Construction Of Lentivirus Vector For Overexpression Of Testin Gene And Study On Modification Of Human Placental Mesenchymal Stem Cell

Objective To construct the recombinant lentiviral vector containing Over-expression human Testin gene,and to check its expression in human embryonic kidney skin cells 293 T,and human placental mesenchymal stem cells modified with p HBLV-Testin Gene,To construct a stable h PMSCs cell line expressing Testin protein.Methods Testin gene amplification was used by real-time polymerase chain reaction(PCR).Gene amplification products were inserted in the lentiviral vector p HBLV-CMVIE-Zs Green-Puro,and constructed lentiviral vector p HBLV-Testin.Then Transformation of Escherichia coli receptive cell DH5α,Polymerase chain reaction analysis and DNA sequencing were used to confirm the constructed vectors whether or not success recombinant vector plasmid and to coinfect 293 T cells with the packaging plasmids including p SPAX2,p MD2 G by Lipofiter TM.The recombiant lentivirus expressing TESTIN was obtained from the cells supematant and the viral titer was tested.The Testin-gene-over-expressed recombiant lentivir-us(p HBLV-Testin)was transfected into 293 T cells and The expression of Testin protein was detected by Western blot.p HBLV-Testin recombinant lentivirus vector was used to transfect h PMSCs cells and the optimal number of infection was determined.The expression of related proteins was detected by western blot,and the proliferative activity of infected h PMSCs cells was detected by CCK-8.The empty vector group(p HBLV-null)and no treatment group were used as control group.Results The result of recombiant plasmids gigestion identification showed that the fragment is about 1266 bp,which is the same size as Testin gene c DNA fragments.The blast results of TESTIN c DNA sequence showed that the sequencing results and expected TESTIN gene sequence is completely consistent.It was confirmed that the TESTIN was correctly insertted into the vector,and that TESTIN-gene-over-expressed lentivirus vectors was successfully constructed.After packing 293 T cells,was successful got recombinant lentivirus p HBLV-Testin with virus drops to 2.0×10~8TU/ml.The result of Western blot showed that the expression of Testin was stably in 293 T cells.Green fluorescent protein was observed directly under fluorescence microscope after transfection of p HBLV-Testin recombinant lentivirus vector into h PMSC,and the optimal MOI value was 50.Western blot assay showed that the expression of Testin protein in infected p HBLV-Testin cells was higher than that in empty vector group(P < 0.05.)CCK-8 results showed that there was no significant difference in cell proliferation between p HBLV-Testin.HPMSCs p HBLV-null.h PMSCs and h PMSCs groups(P > 0.05).Conclusion The recombinant lentivirus over-expression vector of Testin gene was successfully constructed.The packaged lentivirus was successfully transfected into 293 T cells and Testin gene was stably expressed.The cell lines stably expressing the target gene TESIN in h PMSCs overexpression samples were successfully obtained,which laid a foundation for further study of its effect on colorectal cancer bearing mice in vivo.