Construction Of Eukaryotic Expression Vector Of ClfA Gene From Bovine Staphylococcus Aureus

The cow mastitis is one of the most important diseases in the global dairy farms.Staphylococcus aureus is one of the major pathogenic bacteria and differences in milk sample separation to Staphylococcus aureus were reported at different areas.In order to investigate the situation around Gan Su province and to develop the genetic engineering vaccine,we collected 380 milk samples from Lanzhou,Wuzhong and Minhe.Because of Staphylococcus aureus can grow in the medium with high salinity,we has carried on the preliminary screening and found 41 suspected strains.Through oxidation of hydrogen, mannitol, three sugar fermentation and enzyme fermentation Baird-Parker tablet traditional laboratory operations can achieve 37 suspected strains.As Clf A gene is S.aureus bacteria adhesion factor,so used of PCR for further identification and concluded that only 35 strains of S. aureus bacteria strains.Since then,selected MD20 and extracted its DNA.Amplification Clf A,connected Trans T1 cells,the results showed the length of amplification is 1168 bp.The homology with the reported Clf A factor gene sequence（Gene Bank: HQ₄24292）is98%.After the sequencing of accurate,with HindⅢ and XhoⅠenzyme to p GEMT Clf A and pc DNA3.1 His B digestion, recycle purpose.After connecting the purpose of the fragment,with ligased for the night by T4 DNA enzyme,conversion to competent T1 cells,the results showed that:this experiment has been constructed pc DNA3.1B-Clf A.To building the pc DNA3.1 B-Clf A.Transfection to human breast cancer cells,when the cell stable, extracted RNA,collected transfection cells.Western-blot detected the expression of proteins,is about 39 KDa..The foundation for subsequent immune animal experiments,for S.aureus bacteria genetic engineering vaccine research and development laid a solid foundation.