Cloning, Expression Of S.aureus Virulence Factors ClfA, Hla And Sbi And Development Of Eukaryotic Plasmid

The pathogenesis of Staphylococcus aureus attributes to various virulence factors.Researchers have found more than50kinds of virulence factors, which were divided intothree kinds according to the function, adhesion factors, factors that invade and destruct thehost tissue, factors that help Staphylococcus aureus escape from host immune system. ClfAis an important adhesion molecule presents on the surface of Staphylococcus aureus. ClfAbinds to the host extracellular matrix, fibrin and fibrinogen by A region. Hla is a cell lyticprotein secreted by Staphylococcus aureus. Every seven hla monomers compose anoligomer by binding to the specific receptors on target cell surface, and pore the target cells.The mutant H35A was explained without lytic activity on epithelia cells and can obstruct theactivity of wild Hla. In recent years, researchers found immunoglobulin-binding protein Sbi(Second Immunoglobulin-binding protein) play a role similarly to SpA. The region I-IV ofSbi conjuncts specifically with the Fc domain of host IgG, interfering the antibody-mediatedphagocytosis. Sbi also obstruct the deposition of complement system.In this study, Staphylococcus aureus in the milk of goats that in the risk of clinicalmastitis was isolated, and the genome of Staphylococcus aureus was extracted as a templateof PCR. Taking advantage of the PCR engineering, site-directed mutation, overlap PCR andthe prokaryotic expression technology, the recombinant fusion protein CA、Hla and Sbi(I-IV) were expressed in E. coli and were purified. The mutant H35A was obtained and thehemolytic activity of Hla and H35A was detected. Then mice were immunized withvaccines composed of CA, H35A and Sbi (I-IV). The recombinant plasmid CA/H35A/Sbi(I-IV)-pcDNA3.1was developed. The results show that1) the gene ClfA, Hla, Sbi weresuccessfully cloned, and the recombinant protein was expressed. The amino acid sequenceof fusion protein CA, HLA and Sbi (I-IV) was highly identified with other strains(>99.0%).2) the mutant H35A was successfully obtained,the hemolytic activity of Hla washigh (92.843.73%), while the H35A almost without hemolytic activity. the fusion proteinCA, H35A and Sbi(I-IV) can induce high titers of specific antibodies, especially when thethree antigens are co-immunization the antibody titers is the highest(100×28).3) The plasmid CA/H35A/Sbi (I-IV)-pcDNA3.1was constructed successfully.In short, this research successfully cloned and expressed fusion protein CA, HLA, Sbi(I-IV) and H35A. And vaccines composed of CA, H35A and Sbi (I-IV) induced high titersof specific antibodies. Especially the vaccine CA/H35A/Sbi (I-IV) induced the highestantibody titers, laying the foundation for in vitro assessment of recombinant plasmidCA/H35A/Sbi (I-IV)-pcDNA3.1. This research laid the foundation for the development ofDNA vaccines that preventing the infection of Staphylococcus aureus.