Gene Synthesis,Prokaryotic Expression And Antibody Preparation Of Beef Myoglobin

Meat products adulteration seriously affected the consumer's right to know and fair trade rights.The undeclared species in meat product may impose a potential health risk to people with food allergies or poisoning,as well as give consumers a certain economic.Considering beef is a high-risk meat for adulterated flesh,it is very necessary to build a quick and sensitive method for detecting beef adulterationAt present,the method of detecting raw meat has been very mature.The method to detect species adulteration in cooked meats is facing many difficulties,because some protein in the heating process may produce many changes,such as protein denaturation,decreased solubility and loss of antigenicity.The heat-stable muscle protein or unique and heat resistant muscle protein in the species was used as marker to detect adulteration by enzyme-linked immunosorbent assay,which successfully overcome the technical difficulties of enzyme-linked immunoassay.Some literature of aboard have reported that bovine myoglobin?BMb?was identified as a thermostable protein.Hence,the BMb can be used as a specific protein to detect beef adulteration.The aim of this study was to obtain bovine myoglobin?BMb?polyclonal using prokaryotic expression system.BALB/c mice were immuned to produce polyclonal antibody.Specific amino acid fragments of BMb were selected to prepare the monoclonal antibody of mouse anti-BMb.Two specific cell lines were harvested.These results provided a theoretical basis for the establishment of sandwich ELISA or competitive ELISA for the detection of cooked beef.The experimental results are as follows:?1?Expression and purification of recombinant protein:Amino acid sequence of BMb was queried in NCBI.22 primers were designed to obtain the whole gene by SOE-PCR amplification.The gene fragment and the plasmid Pet-28a were digested by NdeI/Xhol and connected via T4 DNA ligase.Then the connected product was transformed into Rosetta?DE3?.After inducted by IPTG,the fusion protein of bovine myoglobin was expressed successfully in Rosetta?DE3?.The weight is 19KD with the analysis of SDS-PAGE.The fusion protein was purified by Ni⁺-IDA column affinity chromatography.?2?Preparation of polyclonal antibody:The obtained fusion protein was immunized into mice,and the titer and sensitivity of antiserum were determined by indirect ELISA and indirect competitive ELISA,respectively.The titer of antiserum was 1:32000,IC₅₀=50.12?g/ml.The antiserum purified by Protein A to obtain a better quality mouse anti-bovine myoglobin polyclonal antibody.?3?Preparation of monoclonal antibody:After comparing the amino acid sequences of myoglobin of beef,pigs,chickens,ducks and rats,the specific peptide was selected in BMb.The peptides with good specificity were conjugated to KLH and then immunized into mice as immunogen.Two hybridoma cell were obtained by cell fusion and screening.The titers of 2 monoclonal antibodies were 1:64000,at least.Monoclonal antibodies were purified by octanoic acid-ammonium sulfate salinate,and their purity was high.