protein,but it is still unclear that it plays role in the growth and development of chlamydia and the specific mechanism of its interaction with host cells.In this study,Liquid Chromatography-Mass Spectrometry（LC-MS）and yeast two-hybrid technology were used to preliminary screen ligands that interact with Cpn0146.Then,GST pull-down technology was applied to further verify the ligand simultaneously involved in both of them.This study may provide experimental basis for further revealing the biological function of Cpn0146.1.Preliminary screening of Cpn0146 interacting molecules by LC-MS technologyAccording to the information provided by STD gene bank,Primer 5.0software was used to design primers containing sequences of restriction enzyme Bam H I and Not I.The target gene Cpn0146 was amplified by PCR,and the PCR products were purified by gel recovery kit.The purified PCR products of Cpn0146 and prokaryotic expression vector p GEX-6p2were respectively digested by restriction enzymes Bam H I and Not I.After ligated by T4 ligase,the recombinated plasmids were transformed into Escherichia coli XL1-Blue and the positive colonies were screened by colony-PCR.The positive colonies verified by sequencing were induced to produce fusion protein GST-Cpn0146 with IPTG.The mixture of the purified fusion protein GST-Cpn0146 with GST beads and HeLa cell lysate were incubated rotately at room temperature,then washed with balance/washing buffer.The mixture was treated with trypsin,then extracted with extraction solution.Through drying and desalination,the extracts were dissolved and conducted to LC-MS under the set conditions.The LC-MS data were analyzed by Maxquant and Xcalibur software.The results indicated that there were 573 molecules that maybe interacted with Cpn0146.With comprehensive analysis both score and kurtosis value,the most likely interaction molecules were nicotinamide adenine dinucleotide phosphate oxidase（NADPH）,heat shock protein 90（HSP90）,ADP ribose polymerase 1（PARP1）,fatty acid synthase（FASN）,actin 10（Myosin-10）,WD repeat protein 44（WDR44）,general transcription factorⅡ-Ⅰ（GTF2I）,eukaryotic translation initiation factor 5B（EIF5B）,glutathione S-transferase P（GSTP1）,and Serine/arginine repeat matrix protein 2（SRRM2）,etc.2.Preliminary screening of Cpn0146 interacting molecules by yeast two-hybrid techniqueTotal RNA was extracted from HeLa cells with Trizol method,and the first strand of c DNA was synthesized with SMART^TMtechnology.Then ds-c DNA was obtained by LD-PCR,and purified by CHROMA SPIN^TM+TE-400 column.The purified ds-c DNA,p GADT7-Rec and Herring Tests Carrier DNA were transferred to yeast strain Y187 for homologous recombination.The culture was performed on SD/-Leu/Amp⁺plate.The results of colony-PCR showed that the library had good polymorphism.The primers containing digestion sequences both Eco R I and Bam H I were designed referring to the above method,and the recombinant plasmid p GBKT7-Cpn0146 was constructed successfully by the way of PCR,enzyme digestion,ligation,transformation,screening and sequencing.The recombinant plasmid p GBKT7-Cpn0146 was transformed into yeast strains AH109 and Y187 respectively,and proved without self-activation and cytotoxicity.Then the AH109 containing p GBKT7-Cpn0146hybrided with Y187 containing HeLa cell c DNA library on the plate.The medium containing yeast zygote was coated on the plate containing adenine,histidine,leucine and tryptophan nutrient deficient medium.The positive colonies were obtained by initial screening on a plate and further screened byβ-galactosidase membrane method.The yeast plasmids were extracted from positive bacteria solution（corresponding to blue colonies）and transformed into competent cells XL1-Blue,which was verified by colony-PCR again.The positive plasmids were tested for backcross and the positive bacterial colonies were obtained.In order to identify ligand molecules,BLAST comparison was performed with human m RNA in genebank after sequencing.The results indicated that molecules interacted with Cpn0146 were as follows:heat shock protein 90（HSP90）,glyceraldehyde-3-phosphate dehydrogenase（GAPDH）,human extension factor 1-α1（EEF1A1L14）,ATP synthase,60s ribosomal protein,23s ribosomal RNA,ribosomal protein RPL36a,Eukaryotic synthetic construct chromosome 14 and PDEST-GADT7.3.The interaction molecules for Cpn0146 identified by Pull-down techniqueThe Pull-down technique was used to verify the molecules interacting with Cpn0146 co-occurred in the above two methods.First,the fusion protein GST-Cpn0146 was fixed to the centrifugal column with GST beads as bait protein,and the supernatant of HeLa cell lysate was then added,the mixture was incubated rotately at 4℃.After washing,the mixture was conducted to WB.Mouse anti-PARP1 monoclonal antibody（1:40 000）and rabbit anti-HSP90AB 1 polyclonal antibody（1:5 000）were incubated with the corresponding secondary antibody of the same species.The results showed that heat shock protein 90（HSP90）and ADP ribose polymerase 1（PARP1）interacted with Cpn0146.In conclusion,Cpn0146 may interact with HSP90 and PARP1 proteins.It is speculated that it may regulate the host cell cycle and inhibit the repair of DNA damage,which is conducive to the growth and development of Chlamydia in host cells,providing an important experimental basis for further study of the biological function of Cpn0146 and revealing the pathogenic mechanism of Chlamydia pneumoniae.