| For the Staphyloccocus aureus generally distributes over the air、the soil and the water, it also distributes over the demic skin follicular、sebaceous gland duc、nasal capsule and the intestinal tract,the opportunity of the pollution about the foods、the meat and the meat products is very big.In recent years, the colloidal gold immunochromatographic for quick detection has a fast development. It has several benefits in specificity、sensibility、expedient operation and rapidress. The preparation of the recombination protein SPA ensures the study of the colloidal gold immunochromatographic test strip about the Staphyloccocus aureus.This study designe and synthetize the selective primer according to the sequence of the SPA registed in the NCBI, and amplify selective SPA gene segment useing the holo-genome of the Staphyloccocus aureus as source. The amplification product is cloned into the plasmid pMD18-T and the recombinant plasmid is transform into the E.coli DH5a, and sequencing the masculine cells; The SPA gene segment digested from recombinant plasmid pMD18-T-SPA by EcoR I and Xho I is purified and cloned into expression plasmid pET-28a (+) to generate the recombinant plasmid pET-28a (+)-SPA. The recombinant plasmid pET-28a (+)-SPA is transformed into the E.coli BL21to generate the system of the prokaryotic expression and the masculine cells are selected. The EcoR I and Xho I digestion confirms the correctness of the clone about the SPA gene segment and the plasmid pET-28a(+). The prokaryotic expression for the recombination protein SPA manifest that the E.coli BL21can express the recombination protein SPA when the inductor IPTG concentration is0.6mmol/L, the time of the induction is4h and the temperature of the induction is37℃. In this study, the recombination protein SPA is purified by Ni-NTA His·Bind Resin and gain the activity by dialysis. The Western Blot and the ELISA manifest that the recombination protein SPA has a high activity of antigen as protein of the following study. |
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