Constructed Lentiviral Expression Vector Of A Novel MPL Exonlo Mutantation

Objective:By constructing lentiviral expression vector of the wild-type and the mutant of MPL exon10gene, packaging lentiviral that contains the target gene fragments,to lay the foundation for the subsequent research of the MPL exon10gene mutant.Methods:1. Constructed the eukaryotic expression vector of MPL MPLA497-L498LVIA ins and control gene.The full-length CDS region of MPL exon1012bp insertion and MPL wild-type is amplified by RT-PCR.Construct eukaryotic expression vector ofpCDH1-MPLA497-L498LVIAIns and pCDH1-MPLWT by restriction enzyme digestion and ligation.pCDH1-MPLWT recombinant plasmid as a template, by site-directed mutagenesis techniques to build MPLW515L expression vector and named as pCDH1-MPLW515L recombinant plasmid.2. Construction of the MPL MPLA497-L498LVIA ins and control gene lentiviral expression systemUsing the best plasmid mixture proportioning to combine the expression vector with the packaging plasmid pPACKH1-GAG,pPACKH1-REV and pVSV-G by the method of calcium phosphate coprecipitation,and transfect them into293T cell.Using laser scanning confocal microscope and flow cytometry to detect the efficiency of the transfaction.3. The lentiviral infection293T cells, determination of virus titerThe lentivirus concentrate infected293T cell by gradient directly. Green fluorescent protein observed under confocal laser scanning microscope in72h and96h respectively, Count the number of positive cells of the the last hole which is able to observe the green fluorescent. According to the formula, Positive cells of the the last hole/Dilution factor×103.Results:1. Identification by restriction enzyme digestion and sequencing, the eukaryotic expression vector of pCDHl-MPLA497-L498LVIA ins、pCDHl-MPLW515L and pCDH1-MPLWT were Successfully constructed.2.Three lentiviral expression system was successfully constructed by the genetic engineering techniques to build Lentiviral expression system of the three purposes gene lentiviral vector. The Laser scanning confocal microscopy and flow cytometry had detected the transfection efficiency.3.The viral titer was determined respectively, MPLA497-L498LVIA ins4×105TU/ml, MPLW515L4×106TU/ml, MPLWT1×106TU/ml, and all the titer are met the requirements of the subsequent experiments.Conclusions:Successfully constructed lentiviral expression system of wild-type and two mutant MPL gene by molecular cloning techniques,laid the foundation for further study about the function of genes.