Target-directed isolation and identification of a serum lectin from lamprey (Lampetra japonica) by chromatographys and MALDI-TOF/TOF
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摘要: 本研究利用阳离子交换和分子筛的方法成功分离鉴定出分子量为105kDa，以多聚体形式存在七鳃鳗凝集素。实验结果表明，首先运用阳离子交换色谱方法能够将七鳃鳗血清混合组分进行初步分离，结合凝集活性鉴定方法，鉴定凝集活性最强组分存在于第二峰中。其次；利用分子筛方法将具有凝集活性组分进一步分离，最终得到分子量为105kDa左右具有凝集活性组分。结合Native-PAGE和SDS-PAGE结果表明，凝集活性组分以三聚体形式存在。同时，飞行时间质谱方法鉴定结果为凝集素（gi:13094239）。最终，体外鉴定结果表明七鳃鳗凝集素对兔红细胞和山羊红细胞均具有较强的凝集活性。七鳃鳗凝集素的分离纯化对研究七鳃鳗先天免疫防御机制和可变淋巴受体介导的适应性免疫防御机制均具有重要意义。Abstract: A 105-kDa polymer lectin was purified from lamprey (Lampetra japonica) serum by chromatography methods including cation ion-exchange chromatography with a SP-SepharoseTM XL column and size exclusion chromatography with a Superdex 200 column. The target fractions were collected according to the direction of hemagglutinating activity. The results revealed that the active fractions could adsorb on SP-Sepharose column and showed a 280 nm UV absorbance peak corresponding to molecular weights of 105 kDa in the following size exclusion chromatography. The target fractions with hemagglutinating activity were further checked by Native-PAGE and SDS-PAGE. Two single bands at around 105 kDa and 35 kDa were displayed by two electrophoresis methods respectively, indicating that the protein exists as a trimer in solution. After Native-PAGE and SDS-PAGE, two bands were excised from the gels respectively and further identified by MALDI-TOF/TOF as serum lectin (gi:13094239). The lectin was able to agglutinate rabbit red blood cells (RRBCs) and sheep red blood cells (SRBCs) in vitro. The lectin isolated from lamprey serum in the current study might be helpful for deeply understanding the innate immune molecules dependent immune defence in jawless vertebrates which have been proved recently that they possess a lymphocyte-based system of anticipatory immunity with variable lymphocyte receptors as mediators.
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Key words:
- Lampetra japonica /
- lectin /
- purification /
- identification /
- MALDI-TOF/TOF
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Cooper M D, Alder M N. 2006. The evolution of adaptive immune systems. Cell, 124(4):815-822 Guo Peng, Hirano M, Herrin B R, et al. 2009. Dual nature of the adaptive immune system in lampreys. Nature, 459(7248):796-801 Guo Xiaonv, Jin Xingkun, Li Shuang, et al. 2013. A novel C-type lectin from Eriocheir sinensis functions as a pattern recognition receptor with antibacterial activity. Fish Shellfish Immunol, 35(5):1554-1565 Kasamatsu J, Sutoh Y, Fugo K, et al. 2010. Identification of a third variable lymphocyte receptor in the lamprey. Proc Natl Acad Sci USA, 107(32):14304-14308 Ogawa T, Watanabe M, Naganuma T, et al. 2011. Diversified carbohydrate-binding lectins from marine resources. J Amino Acids, 2011:838914 Ourth D D, Rose W M, Siefkes M J. 2008. Isolation of mannose-binding C-type lectin from sea lamprey (Petromyzon marinus) plasma and binding to Aeromonas salmonicida. Vet Immunol Immunopathol, 126(3-4):407-412 Rutishauser U, Sachs L. 1975. Cell-to-cell binding induced by different lectins. J Cell Biol, 65(2):247-257 -
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