Cloning Of APXIIA Gene Of Actinobacillus Pleuropneumoniae And Screening For Its Region Involved In Immunity

Actinobacillus Pleuropneumoniae(APP) causes a highly contagious disease Porc- ine Pleuropneumoniae.The disease is widely endemic all over the world and often res- ults in considerable economic losses. The fourteen kind serotype of APP are produced four deffrent kinds of APX named as APXI,APXII,APXIII and APXIV. APXII is of particular interest because it exists in all kinds of serotype except 10 and its operon just composed of active gene APXIIC and structural gene APXIIA. APXIIA has no to- xicity,but there are haematolysis activity and cytotoxicity in existence if it is construct- ed by the way of being added acyl group from APXIIC to. APXIIA is not only one im- portant causative agent of APP but also one of major antigens.The toxin is major viru- lence factors and so most of the study on APP focuses on it.The APXIIA gene which was amplified from APP by PCR , was cloned into the plasmid vector pMD18-T to construct the recombinant plasmid.The constructed reco- mbinant plasmid was analyzed by PCR,restriction enzyme digestion,sequence analysis and analied its homology by biology software. Results showed the gene obtained was 2868 bp,and coded 959 amino acids,and was identified target gene.meanwhile APXIIA gene respectively shares high homology with that from GenBank. Among the total,it shared compele homology.others'homology was about 80%,and there were different regions in 710aa to 956aa in existence. these difference possibelly dued to difference in biological activity.obtained the full-length target gene and settled the base to furture study and research of APP APXIIA gene .This research designed and synthesized 57 bp oligonucleotide sequence and 4 pri- mers and by PCR , ribosome display upstream primers was obtained ,then linked to ri- bosome display downstream primer synthesized and enzyme-digested fragments of APXIIA gene ,a ribosome-display-reconstruction gene was constructed.Then a coupled translation reaction in vitro was proceded and ribosome display DNA was used to sel- ect,then obtained one region involved in immunity of APXIIA and its length was 195 bp. Afrer sequcing and prediction of secondary structure,hydrophilicity and antigeni- city by the DNASTAR software,the results showed that the protein predicted was rich inβ-fold and random coil,it was likely a hydrophilic protein,and had favourable imm- unogeniccity.This is usefuL for the further work.