Protection Efficacy In Mice Induced By DNA Caccination Of Genomic Expression Library Of Actinobacillus Pleuropneumoniae Serotype 1

Porcine contagious pleuropneumonia (PCP) is a highly contagious disease which is caused by Actinobacillus pleuropneumoniae (APP) and characterized by severe fibrinous necrotizing and/or hemorrhagic pleuropneumonia in pigs. PCP can cause acute or chronic infection in any age of pigs, morbidity and mortality of PCP is over 20%, mortality of the acutest type of infection ranges from 80% to 100%, chronic infection is inapparent, which compromised pig growth. Since this disease was discovered in 1957, it spread in the worldwide and caused enormous economic losses in industrialized pig production. Because APP has resistance against many antibiotics, vaccination has been a main measure to prevent PCP. At present, an inactivated whole cell vaccine is mostly applied for PCP prevention and treatment in China. But APP has 15 serotypes, so the protection efficacy of inactivated vaccines against different serotypes of APPs is unfavorable and the development of novel vaccine has become a research hotspot. It had been proved that many attenuated vaccines constructed by molecular biology methods and new ghost vaccine could provide effective protection. But along with the declined virulence of attenuated vaccines, the immunogenicity was also decreased, and moreover, the reversion of virulence might be presented, and ghost vaccine might be inactivated incompletely. Subunit vaccines contain toxic factors had no those disadvantage and could provide effective protection, but bacteriostasis of subunit vaccines is limited because of no whole bacteria contained, inAPParent infected pigs could be carrier without any symptom.The genomic DNA of Actinobacillus pleuropneumoniae(APP) serotype 1 was digested with Sau3A I to generate genomic expression library.The DNA fragments of 500 bp to 3,000 bp were ligated to the eukaryotic expression vector pCDNA3.1(+)and transformed into competent Escoherichia coli TG1 by electroporation.The resultant genomic expression library(about 106 clones)was subdivided into ten pools of clones(about 105 clones/pool)and DNA extracted from each pool was used to inoculate the mice at 100μg per dose.Mice were booster immunized two times and then challenged with virulent APP1.Initially two clone pool was screened by protection that was induced by DNA vaccination.Then these two clone pool were subdivided into 4 clone pool and DNA extracted from each pool was used to inovulate the mice at 100μg per dose.Mice were booster immunized three times and then challenged with birulent APP1.Bcteria in lungs was isolated and pulmonary tissue damage and significant protection were examed.And The dynamic changes of The splenic lymphocytes proliferation in mice induced with protein containing supernatants were prepared by ultrasound lysis of host cells of APP1.Significant protection was induced by DNA vaccination from two clone pools.The result demonstrated that the genomic expression library of APP1 generated in this work could be used to select the protective antigen genes against APP1.