Molecular Cloning And Expression Vector Construction Of The MgrA Gene And Expression And Purification Of The Fusion Protein In E.coli

Objective: Gene mgrA is located in the chromosome of Staphylococcus aureus, including the 444 base pairs, encoding about 147 amino acid. Gene mgrA expression depended on the premise that acquisition to the gene vector to build for the future to further study its expression in the cells preparation. It is proposed to use molecular biology methods, from Staphylococcus aureus whole-genome gene mgrA extracted by the PCR method of gene cloning has been mgrA. MgrA study gene function, the key is its expression in the cells, construct primarily expression vector, and all the problems on the premise that acquisition to the target gene.Preparation for a large number of MgrA, and to explore the biological activity of MgrA, and the realization of large-scale acquisition to create the preconditions for mgrA this study was to use genetic engineering means, in tracking, the principle of innovation, to explore a practical, cost-effective purification, production restructuring program, in order to carry out extensive restructuring mgrA of applied research and industrial production will be the basis of MO.Methods: PCR amplification mgrA genes, and DNA sequence analysis; to the correct sequencing of the gene fusion was cloned into E. coli expression vector pRSET-A; recombinant plasmid pRSET-A was transformed into E. coli BL21 (DE3), with IPTG for After induction, the collection of bacteria, cell lysis, the use of ion-exchange chromatography method for purification of protein expression and SDS-PAGE and Western-blot detection.Results: The primers were designed under the guidance of the application of the United States Ambion company online design tools, http://www.ambion. Com, in accordance with N315 full genome sequence, 20 nt of the design of specific primers. In accordance with the principle of optimization, select the start codon 100 nt, the gene coding region in the CC at the beginning of 20 base pairs in length, G/C content of 30%-52%, and no internal repeat sequences. MgrA was amplified by PCR method of gene sequences, PCR product was confirmed by 1% agarose gel electrophoresis, to be consistent with the expected results of the size (444bp), and no more than a product-specific.Recombinant vector at Hind III and BamH I restriction enzyme analysis after PAGE electrophoresis, the 444bp band size, the initial proof of the success of synthetic insert fragment expression vector. The use of polyacrylamide gel electrophoresis can identify products of digestion. Subsequent DNA sequencing have been able to confirm the sequence of the inserted fragments and synthetic sequence in line with that we have been successfully constructed for mgrA gene expression vector pRSET-A, for further evaluation of pRSET-A vector for gene expression mgrA possible.It can be obtained the expression of recombinant protein by affinity chromatography. By polyacrylamide gel electrophoresis confirmed the preliminary purified MgrA. Identification of protein expression in SDS-PAGE analysis showed that the bacteria induced by IPTG in the relative molecular weight of about 19.2kp appeared a new protein with apparent size of the protein with the theoretical molecular weight with the projection. And detection of luminance contrast between the two values, by statistical analysis, both with statistical significance. Also found 1h after the induction of expression of the protein that is the beginning of the peak, 3-4h.Preliminary purification of the product by ion-exchange chromatography, to obtain a number of protein-eluting peaks by polyacrylamide gel electrophoresis confirmed MgrA was purified recombinant protein, protein purification Gel MgrA more premixed bacterial protein, would be the purpose of recovery from the protein gel, and then carried out SDS-PAGE, the protein bands to be of high purity. Received from the cloning of the 444bp long mgrA genes, the correct sequence; HINDI by E coli and restriction enzyme digestion confirmed, mgrA successfully cloned into the expression vector pRSET-A; constructed expression vector pRSET-A expression in E. coli relative molecular weight 24kD fusion protein, the regular pressure ion-exchange chromatography purified fusion protein reached 90% purity. Successfully cloned and expressed mgrA gene, and its initial purification, for large-scale acquisition to create the conditions mgrA.Conclusion: This study was designed to make use of genetic engineering means, the successful cloning and expression of a gene mgrA of the fusion protein to carry out a preliminary purification, for large-scale access to create the conditions mgrA, in tracking, the principle of innovation, to explore a practical , cost-effective purification, production program of reorganization, for the extensive reorganization of the mgrA applied research and industrial production will be the basis of MO.