Study On The Spleen Transcriptome Influence Of D-type Pasteurella Multocida Infection On The Host

Pasteurella multocida(Pm)is an opportunistic pathogen.This bacterium is widely present in animals.Animals do not cause disease when they are healthy.They can multiply in large numbers when they have reduced immunity,tissue damage or other disease outbreaks.Massive Pm can cause great harm to animals.In this experiment,the following experimental results were obtained by studying the pathogenic mechanism of the HN01strain of Pm isolated from Hainan black goats to goats and the resistance mechanism of the organism when the bacteria is invaded:Experiment 1,using Pm to conduct a mouse challenge experiment,collecting fresh dead mouse spleen tissues from the challenge group and control group,and extracting m RNA from the spleen tissues.Observe and record the state of mice after challenge.It was found that all mice died within 3 days after challenge with Pm dose of 1.58×106(Colony Forming Unit,CFU).The mice showed signs of lethargy and messy coat.After anatomy,the mice spleen showed partial swelling and bleeding.Experiment two,the extracted mouse spleen tissue m RNA was subjected to parametric transcriptome sequencing.Perform bioinformatics analysis on the sequencing results,and use RT-PCR to verify the reliability of the sequencing data.Analyzing the sequencing results:(1)Transcriptome sequencing was performed on the spleen tissues of the challenge group and the control group.A total of 150.22 Mb sequencing sequence and45.39 Gb bases were obtained,of which 74.57 Mb sequence and 22.47 Gb bases were obtained from the control group.The poison group obtained 75.65 Mb sequence and 22.92Gb base.The base recognition accuracy of each sample is above 99.9%,93.93%,and the sequence comparison rate is between 94.96%and 95.96%.(2)Using|log₂FC|?0.5,P<0.01 as the screening conditions,a total of 3 380 differentially expressed genes were screened in the challenge group,including 1,691 up-regulated genes and 1,689down-regulated genes.(3)After cluster analysis of KOG and egg NOG databases,it is found that the function of differentially expressed genes is mainly related to signal transduction mechanism or general function prediction.After GO enrichment analysis,it is found that the DEGs related to immunity are mainly concentrated in Immune response,Cell surface receptor signaling pathway,and Positive regulation of the apoptosis process,Positive regulation of cell migration P and Inflammatory response these 5 pathways.(4)After KEGG enrichment analysis,it was found that differentially expressed genes were significantly enriched in Cytokine-cytokine receptor interaction,Chemokine signaling pathway,HIF-1 signaling pathway and TNF signaling pathway,etc.After protein-protein interaction analysis of immunologically-related differentially expressed genes,it is found that the differentially expressed genes are chemokines as the core,and the chemokine groups are mainly CXC chemokine subfamily and CC chemokine subfamily.(5)After screening 229 immune-related DEGs and annotating with KEGG mapper,30 key DEGs were obtained,among which Socs3 is an important signal inhibitor in the TNF signaling pathway.(6)Randomly select 10 genes(C3,Ccl5,Cd4,Cxcl13,Lck,Gnai1,Grap2,IL-6,Cxcr6 and Serping1)for RT-PCR verification.Fluorescence quantitative results showed that the expression trends of IL-6,Cxcl13,C3,Serping1,Gnai1,Cd4,Lck,Cxcr6,and Grap2 genes were consistent with the sequencing results.Experiment three,extract goat RNA,reverse transcription to synthesize c DNA,amplify the signal suppressor protein Socs3,induce the expression of the target protein,and conduct corresponding bioinformatics analysis.The results showed that the Socs3 gene CDS sequence amplified by PCR using Hainan black goat c DNA as a template was completely consistent with the Capra hircus Socs3 gene sequence submitted in NCBI.By constructing the p ET-28a(+)-Socs3 expression vector to express the protein,the results of SDS-PAGE and Western blot verification experiments showed that a specific band protein appeared at 34.5k D;Solubility analysis found that it was a hydrophobic protein,and the expressed protein was present in the precipitate,which was inconsistent with bioinformatics analysis.In summary,the expression of a large number of immune-related genes in the spleen tissue changes when Pm invades the body,and the chemokine group is the main effect group.