Screening Of Weaken Related Genes Of Brucella RM57 By Comparative Genomics

Brucellosis is a serious zoonotic infectious disease,people,cattle,sheep,pigs,dogs and other mammals can be infected,causing animal miscarriage,genital inflammation,human wave heat.In recent years,with the increasing of livestock in our country,the circulation of animals and their products is frequent,and the brucellosis in some areas is on the rise.It not only seriously affects the production of animal husbandry,but also seriously endangers people's health and public health.Strengthening the prevention and control of brucellosis is of great significance.At present in developed countries to eliminate brucellosis is the main method of direct culling.For brucellosis epidemic in China,the government at all levels is unable to bear the cost of widely culling,so the use of vaccines to prevent and control the brucellosis epidemic is in line with China's national conditions of the only means.Brucella according to phenotypic differences can be divided into smooth and rough type,both in serological no cross-reaction,but there are immune cross-protection.Generally wild virulent brucella is smooth type,while the brucella vaccine is basically smooth too;so vaccine immunization often interfere with clinical diagnosis.The development of rough type Brucella vaccine strain has been a hot spot in the study of brucellosis.United States in the late 90 s of last century,in the wild animals began to use rough vaccine RB51 strain.But its safety and immune protection effect is controversial,and the real effects of domestic animals have no systematic research reports.In particular,RB51 is induced with rifampicin,and rifampicin is a cure for brucellosis,and the widespread use of RB51 poses a potential hazard to human safety.In this study,a strain of Brucella isolates(M1981)was induced to be rough,named RM57,using rough antisera and smooth antiserum cross-induced and repeated screening.Biological characteristics of the study showed that RM57 strain is a virulent stable strain,high safety and good immunization effect.In this study,the integrity of lipopolysaccharide,which caused the rough phenotype of RM57 strain,was identified.At the same time,the gene integrity identification and the change of transcription level were determined according to the related genes that could cause brucella rough phenotype at home and abroad.The results showed that there was a significant difference between the RM57 strain and the lipopolysaccharide of its parental smooth M1981 strain,but the lipopolysaccharide-related genes that had been reported are intact and had no changes in transcription level.It is inevitable that the unknown gene is involved in the brucella LPS biology synthesis process or there is a certain regulatory mechanism to control the Brucella LPS synthesis system.The mutant Brucella RM57 strain and its parental smooth Brucella strain M1981 have a clear genetic background from the same parental strain,which provides valuable evidence for the study of Brucella LPS synthesis by genomics of the fine material.In this study,we used the comparative analysis of genomic analysis of Rleatella roxburghii RM57 strain and its parental smooth Brucella strain M1981,which will broaden the understanding of Brucella LPS synthesis.The whole genome sequence of the strain Brucella RM57 strain and its parental smooth Brucella strain M1981 was sequenced by the NGS sequencing technique.Then,by closing gaps we obtained the complete sequence of the two strains of the genome.The genomic information of two strains was obtained by genetic prediction and repeated sequence prediction.The genomic information including the number of genes,the average length of genes,tandem repeats,small satellite sequences and microsatellite sequences were analyzed.The ORFs of the Brucella genome were predicted and genomic fine maps were finally drawn by gene annotation,gene function classification,virulence gene prediction and so on.According to the genome information of Brucella,the genes related to the growth and metabolism of pathogens were excavated,and the metabolic pathways of brucella were constructed.There were 22 major categories: chromosome structure and kinetics,energy generation and transmission,cell cycle regulation,Cell division,chromosome segregation,amino acid transport and metabolism,nucleic acid transport and metabolism,carbohydrate transport and metabolism,coenzyme transport and metabolism,lipid transport and metabolism,translation,ribosome structure and biogenesis,transcription,replication,recombination and modification,Cell wall / cell membrane biogenesis,cell activity,post-translational modification,protein turnover,partner,inorganic ion transport and metabolism,secondary metabolites biosynthesis,transport and metabolism,only general functional prediction,unknown function,signaling mechanisms,intercellular transport,Secretions and vesicles,defense mechanisms,extracellular structure.According to the genome information of Brucella,the genes related to virulence factors were excavated,including: urease auxiliary protein,urea amide hydrolase,urea amide hydrolase,iron III,ABC transporter,ATP binding protein,cyclic ?1-2 glucan synthase,heme export protein,phosphoglucomutase,presumed protein Rv0981,endopeptidase Clp ATP binding chain C,possible ATP binding component of ABC transporter,possible ATP binding of ABC transporter Components,icl / ace A,lipopolysaccharide biosynthesis protein,hemolysin B,ATP-dependent protease,two-component response regulator,ATP-dependent Clp protease proteolytic subunit,UDP-glucose 4-epimerase,A two-component regulatory system with Pho Q,a transcriptional regulator of low Mg2+ concentration,sod B superoxide dismutase,mannose-6-phosphate isomerase,mannose-1-phosphogluconate Enzyme,man B-mannose-degrading enzyme,mannosyltransferase,perosamine synthase,GDP-mannose 4,6-anhydrase / GDP-4-amino-4,6-dideoxy-D-mannose Transferase,lipopolysaccharide biosynthesis protein,Pano,4'-phosphate pantothenoyltransferase,2,3-dihydro-2,3-dihydroxybenzoic acid dehydrogenase,Isochorismasease,enterobacterium synthase component E,plasma synthetase,Mg2+ transporter,Vir B3 homolog,Vir B8,channel protein Vir B6 homolog,Vir B5,ATPase Vir B4 homolog,Vir B3,Vir B2,adhesion mediator Vir B1 homolog,UDP-Glucose pyrophosphorylase,Kps F protein,flagella motor switch protein,type III secretion system ATPase,flagellar basala protein,flagellar basala protein,flagellum P protein precursor,flagellar biosynthesis protein,glucose / galactose transporter,Hsp60,60 K heat shock protein,nar H,nar G,mannose isomerase / guanosine 5'-diphosphate-D-mannose pyrophosphorylase,GDP-fucose synthase,GDP-mannose 4,6-dehydratase,ferric enterobactin transport ATP binding protein,Gifsy-2 pro-phage: superoxide dismutase precursor Cu-Zn,ahp C,endometrial ABC transporter,prescription type III secretory protein.The results showed that there was a significant difference between the genome length and the amino acid level on the functional gene,and analyzed the effects of the mutant Brucella strain RM57 strain and its parental smooth strain Brucella strain M1981.M1981 strain in the rosy type of antiserum and smooth anti-serum repeated cross-induced process of LPS phenotypic molecular mechanism,and finally excavated 26 genetic mutation sites.