新孢子虫病P43蛋白质(包涵体)的纯化及rELISA方法的建立

Purification of P43 Protein of Neosporosis and Development of rELISA Method

为建立新孢子虫病间接酶联免疫吸附试验(rELISA)检测方法,先诱导表达GST-P43包涵体蛋白,并对其进行提取、纯化、复性处理,再以复性后的P43蛋白作为抗原,优化rELISA检测方法。结果表明,这种包涵体处理方法,可使其纯度达到87%,复性后的目的蛋白具有良好的免疫活性。通过对rELISA各反应条件的优化,确定了最佳反应条件,血清稀释倍数为1∶100,抗原包被浓度为7μg/mL,封闭液为0.5%脱脂乳,酶标抗体的工作浓度为1∶4000,酶标抗体作用时间为1 h,底物作用时间25 min。且该方法不与弓形虫和布鲁氏菌病发生交叉反应。

Development of indirect ELISA for the detection of neosporosis were as follows：First,the fusion protein GST-P43 was induced, which was extracted, purified and treated with renaturation,after which P43 protein were used as the antigen one,and rELISA detection method was optimized. The results showed that inclusion bodies purified by treatment could make the purity reach 87 %. The after renaturation the aim protein has good immune activity. The optimal reaction conditions of rELISA were determined,the antigen coating concentration of the protein was 7μg/mL;The dilution rate of the serum samples was 1 ： 100;Blocking solution was 0.5% skim milk; The optimal dilution of HRP-IgG was 1 ： 4000;The reaction time of coating antigen with serum and serum with HRP-IgG was 1 h;The time of color development reaction was 25 min. The test showed there was no cross reaction between this method,Toxoplasma and brucelliasis.