Construction Of The Eukaryotic Expression Plasmid Of PIRES-Ag85B And Its Expression In CHO Cell

Objective:To construct the Eukaryotic Expression Plasmid of PIRES-Ag85B, which would be expressed in CHO cell after being incised by enzyme and then identified by DNA sequencing and would be tested with Western-blot. With the study, to lay a foundation for developing new-typed TB vaccinums and diagnostic reagent of TB.Methods:Based upon the CDS sequencing of mycobacterium tuberculosis Ag85B in GenBank, a pair of primers were designed with the application of primer and primer5.0. The GGATCC of Nhe I and EcoR I and protective bases were introduced respectively on Primer 5's end. Genes Group of DNA was extracted from H37Rv, the standard strain of mycobacterium tuberculosis and Ag85B gene fragments were amplified by the primer. After the PCR productions were recovered with PCR test kit adhesive and purified, the PCR productions and enzyme-incised PIRES plasmid were combined with T4 DNA ligase. Then DH5 a competent cell was transfected, cloned positively and screened with Ampicillin. The outcome was identified with colony PCR, double enzyme digestion and sequencing. With the application of liposome transfection technique, the recombinant plasmid PIRES-Ag85B was transfected into CHO cell and screened in 48 hours with G418. Western blot was adopted to test Ag85B's gene expression.Results:Ag85B gene of 978bp was amplified from H37Rv, the standard strain of mycobacterium tuberculosis and the Eukaryotic Expression Plasmid of PIRES-Ag85B was successively constructed. The outcomes were incised by enzyme and electrophoresis with Nhe I and EciR I to obtain the stripes of 61,000bp and 978bp, which agreed with experiment's hypothesis. The target gene sequence obtained by sequencing testing was testified in accordance with the sequence published by GenBank. Transfection CHO cell acquired CHO bacterial strain which could stably express Ag85B,whose gene expression was testd in protern byWestern blot.Conclusion:1. Ag85B gene fragments could be amplified from H37Rv, the standard strain of mycobacterium tuberculosis and the Eukaryotic Expression Plasmid of PIRES-Ag85B could be successively constructed. 2. The target gene of Ag85B could find its expression in CHO cell. 3. The successful construction of Eukaryotic Expression Piasmid would lay foundation for developing new-typed TB vaccinums and diagnostic reagent of TB.Figure:14 table:1 references:49...