日本血吸虫钙网织蛋白在Sf9昆虫细胞中的表达及其活化树突细胞的初步分析

EXPRESSION OF SCHISTOSOMA JAPONICUM CALRETICULIN PROTEIN IN SF9 INSECT CELLS AND PRELIMINARY ANALYSIS OF ITS ROLE IN THE ACTIVATION OF DENDRITIC CELLS

为获得在Sf9昆虫细胞中表达的日本血吸虫钙网织蛋白(Schistosoma japonicum calreticulin protein,SjCRT)并分析其活化小鼠骨髓来源树突状细胞的功能,将构建的重组杆状病毒转移载体pFastBacHTA-SjCRT转入DH10Bac细胞,得到重组穿梭质粒reBacmid-SjCRT,再转染到Sf9昆虫细胞,进行重组蛋白的表达。用Western blot和间接免疫荧光对表达蛋白进行鉴定。His柱亲和层析法纯化表达的蛋白,Westen blot鉴定纯化后的蛋白。从BALB/c小鼠产生骨髓来源的树突细胞mDCs,用纯化的重组SjCRT蛋白与mDCs共培养,流式细胞术检测mDCs细胞的表面分子MHCⅡ、CD40和CD86的表达。结果显示,在Sf9昆虫细胞中成功表达了SjCRT蛋白;纯化后的重组SjCRT蛋白既能被感染日本血吸虫42 d的兔阳性血清识别,也能被原核表达的重组SjCRT蛋白免疫鼠的血清所识别。流式细胞术结果显示,与对照组的相比,SjCRT蛋白刺激组mDCs细胞表面分子MHCⅡ和CD86的表达量显著增强(P<0.05)。可见,在Sf9昆虫细胞中表达的SjCRT蛋白能刺激小鼠骨髓来源树突细胞表型的成熟。

To obtain Schistosomajaponicum calreticulin protein （SjCRT） and analyze its function in the activation of mouse bone marrow- derived dendritic cells （DCs）, the recombinant baculovirus transfer vector pFastHTA-SjCRT was transformed into competent cells of E.coli DH10Bac. The purified recombinant shuttle plasmid was then transfected into Sf9 insect cells. The presence of SjCRT was confirmed in Western blot and immunofluorescent assay. The recombinant SjCRT was purified by using His,Tag fusion protein purification method and detected in Western blot. Immature DCs were obtained and sorted by conventional method and stimulated with the recombinant SjCRT.The expression levels of DC surface markers MHC ]] , CD40 and CD86 were assayed by flow cytometry. The results showed that the recombinant SjCRT was expressed in Sf9 insect cells. The purified protein reacted in Western blot and immunofluorescent assay with both rabbit antiserum collected at day 42 post infection with Schistosoma japonicum and mouse antiserum prepared using the recombinant SjCRT protein from the prokaryotic E. coli expression system. In flow cytometry, the recombinant SjCRT significantly promoted up- regulation of the MHC II and CD86 expression （P〈0.05） in DCs.