恶性疟原虫FCC1／HN株环子孢子蛋白的原核表达、纯化及其对肝细胞靶向作用的观察

Expression and purification of circumsporozoite protein from Plasmodium falcipanan FCC1/HN strain and its binding activity in hepatocyte

目的从恶性疟原虫FCC1／HN株基因组中扩增得到环子孢子蛋白(circumsporozoite protein,CSP)的全长编码基因,克隆至原核表达载体pET-32c中进行表达纯化,观察重组环子孢子蛋白对肝细胞的结合能力,探讨其作为原发性肝癌基因治疗的靶向分子的可行性。方法根据恶性疟原虫3D7株环子孢子蛋白的编码序列设计一对引物,利用聚合酶链反应(PCR)技术从恶性疟原虫FCC1／HN株基因组中扩增出CSP的全长编码基因,将其克隆到载体pGEM-T中,通过基因测序加以证实;进一步亚克隆至原核表达载体pET-32c中,在大肠杆菌BL21中用IPTG诱导表达,表达产物用Ni2+螯合柱亲和纯化,采用免疫印迹技术(WB)对纯化的融合蛋白进行免疫反应性检测;采用免疫组化(IHC)技术观察重组环子孢子蛋白对不同组织细胞的结合能力。结果从恶性疟原虫FCC1／HN株基因组中成功扩增到1263 bp的全长CSP基因,该基因在原核系统中经诱导表达出一相对分子质量(Mr)约62×103大小的融合蛋白,表达产物以包涵体的形式存在;通过Ni2+亲和柱纯化获得重组CSP融合蛋白;WB表明,重组CSP融合蛋白能被疟原虫阳性血清特异性识别;免疫组化结果显示重组CSP融合蛋白能够与肝癌和正常肝细胞特异性地结合,与其他组织来源的细胞则未见反应。结论CSP是疟原虫子孢子表面主要的蛋白,重组环子孢子蛋白作为原发性肝癌基因治疗的靶向分子具有一定的潜在应用价值。

Objective To amplify circumsporozoite protein geue from genome DNA of Plasmodium falciparum FCC1/HN strain, to express and purify CSP in prokaryotic vector pET-32c, with evaluation of its immunogenecity and binding activity to hepatic cell. Methods A pair of primers was designed according to cDNA sequence of CSP from Plasmodium falciparum strain 3D7, the CSP geue amplified by PCR was cloned into the vector pGEM-T and proved by DNA sequencing. Then the CSP geue was subcloned into procaryotic expression vector pET-32c and expressed by induction with IPTG. The recombinant CSP was purified with Ni2^＋ chelating HiTrap HP column in denatured condition. Its immunoresponse was evaluated by Western blot, and its binding activity in different tissue sections was detected by immunohistochemistry （IHC）. Results The CSP geue was successfully amplified from genome DNA of Plasmodium falciparum strain FCC1/HN. An approximate Mr 62×10^3 fusion protein was expressed in E.coli and prepared with Ni^2＋ column purification. The result of Western blot showed that rCSP was recognized by Malaria patient serum. The results of IHC showed that rCSP could bind specifically to the hepatoeytes. Conclusion The purified rCSP has strong immunoresponse and binding activity in hepateeytes, which indicates that rCSP has a potential value as a targeting molecule for hepatic geue delivery.