| Porcine Contagious Pleuropneumonia is a respiratory disease of pigs caused by Actinobacillus pleuropneumoniae, the disease can cause severe damage of swine production. Outer membrane protein is one of the important virulence factor of Actinobacillus pleuropneumoniae. This study was successfully constructed a yeast two-hybrid cDNA library of swine lung tissue which is used to screening the positive clones with APP OmpA, this lays a solid foundation for further study pathogenesis of lung infection OmpA. The main work is as follows:1. Construction and identification of the cDNA library from swine lung tissueIn this study, we extracted the total RNA of lung tissue from a 28-day-old piglets by the method of Trizol, then separated and purificated mRNA from total RNA, transcriptase the first strand cDNA by Invitrogen reverse synthesize, then amplified double stranded cDNA by long-distance Polymerase Chain Reaction.I purified out of smaller fragment from double-stranded DNA by BD CHROMA SPIN Columns. Link the double-stranded DNA of swine lung tissue to the yeast expression vector pGADT7-Rec which has the DNA activating domain, transformed these vectors into competent E. coli DH10B by electriction, then harvest the clones as the yeast two-hybrid cDNA library, the fainal total capacity of the cDNA library is 1.1×10 cfu, the average length of insert is about 1.5Kb, the positive rate of Library is 100% after identification which prived that the construction of cDNA library was successful, this lays a foundation about the research of pathogens pathogenic mechanism with pig lung.2. Construction and identification of the yeast bait plasmid with outer membrane protein A of Actinobacillus pleuropneumoniaeWith the full length genomic sequence of Actinobacillus pleuropneumoniae wild strain serotype 5 as template, and specific amplificated the sequence of outer membrane protein A which the signal peptide is removed. Link the fragment with the yeast expression vector pGBKT7-Rec which has the DNA binding domain, the results of identification showed that we received a size about 1,100 bp target band by PCR and double digestion. Transferred the bait vector into yeast AH 109 competent cells by the method of lithium acetate, then harvested transformants named bait pGBKT7-OmpA on SD/-Trp/Kan agar medium. The results about the transcriptional activity and cytotoxicity of the bait shows that:the bait did not grown clones on the media SD/-Trp/-His, SD/-Trp/-Ade, SD/-Trp/-His/-Ade /-Leu, the bait grow only white colonies on SD/-Trp and SD/-Trp/X-a-gal, all these indicated that the bait had no self transcriptional activity;3. Yeast two-hybrid Screen the positive clones with Actinobacillus pleuropneumoniae outer membrane protein A from the cDNA library of swine lung tissueAs the yeast two-hybrid system with a operating platform, the use of a hybrid gene expressed by activating a reporter gene to detect protein-protein interactions, when pGADT7-cDNA which carrying DNA activation domain and pGBKT7-OmpA which carrying the DNA binding domain interact in the yeast strain AH 109, DNA-BD and AD combined with close and activate the reporter gene ADE2, HIS 3, lacZ, MEL1 transcript; this experiment were extracted from the swine lung tissue cDNA library plasmids and bait plasmid pGBKT7-OmpA, conversion the two plasmids co-transformed into yeast AH 109 competent cells by lithium acetate, the transform ants turn coated on QDO medium to obtain the positive clones, then spread the positive clones on TDO medium, obtained repeatedly applied on TDO/X-a-gal/AbA medium, and finally selected 31 yeast positive clones. Expanding the culture-positive clones and extracted plasmid from it. After identification by PCR and sequencing, all of the positive clones are on pig chromosome 16 and some unexplained highly homologous sequences comparative analysis. Then translated the sequence to 3 predicted proteins and did bioinformatics analysis of them. |
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