凋亡蛋白质的原核表达与纯化

Expression, Purification and Activity Detection in vitro of Apoptin

目的构建来自鸡贫血病毒(CAV,chicken anaemia virus)VP3基因编码的凋亡蛋白质表达载体,在大肠杆菌中进行表达,并研究凋亡蛋白质的纯化和稳定性。方法PCR(polymerase chain reaction)获得正确编码VP3基因片断,构建表达质粒pET-28a-VP3在E.coli BL21(DE3)中表达。用Ni-NTA柱纯化可溶性目标蛋白质,并研究不同条件下凋亡蛋白质的稳定性。结果目标蛋白质在大肠杆菌中获得表达,纯化后蛋白质纯度高于90%,卡拉胶可明显提高重组蛋白质可溶状态下的稳定性。结论成功获得高纯度VP3可溶性蛋白质,增加了目标产物的稳定性,为进一步研究该蛋白质的临床应用奠定基础。

Objective To construct an soluble expression system in E. coli for apoptin which was encoded by VP3 gene of chicken anemia virus （CAV）, and study the purification and stabilization of apoptin. Methods The VP3 gene was amplified by PCR, then the segment was inserted into pET-28a （＋） and the expression vector pET-28a- VP3 was constructed in E. coli BL21（DE3）. The recombinant soluble protein was expressed in E. coli BL21（DE3） and purified by Ni-NTA column chromatography. The stability of apoptin was analyzed under different conditions. Results The target protein was expressed in E. coli BL21（DE3）. The purity of apoptin was beyond 90 %after purification. The target protein was better stabilized by carrageenan in E.coli BL21（DE3）. Conclusion Apoptin with high purity and stability can be successfully obtained, which do a base of the further study on clinic application of apoptin.