中国明对虾serpin型丝氨酸蛋白酶抑制剂基因的重组表达及活性分析

Recombinant expression and functional characterization of a Serpin-type serine proteinase inhibitor(Fc-serpin) from the Chinese shrimp(Fenneropenaeus chinensis)

Serpin家族成员作为主要的丝氨酸蛋白酶抑制剂,通过调节一系列丝氨酸蛋白酶和半胱氨酸蛋白酶的活性来调节蛋白酶级联反应,进而参与了包括血液凝结、补体激活、纤维蛋白溶解、炎症反应、肿瘤抑制和激素转运等大量的基本生物过程,在调节机体免疫及其它重要生理功能等方面发挥着重要作用。在前期研究中,从中国明对虾血细胞中克隆得到一个serpin型丝氨酸蛋白酶抑制剂基因(Fc-serpin,GenBank登录号:DQ318857),初步研究显示它在转录水平参与了病原菌和白斑杆状病毒(WSSV)引发的中国明对虾先天免疫应答反应;利用原核重组表达系统,成功获得了重组的中国明对虾serpin型丝氨酸蛋白酶抑制剂(rFc-serpin),纯化复性后得率为0.3 g/L;活性分析显示,重组目的蛋白(rFc-serpin)对多种病原菌的生长均有一定抑制作用,研究结果进一步证实它参与病原微生物引发的对虾防御应答过程,为探讨甲壳动物先天免疫防御应答机制提供了有价值的参考。

Serine proteinase inhibitors （SPI）play an important role in regulating humoral immunity by controlling the activity of serine proteinase （SP） in the process of protease cascade. Serpin family members participate in numerous biological processes by regulating the activity of serine and cysteine proteinases, such as blood clotting,complement activation, fibrinolysis function, inflammation, tumor suppressor and hormone transfer process. In our previous research, we cloned a serpin-type serine proteinase inhibitior gene from hemocytes of Chinese shrimp （ Fc-serpin, GenBank accession number： DQ318857 ）. The transcript of Fc- serpin was induced in response to the infection of bacteria and white spot syndrome virus （WSSV）. In this research,recombinant Fc-serpin（rFc-serpin）was slaccessfully expressed in bacteria E. coli and purified for further research of bioactivity. The concentration of purified target proteins was 0.3 g/L. rFc-serpin showed in vitro antimicrobial activity against main pathogens including Gram-positive, Gram-negative bacteria and fungi. The results suggest that rFc-serpin might play a crucial role in the innate immunity of the shrimp and is expected to be applied in disease control.