The Development Of Nanobody And Construction Of Immunoassay Towards Salmonella Enteritidis

Foodborne diseases are currently one of the most widespread public health problems in the world.Food poisoning cases caused by Salmonella have reached the front of food poisoning incidents around the world.Therefore,establishment of a rapid detection method towards Salmonella is of great significance in the preventing of food safety events.Immunoassays are fast and sensitive,which is suitable for rapid screening of large samples.The variable domain of heavy-chain antibody(VHH)obtained by genetic engineering methods is the smallest known antibody fragment with complete function,also named as nanobody.Nanobodies,with unique superior properties compared to conventional antibodies,including small molecular weight,good water solubility and stability,high affinity,the ability to recognize epitopes between gaps and easy to obtain and express,have been widely used in medicine diagnosis,disease treatment,and food contamination analysis et al.In this study,a phage-displayed nanobody library was constructed by immunizing a bactrian camel with inactivated S.Enteritidis,and nanobodies towards S.Enteritidis were obtained after biopanning.The thermal stability and specificity of the nanobody were preliminarily identified.A sandwich ELISA was established to detect S.Enteritidis,which provided a new idea for the detection method of Salmonella.The main results of this study are as follows:1.A S.enteritidis phage display nanobody library was constructed with good diversity.A bactrian camel was immunized 5 times by using inactivated Salmonella enteritidis.The peripheral bloods of camel was collected after immunization,total RNA was extracted and reverse transcription was performed into cDNA.The VHH gene was amplified by nested PCR.The VHH gene was ligated into the pComb3X vector and then electroporated into E.coli ER2738 competent cells,and the phage display nanobody library was successfully constructed.The library capacity was about 1.78×10~7,and the library insertion efficiency reached 100%;23 colonies were randomly picked and sequenced,and the results showed that the library had good diversity.2.Salmonella enteritidis specific nanobody was developed for the first time with good specificity and high thermal stability.After four rounds of biopanning,positive phages were identified by phage-ELISA.After sequence alignment analysis,three different nanobodies were obtained,named Nb13,Nb16 and Nb22.The plasmids containing these three nanobody VHH gene were transformed into E.coli Top10F'competent cells for induction expression,and three nanobodies were successfully expressed.The thermal stability and specificity of these nanobodies were identified.The results showed that the obtained nanobodies had good thermal stability and specificity.3.A sandwich ELISA immunoassay based on nanobody Nb13 was established for the detection of S.Enteritidis.A double antibody sandwich ELISA method based on nanobody Nb13 was established to detect S.Enteritidis,and the detection sensitivity was 1.4×10~5 cfu/mL.The method was further applied to the detection of milk samples,and the detection sensitivity is up to 1-10cfu/mL after 10 hours of preculture.In summary,this study developed S.Enteritidis-specific nanobodies,and established a nanobody-based sandwich ELISA.The method was successfully applied in milk samples,which paved a new way for the rapid detection of food-borne pathogens.