Sequence And Analysis Of The Complete Sequence Of Plasmid Isolated From The Actinobacillus Pleuropneumoniae

Actonobacillus pleuropneumoniae (APP in brief) was a gram-negative coccobacillibacterium. It was the etiological agent of porcine contagious pleuropneumonia and could induce acute and chronic infection in pigs of all ages. The disease was characterized by necrosis, fibrinous and hemorrhagic. It existed in many countries around the word and caused great economic losses in industrialized pigs raising. A lot of attempts to control and prevent the disease had been ineffective. So it was essential to study A. pleuropnemoniae at the molecular level. The aim of this study was to investigate and analyze the complete sequence of plasmids isolated from the Actonobacillus pleuropneumoniae.We isolated plasmids of APP2 and APP9 from APP2 and APP9, while no plasmid was detected in APP7. The experiment of digestion with single restriction endonuclease such as HaeIII , TaqI, PstI, HindIII, EcoRI and BamHI, respectively, showed that the plasmids could not be digested completely. The restriction endonuclease maps of plasmids had not been constructed by a series of double enzyme digestions. The plasmids were digested with single restriction endonuclease EcoRI, and ligated to the cloning vector pBS digested with the same enzyme. The recombinatant plasmid was isolated and sequenced by primer M13, bidirectionally. Squencing of the complete sequence of plasmid pAPP2 used the primer walking method. The plasmid pAPP2 contain two compatible plasmids pAPP2-7 and pAPP2-8.The plasmid pAPP2-7 was 4722 base pairs in length with a base composition of 31.85% G+C, while plasmid pAPP2-8 was 9659 base pairs in length with a base composition of 32.0%G+C. Three primary open reading frames(ORF) were identified from plasmid pAPP2-7 with coding region for polypeptides of 128, 284 and 315 amino acids respectively. These three putative gene products showed significant homology to MobA ,MobC and Rep functionally characterized proteins in the GeneBank database. And, three primary ORF that coded Mob-Pre,Rep₃ and Transposase₂7 were identified from plasmid pAPP2-8. There were a lot of direct and invert repeats in each plasmid. These six ORF had different orientation on codons of amino acids.Vector NTI and BlastN analysis showed the minimal replication was located in the fragment containing Rep and its upstream sequence. A pair of primers with restriction endonuclease site of EcoRI was designed, and a 2.7kb PCR product was obtained from the plasmid pAPP2-7. After purified, it was subcloned into pMD-18 T vector and transformed into E.coli TG1. The recombinatant plasmid pAPP2-MD was isolated, and then digested by EcoRI.Kan^r gene that has ori of E.coli were cloned through PCR, and then digested by EcoRI. The product was connected with EcoRI digested pAPP2-MD and transformed into E.coli TG1. One recombiant plasmid was isolated by Kan resistance selection and PCR detection, namely pAPK2. The plasmid was purified and transformed into A. pleuropneumonia 7 strain by electroporation. The same plasmid was isolated by Kan resistance selection and PCR detection. It was concluded that the reconstruction of plasmid pAPP2-7 was successful. This result madkes it possible to continue research A. pleuropneumonia in molecular lever.