Cloning And Expression, Purification And Immunogenicity Of Mhpl68₂18、hpl68₃66、hpl68₄77from Mycoplasma Hyopneumoniae

Mycoplasma hyopneumoniae （Mhp） is the primary etiological agent of Mycoplasma pneumoniae of swine, which is a popular chronic respiratory disease around the world. More than95%of piggeries are positive by serum epidemiological survey of Mhp in China. However, the immune effect of Mhp vaccine is poor. So, new-style Mhp vaccine development is one of the core research areas in Mhp research field currently. Outermembrane proteins and extracellular proteins of pathogen can interact with the host cell directly and cause the host’s immune response in disease-causing process of pathogens, so outermembrane proteins and extracellular proteins are often considered as candidate targets of vaccine antigens. In this study, we predicted several outermembrane proteins and extracellular proteins of Mhp by bioinformatics, and the the predicted proteins were expressed in E. coli XL-1Prokaryoticly and purified with glutathione reductase beads.1. Bioinformatics analysis of mhp168₂18, mhp168₃66and mhp168₄77gene fragmentsObjective:Predict subcellular localization of protein Mhp168₂18, Mhp168₃66and Mhp168₄77proteins by Bioinformatics analysis.Method:Nucleotide sequences and its corresponding amino acid sequences of mhp168₂18, mhp168-366and mhp168-477were alimented by BLAST in Mycoplasma hyoneumoniae strains168,232, J,7422and7448and analyzed the conservation of the three genes. SignalP4.1and LipoP1.0were used to predict signal peptide. DAS was used to predict transmembrane domain. CELLO version2.5was used to predict the proteins’ subcellular localization.Result: The conservation of mhp168₂18gene fragment was more than98%, and the conservation of corresponding amino acid sequence was about99%. Mhp168₂18contains a signal peptide and8transmembrane domains, and it is localized in outermembrane and/or extracellular space. The conservation of mhp168₃66gene fragment was more than98%, and the conservation of corresponding amino acid sequence was between96%and99%. Mhp168₃66does not have signal peptide and contains4transmembrane domains, and it is localized in outermembrane. The conservation of mhpl68₄77gene fragment was more than98%, and the conservation of corresponding amino acid sequence was about99%. Mhp168₄77does not have signal peptide and contains4transmembrane domains, and it is localized in outermembrane and/or Periplasmic.Conclusion:Based on the result of bioinformatics analysis, we can conclude that Mhp168₂18, Mhp168₃66and Mhpl68₄77may be outermembrane protein and be used as candidate vaccine protein antigens.2. Expression segmentally and purification of mhp168₂18gene fragmentObjective: Express mhp168₂18gene segmentally in E. coli XL-1and purify the recombinant protein with good natural construction.Method:Mhp168₂18gene was divided into four fragments, named mhp168₂18-1, mhpl68₂18-2, mhpl68₂18-3and mhpl68₂18-4. mhpl68₂18-1, mhpl68₂18-3and mhp168₂18-4fragments which did not contain TGA codon which codes tryptophan were ligated directly to pGEX-6P-2vector and named pGEX-6P-2-mhpl68₂18-1, pGEX-6P-2-mhp168₂18-3and pGEX-6P-2-mhp168218-4. Mhp168₂18-2fragment which contained TGA codon which codes tryptophan was ligated to pMD19-T vector and mutated the TGA codon into TGG. Then, the mutated mhpl68₂18-2fragment was ligated to pGEX-6P-2vector and named pGEX-6P-2-mhp168₂18-2. Four recombinant plasmids were transformed into E.coli XL-1and the targeted recombinant proteins were induced with0.2mM IPTG. After purification of four recombinant proteins with Glutathione-Sepharose4B beads, the fusion proteins were digested by Prescission Protease. The expression and purification results of the recombinant proteins were tested by SDS-PAGE.Result:Sequenceing result showed that mhpl68₂18-2fragment was mutated successfully. The GST-fused recombinant proteins Mhp168₂18-1, Mhpl68₂18-2, Mhp168₂18-3and Mhp168₂18-4were solubly expressed. The molecular weights of the recombinant proteins were41kDa,59kDa,40kDa and58kDa. These recombinant proteins can be purified by glutathione Beads. Mhp168₂18-2recombinant protein fused with GST can be digested with PreScission protease and obtain33kDa protein.Conclusion:Mhpl68₂18-1, Mhp168₂18-2, Mhp168₂18-3and Mhp168₂18-4were solubly expressed in E.coli XL-1with natural construction, and can be purified by glutathione beads. The GST lable in Mhp168₂18-2recombinant protein can be digested with PreScission protease.3Prokaryotic expression and purification of mhp168₃66, mhp168₄77gene fragmentsObjective:Prokaryotic expression of mhp168₃66, mhpl68₄77gene fragments and purify the recombinant protein with good natural construction.Method: Mhpl68₃66and mhpl68₄77gene fragments was ligated to pMD19-T vector and mutated four TGA codons which codes tryptophan from mhp168₃66into TGG codons and mutated three TGA codons from mhpl68₄77into TGG codons Then, the mutated mhpl68₃66, mhpl68₄77fragments were ligated to pGEX-6P-2vector.Two recombinant plasmids were transformed into E.coli XL-1and the targeted recombinant proteins were induced with0.2mM IPTG. After purification of four recombinant proteins with Glutathione-Sepharose4B beads, the fusion proteins were digested by Prescission Protease. The expression and purification results of the recombinant proteins were tested by SDS-PAGE.Result:Sequenceing result showed that mhpl68₃66and mhpl68₄77fragments were mutated successfully. The GST-fused recombinant proteins Mhp168₃66and Mhp168₄77were solubly expressed. The molecular weights of the recombinant proteins were45.1KD and59.8KDa.These recombinant proteins can be purified by glutathione Beads.Conclusion:Mhp168₃66and Mhpl68₄77were solubly expressed in E.coli XL-1with natural construction, and can be purified by glutathione beads.4. Immunological activity studies of6proteinsObjective:Detect immunogenicity and reactogenicity of6proteins.Method:The mice were immunized with the recombinant proteins respectively after purification with beads. The indirect ELISA test were established respectively with recombinant proteins used as coating antigens to detect antibody titer of recombinant protein antiserums. The recombinant proteins were detected with positive serum by Western-blot test.Result:The antiserums of recombinant proteins Mhp168218-1、Mhp168₂18-2、 Mhp168₂18-3、Mhp168₂18-4、Mhp168₃66and Mhp168₄77were detected by indirect ELISA test. The results exhibited that6proteins could generate high titer antibodies,their average value of P/N value were2.74、2.34、2.83、2.40、2.67and2.37in turn. The Western-blot test results showed that Mhp168₂18-1、Mhp168218-2、 Mhp168₃66and Mhp168₄77could react with Mhp positive serum, Mhpl68₂18-3and Mhp168218-4could not react with Mhp positive serum。Conclusion:Mhpl68₂18-1、Mhpl68₂18-2、Mhp168₃66and Mhp168₄77possess good immunogenicity and reactogenicity.