Construction,Packaging And Identification Of Recombinant Lentivirus Vetor Inserted RBM3

RBM3 was first identified from a human fetal brain tissue cDNA library in 1936.RBM3protein is a multifunctional protein that has potential research value.It does work by elevating expression not only in the fight against noxious stimuli,such as hypoxia and cold stress,to protect cells,but also in the prevention of cancer,the occurrence of cancer and the treatment of muscle diseases.Building the lentiviral vector including RBM3 gene which could express in cells could lay the foundation for the study of the physiological mechanism of RBM3 protein and its application in clinical.The method of this study:RBM3 gene was amplified by using the molecular biology techniques,and then was inserted into the lentiviral vector Plenti6/v5-DEST,pLenti6/v5-RBM3was cultured and amplified in the DH5?competent cells and RBM3 gene sequence was identified.The 293T cells were transfected with the pLenti6/v5-RBM3.The lentivirus was harvested,and then was used to infect ST cells,Real-time-PCR assay was used to evaluate the mRNA expression of RBM3 mRNA in the infected ST cells,and western blot was used to evaluate the protein expression of RBM3 in the infected ST cells.Experimental results indicated:(1)The sequence of RBM3 inserted into lentivirus vetor was right.(2)The lentiviral titer pLenti6/v5-RBM3 was 10~7 PFU/mL;The lentiviral titer pLenti6/v5-DEST was 4.7×10~8 PFU/mL;The lentiviral titer pLenti6/v5-GFP was 3×10~8 PFU/mL.The lentiviruses produced by 293T cells were efficient to infect ST cells.(3)RBM3 gene expression was detected at the transcriptional level by quantitative PCR method.Empty vector group compared with the blank control group,RBM3 mRNA expression levels closed to the same;Overexpression group compared with the control group,RBM3 mRNA expression level was significantly increased,they had a significant difference(P<0.05).(4)Protein RBM3 expression was detected at the protein level by Western blot method.The protein RBM3 expression between Empty vector group and the blank control group had no significant difference,The protein RBM3 expression between overexpression group and the control group had a significant difference(P<0.05).Conclusion:RBM3 expression in ST cells can be effectively up-regulated by infected with recombined pLenti6/v5-RBM3 lentivirus(P<0.05).