Establishment Of LAMP Detection Method For Actinobacillus Pleuropneumoniae And Partial Function Of Apx Ⅳ Toxin

Porcine Contagious Pleuropneumonia is caused by Actinobacillus pleuropneumoniae and leads to pleurisy and pneumonia in pigs is characterized by an acute or chronic disease. In recent years, this disease is widely popular in the world of the industrialized countries in the pig, to bring great loss to the pig industry, is listed as one of the five major infectious disease to harm the pig industry worldwide. For the prevention of this disease by cell inactivated vaccine to, but because the serotypes of APP are numerous, and each serotype cross protection between power co., coupled with the inactivated vaccine APP is missing in the growth process secretion of toxins, these toxins have been proved that the antigenic components of APP important protective antigen and inactivated process exists more or less damage and loss, inactivated vaccine in the prevention of this disease is not very prominent effect. Therefore, to study the disease vaccine has become many researchers including foreign, attention, has become a hotspot in the field of infectious diseases of pigs. This study is divided into two parts, as follows:The first part, in order to overcome the existing detection technology in APP operation is complex, expensive, time-consuming issues, the study of sequence alignment software by DNAstar dsbe-like gene on serum 1-12 APP, find out the homologous sequence, design the online primer design software,4 LAMP primers, and the LAMP method of the various reaction conditions were optimized, the reaction system of 25μL. In the specific inspection, testing, several kinds of common disease on pig by LAMP method shows that, only with the APP DNA as the template will appear positive reaction, the LAMP method has a good specificity; In the sensitivity test, by gradient dilution of APP nucleic acid, found that the minimum detection limit of the LAMP method was 5 copies/reaction, is 20 times the method has been reported for PCR. Finally, by bacteria isolation, PCR and the LAMP external surface health pig lung and suspected APP infection was detected in the lungs of pigs of LAMP method in clinical samples detection performance, the results showed, separation, bacteria in apparently healthy pigs in the group of PCR method, LAMP method detection rates were 0%,2%,4%, while the positive rate of suspected infection group of three methods were 6.98%,58.14%,69.77%, and LAMP and isolation of bacteria, PCR positive coincidence rate was 100%, the clinical detection of the LAMP method is more suitable for APP. To sum up, in addition, cost anti have reaction of the LAMP is low, does not need the professional skills and many other advantages, can effectively meet the urgent need of porcine infectious pleuropneumonia was placed detectionfield, to prevent our Porcine Contagious Pleuropneumonia disease occurrenceand control of the epidemic should have important significance.The second part, through the Apx IV gene sequence analysis of APP 5 type,analysis of Apx IV gene may be present in the antibody epitope region and a pair of primers were designed to amplify the Apx IV gene sequence using the software such as DNAstar (463-1261bp) of this fragment, it is connected to the pMD18-T plasmid was constructed successfully cloning vector, and then through the enzyme will build the objective fragments connected to pET 21 expression vector and transforming the expression plasmid BL 21. Optimization of expression conditions through you, successfully expressed the target protein, after crushing bacterium and SDS-PAGE detection, protein expression in the form of inclusion body. The recombinant protein was purified, refolded step, the Western-blot antigenicity analysis showed that the recombinant protein was obtained with high purity and showed good immunogenicity. Then by salting out method to extract the Apx Ⅰ Apx Ⅱ APP5, two kinds of hemolytic toxin. Finally, the inactivated with different doses of recombinant expression of Apx Ⅳ and inactivation of Apx Ⅰ, Apx Ⅱ, inactivated bacteria using white oil adjuvant vaccine preparation for respectively, take back multi-point injection way immune mice, again with the virulent strain of our common serotype 5 to 10 times in LD50 dose of poison attack, results show that, with the increase of each vaccine of recombinant Apx Ⅳ content, the immune protective rate was increased, showed that the recombinant Apx Ⅳ can provide immune challenged mice immune protective effect of some of the same serotype virus bacteria. In summary, the recombinant Apx IV as an additional component added can enhance immunity, subunit vaccine but, as for how to improve vaccine immune protection effect, its mechanism needs further research.