牛蛙凝集素受体及其药物靶向载体的初步研究

Lectins are sugar-binding proteins that specifically recognize sugar complexes.Based on the specificity of protein-sugar interactions,different lectins could be used as carrier molecules to target drugs specifically to different cells which express different glycan arrays. In spite of lectin interesting biological potential for drug targeting and delivery,a potential disadvantage of natural lectins may be large size molecules than 10KD. A lectin-like peptide named catesbeianalectin was identified from the cDNA library of rana catesbeiana. Erythrocyte agglutination activity detection showed that a lectin of Rana catesbeiana with the molecular weight of 1470, which is smaller than any other known lectin from amphibian, has very low homology with the other lectin. catesbeianalectin had very high rabbit erythrocyte agglutination activity with the minimum concentration 12.5μg/mL. It was also indicated that it had significant bacterial agglutination activity by laser scanning confocal microscope for Staphyloccocus aureus, streptococcus suis, Salmonella, Actinobacillus pleuropneu moniae.the small size and single disulfide bridge of catesbeianalectin make it easy to manipulate for developing as drug targeting system.Its structure and function were studied by SPR,125I-catesbeianalectin was stable in mice plasma.these findings identify the smallest lectin so far,and show the application potential of catesbeianalectin for drug delivery and targeting.It also disclosed a new strategy of amphibian anti-infection.

凝集素具有对某一种特异性糖基专一性结合的能力，生物体不同的器官与组织含有不同的糖基，凝集素可以专一地与不同的器官或组织相结合，因此凝集素可以作为研究细胞膜结构的探针、药物靶向输送和定位的载体。目前已知的凝集素分子量都大于10KD，且从两栖动物中发现的凝集素非常有限。本研究通过筛选牛蛙皮肤cDNA文库得到分子量为1470的牛蛙凝集素Catesbeianalectin，多肽合成牛蛙凝集素，测定其对红细胞及临床常见致病菌的凝集活性。圆二色谱测定不同溶液环境对多肽结构的影响，并利用表面等离子共振（SPR）筛选出与牛蛙凝集素特异性结合的糖基配体，确定其分子结构。体内试验以同位素示踪法检测牛蛙凝集素在小鼠体内的稳定性及组织专一性，以牛蛙凝集素为基础的药物设计阻断病原体对受体细胞的粘着与药物靶向提供初步的理论基础。

凝集素是能够凝集红细胞、与糖蛋白或糖链特异性结合的生物活性肽，其中小分子量凝集素由于低毒性和低免疫原性可作为理想的药物靶向载体分子。目前海洋生物及甲壳类动物凝集素报道较多，且大多数分子量大于10kD。牛蛙凝集素Catesbeianalectin是首次从牛蛙皮肤发现的小分子量的活性多肽，分子量为1.47kD，是目前已知的分子量最小的凝集素，但有关Catesbeianalectin的生物学活性及其动物体内的代谢与分布还未见报道。本研究多肽合成牛蛙凝集素Catesbeianalectin，二级结构为聚脯氨酸2型（PPⅡ）螺旋。用Catesbeianalectin、麦胚凝集素（WGA）（阳性对照）、卵清蛋白（OVA）（阴性对照）免疫Balb/c小鼠检测其免疫原性，包被浓度分别为80 μg/mL、0.008 μg/mL、0. 25 μg/mL时，血清效价分别为：1:25、1:1600、1:200，表明Catesbeianalectin免疫原性较低。利用表面等离子共振成像(SPRi) 技术检测了Catesbeianalectin特异性结合的糖基配体，将30种供试单糖以浓度5 mmol/L固定于芯片，与其特异性结合的糖基配体为半乳糖或含有半乳糖基的多糖，即为S-型凝集素。利用125I标记Catesbeianalectin研究其在小鼠体内的代谢与分布，通用尾静脉注射、灌胃、腹腔注射三种方式并于随后既定的时间点动态显像、采血及各组织中放射性强度的测量。其中尾静脉注射125I-Catesbeianalectin，肝脏、脾脏和肺脏中放射性含量最高且主要通过肝脏代谢，灌胃及腹腔注射125I-Catesbeianalectin，胃及脾脏中放射性含量最高且主要通过胃代谢，三种途径给予125I-Catesbeianalectin后，均不能通过血脑屏障，为Catesbeianalectin的药物靶向载体作用研究提供基础。为提高Catesbeianalectin的靶向作用，将其与改造肽Temporin-K(E)G(I)进行偶联设计得到C-TeK(E)G(I)，采用MTT法探究Catesbeianalectin、Temporin-K(E)G(I)、C-TeK(E)G三种多肽对小鼠肝癌细胞Hepa1-6、人肝癌细胞Hepg2和人肺腺癌细胞H460的作用，均呈浓度依赖性且对肿瘤细胞具有较强的抑制作用。