The Prokaryotic Expression And Purification Of Mhp168₁16、Mhp168₃22、Mhp168₃41、Mhp168₅49Proteins Of Mycoplasma Hyopneumoniae Vaccine Candidate Antigens

Mycoplasma pneumoniae of swine （MPS） is a chronic respiratory disease, which caused by Mycoplasma hyopneumoniae （Mhp）. Mhp generally do not kill swine, but can significantly makethe production performance lower, the clinical symptoms of MPS were fever, cough and dyspnea. It is easy to cause secondary infection of other swine diseases, such as Porcine Circovirus Disease, swine influenza, Pseudorabies in virosis and Porcine contagious pleuropneumonia、 porcine pasteurelosis、 Streptococcus suis in bacterial disease, and then they can interact with each other and lead to Porcine Respiration Disease Complex（PRDC） which will cause great economic losses of pig industry. Now the most frequently-used Mhp vaccine candidate antigens are Mhp183（P97）、 Mhp378、 Mhp677（P65） and so on, but the immune effect of vaccines which base on those Mhp vaccine candidate antigens only have little difference with the traditional vaccines. It is necessary to screen and identify Mhp proteins in order to abtain Mhp vaccine candidate antigens which have more immune protection.First, the study use the molecular biology softwaresto screen Mhp vaccine candidate antigens which mainly are secretory proteins and outer membrane proteins, then take the mhp168₁16, mhpl68₃22, mhp168₃41and mhp168₅49gene which are screened into prokaryotic expression and purification, those provide surport for the subsequentidentification which based on the reverse vaccinology strategy. The main contents of study include the following several aspects:1, Bioinformatics prediction of secretory proteins and outer membrane proteins which are regard as a part of Mhp vaccine candidate antigensObjctive: To predict Mhp vaccine candidate antigens by bioinformatics prediction.Method: Base on a part of proteins of Mycoplasma hyopneumoniae168, the bioinformatics analysis tools of SignalP4.1, DAS and HMHMM were used to predict the membrane proteins, CELLO version2.5was used to predict the proteins’subcellular localization. BOMP was used to predict the β-barrel outer membrane protein. Conserved outer rmembrane proteins and secretory proteins were identified by BLAST sequence alignmenting among the proteins screening by online bioinformetics softwares.Result:SignalP4.1predict Mhp168₅49may has signal peptide. DAS predict Mhp168₁16, Mhp168₃22, Mhp168₃41, Mhp168₅49may have transmembrane domains. HMHMM predict Mhp168₁16, Mhp168₃41, Mhp168₅49may exist the outside of cell membrane and33-52of Mhp168₃22N-terminal domain may has transmembrane domain with the most of Mhp168₃22exist the outside of cell membrane. CELLO version2.5predict Mhp168₁16, Mhp168₃41are secretory proteins, Mhp168₃22may be the outer membrane protein、secretory protein, Mhp168₅49may be the secretory protein、 Periplasmic protein or outer rmembrane protein. BOMP predict Mhp168₃22may hasp-barrel. To sum up:Mhp168₁16, Mhp168₃41, Mhp168₅49may be secretory protein or outer rmembrane protein, Mhp168₃22may be the outer rmembrane protein with β-barrel. Those proteins all have good sequence conservation.Conclusion:Mhpl68₁16, Mhp168₃22, Mhp168₃41, Mhp168₅49can be Mhp vaccine candidate antigens.2-.The prokaryotic expression and purification of mhp168₁16、mhp168₃41、mhp168₅49gene of Mhp vaccine candidate antigens without TGA encode TrpObjctive: To obtain purifiedprokaryotic gene expression of Mhp168₁16, Mhp168₃41, Mhp168₅49.Method:Based on Mycoplasma hyopneumoniae168, to clone mhp168₁16, mhp168₃41, mhp168₅49gene framents by PCR, then connect with pGEX-6P-2vector, at last establish pGEX-6P-2-mhp168-116, pGEX-6P-2-mhp168-341, pGEX-6P-2-mhp168-549recombinant plasmids. We transformed the recombinant vector into E.coli XL-1, induced it in the final concentration of0.2mM IPTG at30℃, then purified those recombinant strain with Glutathione-Sepharose4BBeads, at last digested them with Prescission Protease, the expression ratio of targetprotein was detected by SDS-PAGE.Result:Cloning the mhp168₁16, mhp168₃41, mhp168₅49gene fragements, we obtained612bp,726bp,837bp prospective segments. The gene fragments were connected with pGEX-6P-2vector. pGEX-6P-2-mhp168-116, pGEX-6P-2-mhp168-341, pGEX-6P-2-mhp168-549recombination plasmids were confirmed to be constructed as expectation by enzyme digestion. The recombination plasmids expressed in E.coli XL-1and the fusion proteins were purified with Beads. Mhp168₁16、 Mhp168₃41and Mhp168₅49proteins with GST-tag showed48kDa,55kDa,57.5kDa in SDS-PAGE.Conclusion:Mhp168₁16, Mhp168₃41, Mhp168₅49proteins can expressed in E.coli XL-1with a good natural conformation of soluble form.3, The prokaryotic expression and purification of Mhp vaccine candidate antigenmhp168₃22gene with TGA encode TrpObjctive:To obtain purifiedprokaryotic gene expression of Mhp168₃22.Method:Mycoplasma hyopneumoniae168straingenome was used as a template to clone mhp168₃22without signal peptide gene sequence by PCR, then connect with pMD19-T to form pMD19-T-mhp168₃22. Four codon TGA encoding Trp were mutated into TGG by quick-change site-directed mutagenesis, then subcloned the mutated gene into the expression vector pGEX-6P-2to form pGEX-6P-2-mhp168₃22. We transformed the recombinant vector into E.coli XL-1, induced it in the final concentration of0.2mM IPTG at30℃, The fusion protein was purified by Beads and digest by Prescission Protease, the expression ratio of targetprotein was detected by SDS-PAGE.Result:Cloning the mhp168₃22gene fragement, we obtained753bp prospective segment. pMD19-T-mhp168-322recombination plasmid was confirmed to be constructed as expectation by enzyme digestion after gene fragment was connected with pMD-T vector. The rare codon TGA was mutated into TGG in pMD19-T-mhp168₃22. Enzyme identification and Sequencing validation proved that point mutation weresuccessful.The target protein expressed by pGEX-6p-2-mhp168₃22was purified by Glutathione4B and Prescission Protease. Mhp168₃22protein with GST-tag showed55.2kDa in SDS-PAGE. Conclusion:Mhp168₃22protein can expressed in E.coli XL-1with a good natural conformation of soluble form.4. Immunogenicity and reactogenicity analysis of protein Mhp168₁16、Mhp168₃22、 Mhp168₃41and Mhp168₅49.Objective:testing the immunogenicity and reactogenicity of protein Mhp168₁16、 Mhp168₃22、 Mhp168₃41and Mhp168₅49.Methods:In order to generate polyclonal antibodies, the mice were immunized with the recombinant proteins:Mhp168₁16、Mhp168₃41and Mhp168₅49proteins with GST-tag respectively. Western blot test carried out to verify the immunogenicity of target protein. Using Fusion protein as antigen and antiserum antibody titer of ELISA test analysis of its immunogenicity. Using four kinds of protein reaction with positive serum of pig and Western blot analysis of its reactogenicity.Results:There are specific reaction in fusion protein GST-Mhp168₁16, GST-Mhp168₃22, GST-Mhp168₃41, GST-Mhp168₅49and serum antibody. ELISA test of serum antibody titer found that the four kinds of protein with high antibody titer and positive rate was100%. Four kinds of fusion protein had specificity reaction and appeared corresponding strip with positive serum of Pig-Conclusion:Mhp168₁16, Mhp168₃41, Mhp168₅49protein has good immunogenicity and reactogenicity.