CN113995831A - 一种半滑舌鳎半乳糖凝集素-8的应用 - Google Patents

一种半滑舌鳎半乳糖凝集素-8的应用 Download PDF

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CN113995831A
CN113995831A CN202011450529.5A CN202011450529A CN113995831A CN 113995831 A CN113995831 A CN 113995831A CN 202011450529 A CN202011450529 A CN 202011450529A CN 113995831 A CN113995831 A CN 113995831A
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姜帅
张腾飞
孙黎
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Abstract

本发明涉及分子生物学领域，具体的说是一种半滑舌鳎半乳糖凝集素‑8(galectin‑8)的应用。半滑舌鳎galectin‑8用于制备杀细菌的制剂。所述半滑舌鳎galectin‑8为在大肠杆菌中表达获得的重组蛋白。本发明的重组galectin‑8用于杀死致病细菌。

Description

一种半滑舌鳎半乳糖凝集素-8的应用
技术领域
本发明涉及分子生物学领域，具体的说是一种半滑舌鳎半乳糖凝集素-8(galectin-8)的应用。
背景技术
半乳糖凝集素(Galectin)是一类糖结合蛋白，半乳糖凝集素-8(Galectin-8)是其中一个成员。Galectin-8为分泌蛋白或胞内蛋白，其结构中包含两个由约130个氨基酸组成的保守糖识别结构域(carbohydrate-recognition domain)，能特异性识别与结合β-半乳糖苷。Galectin-8能识别细菌表面的糖分子而与细菌结合，其与细菌结合后能激引发一系列的免疫反应发生，如促进细胞吞噬功能增强等，从而帮助机体清除病原菌。因此，Galectin-8在宿主先天免疫反应中发挥着重要作用。目前，鱼类的Galectin-8研究很少，其抗细菌感染的免疫功能亦不清楚。
发明内容
本发明目的在于提供一种半滑舌鳎半乳糖凝集素-8(galectin-8)的应用。
为实现上述目的，本发明采用的技术方案为：
一种半滑舌鳎半乳糖凝集素-8的应用，半乳糖凝集素-8在用于制备杀菌制剂中的应用。
所述半乳糖凝集素-8核苷酸序列如SEQ ID No.1中所示。
所述半乳糖凝集素-8在大肠杆菌中表达获得的重组蛋白。
所述细菌为鳗弧菌(Vibrio anguillarum)或哈氏弧菌(Vibrio harveyi)。
本发明具有如下优点：本发明的galectin-8能够结合并杀死致病细菌鳗弧菌和哈氏弧菌。
附图说明
图1为本发明实施例提供的重组galectin-8蛋白对细菌的杀伤作用。
具体实施方式
下面结合实施例对本发明作进一步说明。实施例旨在对本发明进行举例描述，而非以任何形式对本发明进行限制。
实施例1
本发明中的galectin-8(命名为CsGal-8)为序列表SEQ ID No.1中的氨基酸序列。
序列表SEQ ID No.1为：
MLVSRTRQTFLKPSIPFAGTILGGLLPGEMVLVQGSVPVGADRFQVDLACGSSMKPRADVAFHFNPRFSGSPCVVCNTLQCERWGPEEIQHQNPLKSAHTFELVFLVLQDKFKVAVNGAHLLQYKQRVALERVDTILISGKVAVEVVGVVPSMSNAPLAVSNIQDSQMKSILSTAGNTRVPFSGFLGDGLRVGQSVSIRAETNHNAQSFCVNLCSSESSDIALHLNPRLKSRIFVRNSFLSDSWGSEETALDGFPFMAGQYFEMIVYCDSQHYGVAVNGDHILTYKHRVQDLRRINQVEVQGDVSLLDVKLH
(a)序列特征：
·长度：312
·类型：氨基酸序列
·链型：单链
·拓扑结构：线性
(b)分子类型：蛋白质
(c)假设：否
(d)反义：否
(e)最初来源：半滑舌鳎
序列的特征：半滑舌鳎半乳糖凝集素-8包含两个糖识别结构域，其关键位点是63-67位氨基酸HFNPR，84-89位氨基酸WGPEEI，224-228位氨基酸HLNPR，244-249位氨基酸WGSEET。
实施例2
CsGal-8和硫氧还蛋白(Trx)的重组蛋白的制备
1)表达CsGal-8重组蛋白的质粒pEtCsGal-8的构建：
pEtCsGal-8的构建如下：以半滑舌鳎cDNA为模板，用引物F1和R1进行PCR扩增。PCR条件为：94℃5min预变性模板DNA,然后94℃30s,55℃30s,72℃60s,30个循环。PCR产物用EZNA DNA琼脂糖胶回收试剂盒(购自于“Omega Bio-tek”，美国)纯化后与PCR克隆载体PMD19-T(购自于“大连TaKaRa公司”，大连)于室温连接2－4小时，连接混合液转化到大肠杆菌DH5α后在含有100μg/ml安卡青霉素、40μg/ml 5-溴-4-氯-3-吲哚-β-D-乳糖苷和24μg/ml异丙基-β-D-硫代半乳糖苷的LB固体培养基上培养18－24小时，挑出白色转化子，提取质粒，命名为质粒pMDCsGal-8。以质粒pMDCsGal-8为模板用引物F2和R2进行PCR扩增，PCR条件同上。PCR产物用限制性内切酶Nde I和Xho I酶切后回收0.9kb，同时将表达载体pET28a(购自于“Novagen公司”，美国)用限制性内切酶Nde I和Xho I酶切后回收5.3kb。将上述回收的两片段用T4DNA连接酶于室温连接2－4小时，连接液转化入大肠杆菌DH5α，在含卡那霉素(50ug/ml)的LB培养基上培养18－24小时,筛选转化子提取质粒，即为pEtCsGal-8。DNA测序表明pEtCsGal-8含有编码凝集素CsGal-8序列的基因。
所述LB组成成分按重量百分比计：1.0％蛋白胨,0.5％酵母粉,1.0％氯化钠,97.5％蒸馏水。所述F1为5’-ATGCTAGTGTCACGTACAAGGCAG-3’；R1为5’-TCAGTGGAGTTTTACATCCAGCAGTG-3’；F2为5’-GGAATTCCATATGCTAGTGTCACGTACAAGGCAG-3’；R2为5’-CCGCTCGAGTCAGTGGAGTTTTACATCCAGCAGTG-3’。
2)重组CsGla-8蛋白(rCsGla-8)的诱导表达和纯化
将上述的质粒pEtCsGla-8用常规方法转化大肠杆菌BL21(DE3)(购自于“北京全式金生物公司”，北京)，在含有卡那霉素(50ug/ml)的LB固体培养基上培养18－24小时,挑取转化子，将其命名为BL21/pEtCsGla-8。将BL21/pEtCsGla-8于含有卡那霉素(50ug/ml)的LB液体培养基中过夜培养；取5ml过夜后的培养液,加入500ml新鲜的含有卡那霉素(50ug/ml)的LB液体培养基中，于37℃下转速180rpm摇动培养至OD600为0.6，加入终浓度为1mM的IPTG，37℃继续以转速180rpm摇动培养4-5h，而后以5000g，4℃离心10min，收集菌液，加入20ml裂解液，在室温于摇床上缓慢摇动1-2小时，直至菌悬液变澄清为止。将菌液以10000g，4℃离心30min，回收上清。将上清中的蛋白用Ni-NTA蛋白纯化树脂(购自于“QIAGEN公司”，德国)回收纯化，将纯化的蛋白悬浮于PBS缓冲液，命名为rCsGla-8。
所述裂解液为终浓度的9.5mM NaH2PO4、40mM Na2HPO4、0.5M NaCl和8M尿素,pH9.0。
所述PBS组成成分按重量百分比计：0.8％NaCl,0.02％KCl,0.358％Na2HPO4.12H2O,0.024％NaH2PO4，余量为水。
3)重组Trx蛋白(rTrx)的诱导表达和纯化
将pET32a(购自于“Novagen公司”，美国)用上述方法转化大肠杆菌BL21(DE3)，在含有安卡青霉素(100ug/ml)的LB固体培养基上培养18－24小时,挑取转化子，将其命名为BL21/pET32a。将BL21/pET32a于含有安卡青霉素(100ug/ml)的LB液体培养基中过夜培养；取5ml过夜后的培养液,加入500ml新鲜的含有安卡青霉素(100ug/ml)的LB液体培养基中，于37℃下转速180rpm摇动培养至OD600为0.6，加入终浓度为1mM的IPTG，37℃继续以转速180rpm摇动培养4-5h，而后以5000g，4℃离心10min，收集菌液，加入20ml裂解液，在室温于摇床上缓慢摇动1-2小时，直至菌悬液变澄清为止。将菌液以10000g，4℃离心30min，回收上清。将上清中的蛋白用Ni-NTA蛋白纯化树脂(购自于“QIAGEN公司”，德国)回收纯化，将纯化的蛋白悬浮于PBS缓冲液，命名为rTrx。
实施例3
rCsGal-8的杀菌应用
步骤1)细菌悬液制备
在LB液体培养基中培养致病性鳗弧菌C312(Vibrio anguillarum)和哈氏弧菌T4(Vibrio harveyi)至OD600为0.8,然后离心(5000g，4℃，10min)，收集菌体，将其悬浮于PBS中至终浓度为106cfu/ml，即为鳗弧菌和哈氏弧菌悬液。
所述致病性鳗弧菌C312保存于中国微生物菌种保藏管理委员会普通微生物中心CGMCC，地址：北京市朝阳区北辰西路1号院3号，邮编,100101。保藏编号为：CGMCC No.6250，保藏日期2012.6.21，分类命名为鳗弧菌(Vibrio anguillarum)。
所述致病性哈氏弧菌T4保存于中国微生物菌种保藏管理委员会普通微生物中心CGMCC，地址同上，保藏编号为：CGMCC No.1985，保藏日期2007.3.22，分类命名为哈氏弧菌(Vibrio harveyi)。
步骤2)rCsGal-8杀菌检测
取200μl上述步骤1)的鳗弧菌悬液于1.5ml离心管中，向菌悬液中加入终浓度为1μM的rCsGla-8、rTrx或PBS(对照),每组三个平行。室温孵育1h后，用PBS梯度稀释菌悬液，涂LB固体培养基平板。28℃培养18-20小时后菌落计数。rCsGal-8对哈维氏弧菌的杀伤检测方法同鳗弧菌。结果表明，与rCsGla-8孵育后，鳗弧菌和哈维氏弧菌几乎没有存活，而对照蛋白rTrx对鳗弧菌和哈维氏弧菌的存活没有影响(图1)。
由图1rCsGal-8的杀菌效应检测。鳗弧菌(A1)和哈维氏弧菌(B1)与rCsGal-8、rTrx或PBS(对照)保温1h，随后涂于LB固体培养基平板。28℃培养18-20小时后观察细菌存活情况，同时计算细菌数(colony forming units,CFUs)。根据CFUs做成数据图(A2和B2)。实验数据为三次平行实验结果。**p<0.01；结果表明，本发明的rCsGal-8具有显著的细菌杀伤能力。
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Claims (4)

1.一种半滑舌鳎半乳糖凝集素-8的应用，其特征在于：半乳糖凝集素-8在用于制备杀菌制剂中的应用。
2.按权利要求1所述的半滑舌鳎半乳糖凝集素-8的应用，其特征在于：所述半乳糖凝集素-8的氨基酸序列如SEQ ID No.1中所示。
3.按权利要求2所述的半滑舌鳎半乳糖凝集素-8的应用，其特征在于：所述半乳糖凝集素-8在大肠杆菌中表达获得的重组蛋白。
4.按权利要求1所述的半滑舌鳎半乳糖凝集素-8的应用，其特征在于：所述细菌为鳗弧菌(Vibrio anguillarum)或哈氏弧菌(Vibrio harveyi)。
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