| Syncytin is an envelope protein of human endogenous retrovirus W1(HERVW1),which is located in the chorionic trophoblast at early pregnancy and plays an important role in the development of the fetus.Its abnormal expression can lead to intrauterine growth retardation,pathological embryos,tumors and multiple sclerosis,and the abnormal expression of syncytin is related to hematopoietic tumor,lymphoma,melanoma and precancerous eclampsia,hemolytic-elevated liver transaminase-thrombocytopenia syndrome(hemoysis,elvated liver enzymes and low platelets,HELLP),the current regulation of syncytin expression protein is not fully understood.The main work of this study is to find a protein that interacts with syncytin and explore its function.The main work contents the following four aspects:1.In vitro exploration of the fusion function of syncytinIn the study of membrane fusion function of syncytin,electronic transfer of syncytin eukaryotic expression vector containing the label itself express syncytin Hela.Through the RT-qPCR,double fluorescent staining and Western Blot method to detect protein expression of syncytin effects on Hela endogenous syncytin.Compared with the negative transfected group,Hela Syncytin mRNA level rise obviously,and the cells have obviously promoting cells group,gathered phenomenon.But the different time after transfection,Hela syncytin protein levels rise slowly,until the tenth genius appears significant increase.2.Screening syncytin interaction protein on both cellular and bacteria levelIn order to further explored the syncytin protein function,we use the bacteria double hybridization to screen protein interactions.Build screening the bait plasmid pKT25-Syncytin,and no cytotoxicity and the activation;use library carrier pUT18C build whole blood genomic library.To report the fungus DHM1 together,carries on the observation and statistics.Through to the blue single colony further library plasmid purification and PCR detection sequence inserted into the situation,the sequencing analysis directly.Found that there was 93 base pairs before the terminated,encoding amino acid sequences,we will be named SJ-1 gene sequences,amino acids encoded proteins also named SJ-1.In cell level of the interactions of inquiry,we use the precipitation-mass spectrometry(inmunoprecipitation-mass spectra,IP-MS)combination way of screening protein interactions.Western blot test for three kinds of leukemia cell lines and syncytin expression in cervical cancer cell lines.THP-1,express syncytin U937 and Hela have different level.Through the IP,we detected some can combine with Syncytin protein specificity of protein in the cell.After the examination,can be used in MS detection.MS detection IP group identification to 400 proteins,IgG group identification to 355 proteins,IP group and IgG intersection,IP group identification to 258 unique proteins.3.The identification of the influence on endogenous syncytin about protein SJ-1 on cellular levelIn order to verify whether there is interaction with Syncytin,SJ-1 we built respectively and SJ-1 Syncytin two eukaryotic expression vector containing different labels,transfection cell,validated by Co-IP and WB.Input all show the expected size set of protein bands,IP for HA,reveal the Flag label to pull syncytin,IP as the Flag,can show the HA tag SJ-1.Through the RT-qPCR,double fluorescent staining and Western Blot method to detect protein expression of SJ-1 effects on Hela endogenous Syncytin.After nine days,found transfection SJ-1 the endogenous Syncytin fell In addition,RT-qPCR detection mRNA level have significantly lower.Double fluorescence staining results reveal,SJ-1 cell fusion of transfection group was significantly lower than Syncytin transfection group,with no difference between the control group of cell fusion.In this study,we found that the short peptide SJ-1,which could down-regulate the expression of endogenous Synctin,was screened and identified by cell and bacterial level.A total of 258 unique interacting proteins were identified by IP-MS. |
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