Cloning And Expressing Of Apx Of Actinobacillus Pleuropneumoniae And Study Of Multiplex Real-Time PCR

Porcine contagious pleuropneumonia is a serious contacted infectious disease of porcine caused by Actinobacillus pleuropneumoniae which owns multitude serotype.Based on the deficiency of cross-protection within the serovars of Actinobacillus pleuropneumoniae,exploit an efficiency novel vaccine to reduce the enormous economic loss from this disease was significant.Previous studies revealed that Apx-toxins including ApxⅠ,ApxⅡand ApxⅢ, the major protective proteins of APP,could be able to elevate protection of inactivated vaccine.The recombinant proteins of Apx-toxins were found to have the same efficiencies with nature proteins.ApxⅣwas species specific protein of APP which was secreted only by animals infected naturally.It was not cross reacted with other species of Actinobacillus or Gram-Negative bacillus.The structural protein of ApxⅣtoxin encoded by C-terminal fragment gene was considered to be a nice diagnostic antigen by recent researches.Since the yields of nature Apx-toxins was limited and the extraction procedure was also complex that new ways were seek to obtain recombinant proteins by bio-techniques.Intercepted main antigenic determinant regions fromαρχⅠA andαρχⅡA, amplified them from serovar 1 reference 259 and serovar 7 reference 265 to construct prokaryotic expression vectors named pGEX-4T-1/αρχⅠA and pET32a/αρχⅡA,and eukaryotic expression vector named pPICZαA/αρχⅠA. LigatedαρχⅠA andαρχⅡA gene by using T4 ligase,and constructed the prokaryotic expression vectors named pGEX-4T-1/αρχⅠA-αρχⅡA of fusion gene. The C-fragment ofαρχⅣA from serovar 1 reference 259 was amplified to construct prokaryotic expression vectors named pGEX-4T-1/αρχⅣA.The above recombinant prokaryotic expression vectors were induced by IPTG in E.coli.SDS-PAGE analysis results show that recombinant ApxⅠA, ApxⅡA,ApxⅠA-ApxⅡA and ApxⅣA were expressed in the form of inclusion body,and the recombinant proteins with 32%,23%,19%,40%,respectively, accumulated total amount of bacterial protein and the purity quotients of the fusion proteins were between 92%～98%.Western-blot results revealed that the recombinant proteins have favorable reactionogenicity and were suitable for the clinical diagnosis and a novel vaccine of Actinobacillus pleuropneumoniae infection.The above recombinant eukaryotic expression vectors of ApxⅠA was expressed in Pichia pastoris by methanol-induced.SDS-PAGE analysis revealed it only was tested after concentrating the supernatant of culture,and the mRNA coding the ApxⅠA was tested by RT-PCR in yeast induced to confirm the proceeding of transcription.After analysis,the low expression ofαρχⅠA gene in Pichia pastoris has several reasons.First,the codon bias index of the target gene for Pichia pastoris was very lowly.Second,the low abundance of tRNA for target gene was leaded to bottle neck effect in translation procedure.Third,the high AT content of target gene induced to premature termination of the transcription.Co-infection of Porcine contagious pleuropneumonia and porcine circovirus typeⅡoccurred commonly in clinic,and they both attacked respiratory system mainly.A duplex real-time PCR,based on Taqman hybridization probes technology,were developed and applied to detect anctinobacillus pleuropneumoniae and porcine circovirus type 2 in a reaction system.Two pairs of specific primers and two probes labeled with different report groups were designed for detection a 132bp fragment of APPαρχⅣA gene and a 169bp fragment of PCV2 ORF2 gene.The two fragments were ligated with pMD-18T and transformed to E.coli DH5αfor positive plasmid.Two standard curves were developed,and the sensibility for detection of APP DNA and PCV2 DNA were 1×10~1 copies and 1×10~0 copies.The assay was specific for APP and PCV2,and has no cross-reaction to other organisms.Simultaneously, two single real-time PCR assay for APP and for PCV2 were developed.The diagnostic methods developed were highly specific and sensitive,could be used for rapid diagnosis and screening.