Serotype Identification Of Field Strains Of Actinobacillus Pleuropneumoniae And Expression Of The ApxIA Gene Of Actinobacillus Pleuropneumoniae And Purification Of ApxI Of Actinobacillus Pleuropneumoniae

Rabbits were inoculated with formaldehyde-inactivated bacteria suspensions of Actinobacillus pleuropneumoniae(APP)strains of 12 serotypes with bee glue as an adjuvant.to prepare a set of monospecific antisera against each serotypes of APR.Although there were some cross-reactions between reference strains of different serotypes,any monospecific antiserum gave the highst titers to its homologous APP reference strain bacteria suspension in agglutination test,they reached titers of 6-8 log2 respectively.By use of the set of antisera,the field strains isolated from different areas in Shandong privince in 2003 wre identified for their serotypes,they belong to serotypes 3(2 isolates), 4 (1 isolates), 5 (4 isolates) and 7 (4 isolates).We investigate the epidemic situation of present porcine infectious pleuropneumonia in Shandong province.At the same time,it is very useful to prevent and control the porcine infectious pleuropneumonia.According to the seqence of the the apxIA gene of Actinobacillus pleuropleumoniae (serovar9,HS111) in Gene Bank,three pair of primers were designed. apxIA gene was departed into three fragment that were all about 1000bp by PCR.There were overlaps that were about 100bp between three DNA fragment in order to protect the immunogenicity of the apxIA gene.These amplified DNA fragment were cloned into an expression vetor (pGEX-5X-3) to construct the expression plasmid.Sequencing analysis showed that both of the sequence and ORFof the apxIA gene was right. SDS-PAGE and Western blotting analysis showed that apxIA gene was expressed in E.coli BL21 and the expression product have immunogenicity.At the same time, we separate and purify the Actinobacillus pleuropneumoniae-RTX-toxin(APX) by the seperation and purificationtechnique ofprotein and get pure apxl and apx II. Then, we research their biological characterization primarily.This research established a solid foundation to find new method of detecting APP such as ELISA, PCR etc and produce an Actinobacillus pleuropleumoniae genetic engineering vaccine strain effective.