| Bovine mastitis is an inflammation mainly caused by pathogenic microorganisms invading breast tissues of dairy cows.This disease is difficult to cure and its morbidity is high,it resulted in reducing production and quality of milk,it is one of the important diseases which causes clinical mastitis and death of dairy cows.It has been reported that many kinds of microorganisms can cause bovine mastitis,mainly including Staphylococcus aureus,Streptococcus and Escherichia coli,bovine mastitis which caused by these three pathogens accounts for more than 90% of the total morbidty of the disease.At present,bovine mastitis caused by Escherichia coli is increasing,especially in developed countries,the morbidity has reached28.39%.The proportion of mastitis which caused by E.coli high in China.For example,The incidence of mastitis in Foshan is up to 50% and the incidence of mastitis in Lanzhou is up to 24.44%.Henan province is a big province of animal husbandry,the population of dairy cattle sharply have increased in recent years,.But the morbidity of bovine mastitis is still high.it leads to high elimination of rate of dairy cattle and serious impact on the development of breeding dairy cattle.Many studies have shown quorum sensing(QS)is a common regulatory system in bacteria,and QS is the cell population density-dependent regulation of gene expression by small signaling molecules,called auto-inducer(AI).LuxS/AI-2 system exists in gram-negative and gram-positive bacteria,it plays an important rolein the same kind of bacteria,in the different kinda of bacteria,in intracellular and extracellular information exchange.LuxS/AI-2 density sensor systems can coordinate the function of bacterial populations,regulate the transfer of plassmid,biofilm formation,virulence gene expression and bacteria glow through the signal molecules(Autoinducer,AI)produced by bacteria.The emergence of drug-resistance of E.coli,especially multiple drug resistance has underlined emergency action to deal.Interfering with or blocking the bacterial density sensing system of E.coli,especially the suppression of LuxS/AI-2 density sensing system,can be a new way to prevent and control of mastitis caused by E.coli.At present,there are few studies on the pathogenic mechanism of LuxS/AI-2 in Mastitis caused by E.coli,and this study reports that there is LuxS/AI-2 density sensing system in cow mastitis caused by E.coli.The system has been studied from the following two aspects,in order to lay solid foundation for the synthesis of AI-2 in vitro,effects of AI-2on the E.coli adhesion,the control of virulence factors and the study of new drugs for suppressing AI-2,and provide new ideas for prevention and treatment of Mastitis caused by E.coli.1.Detection of AI-2 of bovine mastitis caused by E.coliIn order to know AI-2 production of the different bovine mastitis caused by E.coli,we analysed the effects of different growth periods and different culture conditions on AI-2 productionof bovine mastitis caused by E.coli,detected the correlation between synthesis of AI-2 and the transcription level of lux S and pfs gene.In this study,we detected AI-2 in E285,E80814,W41,E30707 and EA701205 isolates of E.coli from bovinemastitis,using Vibrio harveyi BB170 bioassay.Based on the original,E285 was used as the detection strain,The levels of lux S and pfs m RNA were analyzed using real-time PCR.The results showed that the E285,E80814,W41,E30707 and EA701205 strains of bovine mastitis caused by E.coli all had produced AI-2,but any of AI-2 production was lower than that of positive control.Activity the test results of AI-2 production in different growth phases showed that AI-2 was not observed in lag phase and early stage of the logarithmic phase.But AI-2 was expressed in the middle of the logarithmic mid-term,AI-2 production reached the peak in the late stage of logarithm,which was 12 times more than negative control,then fallen to3.6 times more than the negative control in stable phase.The results showed that the production of AI-2 and the transcription level of lux S gene were significantly correlation with.The results of different culture conditions showed that the production of AI-2 and the transcription level of lux S gene were increased by supplementation with sucrose,maltose,glucose,mannitol,Na Cl and lactose,and the addition of lactose most largely increased the AI-2 exprssions in E285 strain.The level of AI-2production is consistent with the level of lux S gene in different phases,but he activity of AI-2 was not correlated with the transcription level of pfs gene.The results showed that the production of AI-2 was highly correlated with the transcription level of lux S gene and had no correlation with the transcription level of pfs gene.Sucrose,maltose,glucose,mannitol,Na Cl and lactose can promote the production of AI-2 signaling molecules in E285.2.Cloning,expression and purification of lux S and pfs genesIn the bacterial LuxS/AI-2 density sensing system,LuxS and Pfs proteins are two key enzymes in the process of producing AI-2 signaling molecules.In order tounderstand the genetic variation of LuxS and Pfs proteins to get the recombinant of LuxS and Pfs proteins,a pair of specific primers were designed according to the gene sequence published by lux S(YP-001199965)and pfs(NC-009443)in Genbank.In this study,the DNA of E285 was used as template,The lux S gene and pfs gene were amplified by PCR and were digested by Eco RI and Hind III respectively.They were inserted into the prokaryotic expression vector p ET30 a,and then identified by PCR and restriction enzyme digestion.The p ET30a-lux S and p ET30a-pfs were transformed into BL21,and then the fusion recombinant protein LuxS and Pfs were induced by IPTG.The proteins were purified by His-tag protein,immunized New Zealand white rabbits.The immunogenicity of the recombinant proteins were verified by western-blotting.The results showed that the amplified lux S and pfs genes were highly conserved.,and had more than 98% homology with the nucleotide sequencepublished in Genbank,which indicated that lux S and pfs genes were common in E.coli.The two genes has been efficiently expressed in prokaryotic expression system,.After purified,LuxS protein concentrations were 4.6 mg/m L and Pfs protein concentrations were 5.3 mg/m L in the supernatant.The result of Western blotting showed that the recombinant protein LuxS and Pfs had strong immunogenicity.Through cloning,expressing of lux S and pfs gene,this study provided technical support for the synthesis of AI-2 in vitro. |
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