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目的研究伯氏疟原虫CSP基因的N端蛋白与小鼠肝细胞膜HPSGs受体结合后在体外、体内的肝组织靶向性。方法将伯氏疟原虫CSP基因N端片段(PbCSP-N)克隆入原核表达质粒pET-28α,经IPTG诱导表达重组蛋白,经亲和层析纯化后采用Western blot方法鉴定;采用Pull-down方法检测PbCSP-N蛋白与小鼠肝细胞表面受体硫酸乙酰肝素糖蛋白(Heparan sulfate proteoglycan,HSPG)的结合力;应用免疫组化、放射性碘标记示踪方法检测该蛋白在体外、体内特异性靶向小鼠肝组织作用。结果成功构建PbCSP-N原核表达质粒并表达、纯化出PbCSP-N重组蛋白(15ku),Pull-down方法证实该蛋白可与小鼠肝细胞膜HPSGs受体结合,免疫组化试验显示该重组蛋白定位于正常小鼠肝细胞膜,表明该蛋白可特异结合正常小鼠肝细胞膜;放射性碘(125I)标记示踪显示,标记的重组蛋白在注射1、2、4、6、12h后在小鼠肝脏分布较多,表明该重组蛋白可靶向正常小鼠肝组织。结论伯氏疟原虫CSP基因N端蛋白与肝细胞膜HPSGs受体结合后在体内、外靶向肝细胞,可作为肝细胞靶向性药物候选分子。
Objective To investigate the targeting of hepatic tissue in vitro and in vivo after the binding of N-terminal protein of CSP gene of Plasmodium berghei and HPSGs receptor in mouse hepatocyte membrane. Methods Plasmodium berghei CSP gene N-terminal fragment (PbCSP-N) was cloned into prokaryotic expression plasmid pET-28α. The recombinant protein was induced by IPTG and purified by affinity chromatography. The recombinant protein was identified by Western blot. The binding ability of PbCSP-N to Heparan sulfate proteoglycan (HSPG) was detected by immunohistochemistry and radioiodine labeling method. The specific target of PbCSP-N was detected in vitro and in vivo To mouse liver tissue effect. Results The PbCSP-N prokaryotic expression plasmid was successfully constructed and purified. The recombinant protein PbCSP-N (15 ku) was purified and identified by pull-down assay. The results showed that this recombinant protein could bind to the HPSGs receptor of mouse hepatocyte membrane. Immunohistochemistry showed that the recombinant protein was localized The results of radioactive iodine (125I) labeling showed that the labeled recombinant protein was distributed in the liver of mice at 1, 2, 4, 6 and 12 h after injection More, indicating that the recombinant protein can target normal mouse liver tissue. Conclusion The CSP gene of P. berghei C-terminal N-terminal protein binds to the HPSGs receptor of hepatocyte membrane and targets hepatocytes in vivo and in vitro, which can be used as hepatocyte-targeting drug candidate molecules.