Immuno-molecular study on the role of eosinophil granule basic protein in experimental Angiostrongyliasis cantonensis

我们先前的数据表明，脑脊液中增加的嗜酸性粒细胞参与了广州管圆线虫感染小鼠颅内蠕虫的杀灭。然而，目前还不清楚嗜酸性粒细胞释放出哪些效应分子来杀死这些蠕虫。因此，在本研究中，我们试图生产重组主要碱性蛋白-1(MBP-1)，这是一种含有嗜酸性粒细胞颗粒的碱性蛋白。从感染小鼠或IL-5转基因小鼠的骨髓中提取总RNA，逆转录为cDNA。用聚合酶链式反应扩增小鼠成熟MBP-1基因，编码C-端到N-端(GenBank L46768，Larson等，1995)。将MBP-1基因连接到载体pIVEX2.3和pIVEX2.4a、b、c中，构建了含C端His标签基因和N端His标签基因的融合表达载体pIVEX2.3。序列分析表明，除1个碱基外，其余序列均与GenBank L46768的序列一致。与MBP-1c DNA连接的3个质粒为ta…更多地与大肠杆菌形成，并通过大规模的质粒制备方法进行扩增。将纯化的pIVEX2.3和pIVEX2.4c应用于体外蛋白质表达系统(快速翻译系统RTS 500，罗氏分子生物化学公司)。然而，获得成熟的MBP-1蛋白仍然是不成功的，提示MBP-1可能具有高度的细胞毒性。接下来，我们检查了MBP在颅内蠕虫表面或脑组织中的沉积情况。兔抗鼠MBP多克隆抗体由James Lee博士在美国Mayo Clinic Scottsdale Research提供。雄性C57BL/6小鼠感染24只第3期幼虫，于第8~22天处死。在尸检时，将大脑固定在福尔马林中，并制备石蜡切片。根据李博士实验室的方案，对MBP进行了免疫组织化学染色。蛛网膜下腔内可见明显的嗜酸性粒细胞浸润，嗜酸性粒细胞周围分布有MBP颗粒。有趣的是，发现退化的颅内蠕虫被大量的嗜酸性粒细胞包围，并且MBP沉积在蠕虫的表皮表面。这些结果表明MBP可能是嗜酸性粒细胞杀虫的效应分子之一。较少

Our previous data show that eosinophils increased in cerebrospinal fluid are involved in the killing of intracranial worms in mice infected with Angiostrongylus cantonensis. However, It is still unclear what effector molecules are released from eosinophils to kill the worms. In this study, therefore, we attempted to produce recombinant major basic protein-1 (MBP-1), one of basic proteins containing eosinophil granules. Total RNA was extracted from bone marrow of infected mice or IL-5 transgenic mice and reverse-transcribed to cDNA. Mouse mature MBP-1 cDNA coding from C-terminal to N-terminal (GENBANK L46768, Larson et al., 1995) was amplified by PCR. The MBP-1 cDNA was ligated into the plasmid vectors, pIVEX2.3 fusing the gene with a C-terminal his-tag and pIVEX2.4a, b, c with a N-terminal his-tag gene. Sequencing analysis showed that all sequences, except to one base (silent after translation), were identical with those of GENBANK L46768. Three plasmids ligated with MBP-1 cDNA were ta … More nsformed with E. coli and amplified by large scale plasmid preparation method. Purified pIVEX2.3 and pIVEX2.4c were applied on in vitro protein expression system (Rapid Translation System RTS 500, Roche Molecular Biochemicals). However, It is sill unsuccessful to get mature MBP-1 protein, suggesting that MBP-1 may be highly cytotoxic. Next, we examined a deposition of MBP on the surface of intracranial worms or on the brain tissure. Rabbit anti-mouse MBP polyclonal antibody was kindly provided by Dr. James Lee in Mayo Clinic Scottsdale Research in USA. Male C57BL/6 mice were infected with 24 third stage larvae and killed from days 8 to 22 p.i. At necropsy, brains were fixed in formalin and paraffin sections were prepared. According to the protocol in Dr. Lee's lab, MBP was immunohistochemically stained. Prominent eosinophil infiltration was noted in pia-arachnoid and MBP particles were distributed around eosinophils. Interestingly, degenerating intracranial worms were found surrounded by numerous eosinophils and MBP was deposited on the cuticular surface of the worms. These results indicate that MBP is one of possible effector molecules from eosinophils for worm killing. Less