CN110055257B - 一种雪蚤抗冻蛋白sf-p的制备方法及其应用 - Google Patents
一种雪蚤抗冻蛋白sf-p的制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种雪蚤抗冻蛋白SF‑P的制备方法及应用。所述抗冻肽是通过合成雪蚤抗冻肽基因，构建原核表达载体后表达纯化得到的。首先将人工合成的SF‑P编码基因插入到PET28a‑sumo载体中，构建所述抗冻肽的表达质粒；将重组质粒转化至大肠杆菌感受态细胞，进行蛋白表达，用亲和层析法对蛋白进行纯化后进行酶切，获得SF‑P抗冻肽。大肠杆菌具有遗传稳定性，可以持续分泌表达外源基因，所获重组工程菌株，可以高量、稳定地分泌表达雪蚤抗冻肽SF‑P，在抗冻蛋白领域发挥了重要的作用。
Description
技术领域
本发明属于生物与食品技术领域，具体地本发明涉及一种雪蚤抗冻蛋白SF-P的制备方法其应用。
背景技术
在寒冷环境中，生物体内的细胞环境会发生变化，具体表现为脱水形成结晶，因此造成细胞膜的破坏，进一步导致生物体受到损伤，为了适应极端寒冷环境，一些低温生物体内会产生一类多肽或糖肽，帮助它们抵御寒冻危险。这类多肽或糖肽称作抗冻肽(antifreeze peptide，AFP)，其由DeVries首在南极硬骨鱼中发现，随后证实具有抑制冰晶形成的功能。AFP具有特殊的抗冻活性，主要表现为：(1)能结合到冰晶表面并抑制冰晶进一步生长；(2)能以非依数形式降低溶液的冰点，使溶液的熔点和冰点之间出现差值，表现为热滞活性；(3)具有冰重结晶抑制效应。AFP可以避免细胞受到由冰晶导致的机械性损伤，因而其资源开发及应用引起了广泛关注，在农业低温育种、冷冻食品加工与储藏、医药行业上的广泛的应用发挥了一定的作用。
雪蚤抗冻蛋白(snow flea antifreeze protein,SF-P)首次发现于加拿大，富含甘氨酸。目前经过研究发现的雪蚤抗冻蛋白具有两种同种型模体，分子量分别为6.5kDa和15.7kDa，后者具有更高的抗冻活性。雪蚤蛋白可以通过使体液凝固点降低6℃的方法来抑制冰晶的生成，此功能有助于避免雪蚤在暴露的积雪地面上结冰。经过证实，雪蚤蛋白粗提物的热滞活性高达6℃，比在鱼类及植物中发现的大多数抗冻蛋白高一个或者两个数量级。但是因为自然条件的苛刻，雪蚤体内抗冻蛋白含量稀少，使得人们难以从雪蚤体内直接分离纯化，对其应用价值造成了限制。因此，雪蚤抗冻蛋白SF-P的高效制备成为了研究热点。大肠杆菌是目前广泛采用的进行活性蛋白或多肽生物表达的宿主，通过现代生物技术进行结合基因工程菌，可得到活性较高的雪蚤抗冻蛋白，将其运用于改善冻融产品的质量、产品储存、疾病治疗等领域中，具有极大的应用前景。
发明内容
有鉴于现有技术的上述缺陷，本发明所要解决的技术问题是如何高效表达雪蚤抗冻蛋白SF-P。
为实现上述目的，本发明提供了一种重组雪蚤抗冻肽SF-P的制备方法，其特征在于，包括以下步骤：
步骤一：将人工合成的抗冻肽编码基因构建于克隆载体上；
步骤二：将含有所述抗冻肽表达序列的重组质粒转化至大肠杆菌感受态细胞中，进行蛋白表达；
步骤三：用亲和层析法对表达蛋白进行纯化；
步骤四：使用蛋白酶切开纯化蛋白标签，获得所述抗冻肽。
进一步地，所述步骤一中，合成的的抗冻肽基因长度为252bp，其基因序列如SEQID NO.2所示。
进一步地，所述克隆载体包括标签序列，所述标签序列如SEQ ID NO.1所示。
进一步地，所述步骤一中，所述克隆载体为PET28a-sumo载体。
进一步地，所述步骤二中，所述大肠杆菌感受态细胞为大肠杆菌BL21(DE3)感受态细胞。
进一步地，所述步骤三中，所述亲和层析法中所用层析柱为镍柱。优选参数如下：填料：Ni-Smart 6FF，流速为2mL/min。
进一步地，所述步骤四中，所述蛋白酶为sumo蛋白酶，所切标签为His-sumo标签。
本发明还提供了一种重组雪蚤抗冻肽SF-P的制备方法制备的重组雪蚤抗冻肽在生物和食品抗冻保存中的应用。
进一步地，所述抗冻中生物抗冻中的应用可以具体为菌体冻存中的保证菌体的活力，所述菌体包括但不限于细菌、真菌和细胞的保存。优选地，所述菌体为嗜热链球菌
进一步的所述食品抗冻中的应用可以是低温条件下食品的保鲜。
本发明提供了一种高效表达雪蚤抗冻肽SF-P的方法，可以高效且大量的获得抗冻蛋白。大肠杆菌具有遗传稳定性，可以持续分泌表达外源基因，所获重组工程菌株，可以高量、稳定地分泌表达雪蚤抗冻肽SF-P。本发明生产的雪蚤抗冻肽SF-P在食品保鲜和菌体保藏中有的潜在的应用。
附图说明
图1是目标蛋白SF-P抗冻肽电泳图；
图2是雪蚤抗冻肽对冷冻面团可冻结水含量的影响示意图。
具体实施方式
为了使本发明的目的、技术方案以及优点更加清楚明白，下面结合具体实施例及附图，阐述本发明。应当理解，此处所描述的实施例仅用于说明本发明而不用于限制本发明的范围。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1
一、构建基因基因表达载体
合成252bp的SF-P基因(如下：加粗字体标记)插入PET28a-sumo载体中(如下：下划线字体标记)，将人工合成的SF-P基因构建于PET28a-sumo载体上,构建所述抗冻肽的表达质粒，转化至大肠杆菌感受态细胞。
挑取得到的单克隆菌接种到5mL LB液体培养菌中，37℃培养过夜。第二天加入600mL含卡那霉素抗性的LB液体培养基中，37℃培养3h,至OD600值约0.6时，加入IPTG至终浓度为100μM，37℃诱导4h。4℃，7000rpm离心10min,收集菌体沉淀，弃尽上清，用80mL TBS缓冲液重悬后于-20℃保存。
三、表达产物处理
离心收集菌体，将菌体沉淀于超声细胞破碎仪中超声30min以裂解细胞，超声结束后在4℃条件下10000rpm离心20min，分离上清和沉淀，并将上清液转至干净容器中。
四、分离纯化
1)NI Smart 6FF填料装柱，装柱体积为20mL；
2)用TBS缓冲液平衡5倍柱体积，流速为3mL/min；
3)将80mL细胞破碎上清液0.45μml滤膜过滤，上样，流速为2mL/min；
4)用TBS缓冲液清洗5倍柱体积，流速为3mL/min；
5)用含20mM、50mM、250mM咪唑的TBS缓冲液进行阶段洗脱，流速为3mL/min，收集各阶段洗脱峰。所有蛋白样品均进行SDS-PAGE检测，分析分子量大小和纯度。图1为纯化后的跑胶图，共10个泳道，其中L1和L10为marker，L2为培养上清，L3为流川液，L4、L6和L8分别为20mM、50mM和250mM的咪唑洗脱后跑胶结果，L5、L7和L9分别为20mM、50mM和250mM的咪唑洗脱后酶切跑胶图。
五、酶切纯化融合蛋白
用2mg sumo蛋白酶切开1g融合蛋白的His-sumo标签。然后用TBS缓冲液透析酶切溶液，接着用Ni-Smart 6FF镍柱进行处理，收集穿透峰。
六、雪蚤抗冻肽对冷冻面团可冻结水含量的影响的测定
将制备得到的雪蚤抗冻肽添加于冷冻面团中，观察冻藏4周时间内面团中可冻结水含量变化的情况。如图2所示，冻藏4周后，未添加雪蚤抗冻肽的冷冻面团中可冻结水含量达到82.17％,与初始的68.18％可冻结含水量相比，增幅为20.52％。添加了0.01％雪蚤抗冻肽的冷冻面团可冻结水含量从63.52％增加到74.37％，增幅为17.08％，添加了0.05％雪蚤抗冻肽的冷冻面团可冻结水含量从64.60％增加到70.89％，增幅为9.73％。由此说明，雪蚤抗冻肽的添加，有效地降低了面团中可冻结水含量与水分迁移的情况，其在面团中能与自由水结合，抑制结晶与重结晶。提高了冷冻面团的持水性，减少冻藏过程中冰晶的产生。
实施例2
一、构建基因基因表达载体
合成252bp的SF-P基因(如下：加粗字体标记)插入PET28a-sumo载体中(如下：下划线字体标记)，将人工合成的SF-P基因构建于PET28a-sumo载体上,构建所述抗冻肽的表达质粒，转化至大肠杆菌感受态细胞。
二、诱导表达
挑取得到的单克隆菌接种到5mL LB液体培养菌中，37℃培养过夜。第二天加入600mL含卡那霉素抗性的LB液体培养基中，37℃培养3h,至OD600值约0.6时，加入IPTG至终浓度为100μM，37℃诱导4h。4℃，7500rpm离心10min,收集菌体沉淀，弃尽上清，用40mL TBS缓冲液重悬后于-20℃保存。
三、表达产物处理
离心收集菌体，将菌体沉淀于超声细胞破碎仪中超声15min以裂解细胞，超声结束后在4℃条件下10000rpm离心20min，分离上清和沉淀，并将上清液转至干净容器中。
四、分离纯化
1)NI Smart 6FF填料装柱，装柱体积为2.5mL；
2)用TBS缓冲液平衡5倍柱体积，流速为2mL/min；
3)将40mL细胞破碎上清液0.45μml滤膜过滤，上样，流速为1mL/min；
4)用TBS缓冲液清洗5倍柱体积，流速为2mL/min；
5)用含20mM、50mM、250mM咪唑的TBS缓冲液进行阶段洗脱，流速为2mL/min，收集各阶段洗脱峰。所有蛋白样品均进行SDS-PAGE检测，分析分子量大小和纯度。
五、酶切纯化融合蛋白
用4mg sumo蛋白酶切开1g融合蛋白的His-sumo标签。然后用TBS缓冲液透析酶切溶液，接着用Ni-Smart 6FF镍柱进行处理，收集穿透峰。
六、雪蚤抗冻肽对嗜热链球菌冷冻存活率影响的测定
将制备得到的雪蚤抗冻肽添加于嗜热链球菌液中，观察-20℃条件下冷冻24h后的细菌存活情况。在雪蚤抗冻肽添加浓度分别为0.05mg/mL、0.10mg/mL、0.50mg/mL、1.0mg/mL、5.0mg/mL的情况下，嗜热链球菌的冷冻存活率为60％、80％、60％、62％、38％。与之相较，添加0.2mg/mL PBS缓冲液的嗜热链球菌存活率仅为31％。添加10％甘油的嗜热链球菌存活率为83％；添加1.0mg/mL蔗糖的嗜热链球菌存活率为71％；添加1.0mg/mL脱脂奶粉的嗜热链球菌存活率为69％。说明雪蚤抗冻肽的添加对嗜热链球菌有保护作用，明显提高了嗜热链球菌在冷冻条件下的存活率，并且在添加量合适的情况下，抗冻效果能与市售抗冻剂相媲美。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。
序列表
<110> 上海交通大学
<120> 一种雪蚤抗冻蛋白SF-P的制备方法及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 318
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgggtcacc atcaccatca ccatatgtcg gactcagaag tcaatcaaga agctaagcca 60
gaggtcaagc cagaagtcaa gcctgagact cacatcaatt taaaggtgtc cgatggatct 120
tcagagatct tcttcaagat caaaaagacc actcctttaa gaaggctgat ggaagcgttc 180
gctaaaagac agggtaagga aatggactcc ttaagattct tgtacgacgg tattagaatt 240
caagctgatc agacccctga agatttggac atggaggata acgatattat tgaggctcac 300
agagaacaga ttggtggc 318
<210> 2
<211> 252
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tgtaaaggtg cagatggtgc acatggtgtt aatggttgtc cgggtacagc cggtgcagca 60
ggtagcgttg gtggtccggg ttgtgatggt ggtcatggtg gcaatggcgg taatggtaat 120
ccgggttgcg caggcggtgt tggtggtgct ggcggtgcaa gcggtggtac aggtgttggc 180
ggtcgtggtg gtaaaggtgg tagcggcacc ccgaaaggtg ccgacggtgc tcctggtgca 240
ccgtaactcg ag 252
Claims (5)
1.一种重组雪蚤抗冻肽SF-P的制备方法制备的重组雪蚤抗冻肽在生物和食品抗冻保存中的应用，所述抗冻中的应用具体为菌体冻存中的保证菌体的活力，所述菌体为嗜热链球菌；所述重组雪蚤抗冻肽SF-P采用以下方法制备得到：
步骤一：将人工合成的抗冻肽编码基因构建于克隆载体上；其中，所述克隆载体包括标签序列，所述标签序列如SEQ ID NO.1所示；合成的抗冻肽编码基因长度为252bp，其基因序列如SEQ ID NO.2所示。
步骤二：将含有所述抗冻肽编码基因的重组质粒转化至大肠杆菌感受态细胞中，进行蛋白表达；
步骤三：用亲和层析法对表达蛋白进行纯化；
步骤四：使用蛋白酶切开纯化蛋白标签，获得所述抗冻肽。
2.如权利要求1所述的应用，其特征在于，所述步骤一中，所述克隆载体为PET28a-sumo载体。
3.如权利要求1所述的应用，其特征在于，所述步骤二中，所述大肠杆菌感受态细胞为大肠杆菌BL21(DE3)感受态细胞。
4.如权利要求1所述的应用，其特征在于，所述步骤三中，所述亲和层析法中所用层析柱为镍柱。
5.如权利要求1所述的应用，其特征在于，所述步骤四中，所述蛋白酶为sumo蛋白酶，所述标签为His-sumo标签。
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