Construction Of Recombinant Adenovirus Vector Containing Mycoplasma Hyopneumoniae P97 Gene And The Immunogenicity

Mycoplasma hyopneumoniae?Mhp?was the Infectious agent of Mycoplasmal pneumonia of swine?MPS?,which has already widely existed in our hoggerys and seriously impeded the development of our pig industry.Mhp has a special cellular structure and has no specific medicine,conventional antibiotic may has a limited therapeutic effect to Mhp but the resistance was easy to emerge.So the moral certainty immunization was the only way to prevent MPS attack,control Mhp infect and to reduce the economic losses caused by Mhp.Since the rigorous cultivation conditions and the low growth titer of Mhp,the enough thallus was hard been collected and this leaded to a high vaccine production cost and a weak immunoprotection.So the development of a much stronger immunity and a much cheaper vaccine to Mhp was urgently needed.The present study was designed to construct a recombinant adenovirus vector Ad-P97 which contained Mhp P97 gene and could express Mhp adhesion factor protein P97 in host cell by recombinant adenovirus vector construction technology.The lined recombinant shuttle vector pacAd5 Shuttle-P97 and the adenovirus backbone vector were transfected in to the pakaging cell AD293 by lipofectamine reagent for homologous recombination.The constructed recombinant adenovirus vector Ad-P97 was used to immune performance evaluate.The results of the study were presented as follows:?1?Mhp was cultivated by KM2 culture medium and the thallus was collected for genome DNA extraction.Gene of P97 was amplified by segmented PCR the extracted genome DNA as template.The two amplified DNA segments were connected by digestion and connection operation,and the P97 gene with Xho?and Ban?Restriction Enzyme Cutting Site was amplified by PCR With the connected DNA segment as template.The result shown that the gene P97 was a DNA segment was which about 3.4Kb on the electrophored sepharose gel.?2?Gene P97 and shuttle vector was digested by Xho?and Ban?respectively and the depurated enzyme-digested products were connected by T4 ligase.The connected recombinant shuttle vector transformed into E.coli DH5?for amplification.Extracted the recombinant shuttle vector pacAd5 Shuttle-P97 and authenticated by PCR and digestion,then sent pacAd5 Shuttle-P97 to gene company for DNA sequencing.Sequencing result shown that the construction of recombinant shuttle vector pacAd5 Shuttle-P97 was successful,but three special genetic codon“TGA”which encoded tryptophan in Mhp must be site-directed mutagenesis to general genetic codon“TGG”.?3?Site-directed mutagenesis was operated by PCR.The mutagenesis primers were designed to cover each“TGA”codon and replaced nucleotide“A”to“G”.Four mutagenesis DNA segments were connected by overlapping PCR.The mutagenesis P97 gene was been cloned to shuttle vector again.Sequencing the new recombinant shuttle vector to make sure the validity of mutagenesis and clone.?4?Lined recombinant shuttle vector pacAd5 Shuttle-P97 and the adenovirus backbone vector by restriction endonuclease Pac?and transfected them into the pakaging cell AD293 by lipofectamine reagent for homologous recombination.The recombinant adenovirus vector Ad-P97 was harvested and identified by RT-PCR,Western-blot and immunofluorescence.?5?The identified Ad-P97 was amplificated by infecting AD293 cell and the virus titer was examed as high as 3.16×10¹¹TCID50/mL.?6?The results of mouse immunization shown that the recombinant adenovirus vector Ad-P97 which was immunized by intramuscular injection and intranasal could both induct a significantly higher specific antibody titer to the Ad-GFP control group?P<0.01?.The piglet immunization experiment affirmed that Ad-P97 could activate the immunoreaction and produce a specific antibody against Mhp.In conclusion,the constructed recombinant adenovirus vector Ad-P97 was a high titer and well performence neotype vaccine.