Epidermis Staphylococcus AgrC Protein Prokaryotic Expression Vector Construction And Protein Expression

Objective:To research the role of dissolve staphylococcus enzyme in extract staphylococcus epidermidis genomic DNA. Build epidermis staphylococcus agrC prokaryotic expression vector to express agrC protein. Provide the basis to product the specificity polypeptide drugs which can treat biofilm formation by staphylococcus epidermidis related infection as agrC as targets.Methods:Improve conventional enzymatic hydrolysis. Compare the effect of different doses dissolve staphylococcus enzyme to extract staphylococcus epidermidis genomic DNA by dissolve staphylococcus enzyme wall-breaking together with the lysozyme.Extract the epidermis staphylococcus film-forming positive standard strains RP62a gene. Use PCR to derive agrC gene fragmen. Use T-A clone to build agrC recombinant cloning vector. Connect agrC gene to pET28α(+) prokaryotic expression vector. Use PCR, double enzyme reaction, sequencing primers to verify the recombinant cloning vector and prokaryotic expression vector.Use IPTG to induce agrC protein expression. Use western blot to identify agrC protein.Results:Add dissolve staphylococcus enzyme in conventional enzymatic hydrolysis can help epidermis staphylococcus broken wall. It increases follw dissolve staphylococcus enzyme dose. Get epidermis staphylococcus RP62a whole genome.Get the epidermis staphylococcus agrC gene fragment (about 1290 bp). Build pMD19-agrC Recombinant cloning vector. Build pET28a(+)-agrC prokaryotic expression vector. Express agrC protein(about 43 KD).Conclusion:Add dissolve staphylococcus enzyme in conventional enzymatic hydrolysis can help epidermis staphylococcus broken wall; It can improve the efficiency of epidermis staphylococcus genomic DNA extraction; Through T-A cloning technology to build agrC recombinant cloning vector can be stable and effective preservation agrC genes. pET prokaryotic expression system can effectively express complete agrC proteins Under IPTG induction.