| In order to investigate the effect of using fibronectin- binding protein A to therapy the mastitis that caused by Staphylococcus aureus, we do study about Cloning, Construction of eukaryotic and pronucleus expression vector for fibronectin- binding protein A. These results were expected to lay foundation for further studies on the subunit vaccine ,DNA vaccine of FnBP-A for the prevention of Staphylococcus aureus and producing trangenic animals .The results of research is as following:1. Fibronectin- binding protein A was cloned from Staphylococcus aureus genomic DNA with PCR, which was 3.6 kb . Sequence analysis demonstrated that its homology with the fibronectin- binding protein A of Staphylococcus aureus on GenBank was 99.1 %. Within the open reading frame, six site-mutations occured which could not change protein's function.2. By the strategy of directional cloning, fibronectin- binding protein A was fused with the commonly primary expression vector pBCP and cloned directionally into expression vector pEGFP-C1, the eukaryotic expression vector pEBF for fibronectin- binding protein A was constructed, which included resistant gene and EGFP reporter gene, it could also ensure EGFP and aimed gene express respectively.3. Using T-A cloning technique, the PCR product was cloned into pGEM-T easy Vector successfully and was named plasmid pGEM-fnbA1. pGEM-fnbA1 and pET28a(+) were digested by Hind III and XhoI double enzymes, then the purified fnbA gene was subcloned into the expression Vector pET28a(+) ,and the prokaryotic expression vector pET-fnbA1 was thus constructed successfully.4. The reconstructed plasmid pET-fnbA1 was transformed into E.coli BL21(DE3) competent cells. The bacterium was induced by IPTG(1 mmol/L)and analyzed by SDS-PAGE, approximately 165ku exogenous protein was observed on the SDS-PAGE.5. Western blot analysis indicated the protein is FnBP-A.
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