人血清对氧磷酶1在家蚕杆状病毒表达系统中的表达及活性检测

Expression and Activity Determination of Human Serum Paraoxonase 1 in Silkworm Baculovirus Expression System

对氧磷酶1(paraoxonase 1,PON1)是生物体内存在的一种广谱的非专一性酯酶,在水解有机磷杀虫剂及降解神经毒剂中发挥重要作用。利用已构建的含人血清pon1基因的原核表达载体pET-28a-pon1,在E.coli中诱导表达人血清PON1,通过Ni-NTA亲和层析纯化后制备兔多克隆抗体;利用Bac-to-Bac系统构建重组杆状病毒vBm-pon1,并感染家蚕5龄幼虫,经SDS-PAGE和Western blotting检测鉴定人血清PON1在蚕体内成功表达,并获得了纯度较高的重组人血清PON1。以对氧磷为底物,对2个表达系统获得的重组人血清PON1的酶活性进行检测,结果表明在家蚕幼虫体内表达并纯化的重组人血清PON1的酶活性要明显高于在E.coli中表达的人血清PON1蛋白。推测家蚕杆状病毒表达系统比原核表达系统多了糖基化、磷酸化等蛋白质修饰功能。进一步试验验证家蚕幼虫表达的重组人血清PON1去糖基化后其酶活性丧失,表明糖基修饰对于维持PON1的生物学功能至关重要。

Paraoxonase 1 （PON1） is a kind of non-specific broad-spectrum esterase within the organisms. It plays an important role in the hydrolysis of organophosphorus pesticides and degradation of nerve poisons. The prokaryotic ex- pression vector pET-28a-pon1 containing human serum ponl gene was used to express PON1 in E. coil The target pro- tein purified by Ni-NTA affinity column was used to prepare rabbit polyclonal antibody. Meanwhile, the recombinant bacu- Iovirus vBm-ponl was constructed using Bac-to-Bac system and used to infect the 5th instar silkworm （ Bombyx mori） larvae, SDS-PAGE and Western blotting verified that the target protein was successfully expressed in silkworm larvae andhigh purity recombinant protein was obtained. The biological activity of target protein expressed in these two expression systems were detected using paraoxon as substrate. The results indicated that the biological activity of PON1 expressed in silkworm larvae was obviously higher than that expressed in prokaryotic system, which was presumably due to the glycosylation and phosphoryl- ation in the baculovirus expression system. Further analysis revealed that the deglycosylation of PON1 lead toloss of its catalytic activity, which indicated that glycosylation was essential for maintaining the biological function of PON1.