Construction And Initial Screening Of Signature-Tagged Mutagenesis Libraries Of Actinobacillus Pleuropneumoniae Serotype 1

Porcine contagious pleuropneumonia (PCP) is a highly contagious disease caused by Actinobacillus pleuropneumoniae (APP), characterized by hemorrhagic pleuropneumonia and severe fibrinous necrotizing in swines. This disease is widely spead in the world and caused great economic losses in swines Industry. In this study the libraries of APP1 mutagenesis were Constructed by using Signature-tagged mutagenesis(STM), hoping to obtain the genes which affect APP1 Survival in the animals, which provides essential basis to research the function gene of APP1.In this study the technique of transposon mutagenesis was used to identify genes in pathogens that are required for growth in swine of Actinobacillus pleuropneumoniae serotype 1(APP1)by using a suicide plasmid pUT which included Mini-Tn5 containing signature tags. 12 suicide plasmids pUT-MiniTn5-Km2 which include specific signature tags were constructed,and transformed into E. coliβ2155 respectively. Filter hybridization was used to construct and optimize the conjugation system. Conjugation mutants were screened and get by using culture plates which have resistance and heterotrophia. Those conjugation mutants were identified by PCR using universal primer of resistance and 12 specific tags primer and ApxIV toxins identification primer respectively, and then sequencing. The results showed E. coliβ2155 and APP1 can be transposed and conjugated, and 12 libraries of transposon mutagenesis were got which had 421mutagenesis in all. These results provided essential basis to research the pathogenic mechanism,discover virulence and drug resistance gene ,and develop attenuated live vaccine of APP1.Six libraries of APP1 mutagenesis were choosen randomly from twelve libraries of APP1 mutagenesis, one mutant strain was selected randomly from each of the six libraries, and the mutant strains which selected from the six libraries formed a new library which named"Input pool"(the six mutant strains were 1-32,7-18,8-11,10-23,11-8,12-22),six mice(the experimental group) were infected with the same dose of the mixture of the six kinds of bacterial liquid through the nasal cavity, The infective dose was 1.2~1.5×108cfu/mice; 3 mice in the negative control group, Equivalent volume of PBS was directly injected ,3 mice in the positive control group, equivalent volume of APP1 was infected through the nasal cavity.After infected 3,5,7 days, two mice from the experimental group, one mouse from the positive and one mouse from negative control group were killed and the bacteria from the lungs were collected, and genome was extracted, then identified the properties of APP1 by PCR using specific tags primer ApxIV, and identified the total genome by PCR using the universal primer and six specific primer F, then unknown sequences which in the upstream of the known insert fragment were amplified by genome walking PCR. The results show: Bacteria could be recovered from the lungs of the mice which infected after 7 days, two Negative strains 1-32 and 11-8 were obtained through PCR with total genome by using the universal primer and 6 specific primer F, and the target fragment was obtained by genome walking PCR.