Prokaryotic Expression And Purification Of FimA And PefA Protein Of Salmonella Typhirmurium

Salmonella disease is also known as paratyphoid.Some salmonella serotypes can cause bovine salmonellosis.At present,the long-term abuse of antibiotics leads drug resistance,which not only affects the prevention and treatment,but also threats public health.With the large-scale,intensive feeding and rapid development of dairy industry,salmonella disease has hindered its sustained and healthy development and impacted food quality and safety.Salmonella type I fimbriae is more conservative.fimA encodes the main subunit of type I fimbriae.Pef has good specificity.PefA is the main fimbriae subunit.In this study,the isolation and identification of Salmonella in Ningxia were established by the traditional method.The dominant serotype of Salmonella was determined,Then,the feasibility of rapid detection of four specific primers was explored.In the end,two main subunit of surfaces proteins of Salmonella typhimurium were obtained by prokaryotic expression method.They could be used to analyse antigenicity,and provide reference for diagnosis and prevention of Salmonella typhimurium.Based on the above research,the following was three aspects work:1 Isolation and identification of salmonella from bovine source in NingxiaThis part of experiment was aimed at analyzing the pathogens which caused cows' clinical mastitis,recessive mastitis,calf diarrhea,and bovine miscarriage in Ningxia.The first work was collecting milk and tissue samples from diseased cow.Then the pathogens of the samples were isolated and identified by a series of microbial diagnostic methods and molecular biology methods.It showed that four specific primers were consistent with the expection and they could be used for the subsequent rapid PCR detection of salmonella.In the 627 samples,33 were identified as Salmonella and the isolation rate was 5.26%.7 salmonella serotypes were detected in the milk samples,and 2 salmonella serotypes were detected in the tissue samples.All of them were common serotypes in our country.Salmonella typhimurium is predominant serotype,followed by Salmonella Newport.2 Expression of FimA and PefA proteins from salmonella typhimuriumThis part of experiment was aimed at obtaining Salmonella typhimurium fimbriae FimA,PefA recombinant protein.At the beginning of this phase,the B cells epitope of two fimbriae subunits were analyzed.The primers were designed and synthesized.The expression vector pET-32a(+)and fimA or pefA by T4 ligase formed a recombinant plasmid.The constructed recombinant plasmid was introduced into Escherichia coli and the induction conditions(IPTG concentration,induction time)were optimized.Finally,SDS-PAGE was used to identify and analyze the expression of recombinant protein.The results showed that all the two recombinant vectors were successfully constructed.The two genes in the E.coli expression system were induced by IPTG and expressed in whole.Two recombinant proteins of FimA and PefA were 36 kDa and 33 kDa,respectively,which were consistent with the expected results.It indicats that the expressed proteins were the target proteins.The recombinant proteins FimA and PefA were all expressed in E.coli but FimA was mainly in inclusion bodies form,while PefA was mainly present in soluble form.3 Purification of fimbriae subunit protein FimA and PefAIn order to analyse subsequent antigenicity of fimbriae subunit protein FimA and PefA,it need to obtain purification.Recombinant proteins of FimA and PefA were isolated and purified by Ni-Agarose Resin.Western blot analyse if the construction was success and FimA,PefA fimbriae subunits of Salmonella typhimurium were expression.The isolated and purified recombinant proteins by protein electrophoresis and Western blot showed a single band.The results showed that the size was 36 kDa and 33 kDa,respectively.Recombinant protein FimA and PefA with high purity could provide the raw material for further antigen analysis and the development of vaccine and detection for Salmonella typhimurium.