Establishment Of Double PCR Of Apx?A,Apx?A Gene And Preparation Of Polyclonal Antibody

Porcine contagious pleuropneumonia(PCP)is a respiratory infectious disease characterized by acute lung hemorrhage or chronic fibrous pleural adhesion caused by Actinobacillus pleuropneumoniae(Actinobacillus pleuropneumoniae,APP).Apx exotoxin is the most important pathogenic factor of APP.There are 4 kinds of pathogenic factors,including ApxI,ApxII,ApxIII and ApxIV,among which ApxI can cause typical lung diseases,and also the strongest virulence factor causing porcine contagious pleuropneumonia.The toxicity of ApxIV toxin is relatively weak,but it can be secreted by all APP,and ApxIV is induced only when the animal is in vivo.Therefore,when ApxI and ApxIV two toxins exist at the same time,it is of great clinical value for the diagnosis of porcine contagious pleuropneumonia.By extracting genomic DNA from Actinobacillus pleuropneumoniae ApxIA and ApxIVA,a double PCR method was developed to detect and amplify ApxIA and ApxIVA gene fragments,and TA plasmid was used to obtain recombinant plasmid.Then,bioinformatics analysis was used to analyze the target gene fragment characteristics and further screening,further protein expression,purification and polyclonal antibody preparation.The results are as follows:1?Polyclonal antibodies were prepared from rabbit antisera collected from rabbits immunized with ApxIA and ApxIVA amino acid sequences,and detected by ELIS A and Western Blot.The results showed that the highest potency of ApxIA and ApxIVA polyclonal antibody reached 1:200000 and 1:50000 respectively,and the titer was positive by Western Blot test,providing a good experimental condition for the subsequent study of the haemolytic toxin of Actinobacillus pleuropneumoniae.2 ? Recombinant plasmids pMD18-ApxIA and pMD18-ApxIVA were successfully obtained.Two strong gene fragments of APxIA with 1 209 BP gene fragment length and APxIVA with 972 BP target gene fragment length were screened by genetic bioinformatics analysis.Homology comparison with NCBI gene sequence showed that the similarity was more than 90%.3 ? The results showed that the highest titers of ApxIA and APxIVA polyclonal antibodies were 1:200000 and 1:50000 respectively by ELISA,and the titers were positive by Western Blot test.In conclusion,the double PCR method of ApxIA and ApxIVA gene has been successfully constructed,and its specificity and stability are strong.The polyclonal antibodies of ApxIA and APxIVA genes are obtained,and their titers can reach 1:200000and 1:50000 respectively,providing reference for the diagnosis and control of Porcine Contagious Pleuropneumonia in the future.