Construction Of Macrophage CDNA Library And ApxⅢ-bait Recombinant Plasmid And Development Of Multiplex PCR To Detect Porcine Respiratory Disease-related Bacterial Pathogens

The thesis describes three experiments.1. The construction of the standardized cDNA library of porcine macrophageMacrophage plays an important role in removing foreign substance, production of cytokines and antigen presentation. However, the mechanism of the interaction between macrophage and pathogen is unclear. So, construction of cDNA library of macrophage will provide a platform for exploring the mechanism of interaction by yeast two-hybridization technique.In this experiment, the primary macropahges were isolated from lungs of healthy piglets. The RNA was extracted followed by cDNA synthesis using SMART technology. After normalization, the cDNA was transformed with yeast competent cells to produce the standardized cDNA library. The library quality was evaluated by PCR amplification of house-keeping genes. The size of the gene fragments in the established library was around 1.3kb, and good diversity was showed. The preliminary titer of the transformed yeasts was > 1×10~6 CFU and 1×10~8 CFU after adjusting. Theoretically, this library could meet requirements for yeast two-hybridization.2. The construction of bait plasmid harboring Actinobacillus pleuropneumoniae apxIII geneRTX is the main virulence-related factor of actinobacillus pleuropneumoniae. apxIII was characterized by strong cytotoxcity. To cast the light on the interaction between ApxIII protein and any possible cells, in this study, the fragment, located from 1258 to 2040 nt, was cloned pGBK-T7 vector to construct bait plasmid. The interested ApxIII protein was expressed in yeast cells. No auto-activation and toxic effect to yeast cells were confirmed. The bait plasmid could be used in yeast two hybridization assay in the future.3. Development of the multiplex PCR to detect five pathogens causing respiratory diseases in swine.Recently, more and more respiratory disease outbreaks are being observed, in addition, the co-infection of pathogens makes the disease complicate, becoming a noticeable feature. The known agents which could cause porcine respiratory problems included actinobacillus pleuropneumoniae (App), haemophilus parasuis (HPS), brochititis bortedella (Bb), mycoplasma hyopneumoniae (Mhp) and Pasteurella multicoda (Pm). They could cause the occurrence of corresponding diseases, leading to greatly economic...