Construction And Identification Of Rat Trail Gene Recombinant Lentivirus Vector

Objective: this experiment using the method of PCR amplifiedTrail gene into slow virus vector system, the production of Trail virus particlesand virus titer. Methods: the1.Trail gene was obtained: according to GenBankin rat Trail gene sequence (NM145681.1), to design the gene specific primersof Trail-Agel-F and Trail-Agel-R, and use Agel enzyme cutting site. Using themethod of PCR from rat cDNA Library of the amplified gene.2construction ofrecombinant plasmids: using In-Fusion technology, the enzyme recovery afterPCR products linear switched connection into the Agel restriction of eukaryoticexpression carrier (GV218), generating recombinant plasmid. Connect productplasmid was transformed into E-coli DH5a competent cells to recombinantplasmid, amplification.3connecting product restriction enzyme identification:collected after amplification connected product, enzyme after electrophoresisfragment length are found to be consistent with expectations, gene sequencingresults are correct, thus proving the cloning of the Trail gene has beensuccessfully.4recombinant plasmids were transfected into293T cells byliposome encapsulated Lipofeetamine:2000construction of recombinant plasmidand auxiliary packaging carrier, three plasmid transfected293T cells produce acommon, expressing the Trail protein of Lentivirus virus particles.5virusdetection: the recombinant plasmid transfected into293T cell, fluorescence Real-time identification using quantitative PCR assay for detection of virus titer.Results: the success of Trail gene, recombinant plasmid sequences and theGenBank Trail gene sequence (NM145681.1) Trail gene sequence comparison,proved correct; three plasmid transfected293T cells was observed byfluorescence microscopy of green fluorescent; Western Blot detection wereobserved in58KD near the characteristic strip, its size and purpose gene fusionprotein coincide with; hole dilution method and real time fluorescentquantitative PCR determination of virus titer determination results for2E ten8TU/ml. Conclusion: successful cloning of rat Trail gene and construction oflentiviral vector system and detection of its titer. As a follow-up to the killing oftumor gene (Trail) transduction into neural stem cells to lay the foundation.