抗肿瘤蛋白Onconase的原核细胞表达及细胞毒性检测

本文主要利用pET22b( )表达载体在大肠杆菌中表达一种在两栖类蛙(Ranapipiens)卵细胞内存在的核糖核酸酶-Onconase。通过包涵体复性、蛋白纯化等程序最终获得与天然蛋白活性相似的重组蛋白，并检测Onconase对源于皮肤T细胞淋巴瘤的Hut-78肿瘤细胞的毒性(IC50=0．51μmol／L)，证明Onconase用于抗淋巴瘤的可行性。

Effective expression of ribonuclease-onconase in Escherichia coli and assaying its cytotoxic potential]

Onconase, a ribonuclease purified from Rana pipiens oocytes, has cytotoxic activity against several tumor cell lines in vitro. With the characteristic of containing four pairs of disulfide bonds internal and N terminal sequence attributing mostly to its biological functions, it is difficult to obtain the active Onconase from Escherichia coli expression system with normal strategy. Here, we fused the cDNA coding for Onconase in frame with the pelB signal sequence in pET22b(+) expression vector. Onconase can be effectively expressed in inclusion body without additional residues at N terminal under the proper inducing condition. After refolding and purifying process, we can get the recombined Onconase which has a similar ribonuclease activity as the one isolated from oocytes. The recombined Onconase has a pronounced effect on proliferation of Hut-78 cells (IC50=0.5 micromol/L).