Cloning And Expression Of The Region D Of Fibronectin-binding Protein A Gene Of Staphylococcus Aureus

The staphylococcus aureus is a kind of main contagion cause germ which causes milk cow mastitis.This study using molecular cloning technique and protein expression technique expresses region D of fibronectin-binding protein A gene of Staphylococcus aureus successfully and research its immunologic competence.Region D of fibronectin-binding protein A gene (FnBPA gene)of Staphylococcus aureus is amplified with PCR method, on the basis a pair of specific primers designed according to the relevant nucleotide sequence from GeneBank. We use genome DNA of gene type 3 and gene 11 which separated from Inner Mongolia .After PCR, we complete cloning of FnBPA D region. The positive recombinant clones was sequenced and analysed. The results suggested that FnBPA D region gene was cloned successfully. Then we blast the result with relevant nucleotide sequence on GeneBank. The result shows that the autoploidies of gene type 3 and gene 11 with the sequence on GeneBank are 98% and 94%. And the autoploidy of gene type 3 and gene 11 with is 96.5%.Antigen sites are analysised by the software DNAstar, the result shows gene type 3 has more antigen site than the other two sequences. So we decide to express the gene type 3. The fragment is successfully cloned into pET-32a(+)to construct a recombinant expressed vector pET- FnBPA. The recombinant expressed plasmid is transformed into the host cell E.coli BL21(DE3)and induced by IPTG and then depurated the protein.After experimental animals are immuned by expression product two times, we obtain high potency antibody. Then carry out regulation and anti-binding of staphylococcus aureus experiments, and the results show that the anbody has the contribution of immunoprotection and anti-binding of staphylococcus aureus. This can play a role in research pathogenesis of staphylococcus aureus and preparing vaccine for mastitis.