伯氏疟原虫CSP基因N端蛋白靶向肝细胞的研究

Specific binding of the N terminus of the circumsporozoite protein fromPlasmodium berghei to hepatocytes

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 identify the N terminus of the circumsporozoite protein （CSP） from Plasmodiurn berghei in order to target hepatocytes from normal mice in vitro and in vivo by binding to t-IPSGs on the membrane of hepatocytes. Methods The N terminus of CSP was subjected to PCR, and the product, PbCSP, was cloned into a pET-28a（＋） vector. The recombinant plasmid was transformed into E. coli BL21/DE3. Expression of the fusion protein was then in- duced with IPTG. The fusion protein with a 6× his tag was purified using affinity column chromatography and analyzed using SDS-PAGE and Western blotting. A pull-down assay verified that the fusion protein bound to hepatocytes via heparan sulfate proteoglycan （HSPG）, a receptor on the surface of hepatocytes. Immunohistochemistry and biodistribution studies were used to evaluate the ability of the recombinant protein to target hepatocytes from normal mice in vitro and in vivo. Results The pET28α-PbCSP-N vector was successfully constructed. The fusion protein was purified and pro- duced a single band of about 15 ku in SDS-PAGE. The fusion protein was recognized by anti-His antibodies and anti-PbC- SP-N recombinant protein antibodies. The pull-down assay indicated that the combination of the PbCSP-N recombinant protein and membrane proteins of hepatocytes from normal mice were recognized by both anti-His antibodies and anti- HSPG antibodies, suggesting that the PbCSP-N recombinant protein binds to HSPGs on the surface of hepatocytes. Im- munohistochemistry revealed that the PbCSP-N recombinant protein was markedly localized on the surface of hepatocytes, indicating that the recombinant protein binds to surface of hepatocytes. After the PbCSP-N recombinant protein was la- belled with [125]I-sodium iodine and injected into normal mice, it was found mainly in the liver after 1 h, 2 h, 4 h, 6 h, and 12 h. The PbCSP-N recombinant protein was found to specifically target the liver in vivo. Conclusion The N terminus of CSP targeted hepatocytes in vitro and in vivo by binding to HPSGs on the membrane of hepatocytes, suggesting that the N terminus of CSP may be a hepatocyte-targeting molecule.