Cloning And Expression Of The Region A Gene Of Clumping Factor A Of Staphylococcus Aureus From Bovine Mastitis

Mastitis is a common and frequently-occurring disease in the bovine, it is caused by pathogenic microorganisms. There are more than eighty pathogenic microorganisms which can cause mastitis in nature, such as bacterium, mycoplasma, fungi and viruses. Staphylococcus aureus is the main pathogen in the bacterium, and there are no effective measures to prevent mastitis caused by Staphylococcus aureus. So the Clumping factor A A region gene were cloned and expressed in this article to provide materias for produceing vaccines of mastitis caused by Staphylococcus aureus.According to the ClfA A sequence published in GeneBank of NewMan strain, primers were designed, then four major genotypes of Staphylococcus aureus from Hohhot were amplified. Four Clumping factor A (ClfA) A region genes of Staphylococcus aureus were sequenced and compared with the sequence of standard NewMan in Genbank,the results showed that: homology of gene-3, gene-9, gene-11 with Newman strain were relatively higher than gene-5 with Newman, it were 97.8%, 96.5%, 98.3 % and 85.4% respectively. Meanwhile,Homology among gene-3, gene-9were higher, and lower in gene 5 with the former three genotypes.The epitope were analyzed by DNAstar software and there were one more antigen sites in gene-3, gene-9 than NewMan, so gene-9 were chosen for expression. Then the fragments were inserted into the orientation of the vector pET-32a (+), and a recombinant plasmids,pET-ClfA-A region,were got.Later,the plasmids were transformed into the host of BL21 (DE3), and ClfA-A region protein was produced after induced by IPTG. The target protein was soluble in the cells of host and expressed protein of high purity were purified by His-bind chromatography at last.Rabbits were immuned by the purified protein emulsified with Freund's adjuvant, and we had obtained efficient anti-serum after three times of immunization. It was proved that the protein expressed was from Clumping factor A A region of Staphylococcus aureus by ELASA, haemaglutination test with tube, phagocytosis conditioning test and anti-adhesion of antibodies. At the same time, The results show that the antibody has significant effects of preventing Staphylococcus aureus from adhesiving to fibrinogen of bovine.