新牛蛙凝集素Catesbeianalectin的免疫原性分析及其特异性糖基配体的筛选

Analysis of immunogenicity and screening of specific saccharide ligand of Catesbeianalectin

目的分析新发现的牛蛙凝集素Catesbeianalectin的免疫原性,并筛选其体外特异性结合的糖基配体。方法多肽合成牛蛙凝集素Catesbeianalectin,利用圆二色谱(circular dichroism,CD)检测其二级结构。分别用牛蛙凝集素、麦胚凝集素(wheat germ lectin,WGA)、卵清蛋白(ovalbumin,OVA)免疫BALB/c小鼠,检测血常规与血清效价,分析牛蛙凝集素的免疫原性。表面等离子共振成像(surface plasmon resonance imaging,SPRi)技术筛选30多种与牛蛙凝集素特异性结合的糖基配体。结果牛蛙凝集素Catesbeianalectin为聚脯氨酸2型(PPⅡ)螺旋结构。OVA包被浓度为0.25μg/ml时,小鼠平均血清效价为1∶200;WGA包被浓度为0.008μg/ml时,小鼠平均血清效价为1∶1 600,牛蛙凝集素Catesbeianalectin包被浓度为80μg/ml时,小鼠血清平均效价为1∶25。当30种单糖以浓度5 mmol/L固定于芯片,牛蛙凝集素Catesbeianalectin以浓度0.4 mg/ml通过固定于芯片的糖表面时,主要与半乳糖基和含有半乳糖基的多糖作用。结论牛蛙凝集素Catesbeianalectin免疫原性较低,为S-型凝集素,体外可与带有半乳糖基的多糖结合,为进一步研究牛蛙凝集素Catesbeianalectin的药物靶向载体作用奠定了基础。

Objective To analyze the immunogenicity of Catesbeianalectin, a novel bullfrog lectin, and screen its specific saccharide ligand in vitro. Methods Catesbeianalectin was synthesized, of which the secondary structure was determined by circular dichroism（CD）. BALB / c mice were vaccinated with Catesbeianalectin, wheat germ lection（WGA） and ovalbumin（OVA） respectively, subjected to routine blood test and determined for serum antibody titer to analyze the immunogenicity of Catesbeianalectin. More than 30 kinds of saccharide ligands specifically binding to Catesbeianalectin were screened by surface plasmon resonance imaging（SPRi）. Results The secondary structure of Catesbeianalectin was polyproline type 2（PPⅡ）. The mean serum antibody titer was 1 ∶ 200 when the coating concentration of OVA was0. 25 μg / ml, while was 1 ∶ 1 600 when the coating concentration of WGA was 0. 008 μg / ml, and 1 ∶ 25 when the coating concentration of Catesbeianalectin was 80 μg / ml. Catesbeianalectin at a concentration of 0. 4 mg / ml mainly interacted with galactosyl and polysaccharide containing galactose when it passed though the 30 kinds of monose fixed at a concentration of 5 mmol / L on the biochip. Conclusion Catesbeianalectin showed low immunogenicity, which was a Slection binding to the polysaccharide containing galactosyl in vitro. It laid a foundation of further study on the Cates-beianalection as a drug-targeting carrier.