Feasibility Of Using Signal Peptidase ? As An Antigen Of Mycoplasma Hyopneumoniae Vaccine

Mycoplasma is a mollicute genus of bacteria that lack a cell wall around their cell membranes.It can contort into a broad range of shapes.Mycoplasma is widely distributed in humans and animals.Mycoplasma pneumoniae is a typical pathogenic mycoplasma,but most of the vaccines against Mycoplasma pneumoniae can`t work well.It has been reported that mycoplasma can escape the host`s immune response by altering its surface antigen,which is determined by the genetic variation of mycoplasma.In this process,the signal peptidase recognizes and cleaves the signal peptide of the mutant membrane protein,so the mutant membrane proteins can be transferred to the cell membrane,causing variation in surface antigens.Signal peptidase I is also present in Mycoplasma pneumoniae.Based on the study of E.coli singal peptidase I(Ec SPase I),we surppose that: Mycoplasma hyopneumoniae signal peptidase I(Mh SPase I)may be a target antigen against Mhp.Antibodies induced by Mh SPase I may inhibit the variation of surface antigen in Mycoplasma.The antibodies can specifically recognize Mh SPase I on Mhp and inhibit the activity of Mh SPase I,so that the variant protein in Mhp cannot be cleaved by the signal peptidase and can not be transferred to the cell membrane,and the variation of surface antigen is inhibited.Antibodies induced by Mh SPase I may also inhibit the adhesion of mycoplasma and the release of virulence factors.After the infection by M.pneumoniae,cell adhesion protects M.pneumoniae from removal by the host's mucociliary clearance mechanism and allows it to produce a variety of local cytotoxic effects.For example,M.pneumoniae can release the Community-Acquired Respiratory Distress Syndrome Toxin(CARDS toxin).If the activity of Mh SPase I is inhibited during the synthesis of adhesion proteins and toxins,these proteins can not be recognized and cleaved by thesignal peptidase,so they can not be transported to the cell membrane or released out of the cell to play their roles.In this study,we analyzed the structures of Ec SPase I and Mh SPase I,There are many similarities between Ec SPase I and Mh SPase I,Mh SPase I may have the same function as Ec SPase I in recognizing and cutting signal peptide.And then we wrote the sequence of M protein which was the full-length of Mh SPase I,expressed the M protein in E.coli,but M protein expressed in the form of inclusion body.After that,we removed the transmembrane segment of Mh SPase I,wrote a new sequence of MHS protein without the transmembrane region of Mh SPase I,MHS didn't expressed in the form of inclusion body.Then we respectively injected the mice with MHS protein and inactivated Mhp,and detected the level of specific antibodies in sera by ELISA.We also stimulated the spleen cells of mice with MHS protein and detected the level of activated B cells and T cells by flow cytometry.The results of ELISA showed that MHS protein could be recognized by the antibody induced by inactived Mhp.The results of flow cytometry confirmed that MHS protein could stimulate the spleen cell of the mice,significantly activate B cells and T cells.The antibodies induced by Mh SPase I worked well,which proved the feasibility of Mh SPase I as an antigen.