Research On The Cloning,Expression And Immunity Of Mo P113/Mmc LppA Gene

Mycoplasmal pneumonia of sheep and goats（MPSG）is one of serious contagious diseases,which is prevalent with the clinical symptoms of fibrinous pneumonia and pleurisy.MPSG is mainly caused by Mycoplasma mycoides subsp.Capri（Mmc）,Mycoplasma ovipneumoniae（Mo）and Mycoplasma capricolum subsp.Capricolum.The preliminary investigation data shows that Mo and Mmc are the main kinds of pathogenic in Guizhou Province and cause high death rates and serious economic losses.There are many difficulties for the prevention and control of MPSG.In this article,molecular cloning technology,biological information science technology and gene engineering technology were used to research the P113 gene of Mo Guizhou strains and LppA gene of Mmc Guizhou strains.We hope to find new vaccines or technology for prevention and control of MPSG and provide the basic research data for the disease early detection,evaluation of immune and new-type vaccines.1.Cloning and sequence analysis of Mo P113/Mmc LppA gene:The primers were designed based on the Mmc LppA and Mo SC01 gene sequence information published in the Gen Bank,and the Mo P113/Mmc LppA gene of Guizhou trains were studied by methods of PCR,molecular cloning and bioinformatics analysis.The results showed that the full-length of Mo Guizhou strain（GZ-QX1）P113 gene was3240bp which encoded a protein about 1079 amino acids and the full-length of Mmc Guizhou strain（GZ-DF1）LppA gene was 1572bp which encoded a protein about 523amino acids.Homology of P113 gene sequences between Mo Guizhou strain and Mo Y98 reference strains were 99.9%and less than 39.4%with reference strains of other kinds of sheep mycoplasma pneumonia.Homology of LppA gene sequences between Mmc Guizhou strain and PG3 reference strains were 100%and less than72.8%with reference strains of other kinds of sheep mycoplasma pneumonia.The results also showed that the Mo P113 gene and Mmc LppA protein had high sequence conservation and large differences between different species.Bioinformatics analysis results showed Mo P113/Mmc LppA protein have high antigenicity with many dominant epitopes structure,especially the sequence of C-Terminal of the Mo P113protein and N-Terminal of the Mmc LppA protein that can be used as target protein segments in the study of immunology.2.Prokaryotic expression of Mo P113/Mmc LppA protein and development of indirect ELISA method:The Mo P113 gene/Mmc LppA gene sequences were amplified by PCR and cloned into p ColdⅠfor constructing recombinant expression vector p ColdⅠ-P113 and p ColdⅠ-LppA.Then the target proteins were expressed and the indirect ELISA methods were developed.Results show that recombinant prokaryotic expression vectors for Mo P113 gene/Mmc LppA gene were constructed.The results of SDS-PAGE and Western-Blotting indicated the expressed protein of Mo P113 gene fragment was 33KDa and Mmc LppA gene fragment was 23KDa.The best condition for protein expression of P113 gene were 28℃,0.8mmol/L IPTG,3 h and LppA gene were 30℃,0.8mmol/L IPTG,3 h.The western blotting assay and AGP showed the purified protein has good reactionogenicity.The indirect ELISA was developed with the purified P113 protein and LppA protein.The optimum reaction conditions of indirect ELISA were as follows:Mo P113 fusion protein concentration was 1.5μg/100μL,serum dilution was 1:100,blocking buffer is 1%gelatin,blocking time is 1h,the critical OD₆₃₀value of negative and positive serum is 2.325,Mmc LppA fusion protein concentration is 2.0μg/100μL,serum dilution is 1:50,blocking buffer is 3%BSA,blocking time of 1h,the critical OD₆₃₀value of negative and positive serum is 2.738.Performance evaluation of the ELISA methods showed that they all had better specificity and repeatability and can be used for detection specificity serum antibody of Mo P113 protein/Mmc LppA protein.3.Eukaryotic expression and immunity of Mo P113/Mmc LppA protein:The Mo P113/Mmc LppA genes were amplified by PCR and cloned into p VAX1 to construct recombinant expression vector p VAX1-P113,p VAX1-LppA and p VAX1-P113-LppA,then transfected to MDBK cells.The vitro expression efficiency had been evaluated by PCR and indirect immunofluorescence.And the humoral immune responses and cellular immune responses of immunized BABL/C mice induced by recombinant plasmid were detected with the methods of indirect ELISA,MTT lymphocyte proliferation test,histopathology techniques,flow cytometry and immunoprotection test.The results showed that recombinant eukaryotic expression vectors which contained the Mo P113 gene/Mmc LppA gene were constructed.The recombinant plasmid could transcribe m RNA and express the target protein in transfected MDBK cells.Animal immune experiments showed that recombinant plasmid chould induce specific serum antibody in mice.The value of a trend of increase by a big margin compared with the control group,tended to be stable at the end of the experiment,indicating that recombinant plasmid could induce strong humoral immune response.And the highest average of OD₆₃₀were the group PVAX1-P113（100μg）and p VAX1-LppA（100μg）.It indicated the recombinant plasmid could induce BABL/C mice to produce strong humoral immune response.The detection results showed the serum cytokines level of of IL-2,IL-4 and IFN-γwere higher in recombinant plasmid groups than control group.The results indicated that the recombinant plasmid could induce BABL/C mice to produce good cellular immune response.The results of Flow cytometry for CD4⁺T cells and CD8⁺T cells percentage showed that recombinant plasmid induced Th1/Th2 type immune response,and the Th1 was the chief type.Animal immune experiment showed that the p VAX1-P113,p VAX1-LppA and p VAX1-P113-LppA plasmids were safe for mice.They could provide effective immune protection for Mo and Mmc infection and pneumonia symptoms of the recombinant plasmid groups were reduced.In conclusion,Mo P113/Mmc LppA protein had high sequence conservation and good antigenicity.The sequences of C-Terminal of the Mo P113 protein and N-Terminal of the Mmc LppA protein had good dominant epitopes structure.The target protein were obtained from recombinant plasmid p ColdⅠ-P113 and p ColdⅠ-LppA which had good antigenicity.Indirect ELISA based on the purified protein had good specificity and repeatability.The eukayotic expression plasmid could induce good humoral immune response and cellular immune response for immunized mice and provide effective immune protective for.The results for this study could The results of this article may provide new ideas and technologies for Mo and Mmc preventionl and theoretical basis for the feasibility study of new vaccines.