张志强,刘鑫潮,牛金中,黄瑜,王蓓,简纪常

• 1:广东海洋大学水产学院//广东省水产经济动物病原生物学及流行病学重点实验室//广东省水产经济动物病害控制重点实验室

摘要(Abstract)：

【目的】研究尼罗罗非鱼(Oreochromisniloticus)Galectin-4基因(记作OnGal-4)的原核表达,为大量获取尼罗罗非鱼Galectin-4蛋白(OnGal-4)和后续功能研究奠定基础。【方法】根据NCBI上公布的尼罗罗非鱼Galectin-4基因序列(Genbank:XM-019345120)设计引物,构建原核表达载体,诱导Galectin-4重组蛋白(r OnGal-4)在BL21原核表达系统中表达,并优化其诱导条件。【结果与结论】成功扩增出OnGal-4基因,并构建原核表达载体pGEX-4T-On Gal-4。rOnGal-4最佳诱导条件是温度28℃,IPTG浓度0.4 mmol/L,诱导时间4 h。在此条件下,rOnGal-4在可溶性蛋白中大量表达。Western-blot结果显示,rOnGal-4可与抗GST标签的单克隆抗体发生特异性反应。

关键词(KeyWords)： 尼罗罗非鱼;Galectin-4;基因克隆;原核表达;条件优化

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金青年基金项目(31702386);; 广东省科技厅国际科技合作领域项目(2017A050501037);; 广东省普通高校青年创新人才类项目(2018KQNCX105)

作者(Author): 张志强,刘鑫潮,牛金中,黄瑜,王蓓,简纪常

参考文献(References)：

• [1]甘桢.尼罗罗非鱼T细胞激活相关基因的功能研究[D].湛江：广东海洋大学,2015.
• [2]EL-SAYED A F M.Total replacement of fish meal with animal protein sources in Nile tilapia,Oreochromis niloticus(L.),feeds[J].Aquaculture Research,1998,29(4):275-280.
• [3]LIU L,LU D Q,XU J,et al.Development of attenuated erythromycin-resistant Streptococcus agalactiae vaccine for tilapia(Oreochromis niloticus)culture[J].Journal of Fish Diseases,2019,42(5):693-701.
• [4]WANG Y Y,WANG Q,ZENG W W,et al.Establishment and characterization of a cell line from tilapia brain for detection of tilapia lake virus[J].Journal of Fish Diseases,2018,41(12):1803-1809.
• [5]LIU F T,RABINOVICH G A.Galectins:regulators of acute and chronic inflammation[J].Annals of the New York Academy of Sciences,2010,1183(1):158-182.
• [6]MAHLA R S,REDDY M C,PRASAD D V R,et al.Sweeten PAMPs:role of sugar complexed PAMPs in innate immunity and vaccine biology[J].Frontiers in Immunology,2013,4:248.
• [7]HUGHES R C.Secretion of the galectin family of mammalian carbohydrate-binding proteins[J].Biochimica et Biophysica Acta(BBA)-General Subjects,1999,1473(1):172-185.
• [8]CHEN C C,WANG G X,HUANG X H,et al.Overexpression of galectin-4 promotes cell growth of hepatocellular carcinoma cells in vitro and in vivo[J].International Journal of Clinical and Experimental Pathology,2017,10(10):10233-10242.
• [9]HONG S H,SHIN J S,CHUNG H,et al.Galectin-4interaction with CD14 triggers the differentiation of monocytes into macrophage-like cells via the MAPKsignaling pathway[J].Immune Network,2019,19(3):e17.
• [10]STURM A,LENSCH M,ANDRéS,et al.Human galectin-2:novel inducer of T cell apoptosis with distinct profile of caspase activation[J].Journal of Immunology,2004,173(6):3825-3837.
• [11]BRITTOLI A,FALLARINI S,ZHANG H,et al.“In vitro”studies on galectin-3 in human natural killer cells[J].Immunology Letters,2018,194:4-12.
• [12]MICHALAK M,WARNKEN U,SCHN?LZER M,et al.Detection of malignancy-associated phosphoproteome changes in human colorectal cancer induced by cell surface binding of growth-inhibitory galectin-4[J].IUBMB Life,2019,71(3):364-375.
• [13]NIEPCERON E,SIMIAN F,LOUISOT P,et al.Expression of galectin 4 in the rat small intestine during postnatal development[J].Biochimie,2004,86(2):115-118.
• [14]ARASU A,KUMARESAN V,GANESH M R,et al.Bactericidal activity of fish galectin 4 derived membrane-binding peptide tagged with oligotryptophan[J].Developmental&Comparative Immunology,2017,71:37-48.
• [15]CHEN J H,ZHANG L,YANG N,et al.Expression profiling and microbial ligand binding analysis of galectin-4 in turbot(Scophthalmus maximus L.)[J].Fish&Shellfish Immunology,2019,84:673-679.
• [16]WOOTERS M A,HILDRETH M B,NELSON E A,et al.Immunohistochemical characterization of the distribution of galectin-4 in porcine small intestine[J].Journal of Histochemistry&Cytochemistry,2005,53(2):197-205.
• [17]POROWI?SKA D,WUJAK M,ROSZEK K,et al.Prokaryotic expression systems[J].Post?py Higieny i Medycyny Do?wiadczalnej(Online),2013,67:119-129.
• [18]BRIAND L,MARCION G,KRIZNIK A,et al.Aself-inducible heterologous protein expression system in Escherichia coli[J].Scientific Reports,2016,6:33037.
• [19]GILEADI O.Recombinant protein expression in E.coli:A historical perspective[M]//Methods in Molecular Biology.New York,NY:Springer New York,2017:3-10.DOI:10.1007/978-1-4939-6 887-9_1.
• [20]赵苗鑫,陈华谱,刘金磊,等.罗氏沼虾雌激素相关受体(ERR)基因原核表达与纯化[J].广东海洋大学学报,2017,37(1):108-112.
• [21]马少鸿,黄郁葱,简纪常,等.哈维氏弧菌Psp F基因的克隆及原核表达分析[J].广东海洋大学学报,2019,39(5):1-7.
• [22]DVORAK P,CHRAST L,NIKEL P I,et al.Exacerbation of substrate toxicity by IPTG in Escherichia coli BL21(DE3)carrying a synthetic metabolic pathway[J].Microbial Cell Factories,2015,14(1):1-15.
• [23]牛金中,黄瑜,汤菊芬,等.尼罗罗非鱼补体3基因片段的原核表达及条件优化[J].广东海洋大学学报,2018,38(2):80-84.
• [24]罗国玲,牛金中,黄瑜,等.尼罗罗非鱼Galectin-3基因的原核表达与条件优化[J].广东海洋大学学报,2019,39(4):35-41.
• [25]黄瑜,牛金中,汤菊芬,等.尼罗罗非鱼NCCRP-1基因的原核表达及条件优化[J].基因组学与应用生物学,2018,37(9):3753-3758.
• [26]矫丽媛,才蕾,任艳娜,等.人β2微球蛋白原核表达条件优化及纯化[J].生物医学工程学杂志,2015,32(5):1050-1055.
• [27]马德昭,田菲,吴其中,等.青海湖裸鲤IL-8的原核表达及诱导条件优化[J/OL].基因组学与应用生物学:1-7.[2020-04-08].https://kns.cnki.net/KCMS/detail/45.1369.Q.20190508.1427.005.html?uid