以提取的无乳链球菌ATCC51487菌株全基因组为模板，通过PCR扩增出srtAΔN82基因，经EcoRⅠ和XhoⅠ 双酶切、抗性筛选等方法构建重组表达载体，测定不同诱导温度、诱导剂浓度及诱导时间对表达产物的影响，利用亲和层析和离子交换层析纯化蛋白，并使用荧光共振能量转移法测定蛋白活性。结果显示,试验构建的 pGEX-6p-1-srtAΔN82载体转化至 BL21（DE3）后,在37℃条件下经1 mmol/L IPTG诱导表达6 h 后可得到大量的可溶性蛋白，且纯度大于 85%。纯化后的SrtAΔN82与底物作用后，可显著提高反应体系的荧光强度，表明得到的SrtAΔN82具有较好的生物学活性。
Recombinant expression vector of the Streptococcus agalactiae ATCC51487 gene was established by using PCR amplification,double digestion of EcoRⅠand XhoⅠ,and resistance screening,etc. Effects of induction temperature,concentration and time on the expression products were examined. The protein was purified by affinity chromatography and ion exchange chromatography,and the activity of SrtA was determined using energy transfer resonance. The results showed that after the srtAΔN82-pGEX- 6p - 1 vector was transformed into BL21 (DE3),a large amount of soluble protein were obtained by inducing at 1 mmol/L IPTG at 37℃ for 6 h. The purity of SrtA was higher than 85%. Moreover,the purified SrtA could significantly increase the fluorescence intensity when co-incubated with the substrate,indicating that the recombinant SrtA had good biological activity.