T-PA Lentivirus Vectors Construction And Lentivirus Production

Tissue-type plasminogen activator(t-PA) is a key enzyme in fibrinolysis system, the physiological role of which is to convert zymogen plasminogen into an active form of serine proteinase plasmin,which then initiates or accelerates the process of fibrinolysis and degrades the fibrin network of thrombi and blood clots, is the medicine for the therapy of thrombotic diseases. t-PA is widely distributed in the normal tissue. With the development of science technology, the manufacture of t-PA goes through many stages of development which included the original tissue extracting, prokaryotic expression, eukaryotic expression and animal mammary gland bioreactor expression. At present, although the manufacture of t-PA by animal mammary gland bioreactor expression has achieved industrialization, there were certain defect, for example the problem of the low rate of integration, transgenic animal genetic instability, genetic instability of transgenic animal, growth cycle length of transgenic animal and so on.Now optimal means for transgenic are the usage of virus vectors. As one of them, lentivirus provides effective means for expression of exogenous genes in mammalian cells, which had proved efficient and achieced stable long-term expression of the transgenes, so it was developed as an important transgenic vector for transgenic animals and gene therapy. In this research, we cloned the activity region of human t-PA cDNA, constructed t-PA lentiviral vector and produced high-titer lentivirus by four-plasmid system in 293T cells. These works provide the basis for generating t-PA transgenic animal.The t-PA activity regional cDNA was obtained from melanoma cell through RT-PCR and confirmed by DNA sequence analysis. In order to get strong promoter,we firstly construced pEGFP-N3-t-PA vectors. And then the t-PA lentiviral vectors were generated by subcloning recombinant technology and confirmed by restricition endonuclease analysis.In order to package virus, we extracted and purified recombinant plasmid and the else three packaging plasmids and measured the density of plasmids. The four-plasmid system was co-transformed into 293T cell for lentivirus production. The lentivirus in cell culture medium were collected, concentrated by PEG and quantified. The generated recombinant virus was used to infect 293T cell, detecting the number of positive clone of EGFP to calculate the virulence,which grossly was 1.5×107 TU/mL. The expression of t-PA in 293T cells was detected by RT- RCR and Western blot, and the molecular weight of the recombinated protein was 35.93kDa as our forecast. As a conclusion, the t-PA cDNA was successfully cloned using RT-PCR method from melanoma cell.The recombinant t-PA lentiviral vectors were successfully constructed by the method of molecular cloning and recombinant technics.The GFP protein was dectected by fluorescent microscope and t-PA protein was dectected by RT-PCR and Western blotting analysis in 293T cell.High-titer lentivirus was successfully produced and identified to be able to generate the transgenic animal.