Cloning, Prokaryotic Expression And Immunogenicity Analysis Of Outer Membrane Lipoprotein E Gene Of P.Multocida From Procine

Pasteurellosis which caused by Pasteurella multocida is an acute and thermal infectious disease. Acute type often in sepsis and hemorrhagic inflammation as the main characteristics and chronic type often presents with subcutaneous connective tissue, the joints and the internal organs of the fester sex lesions. The sera types of Pasteurella multocida are so many that the cross-protective is weak or not between different bacterial strains. According to reports of Jin-Ru Wu in Taiwan that plpE gene of Pasteurella multocida from chicken can code an outer membrane lipoprotein antigen having good cross-protective function. In this study,the pasteurella multocida of sera type A from swine were used to clone amplified a section of plpE which coding a mature protein,then analysising the sequence of the obtained Purpose gene, construction of the Prokaryotic expression vectors,Prokaryotic expression and cross-protective study of plpE protein.Ⅰ.Isolation and identification of pasteurella multocida from swineInoculating the well grout composed of lungs, liver and lymph nodes organization from swine suspected of being affected by pasteurella multocida into fluid nutrient medium and being cultured for overnight, coating them onto solid medium, then picking the single bacteria similar with the pasteurella multocida onto solid medium again for subculturing and purification for three times. At last observed by electron microscope, tested by biochemistry, HDRA, half lethal dose and species and serotype identification of pasteurella multocida.Ⅱ.Cloning and sequence analysis of plpE geneAccording to the reference the plpE gene nucleotide sequence of pasteurella multocida recorded by GenBank (GQ202241.1), We designed and synthesized a Pair of specific Primers by PrimerPremier5.0(adding two restriction sites of BamH I and Sal I),and extracted the total DNA of sera type A pasteurella multocida separated in Sichuan which can be cultured on TSA solid medium as a template, amplified the purpose fragments by PCR, recovered the Product of PCR, then successfully transfer to E.coli DH5α.The gene fragment was cloned in the pMD19-T Simple Vector by TA cloning method to construct recombinant clones plasmid pMD-plpE,The Positive recombinant Plasmid was send to the company for identifying sequence after the correct digestion by PCR and restriction enzyme. Sequencing revealed that the length of the cloned gene fragment in plpE is951bp,and encoding317amino acids. Carrying out bio-informatics analysis using these sequencing results and amino acids sequence with the different isolates recorded by GenBank..Sequence analysis shows that the nucleotide homology is96.4%～99.8%by the obtained purpose gene compared with All Pasteurella multocida separation plants. As proved that this genetic fragment is right and highly conservative in different serotypes.Prokaryotic expression of plpE geneⅢ. Prokaryotic expression of plpE gene and plpE protein purificationAccording to the obtained recombinant Plasmid pMD-plpE and the prokaryotic expression vector PET-32a(+), then both of them which digested by the two enzymes will be connected for constructing the prokaryotic expression plasmid PET-32a(+)-plpE. The positive plasmid should be chosen and transformed into BL21(DE3),induced to express by0.5mmol/L IPTG and optimized expression conditions..The expression Products is verified by sodium dodecyl sulfate Polyacrylamide gel electrophoresis aroylamino(SDS-PAGE) and immunoblotting experiments (Western blot))that the correct size of the product is the immuno-reactive of the Protein, while detecting it’s soluble and pET-plpE recombinant protein. Results show that the prokaryotic expression plasmid PET-32a(+)-plpE existed in form of the inclusion body and is about the size of36.3kD, the Percent is18.5%in total bacterial Protein.The expressed products is high test at37℃,2-6h.Ⅳ. The cross-protective immune study in mice of pET-plpE recombinant proteinThree experiments were conducted in BALB/c mice. In experiments1and2(50mice per group),groups of mice were immunized subcutaneously with20μg purified pET-plpE recombinant protein either alone or together with a bacterin composed of 1.0×107CFU or2.0×108CFU of pasteurella multocida of sera type A from swine. Two weeks after second immunization, mice were challenged with subcutaneous injection of10-20LD50of pasteurella multocida of sera type A. In experiment3,all mice were immunized with10μg purified pET-plpE recombinant protein, then two weeks after second immunization, challenged with subcutaneous injection of10-20LD50pasteurella multocida of serotype B or D. all mice challenged were observed for10days and their survival rates were recorded. Results demonstrated that the cross-protective immunity by immunized with20μg purified pET-plpE recombinant protein alone is good and better than bacterin and when mice immunized with them together,the pET-plpE recombinant protein can significantly enhance the protective efficacy of thebacterin...