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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 clearly delete the central repeat of CSP gene of Plasmodium berghei does not affect the membrane localization of the protein and the characteristic of specific binding to hepatocytes. Methods The CSP fragment of Plasmodium berghei (PbCSP) with central repeats was cloned into prokaryotic expression plasmid pGEX-4T-1 and induced in E. coli BL21 / DE3. The recombinant protein was purified and the corresponding antibodies were obtained from the immunized rats. The Hela cells expressing EGFP-PbCSP ’were screened by flow cytometry. Confocal microscopy and immunohistochemistry were used to observe whether the expressed protein was located in the cell membrane and could specifically bind to mouse hepatoma cells (H22). Results The prokaryotic expression and purification of pGEX-PbCSP ’were carried out. The antibodies were obtained from the immunized rats. The EGFP-PbCSP’ protein expressed in Hela cells localized on the cell membrane and specifically bound to mouse hepatoma cells (H22). Conclusion The CSP fragment of Plasmodium berghei, which removes the central repeats, specifically binds to hepatocytes, which lays the foundation for further study on whether it can serve as a target molecule for hepatocytes.