Prokaryotic Expression And Development Of Monoclone Antibody Against Troponin Ⅰ Of Porcine Skeletal Muscle

Fraudulent adulteration of higher value meat with less valuable flesh in meat products is contrary to the enforcement of food labeling regulations, fair trading and consumer protection laws. Meat products containing undeclared species also may impose a potential health risk to people with allergies to specific proteins. Meat products containing undeclared species also may impose apotential health risk to people with allergies to specific proteins. Meat species adulteration in ground and comminuted products has been a widespread problem in retail markets; the incidence of mixing undeclared species appeared to be higher in cooked meat products than in raw products. Because of lower value and similarity in color and texture, pork is a potential source for adulteration of higher value meat such as beef and veal.Methods for identification of raw meat based on ELISA have been well established. Detection of species adulteration in cooked meats appears to be more complicated than in raw meats because heat induces denaturation of most immunogenic proteins, degrades the solubility of immunogenic proteins and diminishes the antigenicity. Therefore, any antigens for cooked meat identification should withstand cooking or be renatured after heating. The use of a defined antigen with known antigenic determinants, a so-called species marker, would substantially increase the chance of eliciting specific antibodies. The selected species marker needs to possess a unique antigenic region for a given species that is not present in the counterpart molecules of other species. In addition, thermal stability of the species marker antigen is indispensable in developing immunoassays for the detection of species origin of heat-processed meats.Many literatures of abroad have reported, porcine skeletal muscle troponin I (PsTnI) was identified as porcine specific thermostable muscle protein (TSMP). The antigenic specificity and thermal stability of PsTnI indicate its potential as a thermostable species marker protein (TSSMP) for the identification of the origin of meats in severely heated products. So following researchs were explored:1. Cloning and expression of Procine Skeletal Muscle TnI-fast/slow protein geneAccording to published Sus scrofa troponin I (TNNI2) cDNA sequence (DQ091303) and Sus scrofa troponin I (TNNI1) cDNA sequence (AY282922) in GenBank, specific primers (TnI-FP1/ TnI-FP2 and TnI-SP1/ TnI-SP2) were designed and synthesized. TnI-fast protein gene and TnI-slow protein gene were amplified from total RNA of porcine skeletal muscle by RT-PCR and cloned into the pMD18-T vector. The recombinant plasmid was proved to be true by restriction-enzyme analysis and sequencing. The sequence BLAST showed that TnI-fast protein gene have the most homology 99% , TnI-slow protein gene have the most homology 99%. The prokaryotic expression plasmid (pKG-TnI-fast, pKG-TnI-slow and pET-32a-TnI-fast) were construced and expressed in E.coli BL21 (DE3) successfully. The expressed GST-TnI-FAST fusion protein in E.coli BL21 (DE3) is an about 47KDa protein in the analysis of SDS-PAGE, the expressed GST-TnI-SLOW fusion protein in E.coli BL21 (DE3) is 47KDa protein and the expressed HIS-TnI-SLOW fusion protein in E.coli BL21 (DE3) is 41KDa protein in the analysis of SDS-PAGE and Western-blot with immunogenicity.2. Preparation and purification of of Rabbit Anti-Porcine Skeletal Muscle TnI-SLOW protein polyclonal antibodyRabbit were immunized with purified GST-TnI-SLOW fusion protein emulsified with Frund's adjuvant. Rabbit anti-GST-TnI-SLOW fusion protein were prepared successfully. The depurated antibodies (anti-GST-TnI-SLOW IgG) were obtained after these sera were purified by saturation (NH₄)₂SO₄ precipitation and Sephadex G-200 respectively.3. Preparation and purification of of Rabbit Anti-Porcine Skeletal Muscle TnI-SLOW protein monoclonal antibodyAfter immunization of BALB/C mice with the purified GST-TnI-SLOW fusion protein, the stimulated splenocytes were routinely fused with SP2/0 myeloma cells to produce hybridomas. After three cycles of cloning, 3 stable hybridoma clones (3C8, 3H10, 4H9) producing monoclonal antibodies to TnI-SLOW protein. All the hybridoma cells were cultrured in RPMI-1640 with 5% FCS, harvested and injected into nud mice, and then mouse ascetic fluids were harvested 10 days later. The ELISA titers of 3 monoclonal antibodies for TnI-SLOW protein were 1:6.4×10⁵ at least. Selected monoclonal antibodies with higher titers were purified from mouse ascetic fluids using sequential precipitation with caprylic acid and ammonium sulfate.The species specificity of the porcine sandwich ELISA using Porcine Skeletal Muscle TnI-SLOW McAb as the capture antibody and Porcine Skeletal Muscle TnI-SLOW PcAb as the detection antibody is presented. The results showed that the optimum coating concentration of TnI-SLOW McAb (Ab₁) were 5μg/mL, and the optimum working concentration of rabbits anti-TnI-SLOW(Ab₂) were 4μg/mL. The porcine assay exhibited a positive reaction in cooked (100℃for 30 min) pork extracts, with no cross-reaction with extracts from any of the other species tested, including bovine, ovine, chicken and duck meat extracts. So this methed have high specificity and sensitivity.