广州管圆线虫半乳糖凝集素-1基因的克隆、表达和凝集反应

摘要/Abstract

摘要：

目的克隆、表达广州管圆线虫（Angiostrongylus cantonensis）半乳糖凝集素-1（galectin-1）基因并分析其凝集反应。方法用Swiss Model分析galectin-1的三维结构。提取广州管圆线虫雌性成虫总RNA,根据galectin-1基因编码序列（GenBank登录号为JN133961.1）设计引物,RT-PCR扩增目的基因,亚克隆至pCold Ⅲ质粒,转化大肠埃希菌（Escherichia coli）BL21,取重组质粒进行双酶切、PCR鉴定和测序。阳性质粒经0.1 mmol/L异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达,镍柱亲和层析法纯化galectin-1重组表达蛋白后,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析重组蛋白表达情况。以广州管圆线虫全虫免疫的小鼠血清为一抗,蛋白质印迹（Western blotting）验证目的蛋白。将重组蛋白10倍稀释为5.55 × 10^-1~5.55 × 10^-5 ng/μl,显微镜观察其与新鲜的ICR小鼠血液中红细胞的凝集反应。结果 Swiss Model分析结果显示,galectin-1以非二聚体形式发挥作用。galectin-1基因RT-PCR扩增产物约为850 bp,与预期结果一致。经双酶切、PCR和测序结果显示,重组质粒pCold Ⅲ-galectin-1构建成功。SDS-PAGE结果显示,经IPTG诱导获得相对分子质量约36 000的可溶性重组融合蛋白。Western blotting分析结果显示,galectin-1蛋白能被全虫免疫的小鼠血清识别。凝集反应结果显示,galectin-1蛋白与小鼠红细胞发生明显凝集反应的最低浓度为5.55 × 10^-4 ng/μl。结论克隆的galectin-1基因能在pCold Ⅲ原核表达系统中呈可溶性表达,并与小鼠红细胞发生凝集反应。

关键词: 广州管圆线虫, 半乳糖凝集素-1, 原核表达, 凝集反应

Abstract:

Objective To clone and express the galectin-1 gene of Angiostrongylus cantonensis, and test the agglutination property of its protein. Methods The three-dimensional structure of galectin-1 was analyzed with Swiss Model. Total RNA was extracted from male worms of A. cantonensis. Primers were designed for galectin-1 based on its coding region (GenBank Accession No. JN133961.1). RT-PCR was performed, and the PCR products were subcloned to pCold Ⅲ plasmid and transduced into Escherichia coli BL21 strain. The recombinant plasmid was extracted from positive clones on LB plate containing 100 μg/ml Kanamycin, and validated with double digestion, PCR identification and sequencing. The confirmed positive clones of E. coli BL21 with the recombinant plasmid were grown in LB medium containing ampicillin (100 μg/ml, 100 μl). IPTG was added to induce expression of the plasmid. The galectin-1 recombinant protein was purified with Ni-NTA beads, and analyzed with SDS-PAGE and Western blotting using anti-serum of mouse immunized with whole worms of A. cantonensis. The agglutination reaction with red blood cells in fresh blood of ICR mouse was observed for the 10-fold serial dilutions of recombinant proteins (5.55 × 10^-1-5.55 × 10^-5 ng/μl). Results The Swiss Model analysis showed that the functional galectin-1 had a non-dimeric form. As was expected, the RT-PCR products had a size of 850 bp. Results of double digestion, PCR and sequencing showed successful construction of the pCold Ⅲ-galectin-1 plasmid. SDS-PAGE revealed expression of soluble recombinant fusion protein with molecular weight of ~36 000. Western blotting showed that the galectin-1 protein was recognized by mouse anti-serum. In addition, the minimun concentration of galectin-1 that showed significant agglutination reactions with mouse red blood cells was 5.55 × 10^-4ng/μl. Conclusion The galectin-1 clone can be expressed in the pCold Ⅲ plasmid, and its protein product has agglutination property.

Key words: Angiostrongylus cantonensis, Galectin-1, Prokaryotic expression, Agglutination

中图分类号:

R383.19

李星潘, 赵梦静, 史晓萌, 闫兰竹, 闫宝龙, 黄慧聪. 广州管圆线虫半乳糖凝集素-1基因的克隆、表达和凝集反应[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(1): 48-52.

Xing-pan LI, Meng-jing ZHAO, Xiao-meng SHI, Lan-zhu YAN, Bao-long YAN, Hui-cong HUANG. Cloning of galectin-1 gene of Angiostrongylus cantonensis and testing the agglutination property of the galectin-1 protein[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(1): 48-52.

图/表 6

图1

图2

图3

图4

图5

图6

参考文献 30

[1]	张仪, 吕山, 杨坤, 等. 我国广州管圆线虫自然疫源地分布首次调查[J]. 中国寄生虫学与寄生虫病杂志, 2009, 27(6): 508-512.
[2]	薛大燕, 阮云洲, 林宝楚, 等. 温州市一起广州管圆线虫病暴发流行的调查[J]. 中国寄生虫学与寄生虫病杂志, 2000, 18(3)176-178.
[3]	李华, 陈晓光, 沈浩贤, 等. 不同发育阶段广州管圆线虫的抗原分析[J]. 中国寄生虫学与寄生虫病杂志, 2005, 23(1): 36-39.
[4]	Leffler H, Carlsson S, Hedlund M, et al. Introduction to galectins[J]. Glycoconj J, 2004, 19(7-9): 433-440.
[5]	Cumpstey I, Salomonsson E, Sundin A, et al. Studies of arginine-arene interactions through synthesis and evaluation of a series of galectin-binding aromatic lactose esters[J]. Chembiochem, 2007, 8(12): 1389-1398.
[6]	Hasan SS, Ashraf GM, Banu N.Galectins-potential targets for cancer therapy[J]. Cancer Lett, 2007, 253(1): 25-33.
[7]	Elola MT, Wolfenstein-todel C, Troncoso MF, et al. Galectins: matricellular glycan-binding proteins linking cell adhesion, migration, and survival[J]. Cell Mol Life Sci, 2007, 64(13): 1679-1700.
[8]	Barrow H, Rhodes JM, Yu LG.The role of galectins in colorectal cancer progression[J]. Int J Cancer, 2011, 129(1): 1-8.
[9]	Delgado VM, Nugnes LG, Colombo LL, et al. Modulation of endothelial cell migration and angiogenesis: a novel function for the “tandem-repeat” lectin galectin-8[J]. FASEB J, 2011, 25(1): 242-254.
[10]	沈歆, 曹蔚, 王四旺. 半乳糖凝集素抑制剂及其抗肿瘤作用研究进展[J]. 西北药学杂志, 2013, 28(6): 653-655, 656.
[11]	贺雪明, 唐丽, 贺福初. 半乳糖凝集素1的免疫功能[J]. 生物技术通讯, 2012, 23(6): 896-899.
[12]	Banh A, Zhang J, Cao H, et al. Tumor galectin-1 mediates tumor growth and metastasis through regulation of T-cell apoptosis[J]. Cancer Res, 2011, 71(13): 4423-4431.
[13]	Amano M, Suzuki M, Andoh S, et al. Antiangiogenesis therapy using a novel angiogenesis inhibitor, anginex, following radiation causes tumor growth delay[J]. Int J Clin Oncol, 2007, 12(1): 42-47.
[14]	Huang HC, Yao LL, Song ZM, et al. Development-specific differences in the proteomics of Angiostrongylus cantonensis[J]. Plos One, 2013, 8(10): e76982.
[15]	黄慧聪, 姚丽丽, 周子博, 等. 广州管圆线虫Ⅴ期幼虫具抗原表位功能肽段的筛选及鉴定[J]. 中华医学杂志, 2014, 94(24): 1905-1908.
[16]	郝丽, 吴焜, 陈晓光, 等. 广州管圆线虫半乳凝素基因的克隆表达、蛋白纯化及免疫反应性研究[J]. 南方医科大学学报, 2007, 27(5): 584-587.
[17]	Chung LY, Tang SJ, Sun GH, et al. Galectin-1 promotes lung cancer progression and chemoresistance by upregulating p38 MAPK, ERK, and cyclooxygenase-2[J]. Clin Cancer Res, 2012, 18(15): 4037-4047.
[18]	Masutani K, Akahoshi M, Tsuruya K, et al. Predominance of Th1 immune response in diffuse proliferative lupus nephritis[J]. Arthritis Rheum, 2001, 44(9): 2097-2106.
[19]	Baeuerle PA, Gires O.EpCAM (CD326) finding its role in cancer[J]. Br J Cancer, 2007, 96(3): 417-423.
[20]	Cumpstey I, Sundin A, Leffler H, et al. C2-symmetrical thiodigalactoside bis-benzamido derivatives as high-affinity inhibitors of galectin-3: efficient lectin inhibition through double arginine-arene interactions[J]. Angew Chem Int Ed Engl, 2005, 44(32): 5110-5112.
[21]	Brandwijk RJ, Dings RP, van der Linden E, et al. Anti-angiogenesis and anti-tumor activity of recombinant anginex[J]. Biochem Biophys Res Commun, 2006, 349(3): 1073-1078.
[22]	Chow SN, Chen RJ, Chen CH, et al. Analysis of protein profiles in human epithelial ovarian cancer tissues by proteomic technology[J]. Eur J Gynaecol Oncol, 2010, 31(1): 55-62.
[23]	Chiariotti L, Salvatore P, Frunzio R, et al. Galectin genes: regulation of expression[J]. Glycoconj J, 2002, 19(7): 441-449.
[24]	Barrionuevo P, Beigier-Bompadre M, Ilarregui JM, et al. A novel function for galectin-1 at the crossroad of innate and adaptive immunity: galectin-1 regulates monocyte/macrophage physiology through a nonapoptotic ERK-dependent pathway[J]. J Immunol, 2007, 178(1): 436-445.
[25]	Zúñiga E, Gruppi A, Hirabayashi J, et al. Regulated expression and effect of galectin-1 on Trypanosoma cruzi-infected macrophages: modulation of microbicidal activity and survival[J]. Infect Immun, 2001, 69(11): 6804-6812.
[26]	Kikawada E, Lenda DM, Kelley VR.IL-12 deficiency in MRL-Fas(lpr) mice delays nephritis and intrarenal IFN-gamma expression, and diminishes systemic pathology[J]. J Immunol, 2003, 170(7): 3915-3925.
[27]	Hotta K, Funahashi T, Matsukawa Y, et al. Galectin-12, an adipose-expressed galectin-like molecule possessing apoptosis-inducing activity[J]. J Biol Chem, 2001, 276(36): 34089-34097.
[28]	Almkvist J, Dahlgren C, Leffler H, et al. Activation of the neutrophil nicotinamide adenine dinucleotide phosphate oxidase by galectin-1[J]. J Immunol, 2002, 168(8): 4034-4041.
[29]	Garín MI, Chu CC, Golshayan D, et al. Galectin-1: a key effector of regulation mediated by CD4+CD25+ T cells[J]. Blood, 2007, 109(5): 2058-2065.
[30]	Lefranc F, Mathieu V, Kiss R.Galectin-1-mediated biochemical controls of melanoma and glioma aggressive behavior[J]. World J Biol Chem, 2011, 2(9): 193-201.