Cloning And Expression Of Parts Gene Of Actinobadllus Pleuropneumoniae And Establishment Of An Indirect ApxⅣA-ELISA

Actinbacillus pleuropeumoniae (APP) is the causative agent of porcinepleuropeumoniae, a highly contagious disease which causes respiratory illness in pigs.Plenty of experiments showed that Apx toxin is an important virulence factor as well as amajor protective antigen of APP, all15serotypes of APP can secrete it, so it is signalityto study on it.This research is with the purpose of establishing PCR method of APP thenexpressing parts of Apx ⅣA gene, and also establishing the indirect method ofApx ⅣA-ELISA.We designed a pair of primers according to Genbank (Accession number:AX002405), the size of fragment is1284bp, and we insert EcoRⅠ to the forward primeras well as Sal Ⅰ to the reverse primer.We confirmed the concentration and Tm value ofthe primers. In our experiment, the best concentration of primers is at5mmol/L and thebest Tm value is at59℃. Also, Escherichia coli, staphylococcus aureus,Haemophiluspleuropneumoniae，riemerella and pasteurella can not be amplified by our PCR method.The least concentration of bacterial that can be detected is about3000CFU. We alsoanalyzing the alignment compared to other genes in NCBI, the homology is about94.9%～97.2%. All these experiments showed that the primer is sensitive and specific.By purifying the product of PCR, we insert the fragment into plasmid pMD18-T,then transform it into DH5α，by extracting the plasmid and enzyme digestion, we insertthe fragment into pET30a and transform it into DE3, Under the induction of IPTG, theprotein is successfully expressed in DE3through the method of SDS-PAGE, and theprotein is about48KD.We use the protein for ELISA as antigen to coating the plate and aimed atestablishing the method of Apx ⅣA-ELISA. We confirmed the best coating concentrationof protein is at5ug/ml，the best dilution ratio of serum is1:100, and the best IgG-HRP is1:2500.