CN116270973A - 克氏原螯虾甘露糖结合蛋白在抑制细菌中的应用 - Google Patents
克氏原螯虾甘露糖结合蛋白在抑制细菌中的应用 Download PDFInfo
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- CN116270973A CN116270973A CN202310090936.7A CN202310090936A CN116270973A CN 116270973 A CN116270973 A CN 116270973A CN 202310090936 A CN202310090936 A CN 202310090936A CN 116270973 A CN116270973 A CN 116270973A
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- Food Science & Technology (AREA)
Abstract
本发明公开了克氏原螯虾甘露糖结合蛋白在制备具有抑制金黄色葡萄球菌、藤黄微球菌、枯草芽孢杆菌、苏云金芽孢杆菌、副溶血弧菌、哈氏弧菌、嗜水气单胞菌功效的制剂中的应用,在制备促进克氏原螯虾、日本沼虾或中华绒螯蟹机体对细菌的清除速率、提高存活率的制剂中的应用,其氨基酸序列如SEQ ID NO.1所示。本发明首次构建了克氏原螯虾甘露糖结合蛋白基因的原核表达载体并研制了重组蛋白,该表达载体在大肠杆菌内表达稳定;研究了重组蛋白在免疫防御功能中的用途,本发明为克氏原螯虾的病害防治奠定了理论基础,在开发抗菌药物、免疫增强剂、饲料添加剂生产等方面具有潜在的应用价值。
Description
技术领域
本发明涉及克氏原螯虾甘露糖结合蛋白在抑制细菌中的应用,属于凝集素的活性研究技术领域。
背景技术
在病原体感染脊椎动物早期,机体会产生一种急性期甘露糖结合蛋白(mannose-binding protein,MBP),它主要由肝脏合成并分泌,是一种C型(Ca2+依赖型)血清凝集素,属于胶原凝集素家族,因此又被称为甘露糖结合凝集素(mannose-binging lectin,MBL)。作为模式识别受体,MBP能够特异识别并结合细菌、病毒等病原体表面的甘露糖、葡萄糖、岩藻糖和N-乙酰-d-半乳糖胺等糖类物质,从而启动凝集素补体活化途径,在免疫防御和免疫监视中具有重要意义。另外,MBP还可以识别完整的糖分子或部分糖分子,甚至可以识别糖苷键。除了具有识别及结合功能外,MBP作为一种调理素还可调控巨噬细胞的吞噬作用,还参与了一些重要的生物学现象,如某些病原体识别、蛋白质合成和运输、信号转导和细胞间相互作用等。成熟的MBP肽段包含一个信号肽、碳水化合物识别区域(carbohydraterecognition domain,CRD)和胶原疏水区域,其中,胶原疏水区域能够与MBL相关丝氨酸蛋白酶MASP结合形成复合体,而CRD在钙离子参与下能够参与对微生物的识别,特别是CRD中的EPN(Glu-Pro-Asn)基序仅特异性的识别甘露糖,从而引发补体激活、吞噬作用或炎症反应来清除细菌。MBP作为天然免疫分子之一,其结构、生物学功能、分子病理机制等研究备受关注。
克氏原螯虾(Procambarus clarkii),俗称小龙虾,分类学上隶属于节肢动物门、甲壳纲。因其适应能力强、营养价值高,现已成为我国最具特色的淡水虾类养殖品种。然而,随着养殖密度的增加,弧菌、嗜水气单胞菌等病原微生物引发的病害问题愈发严重,造成了巨大的经济损失。作为无脊椎动物,克氏原螯虾缺乏真正的获得性免疫,主要依赖先天免疫抵御病原体的入侵。因此迫切需要从螯虾自身的免疫防御因子入手研究其免疫防御机制和开发新型的抗菌药物。目前关于螯虾MBP的研究尚少,开展MBP的研究对理解螯虾的抗病菌机制、进行病害防治具有重要的理论和实践意义。
发明内容
针对上述现有技术,本发明研究了克氏原螯虾甘露糖结合蛋白在宿主抗细菌先天免疫防御中的作用,发现了克氏原螯虾甘露糖结合蛋白在抑制某些细菌方面的用途。
本发明是通过以下技术方案实现的:
克氏原螯虾甘露糖结合蛋白,其氨基酸序列如SEQ ID NO.1所示。
克氏原螯虾甘露糖结合蛋白在制备具有抑制金黄色葡萄球菌功效的制剂中的应用。
克氏原螯虾甘露糖结合蛋白在制备具有抑制藤黄微球菌功效的制剂中的应用。
克氏原螯虾甘露糖结合蛋白在制备具有抑制枯草芽孢杆菌功效的制剂中的应用。
克氏原螯虾甘露糖结合蛋白在制备具有抑制苏云金芽孢杆菌功效的制剂中的应用。
克氏原螯虾甘露糖结合蛋白在制备具有抑制副溶血弧菌功效的制剂中的应用。
克氏原螯虾甘露糖结合蛋白在制备具有抑制哈氏弧菌功效的制剂中的应用。
克氏原螯虾甘露糖结合蛋白在制备具有抑制嗜水气单胞菌功效的制剂中的应用。
克氏原螯虾甘露糖结合蛋白在促进克氏原螯虾、日本沼虾或中华绒螯蟹机体对细菌的清除速率、提高存活率中的应用,所述细菌选自副溶血弧菌、金黄色葡萄球菌。
所述制剂可以是菌抑制剂、抗菌剂、杀菌剂、药物制剂、免疫增强剂、饲料添加剂等。
本发明克隆了克氏原螯虾甘露糖结合蛋白的编码基因,并导入大肠杆菌进行异源表达,纯化得到了重组蛋白,然后通过细菌结合、糖直接结合、细菌抑制、检测细菌生物被膜、细菌清除和存活率实验研究了重组蛋白的抗细菌作用。研究结果表明MBP重组蛋白能够结合四种革兰氏阳性菌(金黄色葡萄球菌、藤黄微球菌、枯草芽孢杆菌和苏云金芽孢杆菌)、三种革兰氏阴性菌(副溶血弧菌、哈氏弧菌和嗜水气单胞菌),直接结合三种糖类(脂多糖、肽聚糖和甘露糖),抑制副溶血弧菌和金黄色葡萄球菌生物被膜的生长及细菌的生长,促进克氏原螯虾、日本沼虾和中华绒螯蟹机体对细菌的清除速率、提高螯虾、沼虾和河蟹的存活率。
本发明首次构建了克氏原螯虾甘露糖结合蛋白基因的原核表达载体并研制了重组蛋白,该表达载体在大肠杆菌内表达稳定;研究了重组蛋白在免疫防御功能中的用途,本发明为克氏原螯虾的病害防治奠定了理论基础,在开发抗菌药物、免疫增强剂、饲料添加剂生产等方面具有潜在的应用价值。
本发明使用的各种术语和短语具有本领域技术人员公知的一般含义。
附图说明
图1:细菌结合实验结果示意图。
图2:糖直接结合实验结果示意图,其中,A:脂多糖;B:肽聚糖;C:甘露糖。
图3:细菌生物膜检测实验结果示意图,其中,A:副溶血弧菌;B:金黄色葡萄球菌。
图4:细菌抑制实验结果示意图,其中,A:副溶血弧菌;B:金黄色葡萄球菌。
图5:细菌清除实验结果示意图,其中,A:螯虾对副溶血弧菌的清除效率;B:螯虾对金黄色葡萄球菌的清除效率;C:沼虾对副溶血弧菌的清除效率;D:沼虾对金黄色葡萄球菌的清除效率;E:河蟹对副溶血弧菌的清除效率;F:河蟹对金黄色葡萄球菌的清除效率。
图6:存活率检测实验结果示意图,其中,A:副溶血弧菌刺激后螯虾的存活率;B:金黄色葡萄球菌刺激后螯虾的存活率;C:副溶血弧菌刺激后沼虾的存活率;D:金黄色葡萄球菌刺激后沼虾的存活率;E:副溶血弧菌刺激后河蟹的存活率;F:金黄色葡萄球菌刺激后河蟹的存活率。
图7:SDS-PAGE胶结果示意图,其中,M:蛋白分子量标准;1:包含pET30a-MBP重组质粒的Escherichia coli Transetta(DE3)菌株诱导前;2:包含pET30a-MBP重组质粒的E.coli Transetta(DE3)菌株0.5mM IPTG诱导后;3:纯化的rMBP蛋白。
具体实施方式
下面结合实施例对本发明作进一步的说明。然而,本发明的范围并不限于下述实施例。本领域技术人员能够理解,在不背离本发明的精神和范围的前提下,可以对本发明进行各种变化和修饰。
下述实施例中所涉及的仪器、试剂、材料,若无特别说明,均为现有技术中已有的常规仪器、试剂、材料,可通过正规商业途径获得。下述实施例中所涉及的实验方法、检测方法,若无特别说明,均为现有技术中已有的常规实验方法、检测方法。
本发明的克氏原螯虾甘露糖结合蛋白重组蛋白(MBP重组蛋白),是通过常规方法制备得到的(克隆克氏原螯虾甘露糖结合蛋白的编码基因,导入大肠杆菌进行异源表达,纯化,得到重组蛋白),具体的制备过程详见实验7。
实验1细菌结合实验
选用金黄色葡萄球菌(Staphylococcus aureus)、藤黄微球菌(Micrococcusluteus)、枯草芽孢杆菌(Bacillus subtilis)、苏云金芽孢杆菌(Bacillusthuringiensis)、副溶血弧菌(Vibrio parahemolyticus)、哈氏弧菌(Vibrio harveyi)和嗜水气单胞菌(Aeromonas hydrophila)作为实验菌株,检测MBP重组蛋白的细菌结合作用。过夜培养的实验菌株经离心后,用灭菌PBS洗涤并重悬至菌液浓度为2×108个/mL,MBP重组蛋白用灭菌PBS稀释至浓度为500μg/mL,室温静置待用。实验组于灭菌离心管中混匀500μL细菌重悬液和500μL重组蛋白样品,室温孵育30min;对照组则使用ADP-核糖基化因子(ADPribosylation factor,Arf)重组蛋白代替MBP蛋白与细菌孵育。结束后离心混合液,并用1mL灭菌PBS洗涤沉淀四次,取洗涤液经蛋白高温变性处理后,进行12% SDS-PAGE电泳和Western blot检测。结果如图1所示。
由图1可见,MBP重组蛋白能结合金黄色葡萄球菌、藤黄微球菌、枯草芽孢杆菌、苏云金芽孢杆菌、副溶血弧菌、哈氏弧菌和嗜水气单胞菌。
实验2糖直接结合实验
采用酶联免疫吸附实验ELISA确认MBP重组蛋白对糖的直接结合活性。选择脂多糖、肽聚糖和甘露糖进行实验,步骤如下:
a)将糖用灭菌蒸馏水稀释至80μg/mL,超声破碎(3×15s);
b)将超声后的糖加入96孔板中,每孔50μL;将板于37℃放置过夜待水分蒸发完全后,置于60℃30min,使糖包被于孔中;
c)每孔加入200μL BSA(1mg/mL,TBS配制),于37℃封闭2h;
d)吸去封闭液,每孔加入200μL TBS洗涤四次;
e)向每孔中依次加入梯度稀释的MBP或Arf蛋白溶液(0~50μg/mL,溶于含0.1mg/mL BSA的TBS中),每孔50μL;室温放置3h;
f)TBS洗涤四次,每孔加入100μL抗His-tag的兔单克隆抗体(1:1000稀释于含0.1BSAmg/mL的TBS中),37℃孵育1h;
g)TBS洗涤四次,每孔加入辣根过氧化酶标记的羊抗兔IgG(1:5000,TBS配制),37℃孵育1h;
h)TBS洗涤四次,加入100μL含0.01%3,3’,5,5’-四甲基联苯胺(sigma公司)的柠檬酸-磷酸氢二钠缓冲液于室温发色,待发色适中时,用50μL 2M硫酸终止显色反应;
i)使用酶标仪在吸光度450nm读取吸光值;实验重复三次。结果如图2所示。
由图2可见,重组蛋白能直接结合脂多糖、肽聚糖和甘露糖且呈剂量依赖性,在8-50μg/mL时具有较高的亲和力。重组蛋白对脂多糖和肽聚糖的结合亲和力相似,对甘露糖的结合亲和力更强。
实验3结晶紫染色检测生物被膜
选用V.parahemolyticus和S.aureus作为实验菌株。首先向无菌96孔聚苯乙烯培养板中加入160μL LB培养基,然后加入20μL待测细菌培养液(浓度约为106CFU/ml),最后加入20μg重组蛋白,轻摇混匀后在37℃下孵育24h~48h。对照组加入相同浓度的Arf溶液。轻轻弃去培养基,用无菌PBS溶液洗涤96孔板,以除去多余未吸附菌体;用移液枪轻轻吸取吸净孔底的残余液体,加入200μL无水甲醇固定15min;弃去甲醇,倒置96孔板晾干孔底液体,然后向孔中加入200μL的0.1%结晶紫溶液(上海生工)进行染色30min;染色结束后使用无菌水洗漆残留染色溶液,用移液枪吸取剩余液体,晾干后在通风橱中向每孔中加入150μL的30%冰醋酸静置30min,充分溶解结晶紫染液;将培养板放入BioTek酶标仪测量600nm波长的吸光度。结果如图3所示。
由图3可见,重组蛋白能够抑制副溶血弧菌和金黄色葡萄球菌生物被膜的生长。
实验4细菌抑制实验
选用V.parahemolyticus和S.aureus作为实验菌株。将纯化的MBP重组蛋白进行细菌抑制实验,选用副溶血弧菌和金黄色葡萄球菌作为实验菌株,并设置对照组。取过夜培养的新鲜菌液,按1:100转接至LB培养基,再向培养基中加入MBP重组蛋白至终浓度为200μg/mL,对照组则加入灭菌PBS。设置测样时间点为0h、1h、2h、3h、4h、5h和6h,每个时间点用酶标仪测定菌液在波长为600nm时的吸光度值,进行三次独立重复实验。结果如图4所示。
由图4可见,重组蛋白能够抑制副溶血弧菌和金黄色葡萄球菌的生长。
实验5细菌清除实验
选用V.parahemolyticus和S.aureus作为实验菌株。步骤如下:
a)将随机挑选的克氏原螯虾/日本沼虾/中华绒螯蟹分成6组,每组10只;
b)PBS洗涤副溶血弧菌或金黄色葡萄球菌三次并重悬,调整浓度至2×108细胞/mL,待用;
c)将500μL纯化的MBP重组蛋白(500μg/mL)与500μL副溶血弧菌或金黄色葡萄球菌混合后,置于30℃轻摇30min,Arf和PBS作为蛋白的对照,也进行同样的处理;
d)将每组中蛋白与细菌的混合物50μL分别注射入螯虾/沼虾/河蟹体内;
e)在注射后20min时间段,每组10只螯虾/沼虾/河蟹分别抽取500μL血淋巴,将其与等体积抗凝剂混匀;
f)将混合物以灭菌PBS稀释100倍或1000倍,取30μL涂布于LB平板上,37℃培养过夜;
g)对每个平板进行计数,并计算细菌残留数量。结果如图5所示。
由图5可见,螯虾MBP重组蛋白能促进螯虾对副溶血弧菌和金黄色葡萄球菌的清除作用,也能促进沼虾和河蟹对副溶血弧菌和金黄色葡萄球菌的清除作用。
实验6存活率检测实验
选用V.parahemolyticus和S.aureus作为实验菌株。随机挑取120只健康的克氏原螯虾/日本沼虾/中华绒螯蟹,分为四组,每组30只。两组实验组每只螯虾/沼虾/河蟹注射50μL副溶血弧菌或金黄色葡萄球菌(约2×108个细菌)和MBP重组蛋白(50μg)混合物,随后放回水族箱。每天记录螯虾/沼虾/河蟹死亡的数量,记录周期约6天。对照组注射同剂量的副溶血弧菌或金黄色葡萄球菌和Arf蛋白;用同样的方法进行统计,使用GraphPad软件对数据进行统计分析。结果如图6所示。
由图6可见,螯虾MBP重组蛋白能够提高克氏原螯虾(感染了副溶血弧菌或金黄色葡萄球菌)的存活率,也能提高沼虾和河蟹(感染了副溶血弧菌或金黄色葡萄球菌)的存活率。
实验7重组甘露糖结合蛋白的表达和纯化
克氏原螯虾甘露糖结合蛋白的氨基酸序列如下所示(如SEQ ID NO.1所示)(下划线部分是去掉信号肽后的序列):
MKGVLAVLSVVVGVSQCQLPYGSYGGGGGFPQRHNNFPGRPGIFPGRPGGGGVGGGGPIKFPSSVAGS VHGKPGGVGGFGNAGGIGGAGIIGGFGGAVGGGQVQERPLSTQFCPAYISPLVHVSVSGSNYHFSWCADGGQKYVW EQAKNYCKKLGPGWSSVSIETPTENQFISSIIDKHGLPYIWTSGNRLGGGPKGWKWATGQPLTYNNWALTGFTPGK PQPDNQEDNNEQCLSVLNRFYPNDGITWHDVGCHHVKPTICEYTNVQSYVG。
表达克氏原螯虾甘露糖结合蛋白的核苷酸序列如下所示(5’-3’)(如SEQ ID NO.2所示,)(包含了非编码区)(表达克氏原螯虾甘露糖结合蛋白的核苷酸序列已由他人上传至NCBI数据库,ACCESSION FJ410911,但本发明扩增得到的序列非编码区与上传的序列有不同):
GTCAAGATGAAGGGTGTGTTGGCTGTGCTGAGTGTGGTGGTGGGGGTGTCTCAGTGCCAGCTCCCTTACGGTAGCTACGGAGGAGGCGGCGGCTTCCCACAGAGGCACAATAACTTCCCTGGAAGACCCGGTATTTTCCCCGGGAGACCTGGTGGCGGCGGCGTCGGCGGCGGCGGACCGATAAAGTTCCCCAGCTCTGTTGCCGGGTCTGTACATGGGAAGCCGGGAGGGGTTGGTGGATTTGGCAATGCAGGAGGGATCGGTGGTGCTGGAATAATTGGTGGATTTGGAGGTGCTGTTGGCGGAGGCCAGGTCCAGGAACGGCCTCTCTCAACTCAGTTCTGTCCTGCATACATCAGTCCCCTGGTTCACGTATCTGTGAGTGGAAGCAACTATCATTTCTCCTGGTGCGCTGACGGGGGGCAGAAGTACGTGTGGGAGCAGGCCAAAAATTACTGTAAGAAACTGGGTCCTGGGTGGAGTAGTGTGAGCATAGAGACCCCGACTGAGAACCAGTTCATCTCTTCCATCATTGACAAACACGGTCTACCATACATCTGGACGAGCGGGAACCGTCTGGGCGGCGGTCCCAAAGGCTGGAAGTGGGCCACAGGTCAGCCCCTCACCTACAACAACTGGGCTCTCACCGGATTCACTCCCGGCAAGCCTCAGCCGGACAACCAGGAAGACAACAATGAACAATGCCTCTCAGTGCTCAACCGCTTCTACCCCAACGACGGCATCACCTGGCACGACGTGGGTTGCCACCATGTCAAGCCTACCATCTGCGAGTATA CCAATGTCCAAAGCTATGTTGGATAGACCTGTCGAGCGGTATAACTTATTTGATTGACAAATTTCCTATAATTTAGCGATCTGTCCATTAAGAATTATAATTTTCCGACTAGTGTTTATTCACTTAAATGCAGATCACTGACACTAGCAGTTAAGGTGTAATGTTTTACCATGGAAGTATTTATGTTGTAGCAATTATGATCAAATTTTACTTCCAATACACTTCCACACTAATTACCACGTGAGTGCCTCAAGTTACAAAGAAAATTAACGTTCAAGTTAGCTTAAAATTGTATGTAACCCTTCAAAACAGAAAACGGACATTCCTTCAAATGCCGTTCTCTCTCATTGATGCTGTATACATATAGAAAACGTTATGATGGGATATGATACTGTTCGATGCTAGAAAAAGATAAGCAACCAATTTTTCAAAAATATATAGCTAGTAGGGATAGATTGTACAAAATATGGATTGTTGTAAAAAAAAAAAGTCAACGTTTTTGTACTATTTTAAATATATATGGCAGCCTAGCTACAATCGTTATCCATCGTGTCCTACAGGTAAACAGGCCATCCTTGCCCTAACATACTCAATATTAGTCCTAATTTAGTACACAGCTGTGCAAAATTTGTTTTTGTTTGTTGCCTTCTAGCTCAGGCCTCTATAAATGGAATGGTGCAAAACAAGGGTTATTTTTTGTAAATACAGCAGTCCATATTTTGAACATTTCACTCATAAGTTTAAAACTGTTGTGGGCCTATTTTTCAACCACTAGTACTATAAAAAAAACTATTATATACTTTAAGCTGTGCAACTAGCTTATTAAAGATTTTTACTTACTCAGCTAAGTAAATTTTTGGGGTTCAGTTCCTGTACGCAATATGTGCCTCAGTAACCTTTCCTACTCCTGCCCACGAGATGAGTATGGGGCCCACGACCCCCCACGAGATGAGTATGGGGCCCACGACCCCCCACGAGATGAGTATGGGGCCCACGACCCCCCACGAGATGAGTATGGGGCCCACGACTCCCACGAGAAGGGTATGGGGCCCACGACTCCCACGAGAAGGGTATGGGGCCCACCACTCCCACAGGATGGTTATAGGGACAACGAACGCCGATATAAGTCTGAATATTATTAAGACATTTCGAAAAGCTTTCAGGATTGTGATACAGGATCGAAAAGTGGCATTATCTCTCTCTCTGTCTCTGTTTATTTAACTCCTAACTACTTAGTGTGAAAGTTAATTGTAATAAAATTTATTCTTCA。
1.MBP基因克隆
根据MBP编码区cDNA序列,设计含有限制性内切酶KpnⅠ和EcoRⅠ酶切位点的特异性引物:
MBP-exF:5’-CGGGGTACCCAGCTCCCTTACGGTAGCTACG-3’,如SEQ ID NO.3所示;
MBP-exR:5’-CCGGAATTCCTATCCAACATAGCTTTGGAC-3’,如SEQ ID NO.4所示。
通过PCR技术扩增编码MBP成熟肽的基因片段(去除信号肽序列)。反应体系共50μL,包含25μLMax Premix(2×);1μL上游引物;1μL下游引物;2μL cDNA;21μLddH2O。反应条件为:98℃变性10s,55℃退火5s,72℃延伸10s,30个循环后,72℃延伸5min。将PCR产物进行1.2%的琼脂糖凝胶电泳,利用胶回收得到MBP表达片段。
2.目的基因原核表达载体的构建
a)按照下列反应体系进行pET-30a质粒双酶切:
载体pET-30a/DNA片段,1μg;
10×M Buffer,2μL;
KpnⅠ,1μL;
EcoRⅠ,1μL;
RNase free H2O,至20μL;
酶切反应条件:37℃水浴3h。
b)通过核酸凝胶电泳检测限制性内切酶切割效果,回收酶切样品并测定其浓度;
c)将回收的酶切后载体与片段进行连接,体系(10μL)为:
10×T4 DNA Ligase Buffer,1μL;
pET-30a质粒酶切产物,2μL;
DNA片段酶切产物,6μL;
T4 DNA连接酶,1μL;
16℃连接过夜。
3.连接后的重组质粒转化至克隆感受态细胞
a)取5μL的载体和目的片段连接产物加入50μL冰上融化的克隆感受态细胞E.coliTrans1-T1(TransGen,北京)混匀,冰浴30min;
b)42℃水浴热激45~60s,冰上静置2min;
c)向离心管中加入500μL灭菌且无抗生素的LB培养基,放入37℃摇床,200rpm,培养1h;
d)取100μL菌液均匀的涂在LB平板上(含卡那霉素),将平板倒置放于37℃培养箱过夜。
4.菌落PCR筛选阳性单克隆及测序分析
自平板中随机挑取5个单克隆菌株作为PCR反应模板,冰上配制25μL PCR反应体系,包括1μL菌液;1μL上游引物(10μM);1μL下游引物(10μM);12.5μL Easy DNAPolymerase;9.5μL灭菌ddH2O。反应条件:94℃预变性3min,94℃变性30s,56℃退火30s,72℃延伸1min,34个循环后,72℃延伸5min,4℃冷却5min。用1.2%琼脂糖凝胶进行电泳检测,取阳性克隆菌株测序。测序结果返回后,利用NCBI比对序列信息,并选出测序结果良好的返样质粒,转化E.coli Transetta(DE3)感受态细胞中,用含Kana的LB平板于37℃过夜培养并挑单克隆,取菌液PCR阳性单克隆进行下一步实验。
5.MBP重组蛋白的试表达
a)按1:100将培养的菌液接种至5mL灭菌的LB液体培养基(含卡那霉素)中,37℃,200rpm,培养3h,测定OD600为0.6~0.8;
b)加入诱导剂异丙基-β-D-硫代半苷(IPTG)至终浓度0.5mM,37℃,200rpm诱导4-5h;
c)10,000rmp离心1min,弃掉上清,重复收集全部细菌于离心管中;
d)用PBS+0.2% Triton X-100重悬收集的细菌,超声破碎(200W,6min,超声5s,间隔5s)重悬的菌液;
e)将破碎后的菌液10,000rpm,4℃离心10min,将分离得到的上清置于新的离心管中并重悬沉淀,将分离的上清和沉淀进行12% SDS-PAGE凝胶电泳和考马斯亮蓝染色检测,观察蛋白表达情况。
6.MBP重组蛋白的大规模表达和纯化
按试表达的方法将表达体系扩大至300mL Kana+LB液体培养基,并取IPTG诱导前的菌液用于后续检测。菌液经离心后,利用20mL灭菌后的1×PBS+0.2% Triton X-100重悬菌体。使用超声细胞粉碎机于冰浴中超声破碎菌体细胞,变幅杆2,功率200w,超声5s、间歇5s,工作总时间50min。菌体破碎后离心分离上清和沉淀,将上清过柱纯化,具体步骤如下:
a)组装His纯化柱:取含20%酒精的Ni柱进行柱子填装(试剂体积为2mL);
b)用6mL的无菌水洗涤填装后纯化柱(7~8s/滴);
c)用10mL的1×Charge Buffer(50mM NiSO4)洗涤柱子(7~8s/滴);
d)用6mL的Binding buffer缓冲液(0.5M NaCl,5mM imidazole,20mM Tris-HCl,pH 7.9)洗涤柱子(7~8s/滴);
e)把蛋白样品加入柱子中,收集穿透液(冰上收集,15s/滴);
f)用20mL的Binding buffer缓冲液洗涤柱子(7~8s/滴);
g)用Wash Buffer缓冲液(0.5M NaCl,60mM imidazole,20mM Tris-HCl,pH 7.9)洗涤柱子,分别收集洗脱样品并做好标记(冰上收集,15s/滴);
h)用10mL的Elute buffer缓冲液(0.5M NaCl,1M imidazole,20mM Tris-HCl,pH7.9)洗脱Ni柱上结合的MBP蛋白;
i)用8mL的1×Strip Buffer缓冲液(0.5M NaCl,0.1mM EDTA,20mM Tris-HCl,pH7.9)洗涤柱子(7~8s/滴);
j)用20mL的无菌水洗涤柱子(7~8s/滴);
k)用10mL的20%的酒精洗涤柱子,重悬填料,回收柱子,4℃保存;
l)分别对穿透液、洗涤液及洗脱液进行12% SDS-PAGE凝胶电泳检测并用考马斯亮蓝染色液检验(结果如图7所示),得到目标重组蛋白。
m)用TBS缓冲液对纯化后的重组蛋白进行透析,将最终得到的重组蛋白运用考马斯亮蓝蛋白定量测试盒(建成,南京)测定其浓度。
本发明成功地构建了MBP基因的大肠杆菌表达载体,并进行了重组表达和蛋白质纯化。
给本领域技术人员提供上述实施例,以完全公开和描述如何实施和使用所主张的实施方案,而不是用于限制本文公开的范围。对于本领域技术人员而言显而易见的修饰将在所附权利要求的范围内。
Claims (10)
1.克氏原螯虾甘露糖结合蛋白在制备具有抑制金黄色葡萄球菌功效的制剂中的应用,所述克氏原螯虾甘露糖结合蛋白的氨基酸序列如SEQ ID NO.1所示。
2.克氏原螯虾甘露糖结合蛋白在制备具有抑制藤黄微球菌功效的制剂中的应用,所述克氏原螯虾甘露糖结合蛋白的氨基酸序列如SEQ ID NO.1所示。
3.克氏原螯虾甘露糖结合蛋白在制备具有抑制枯草芽孢杆菌功效的制剂中的应用,所述克氏原螯虾甘露糖结合蛋白的氨基酸序列如SEQ ID NO.1所示。
4.克氏原螯虾甘露糖结合蛋白在制备具有抑制苏云金芽孢杆菌功效的制剂中的应用,所述克氏原螯虾甘露糖结合蛋白的氨基酸序列如SEQ ID NO.1所示。
5.克氏原螯虾甘露糖结合蛋白在制备具有抑制副溶血弧菌功效的制剂中的应用,所述克氏原螯虾甘露糖结合蛋白的氨基酸序列如SEQ ID NO.1所示。
6.克氏原螯虾甘露糖结合蛋白在制备具有抑制哈氏弧菌功效的制剂中的应用,所述克氏原螯虾甘露糖结合蛋白的氨基酸序列如SEQ ID NO.1所示。
7.克氏原螯虾甘露糖结合蛋白在制备具有抑制嗜水气单胞菌功效的制剂中的应用,所述克氏原螯虾甘露糖结合蛋白的氨基酸序列如SEQ ID NO.1所示。
8.克氏原螯虾甘露糖结合蛋白在制备促进克氏原螯虾、日本沼虾或中华绒螯蟹机体对细菌的清除速率、提高存活率的制剂中的应用,所述克氏原螯虾甘露糖结合蛋白的氨基酸序列如SEQ ID NO.1所示。
9.根据权利要求8所述的应用,其特征在于:所述细菌选自副溶血弧菌、金黄色葡萄球菌。
10.根据权利要求1~9中任一项所述的应用,其特征在于:所述制剂选自菌抑制剂、抗菌剂、杀菌剂、药物制剂、免疫增强剂、饲料添加剂。
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