伯氏疟原虫CSP片段与肝细胞特异结合的生物学特性

Binding of the liver cells specially by Cirumsporozoite protein fragment from P.berghei as a membrane protein expressed in Hela cells

目的明确去除伯氏疟原虫CSP基因的中央重复序列不影响该蛋白的膜定位及与肝细胞特异结合的特性。方法将去除中央重复序列的伯氏疟原虫CSP基因片段(PbCSP’)克隆入原核表达质粒pGEX-4T-1,在大肠杆菌BL21/DE3中诱导表达,纯化重组蛋白,免疫大鼠获得相应抗体;用流式细胞仪筛选表达EGFP-PbCSP’的Hela细胞,应用Confocal显微镜及免疫组化方法观察表达的蛋白是否定位于细胞膜及能否与小鼠肝癌细胞(H22)特异结合。结果 pGEX-PbCSP’的原核表达及纯化、免疫大鼠获得抗体,在Hela细胞表达的EGFP-PbCSP’蛋白定位于细胞膜,并能与小鼠肝癌细胞(H22)特异结合。结论去除中央重复序列的伯氏疟原虫CSP片段与肝细胞特异结合,为进一步研究其是否可作为肝细胞的靶向分子奠定了基础。

Objective To identify CSP（cirumsporozoite protein）without central repeat region from Plasmodium berghei being expressed and localized on the membrane of Hela cells and bind the liver cells from mouse specially. Methods The PCR products of PbCSP＇ without central repeat region were cloned into pGEX-4T-1 vector and the fusion protein was purified. The antibody of GST-PbCSP＇ was obtained from immuned the rats. pEGFP-PbCSP＇ vecor was transfected into Hela cells and the localization of the fusion protein was identified by confocal microscope and immunohistochemistry. The Hela cells expressing EGFP-PbCSP＇ protein were separated and incubated with H22 cells, spleen cells and marrow cells of mouse to observe their binding,respectively. Results pGEX-PbCSP＇的原核表达及纯化、免疫大鼠获得抗体,EGFP-PbCSP＇ protein was expressed and localized on the membrane of Hela cells. The Hela cells could bind H22 cells specially. Conclusions PbCSP fragment without central repeat region could target to the liver cells.