泰勒虫重组抗原Tams1-spag基因原核表达与纯化

Prokaryotic Expression and Purification of Theileria annulata Recombinant Antigen Tams1-spag Genes

为了在低成本的情况下,得到高浓度、高纯度的环形泰勒虫表面重组抗原的融合蛋白,设定表达过程中不同的OD600nm、IPTG浓度与诱导时间,探索最佳表达条件。通过KCl染色法切胶后分别经透析袋电洗脱法与快速离心法回收目的蛋白。对上述试验的蛋白样品进行SDS-PAGE和Western blot检测。结果表明,在OD600nm=1时加入浓度为1mmol/L IPTG,过夜诱导时,重组蛋白的表达量最大。在KCl染色法切胶回收目的条带的基础上,电洗脱法与快速离心法均能得到同等浓度的高纯度目的蛋白,经Western blot检测重组蛋白的生物活性没有改变,仍具有良好的反应原性。

To study the purification method of the of fusion protein of Theileria annuLata surface recombi- nant antigens and to obtain high purity products at low cost, different parameters were set in order to ob- tain the optimum of expression. The experimental parameters included OD600 nm,IPTG concentration in- duction time. Firstly, the target proteins were isolated by cutting the gel slices that contained the right bands which were stained by KC1 solution. Secondly, the recycle protein were recovered by electrical elu- tion and high centrifugation. Through SDS-PAGE and Western-blot, the experimental results indicated that the maximal quantity of expression of recombinant proteins was confirmed when IPTG concentration was 1 mmol/Land OD600 nm= 1, the induced time was stay overnight. The two methods can obtain the same purified products and high purity. Western-blot assay indicated that the recombinant protein were not denaturated,and had good antigencity.