利用家蚕杆状病毒表达家蚕肌质网型钙离子ATP酶蛋白

Expression of sacro/endoplasmic reticulum Ca2+-ATPase from Bombyx mori by baculovirus expression system

摘要:

肌质网型钙离子ATP酶 (Sacro/endoplasmic reticulum Ca2+-ATPase, Serca) 负责将细胞中多余的Ca2+转运并存储于内质网中，从而维持细胞内适宜的Ca2+环境。家蚕Serca创造的细胞内及细胞外Ca2+平衡对家蚕正常生命活动的维持具有重要作用。由于Serca分子量较大并具有10次跨膜结构，很难在大肠杆菌表达系统中表达。为了获得具有生物学活性的重组Serca蛋白，利用pFastBac Dual载体构建了用于表达egfp和serca的双元杆状病毒表达载体，转染细胞后获得重组病毒，将重组病毒感染细胞后，成功地在细胞中表达了EGFP和Serca。通过荧光观察及Western blotting分析表明，感染后细胞中Serca和EGFP表达模式一致，从感染后48 h开始表达，在感染后96 h表达量最大。对获得的重组蛋白进行酶活分析，发现感染后48 h至120 h的细胞Serca酶活显著提高。表明具有生物学活性的Serca在此系统中成功获得表达，为深入研究Serca蛋白的功能奠定了基础。

Abstract:

Sacro/endoplasmic reticulum Ca2+-ATPase (Serca) is responsible for transporting Ca2+ into the endoplasmic reticulum and maintaining a suitable calcium environment in cells. The suitable calcium environment created by BmSerca is vital for the growth and development of silkworm. With a large molecular weight and 10 transmembrane domains, Serca is very difficult to express in Escherichia coli expression system. In order to obtain recombinant Serca with biological activity, pFastBac Dual vector was used to construct a binary baculovirus expression vector for expressing egfp and serca in cells. After transfection and infection, EGFP and Serca were expressed successfully in BmN-SWU1 cell line. Fluorescent observation revealed that the expression patterns of EGFP and Serca in infected cells were the same. Western blotting analysis showed that the recombinant proteins were about to express in cells 48 h post infection and highly expressed 96 h post infection. Ca2+-ATPase activities assays were used to evaluate the enzyme activities of recombinant Serca and found that the enzyme activities increased significantly after infection. The obtained data showed that this binary baculovirus expression system can be successfully used to express Serca with biological activity. The expression of Serca protein with this system is useful for further research on the function of Serca.