新孢子虫NcSAG1与NcSRS2基因的原核表达与纯化

Prokaryotic expression and purification of NcSRS2 and NcSAG1 genes of Neospora canium

PCR扩增新孢子虫NcSAG1与NcSRS2基因,将其构建至原核表达载体pET28a(+),转化大肠杆菌BL-21菌株。对重组表达质粒pET28a/SRS2与pET28a/SAG1IPTG的诱导浓度和诱导温度等条件进行了摸索,确定蛋白最佳诱导时间分别为5、3h,IPTG浓度均为0.8 mmol/L。经组氨酸标记Ni 2+柱纯化后获得的蛋白纯度均大于93.5%,蛋白质量浓度分别为1.23、1.1g/L。经Western blotting检测证明表达的蛋白具有较好的特异性。用纯化后pET28a/SRS2、pET28a/SAG1和二者等量混合物包被酶标板,经ELISA检测表明,表达的蛋白具有较强免疫活性,2种蛋白等量混合物包被酶标板与各蛋白单独包被酶标板检测比较,并未见D值升高。

NcSAG1 and NcSRS2 gene of Neospora canium were amplified by PCR and cloned into prokaryotic expression vector pET28a（＋ ）, the recombinant plasmids were transformed into E. coil BL-21. The best concentration of IPTG was 0.8 mmol/L,and the inducement time was 5 h or 3 h to recombinant plasmids pET28a/SRS2 and pET28a/SAGl,respectively. The concentration of NcSAG1 and NcSRS2 fusion proteins after purification were 1.23 g/L and 1.1 g/L,respectively, that is more than 93. 5% to the total protein. The western blotting assays demonstrated the ex- pressive products shared a good specificity. The indirect ELISA method was developed by using the purified fusion pET28a/SRS2, pET28a/SAG1 proteins and two proteins equivalence mixture as coating antigen. The results indicated that the purified proteins have a good immunogenicity. Co immobilized protein did not cause the higher D value compare with the single immobilized group.