Expression Of SodA Gene Of Pasteurella Multocida From Goat And Preliminary Study On Its Infection Mechanism And Diagnosis Method

When goats infected with Pasteurella multocida?Pm?,it will trigger the immune response.In order to resist the respiratory storm caused by phagocytosis,the pathogen usually catalyzes the reaction through the superoxide dismutase?SOD?to eliminate free radicals,so the bacterias can reduce self injury and survive,even reproduce in host.Because Pm contains many virulence factors,it has multiple pathogenicity.Tox A gene is one of them.This gene can encode pasteurella multocida toxin?PMT?.The N-terminal of PMT has the function of receptor binding and transporting.In this experiment,type A and type D Pm?named HN02 and HN01,respectively?,which were isolated from goats,were used as materials.The following were the main experiments of the study.In experiment 1,the genome of HN01 strain was used as template to amplify the sod A gene.Constructed the recombinant plasmid named p ET-28a-sod A,and transferred it into E.coli for cloning and expression.Then,extracted the plasmid for sequencing.The induction process of the expression competent cells,which contained the plasmid,was optimized.And the induced SOD protein was identified.Bioinformatics and phylogenetic analysis were performed on the sequencing results.The results showed that there was a band at 645bp,and the protein about 28 ku was identified by SDS-PAGE and Western Blot.The protein was stable and acidic,and it included two main secondary structures.And based on the amplified gene,the three strains of HN01,HN07 and Pm70 were closely related.In experiment 2,goat bronchial epithelial cells were infected with the infection solution prepared by HN02 strain,and different infection conditions were set to establish the infected cell model.The cell lysates in different treatment groups were sent to the company for m RNA and mi RNA sequencing.The results were as follows:?1?The growth curve of HN02 strain was drawn.The linear equation between colony forming units and OD_600nmwas established.And the infection model was established with MOI=100.?2?The results of bioinformatics analysis showed that the SDEGs of m RNA and mi RNA had 16kinds of expression trends,respectively,among which the m RNA were significantly enriched to 4 kinds and the mi RNA were significantly enriched to 3 kinds.In the earlier infection stage,the SDEGs of m RNA enriched in transcriptional regulation and endocytosis.And in the later infection stage,most of them enriched in metal ion binding,MAPK signaling pathway and focal adhesion.During the infection,the target genes of mi RNA were more gathering in MAPK signaling pathway and endocytosis pathways.SOD2 and SOD3 were the target genes of 18 SDEGs of mi RNA.SOD2 was significantly enriched in 2 GO terms?GO:0006357,GO:0030145?and 2 KEGG pathways?ko04068,ko04146?,SOD3 was significantly enriched in 5 GO terms?GO:0005737,GO:0008270,GO:007062,GO:0005802,GO:0001666?and 1 KEGG pathway?ko04146?.In experiment 3,the positive standard plasmid named p ET-28a-tox A-N from HN01was used as template.And with optimizing the reaction conditions,the method of RPA LFD,which could detect Pm in a very short time,was preliminary established.The following were results.A pair of primers was screened out from 4 pairs,which could amplify 251 bp fragment from the tox A-N gene.This method can detect Pm at 37?in 20min,and its sensitivity was up to 4 copies/reaction.