弓形虫MIC1蛋白在杆状病毒系统中的表达及活性分析

Purification and activity analysis of Toxoplasma gondii MIC1 protein expressed in baculovirus system

为构建可溶性表达弓形虫MIC1蛋白质的重组杆状病毒株并分析重组蛋白质的活性,通过PCR扩增弓形虫mic1基因的编码序列并连接到pFastBac 1质粒中,将重组的转移质粒转化DH10Bac感受态细胞,通过蓝白斑筛选获得重组杆粒,转染Sf9细胞后连续培养3代获得重组杆状病毒株。结果显示,Sf9细胞在感染后的第3天出现典型的细胞病变;间接免疫荧光和Western blot试验结果表明MIC1重组蛋白质在感染的Sf9细胞中成功可溶性表达;纯化的MIC1重组蛋白质不仅具有结合唾液酸乳糖的能力,同时能够刺激Balb/c小鼠产生较高水平的特异性抗体(>1∶25600)。以上结果表明,通过杆状病毒表达系统可获得具有生物学活性的弓形虫MIC1重组蛋白质。

The purpose of this study was to construct a recombinant baculovirus strain expressing soluble Toxoplasma gondii MIC1 protein and identify the biological activity of the recombinant protein.The CDS sequence of mic1 gene was amplified by PCR,ligated into the pFastBac 1 vector,and then transformed into DH10Bac cell.To obtain recombinant baculovirus,recombinant Bacmid was extracted and transfected into Sf9 cells.The transfected Sf9 cells exhibited typical lesion on the 3rd day after transfection.The results of indirect immunofluorescence assay and Western blot showed that the MIC1 recombinant protein was successfully expressed in transfected Sf9 cells.The purified MIC1 recombinant protein not only possessed the ability to bind to sialyllactose,but also could stimulate Balb/c mice to produce a high level of specific antibodies(>1∶25600).These results showed that the biologically active MIC1 recombinant protein was successfully obtained through the baculovirus expression system.