Establishment And Identification Of Eukaryotic Expression Vector Stably Expressing Syncytin Gene And EL4-syncytin Cell Line

Human endogenous retrovirus?human endogenous retroviruses,HERV?are remnants of germline infections of the human ancestor by retroviruses millions of years ago and occupy about 8%of the human genome.Most of such retroviral genes have lost their coding capacities due to mutations and deletions.However,some open reading frames express in specific tissues or at specific stages of development,suggesting the possible involvement in certain disorder'pathogenesis.HERVs have been indicated the association with a variety of tumor formation,such as breast cancer,myelbma,chronic leukemia.Syncytin mRNA and protein were expressed in nine kinds of leukemia or lymphoma cell lines.Syncytin expression were detected in lymphoma cell lines,normal human peripheral blood mononuclear cells?PBMC?,peripheral blood of leukemia patients with real-time RCR,the results show that no syncytin expression in normal PBMC but high expression in the eight kinds of lymphoma cell lines,the expression level in the peripheral blood of leukemia lymphoma cell lines compared to the C8166,ranging between 0.8-21.7 times.In addition,clinical data indicate that syncytin mRNA expression levels were related to the leukemia patients and fall after the leukemia patients were cured.Those reveal that the syncytin were related with the development of leukemia.The protein of Syncytin residues 373-397 is a polypeptide which having immunological activity of inhibiting escape immune,it highly expression of cancer cell surface form immune attack,while it has the fussion of activity is conducive to cell migration.The result show that the syncytin genes involved in the leukemia immune escape.The correlation with leukemia immune escape still needs more study.In the present study,two pair of primers was designed based on the sequence of human syncytin gene?nucleotide library number:NM₀01130925.1?.The total RNA of C8166 cells as the template for the reverse transcription cDNA.The whole length of syncytin gene ORF,was amplified by nested PCR.The PCR products and PIRES2-EGFP vector were digested respectively with restriction enzymes EcoR I and BamH I,and the purified with DNA Fragment Purification Kit.The purified DNA fragments were incubated at 16? metal bath overnight added with T4 ligase.The ligated products were transformed into Trans1-T1 phage resistant chemical competent cells,then inoculated on Ka+ resistance agar dish and the positive clones were picked out The positive clones were determined with PCR and then were purified and digested with restriction endonuclease EcoR I and BamH I and finally sequenced.The coincidence between the amplified sequences and the target sequence?NM₀01130925.1?is 100%according to Clustalw analyses.Successfully constructed the eukaryotic expression plasmid whose were named PIRES2-syncytin-EGFP.The eukaryotic expression plasmid may provide tolls for the study of the function of syncytin genes.In order to understand the function of syncytin genes how to participate in immune escape in human leukemia,this study intends to establish stable expressing the Syncytin protein cell lines for exploring the role of Syncytin protein in tumor cells.Cells were transfected with Roche's X-tremeGENE the HP DNA Transfection Reagent according to the manufacturer recommendation.Cells were transfected with PIRES2-EGFP and PIRES2-syncyttni-EGFP,after transfected 48 hours the cells were observed under the fluorescence microscope and selected by G418.Maintain screening concentration cell can fluoresce under microscopy by using limited dilution method chooses monoclonal cell lines,the selection ofmonoclonal cell line to expand training and selection.Syncytin stably expressing EL4 cells line were successfully established.The syncytin mRNA level of EL4-syncytin cells were measured with real-time fluorescence quantitative PCR using GAPDH as an internal control The results of real time PCR demonstrated that the syncytin mRNA level of EL4-syncytin cell lines was significantly higher than the EL4 cells transfected with empty vector.The western blot show that the syncytin protein level of EL4-syncytin cell lines was higher than the EL4 cells transfected with empty vector.The syncytin mRNA levels were measured with real-time fluorescence quantitative PCR using GADPH as an internal control in EL4-syncntin and other cell lines.The results of real time PCR demonstrated that the syncyin mRNA level of EL 4-syncytin cell lines was significantly higher than that of the cells transfected with empty vector.Then the Western blot were used to measure the syncytin protein level using-action as an internal control.The result of WB show that the syncytin protein of EL4-syncytin was more highly than the control cell lines.All of that,we can say that the syncytin stably expressing EL4-syncytin cell lines were successful establishmented.In summary,we successfully cloned the full length of human syncytin coding region from the C8166 cells,and the sequence successfully inserted into the eukaryotic expression vector PIRES2-EGFP.The bacteria PCR?restriction enzyme digestion and sequencing were used to identify the vector,the result show that the eukaryotic expression vector PIRES2-syncytin-EGFP were successfully constructed.By liposome transfection method recombinant expression vector transfected into EL4 cells and using G418 selection of stably expressing syncytin cell lines.The real-time quantitative PCR and Western blot were used to measured the mRNA and protein levels in EL4-syncytin cells and the control cells.The results showed that the EL4-syncytin cells can stably expressing syncytin.Syncytin stably expressing EL4 cell line may provide a cell model for further research on Syncytin function and its relationship with leukemia immune escape.