蜱组胺结合蛋白与渗透肽TAT在杆状病毒表达体系中的融合表达和功能分析

Bac-to-Bac expression and biology function analysis of TAT-fused recombinant histamine-binding proteins of ticks

为真核融合表达亚洲璃眼蜱(H.asiaticum)唾液腺中的组胺结合蛋白(HBP)与渗透肽TAT,并研究其生物学活性,本研究将编码HBP的基因片段与编码渗透肽TAT基因片段通过酶切位点Eco RⅠ酶切连接后克隆至真核表达载体p Fast Bac HTa中,构建重组杆粒r Bac-HBP-TAT,转染至sf9昆虫细胞中,制备重组病毒并进行融合蛋白的表达。经SDS-PAGE、western blot和间接免疫荧光试验检测结果表明,HBP-TAT在Bac-to-Bac杆状病毒系统中获得稳定表达,融合蛋白约为31 ku。此外,组胺结合试验证明HBP-TAT具有与组胺结合的能力。本研究为研制新型蜱源抗组胺药物提供了实验依据。

In order to investigate the function of histamine binding protein （HBP） of Hyalommaasiaticum and transcriptional activator protein （TAT）, HBP and TAT were ligateed after EcoR I digestion and the product was cloned into the vector pFastHTa, which was transformed into the comptent cells of E.coli DH10Bac to construct the recombinant bacmid of rBacmid-HBP-TAT. Then, rBacmid-HBP-TAT was transfected into insect sf9 cells to generate the recombinant baculovirus, and the fusion protein of HBP-TAT was proved to be efficiently expressed in st9 cells by SDS-PAGE, western blot and immunofluorescence assay. The molecular weight of HBP-TAT was approximately 30 ku, which was consistent with the predicted size. Histamine binding assay demonstrated that HBP-TAT had activity to bind with histamine.