Protein Of Recombinant Escherichia Coli Heat-stable Enterotoxin,Preparation And Application Of Its Monoclonal Antibody

The heat-stable enterotoxigenic(STa)of Enterotoxigenic E.coli(ETEC)was one of the important causative factors of diarrhea or death in neonatal animals,humans,partic?larly among visitors,or children in developing countries.It co ? Id not only threat to human health,but also cause serious economic losses to the pig industry.STa was a low-molecular-weight(about 2Ku),nonimmunogenic and difficult to obtain its pure natural product,so it could not be dyed and be directly coated to the ELISA plates,and therefore could not use the SDS-PAGE and ELISA to detect.Conventional detection methods are fed neonatal rat test,but it co ? ld not only need the specific animals but also not sensitive eno?gh.Thus,preparation of the immunogenic recombinant protein STa and anti-STa monoclonal antibodies had a important theoretical and practical significance for establishing STa rapid detection methods and immune antibodies detected.STa falls into STh(human)and STp(porcine or bovine origin),two subtypes.STp was researched in this study,expressed recombinant protein STp5-His,prepared anti-STp monoclonal antibodies,initially established indirect competitive ELISA method for detecting STa.Expression of recombinant protein STp5-His and MBP-5STp.STp5-His and 5STp gene fragments were constructed by estp series in four times.The STp5-His and 5STp gene were cloned into pGEX-6P-1 and pMAL-c4x vector,to construct recombinant E.coli BL21(PlySs)/pGEX-6p-1-STp5-His and Rossetta/pMAL-c4x-5STp,expressed recombinant proteins.The results showed,recombinant protein STp5-His(38Ku,precipitation)and MBP-5STp(58Ku,supernatant)were acquired after IPTG induction,Western blot showed both proteins were reacted with MAb-His and STa yolk antibody,respectively.Preparation of monoclonal antibodies.Recombinant protein STp5-His was used to immunized the BALB/c mice.The spleen cells from BALB/c mice immunized were fused with SP2/0.The supernatants of hybridoma cells were selected by indirect ELISA.The results showed four hybridoma series secreting monoclonal antibodies(MAbs)against recombinant protein STp5-His were obtained,named C3(MAb-C3,IgG1),G1(MAb-G1,IgM),F3(MAb-F3,IgG2b),G9(MAb-G9,IgG2b),respectively.The supernatant titers of the four hybridoma cells' were from 1:6400 to 1:12800.The titers of purified ascites were 1:105 to 1:106.Western blot showed that the all MAbs were able to reacted with MBP-5STp.Competitive ELISA showed that all MAbs with good specificity could competitively reacted with natural STa,rather than reacting with LT,STb,Stx2e,Stx1,Stx2.C3 was showed to neutralizing with natural STa by suckling mice fed orally.Establishment of an indirect competitive ELISA.Recombinant protein MBP-5STp as coating antigen and MAb-C3 as the detection antibody,were used to initially established indirect competitive ELISA method for STa.The res?lts showed that optimal concentration of antigen was 1.5mg/ml,detection antibody MAb-C3 optimal dilution was 1:160,the minimum detectable amount of toxin 1.5mg/ml,the best HRP-goat anti-mouse was 1:8000,the optimal concentration of the samples were 1.5 to 3mg/ml.It was concluded that the immunogenic recombinant protein STp5-His and MBP-5STp for antibody screening and detection were acquired,respectively.Four monoclonal antibodies reacted with recombinant protein STp5-His and natural STa were prepared.Development of an indirect competitive ELISA for detecting STa was initially established.These provided material basis for STa detection assay and antibody monitored.