Preparation And Application Of Different Types Of Antibodies Against Salmonella PhoN Protein

Salmonella is an important food-borne zoonotic pathogen.It infects animals and humans to cause salmonellosis,which seriously harms the development of the livestock and poultry industry and human health,and has important public health significance.According to relevant statistics,food poisoning caused by Salmonella ranks first among the bacterial food poisoning worldwide.So far,more than 2,600 Salmonella serotypes have been discovered.At present,the standard detection for Salmonella is still the microbial culture method.However,this operation is complicated and requires at least 3 days of selective enrichment culture.Therefore,the establishment of rapid enrichment techniques such as immunomagnetic beads based on Salmonella specific targets is of great significance to improve the efficiency of Salmonella detection.Salmonella outer membrane proteins can stimulate host body to produce a strong humoral immune response with higher level of antibodies which plays an important role in the pathogenic process of bacteria.PhoN protein is one of the outer membrane proteins of Salmonella,a non-specific acid phosphatase,which can stimulate host to produce humoral immunity and cellular immunity.More importantly,phoN gene present in most serotypes of Salmonella,but does not exist in non-Salmonella enterobacteriaceae such as Escherichia coli.It has shown great potential as a detection target for Salmonella based on its distribution characteristics which has not been reported.Monoclonal antibody(McAb)has the advantages of strong specificity,high sensitivity and high purity.The application of this technology has improved the specificity,sensitivity and stability of Salmonella serological methods.McAb-based detection and diagnostic applications,such as immunomagnetic beads,can quickly detect and diagnose Salmonella.In recent years,miniaturized antibodies have become one of the main research goals of antibody technology in the field of disease diagnosis and treatment.There is a special antibody called heavy chain antibody(HcAb)that naturally lacks the light chain and heavy chain constant region 1 in camels.Nanobody(Nb)is a single domain heavy chain antibody obtained by cloning the variable region of heavy chain of HCAb(VHH),which is the smallest fragment of antibody that can effectively bind to the antigen.Nb has a broad prospect in the fields of anti-tumor,anti-virus,pathogenic microorganism detection and other fields due to its series of advantages compared with traditional McAb.The current more mature method for preparing Nb is phage display technology,which can screen antibody libraries with larger capacity in high throughput.In this study,the Salmonella outer membrane protein expression gene phoN was used as the target,and the phoN prokaryotic expression vector was successfully constructed.After induced expression and purification,a soluble recombinant PhoN protein with high purity was obtained.The recombinant protein was immunized in mice,and PhoN-specific McAbs were screened and coupled with magnetic beads to prepare immune enrichment magnetic beads,which can successfully enrich Salmonella,At the same time,the recombinant protein was immunized to alpaca,and the PhoN-specific Nbl and Nb2 sequence was obtained by constructing a phage display library and screening.Select Nb2 to design primers,construct prokaryotic expression vector,and successfully obtain Nb,which provided an important material basis for the rapid enrichment and detection of Salmonella.1.Preparation and application of traditional McAb to PhoN protein of SalmonellaUsing the genome of Salmonella typhimurium standard strain ATCC14028S as template,the outer membrane protein coding gene phoN was cloned and constructed into the prokaryotic expression vector pCold I and pGEX-6P-1 by one-step method to obtain recombinant plasmids of pCold I-phoN and pGEX-6P-1-phoN,after being verified by PCR and sequencing.The plasmids were transformed into engineering bacteria Escherichia coli strain BL21(DE3)to obtain recombinant expression strains of BL21(DE3)-pCold-phoN and BL21(DE3)-pGEX-6P-1-phoN.The recombinant His-PhoN and GST-PhoN protein was mainly expressed in the form of soluble protein after induced by IPTG with the expected molecular weight of 28 kDa and 54 kDa respectively.The recombinant PhoN proteins with high purity was obtained after purification,and showed well immunoreactivity to McAbs against His and GST tag analysized by Western Blot.The His-PhoN protein was combined with Freund's adjuvant to immunize female BALB/c mice aged 6-8 weeks to generate traditional McAb to PhoN protein.After 3 immunizations,the B lymphocyte hybridoma was prepared by cell fusion and the positive cell clones were identified by an indirect ELISA method using GST-PhoN as the detection agent.After 3 times of sub-cloning,5 hybridoma cell lines that stably secrete McAbs against PhoN protein were finally obtained which was named 3B2,4B2,8F2,8H2,11E5,respectively.All the 5 McAbs showed a subtype of IgG2a and displayed specific reactivity to the target PhoN protein but not other recommbinate proteins of Salmonella such as His-PdgL,His-OmpC,and His-OmpF detected by ELISA analysis.The ascites of 4B2 was prepared and purified to further analyze the specificity of the McAb.Western Blot results showed that 4B2 McAb can compatible with Salmonella Enteritidis,Salmonella Typhimurium,Salmonella Derby,Salmonella Infantis,Salmonella Indiana,Salmonella Indiana,Salmonella Indiana Salmonella London,Salmonella Corvallis,Salmonella Rissen specifically.Meanwhile,the McAb does not react with non-salmonella bacteria such as Escherichia coli,Shiga bacillus,Campylobacter jejuni and Listeria monocytogenes.These results suggest the ability of 4B2 McAb to specifically distinguish Salmonella which indicationg excellent application potential for Salmonella detection.Furthermore,the immunomagnetic beads against PhoN was prepared by coupling 4B2 McAb with Fe3O4 carboxyl magnetic nanospheres to evalute their potential in the enrichment of samples for Salmonella detection.The immune enrichment results showed that these beads can enrich Salmonella Typhimurium,Salmonella Enteritidis,Salmonella Derby,Salmonella Infantis,Salmonella Corvallis and Salmonella Rissen.Among them,the capture efficiency of Salmonella Typhimurium is the highest with enrichment rate of 80%,which provides an important materials reserve for the pre-treatment of sample in Salmonella detection.2.Preparation and application of Nanobody of Salmonella PhoN proteinAdult alpacas were immunized by subcutaneous injection of recombinant His-PhoN protein emulsified with Freund's complete adjuvant for the first time and Freund's incomplete adjuvant for the next three time.After 4 immunizations,the titer of PhoN-specific antibodies in alpaca serum can reach 1:3276800.The peripheral blood lymphocytes were separated from alpaca peripheral blood using lymphocyte separation solution kit for extraction of total RNA reverse transcription of cDNA.VHH genes were amplified using VHH specific primers and cDNA as model by nested PCR.The fragment of 700 bp in the first round of PCR was selected and recovered,which was used as a template for the second round of PCR.The product of 450 bp length in the second round of PCR was selected and recovered,which is the target fragment of the nanobody.The target fragment of the Nanobody was ligated to the pCANTAB5E vector and transformed into TG1 competent cells,coated on ampicillin-resistant LB medium,and verified by counting the coated plates and amplification of VHH specific primers.The final library capacity was 4.6×108 CFU/mL with an insertion rate of 91.6%.After the rescue of the phage library,3 rounds of specific biopanning were performed using the phage ELISA method to obtain specific VHH clones.96 clones were randomly selected,cultured overnight,and IPTG was added to prepare the crude nanoantibody extract after expanded culture.The crude nanobody extracts were detected by M13 and E-tag using indirect ELISA coated with GST-PhoN recombinant protein,and positive clones were taken and sent to Beijing Kinco Biology Company for gene sequencing.was used to analyze the obtained sequence.Two PhoN-specific Nanobodies named Nbl and Nb2 were screened from the amino acid sequences analyzed by DNAMAN software.Nb2 was expressed by the E.coli prokaryotic expression system in soluble form analyzed by SDS-PAGE.Western Blot result showed that the Nb2 protein can specifically recognize Salmonella PhoN protein,indicating the specific activity of antibody.Furthermore,Nb2 is coupled with Fe3O4 carboxyl magnetic nanospheres to prepare immunomagnetic beads.The results of immunoenrichment test show that it can enrich S Typhimurium,S Enteritidis,S Derby,S Infants,S Corvallis and S Rissen.The capture efficiency is similar to traditional immunomagnetic beads,but with higher specificity.The enrichment efficiency of Salmonella in pork samples can reach 79%,which provides important biological material reserves for the pretreatment of immunoenrichment of samples in Salmonella detection.