CN114381456B - 一种人工合成的纳米银合成蛋白基因及其表达的蛋白和应用 - Google Patents
一种人工合成的纳米银合成蛋白基因及其表达的蛋白和应用 Download PDFInfo
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Abstract
本发明公开了一种人工合成的纳米银合成蛋白基因及其表达的蛋白和应用，根据铁硫蛋白家族蛋白序列共有特征，依靠蛋白质工程的相关技术支撑，人工设计了出具有一定还原性的纳米银合成蛋白。所述蛋白的DNA序列如SEQ NO:1，氨基酸序列如SEQ NO:2所示。本发明的到的蛋白质能够高效合成纳米银，合成效率高，合成的纳米银溶液呈褐色，具有良好的广谱抗菌性且抗菌效果显著。
Description
技术领域
本发明属于生物工程技术领域，涉及一种纳米银合成蛋白，具体涉及一种人工合成的纳米银合成蛋白基因及其表达的蛋白和应用。
背景技术
纳米银是粒径达到纳米级别的金属单质，其颗粒微小，比表面积大，具有超强的渗透性。纳米银通过释放阴离子使细胞膜结构发生变化，造成细胞液渗漏；还可以与细胞内部分重要酶中的硫氢基团反应，使细胞无法分裂；还可以阻止DNA复制，进而引起细胞凋亡。由于其物理抑菌能力，纳米银有着传统无机抗菌剂不具备的广谱抗菌性、极低的耐药性。
目前，主流纳米银合成方法分三类：1.物理法，使用机械研磨或者激光烧蚀大块的银材料得到纳米银粉末。物理法合成纳米银得到的产品容易团聚，在合成过程中存在许多难控制的步骤，使得能耗大、对设备要求高。2.化学法，在银盐溶液中还原剂、保护剂、分散剂，在适当的条件下将银盐还原为银单质。这类的工艺过程简单，但是过程中使用了较多的化学试剂，对环境污染大，后续废液处理困难。3.生物法，通过生物细胞内外的生物学功能进行纳米银的制备。利用自然界中某些生物，如一些具有较强还原性如产甲烷菌或真菌、植物中的含有活性成分的提取液制备纳米银，制备条件温和，无需任何不利于环境保护的化学试剂，充分利用生物资源，能够达到可持续发展的绿色环保效果。
发明内容
针对现有技术的不足，本发明的目的在于提供一种人工合成的纳米银合成蛋白基因及其表达的蛋白和应用。本发明的人工合成蛋白基因锁表达的蛋白属于铁硫蛋白家族蛋白，可用于高效合成纳米银。
本发明是通过以下技术方案实现的：
一种人工合成的纳米银合成蛋白基因，所述基因的DNA序列如SEQ NO:1所示。
本发明还提供了如上述人工合成的纳米银合成蛋白基因编码得到的纳米银合成蛋白。
进一步的，所述纳米银合成蛋白为如下（1）或（2）的蛋白：
（1）具有如SEQ NO:2所示氨基酸序列的蛋白；
（2）如SEQ NO:2所示氨基酸序列中的氨基酸经过一个或多个氨基酸残基的取代、缺失及添加形成的具有纳米银合成能力的蛋白质。
本发明还提供了一种重组载体，由空载体和插入该空载体的目的基因组成，所述目的基因为具有DNA序列如SEQ NO:1所示的基因。
进一步的，所述空载体为pET-28a。
进一步的，本发明还提供了一种重组菌，所述重组菌包具有DNA序列如SEQ NO:1所示的基因。
本发明的进一步改进方案为：
上述纳米银合成蛋白在合成纳米银中的应用。
进一步的，合成纳米银时，硝酸银底物浓度为5-50mM,合成反应温度为20-70℃，pH为11，反应时间为12-24h。
与现有技术相比，本发明的有益效果为：
1.根据产甲烷菌铁硫蛋白家族的共有特征，依靠蛋白质工程的相关技术支撑，结合各种氨基酸的还原性、水溶性等相关参数，通过分子配对模拟与银离子结合的各种可能情况，设计并筛选出如SEQ NO:2所示的氨基酸序列。本蛋白在碱性条件下，无需其他试剂即可合成纳米银。合成条件简单，设备要求低，对环境友好。
2.本发明中的人工设计的铁硫蛋白家族蛋白在高效合成纳米银时，pH范围在10-11，温度范围20-70℃，合成时间12-24h。得到的纳米银溶液为褐色，性质稳定，室温储存一个月均有抑菌效果。
3.本发明的蛋白合成的纳米银，对金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌、酵母均有良好的抑菌效果，具有及其广阔的应用前景和工业价值。
附图说明
图1为人工设计的铁硫蛋白家族蛋白质Mcy11 SDS-PAGE蛋白电泳图；
图2为合成纳米银溶液稀释5倍后的全波长扫描图；
图3为使用本发明的蛋白合成纳米银进行平板抑菌实验的结果，其中，a为酵母，b为金黄色葡萄球菌、c为大肠杆菌。
具体实施方式
下面结合具体实施例对本发明做进一步的说明。
以下实施例中所使用的的材料、试剂等，若无特殊说明，均可从商业途径获得。
实施例1 人工设计的铁硫蛋白家族蛋白的基因的制备
通过数据库比对分析，总结出产甲烷菌铁硫蛋白家族蛋白质氨基酸偏好性和结构共性，依靠蛋白质工程技术设计出氨基酸序列骨架；使用Alphafold2模拟出氨基酸序列骨架的三维结构；使用AutoDock模拟银离子与蛋白质相互作用情况，对氨基酸骨架进行优化，优先选择天冬氨酸，组氨酸，丝氨酸等活性较高的氨基酸作为纳米银合成中的电子供体；搭配脯氨酸、异亮氨酸、精氨酸等耐热蛋白中高频氨基酸补充，提高蛋白质的结构强度；同时还要考虑蛋白质的可溶性，预测蛋白质溶解性。得到了如SEQ NO:2所示的氨基酸序列；再根据表达宿主的密码子偏好性，设计出如SEQ NO:1所示的DNA序列，人工合成该基因片段。
实施例2 重组克隆、表达载体的构建与验证。
将合成的目的基因片段（实施例1制备）、pET-28a（Novagen）用分别Xba Ⅰ和Xho Ⅰ双酶切，琼脂糖电泳回收酶切PCR及载体大片段。割胶回收后的目的片段与载体，加入1 μL10 X Ligase Buffer和1 μL Ligase，于16 ℃连接过夜。用连接反应产物转化大肠杆菌DH5α，然后涂于含100 μg/mL Kana（卡那青霉素）的培养皿，37 ℃培养10-15 h。
从转化平板上挑取多个单菌落，采用BIOMIGA的质粒小量提取试剂盒提取质粒。对获得的质粒双酶切验证并对获得的重组质粒进行测序。测序结果显示，pET-28a载体中插入了所克隆的目的片段（核苷酸长度为432bp），进而得到重组克隆、表达载体pET-28a-Mcy11，其表达蛋白基因的DNA序列如SEQ NO:1所示，其表达的蛋白的氨基酸序列如SEQ NO:2所示，将该蛋白命名为Tp Mcy11。
实施例3 重组纳米银合成蛋白Tp Mcy11的表达与纯化
将重组克隆、表达载体pET-28a- Mcy11（实施例2制备）热激转化至宿主菌E. coliBL21（DE3）（Novagen），获得含有重组质粒的重组菌。将单菌落的重组菌接种于5 mL含有100μg/mL卡那青霉素的Luria-Bertani broth（LB）培养基，在37 ℃温度下，200 rpm震荡培养4h。将上述4 mL菌液接种于含800 mL培养基的2000 mL摇瓶中，37 ℃温度下，200 rpm振荡培养，当吸光度达到0.4-0.6时，加入800 μL的0.1M IPTG，并在22 ℃温度下，150 rpm诱导表达15 h。用高速冷冻离心机将培养液在4 ℃下以6000 rpm离心10 min，收集菌体。用50 mL超纯水洗涤并在4 ℃下以6000 rpm离心10 min，回收菌体，接着用20 mL Binging Buffer重悬（0.5M NaCl，20mM Tris-HCl，5mM Imidazole，pH 7.4），在冰水浴中，用超声波破碎机破碎细菌细胞，并在4℃下以10000 rpm离心50 min，得到重组纳米银合成蛋白Tp Mcy11的粗提液。
粗提液使用Ni-NTA亲和层析柱进行纯化（方法见His-Band Kits，Novagen）。纯化蛋白纯度的鉴定和分子量的测定采用SDS-PAGE方法进行，结果见图1，1表示200 mM咪唑洗脱的纯化Mcy11蛋白。分子量约为16 kDa，与理论值相近。
实施例4 使用重组纳米银合成蛋白Tp Mcy11合成纳米银
将实施例3得到的纯化后的蛋白溶液透析除去咪唑和Nacl，超滤浓缩至15mg/ml，按照硝酸银浓度（mM）：蛋白质终浓度（μg/ml）=1:2的比例配置反应溶液30ml，使用氢氧化钠调节pH至11，在30℃、150rpm转速的摇床震荡20h后得到深褐色的纳米银溶液，其全波长扫描如图2所示。纳米银溶液在全波长扫描下，在410mm-440mm下通常出现一个吸收峰，在一定浓度范围内，纳米银浓度越高，吸收峰越高。低于纳米颗粒来说，随着粒径的减小，由于量子尺寸效应，纳米材料的吸收波长向短波方向移动；表现为合成纳米银颗粒的粒径越小，峰越向400mm方向移动。本蛋白合成的纳米银颗粒全波长扫描结果显示，得到的纳米银粒径均一且粒径较小，通过动态光衍射分析粒径分布在20mm左右。
实施例5 纳米银抑菌圈实验
病原菌平板的制备：每100ml添加2%营养琼脂的LB培养基中加入2ml过夜培养的病原菌（金黄色葡萄球菌ATCC6538、大肠埃希氏菌 ATCC25922、球拟酵母菌）培养基，混匀倒入培养皿。在每个滤纸片上分别吸附6μl 50μg/ml的氨苄青霉素或者硫酸卡那霉素、未加入蛋白的纳米银合成溶液上清、透析除去氯化钠的蛋白溶液、使用Tp Mcy11合成的纳米银溶液。置于37℃培养箱培养24小时。通过抑菌圈情况发现：未添加蛋白质的纳米银合成溶液上清与蛋白质溶液均没有抑菌效果，而添加了蛋白质的纳米银合成溶液上清具有明显的抑菌圈，证明此蛋白可以合成纳米银，且纳米银对真菌（球拟酵母菌）、革兰氏阳性（金黄色葡萄球菌ATCC6538）、革兰氏阴性菌（大肠埃希氏菌 ATCC25922）均有一定的抑菌效果。
序列表
<110> 淮阴工学院
<120> 一种人工合成的纳米银合成蛋白基因及其表达的蛋白和应用
<130> 2021
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<213> 人工序列(Artificial sequence)
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aaactgattg atcgcgcggg caaagaaagc cagcaggtga ccccggatct ggatgatacc 240
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Lys Gly Arg Leu Gly Asn Met Ile Glu Lys Leu Glu Ala Ile Gln Thr
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Asp Glu Ala His Val His Lys Leu Glu Leu Phe Ile Arg Glu Thr Leu
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Claims (6)
1.一种人工合成的纳米银合成蛋白，其特征在于，由SEQ NO:1所示的基因编码得到。
2.一种重组载体，其特征在于，由空载体和插入该空载体的目的基因组成，所述目的基因为权利要求1中所述的基因。
3.根据权利要求2所述的一种重组载体，其特征在于：所述空载体为pET-28a。
4.一种重组菌，其特征在于，所述重组菌包含权利要求1中所述的基因。
5.如权利要求1所述的纳米银合成蛋白在合成纳米银中的应用。
6.根据权利要求5所述的纳米银合成蛋白的应用，其特征在于：合成纳米银时，硝酸银底物浓度为5-50mM,合成反应温度为20-70℃，pH为11，反应时间为12-24h。
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