【摘要】 为建立利用重组李氏杆菌溶血素(LLO)检测单核细胞增生李氏杆菌的ELISA方法,试验首先优化了重组李氏杆菌溶血素的表达条件,获得纯度较高的包涵体之后,用亲和层析法纯化重组李氏杆菌溶血素蛋白,并用纯化蛋白免疫试验兔获得诊断抗体,然后建立阻断ELISA检测单核细胞增生李氏杆菌的方法,并分析最低检测菌数。结果表明:在振摇培养条件下,当IPTG浓度为0.6 mmol/L、诱导温度为37℃、菌液OD600值为0.25时开始诱导,诱导时间为150 m in,重组李氏杆菌溶血素的表达量最大;纯化蛋白浓度为480μg/mL,具有免疫活性。说明建立的检测方法特异性好,最低检测菌数为4.8×106个/mL。提示利用重组李氏杆菌溶血素检测单核细胞增生李氏杆菌的ELISA方法是可行的,具有潜在的应用价值。更多还原

【Abstract】 To establish an blocking ELISA based on the recombinant LLO protein expressed in E.coli for detecting Listeria monocytogenes,E.coli habouring the recombinant expression plasmid pGEX-6P-hly induced by IPTG,and the induction conditions were optimized.Experimental results indicated that under shake incubator,LB medium and inducing 2.5 hours with 37 ℃,hly expression quantity was the most while concentration of IPTG was 0.6 mmol/L.After the purification of recombinant LLO,immunized the rabbits with the recombinant LLO to acquire anti-recombinant LLO antibody which used as a diagnostic antibody.And then a blocking ELISA was established to detect the LMO,and the sensitivity and specificity was well.The result indicated that the blocking ELISA could be used for detecting LMO.更多还原