CN109134663B - 一种宽吸收光谱重组光合蛋白分子及其构建方法 - Google Patents
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Abstract
本发明公开了一种宽吸收光谱重组光合蛋白分子，由重组叶绿素结合蛋白、藻胆蛋白融合蛋白与叶绿素a在体外进行结合得到，首先，利用分子生物学和基因工程的手段，结合藻胆蛋白色基体内组合催化技术，构建叶绿素结合蛋白、全色基藻胆蛋白融合分子，并在大肠杆菌工程菌株BL21中进行异源表达，再将叶绿素a与叶绿素结合蛋白、藻胆蛋白融合分子进行体外重组，得到既包含LHCⅡ又包含藻胆蛋白吸收光谱的宽吸收光谱光敏剂，为大规模制备重组藻胆蛋白及LHCⅡ色素蛋白分子提供了成熟的方法，并为探究生物基染料敏化太阳能电池的应用以及拓宽染料敏化剂吸收光谱范围提供了新的思路。
Description
技术领域
本发明涉及生物材料领域，涉及一种光合蛋白分子，具体涉及一种宽吸收光谱重组光合蛋白分子及其构建方法。
背景技术
能源危机和环境问题都是当前人类面临的日益紧迫的重要问题，随着不可再生资源的日渐减少和环境污染的不断加剧，人们开发新型清洁能源的任务迫在眉睫。太阳能作为一种取之不尽、环境友好和分布广泛的可再生资源，各国研究者一直都非常热衷于将太阳能进行开发利用，应用于人类生活的各个方面。
染料敏化太阳能电池(Dye Sensitized Solar Cell,DSSC)继硅系列太阳能电池和化合物薄膜太阳能电池之后出现，是典型的第三代太阳能电池，相比于单晶硅太阳能电池，染料敏化太阳能电池具有制备工艺简单、成本低廉以及环境友好等优点。DSSC的基本结构主要包括：透明导电玻璃、TiO2纳米晶粒薄膜、光敏染料、电解液以及对电极。染料敏化剂吸收太阳光后，激发态的染料分子会将电子转移到TiO2纳米晶粒上，然后形成电子传输通道，电子将会在透明导电玻璃上进行收集，染料失去电子变为氧化态，并在电解液中还原态物质的作用下便变回基态，最后电解质在对电极处得到还原，从而完成整个太阳能电池反应的全过程。DSSC研究中一个至关重要的问题就是染料敏化剂。
对于敏化剂的选择，相对于化学染料来说，天然生物染料分子更加天然环保，不污染环境，来源广泛。而作为最理想的敏化剂应该具备的最重要的两个特征：一、染料分子的吸收光谱可以覆盖整个可见光区。二、染料分子的光量子产率尽可能高。但是目前天然生物染料敏化太阳能电池光敏剂普遍存在吸收光谱范围窄的问题。
发明内容
有鉴于此，本发明提供了一种宽吸收光谱重组光合蛋白分子及其构建方法，旨在解决现有技术中生物染料敏化剂吸收光谱范围窄的问题。
为了实现上述目的，本发明采用如下技术方案：
一种宽吸收光谱重组光合蛋白分子，由叶绿素结合蛋白、全色基藻胆蛋白融合蛋白与叶绿素a在体外进行结合得到，
其中，融合蛋白为藻蓝蛋白α亚基cpcA基因与叶绿素结合蛋白LhcⅡ基因经重叠PCR融合后分别与本实验前期构建并保存的重组质粒pCDFDuet-cpcA-cpcEF-hox1-pcyA和pCDFDuet-cpcA-cpcEF-hox1-pebS连接所构建表达的蛋白分子。
重组表达的LHCⅡ/PCA融合蛋白在体外可以结合8个叶绿素a分子。在AM1.5，光照强度为100mW/cm2的条件下，测得重组后LHCⅡ复合物敏化电池效率η为0.179％；相同浓度下，叶绿素a敏化电池的效率η为0.198％。相较之下，叶绿素a比人工体外重组的LHCⅡ的光电效率高，分析其原因，是由于同样的叶绿素a，其吸收光谱相同，但是叶绿素a比重组LHCⅡ的分子质量小，在染料敏化太阳能电池的光电阳极上的吸附效果好，所以表现出相对较好的光电性能。本发明构建了藻胆蛋白与叶绿素结合蛋白的融合分子，并且将融合蛋白LHCⅡ-PCA-PEB与叶绿素a在体外重组成功。
本发明的宽吸收光谱重组光合蛋白分子有益效果在于：
(1)天然有机染料来源广泛，且吸收光谱响应范围比无机材料的吸收范围宽。
(2)作为光敏剂，天然色素分子不仅能大幅度提高对光的捕获能力，而且其量子产率一般也在一个较高的水平上，捕获的大部分光子基本都能形成有效的电荷分离。
(3)天然色素染料分子环保，无污染，不会像化学染料合成过程中毒性比较大，污染环境。
本发明还提供了一种宽吸收光谱重组光合蛋白分子的构建方法，包括以下步骤：
(1)分别以pUC19-LhcⅡ为模板，LHCⅡ-F和LHCⅡ-PCA-R为引物；以pCDFDuet-cpcA-cpcEF-hox1-pcyA质粒为模板，LHCⅡ-PCA-F和PCA-R为引物配制PCR体系，进行将藻蓝蛋白α亚基cpcA基因与叶绿素结合蛋白LhcⅡ基因中目的片段LhcⅡ和cpcA的扩增进行PCR扩增延伸，然后切胶，进行胶回收；
(2)以胶回收得到的LhcⅡ片段和cpcA片段为模板，LHCⅡ-F、PCA-R为引物进行重叠PCR，得到融合片段LhcⅡ-cpcA，将PCR产物进行回收，得到融合基因片段，-20℃冰箱保存；
(3)将融合基因片段与T载体进行连接，连接体系由体积比为4:1:5的LhcⅡ-cpcA、pMD19-T和SolutionⅠ组成，得到pMD19-T-LhcⅡ-cpcA质粒，再取实验前期构建并保存于超低温冰箱的pCDFDuet-cpcA-cpcEF-hox1-pcyA和pCDFDuet-cpcA-cpcEF-hox1-pebS质粒，分别使用限制性内切酶BamHⅠ、SacⅠ，37℃恒温水浴锅中分别对三种质粒进行双酶切，时长为16h，体系中DNA、BamHⅠ、SacⅠ、10×Cut Smart Buffer、H2O的体积比为30:1:1:5:13，结束后进行胶回收，得到目的片段LhcⅡ-cpcA和相应载体片段；
分别将藻胆色素合成相关基因cpcEF、hox1、pcyA以及pebS等基因经适当酶切后连接到同一载体上，构建得到可表达LHCⅡ-PCA-PCB及LHCⅡ-PCA-PEB的重组质粒(pCDFDuet-LhcII-cpcA-cpcEF-hox1-pcyA和pCDFDuet-LhcII-cpcA-cpcEF-hox1-pebS)，并将其分别转化进大肠杆菌工程菌株BL21感受态细胞中，将重组质粒进行表达制备；
(4)将收集的菌体进行悬浮、破碎、离心、过滤、上样、咪唑梯度洗脱，纯化出的蛋白进行脱盐后保存在-80℃冰箱；
(5)将纯化成功的融合蛋白与叶绿素a在体外经超声处理、反复冻融以及脱盐等结合纯化步骤，即得宽吸收光谱重组光合蛋白分子。
上述所涉及的引物LHCⅡ-F序列为
5’-GCGGATCCGGGTAATGATCTGTGGTATGGTC-3’，
引物PCA-R序列为
5’-ATGAGCTCCTACTAGCTTAGGGCGTTGATC-3’，
引物LHCⅡ-PCA-F序列为
5’-CAACCAAATTTGCTCCGGGCAGCGGCGGCGGCAGCGGCGGCGGCAGCATGAAAACCCCCCTAACCGAAG-3’
引物LHCⅡ-PCA-R序列为
5’-CTTCGGTTAGGGGGGTTTTCATGCTGCCGCCGCCGCTGCCGCCGCCGCTGCCCGGAGCAAATTTGGTTG-3’。
本发明利用分子生物学和基因工程的手段，结合藻胆蛋白色基体内组合催化技术，构建叶绿素结合蛋白/藻胆蛋白融合分子，并在大肠杆菌工程菌株(BL21)中进行外源表达，再将叶绿素a与叶绿素结合蛋白/全色基藻胆蛋白融合分子进行体外重组，得到既包含LHCⅡ又包含藻胆蛋白吸收光谱的宽吸收光谱光合蛋白光敏剂。为大规模制备重组全色基藻胆蛋白和LHCⅡ及其与叶绿素a的体外重组提供了成熟的方法，为探究生物基染料敏化太阳能电池的应用及拓宽生物染料敏化剂吸收光谱范围提供了新的思路。
附图说明
图1为本发明实施例1中PCR产物1％琼脂糖凝胶电泳结果；
图2为本发明实施例2中LhcⅡ-cpcA连接产物1％琼脂糖凝胶电泳结果；
图3为本发明实施例3中双酶切产物1％琼脂糖凝胶电泳结果；
图4为本发明实施例4中LHCⅡ-PCA-PCB和LHCⅡ-PCA-PEB的菌体PCR电泳图；
图5为本发明实施例5中LHCⅡ-PCA-PCB和LHCⅡ-PCA-PEB包涵体纯化蛋白电泳；
图6为本发明实施例5中LHCⅡ-PCA-PEB的SDS-PAGE电泳及锌离子检测；
图7为本发明实施例7中融合蛋白LHCⅡ-PCA-PEB与叶绿素a体外结合的紫外吸收光谱。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
PCR扩增目的基因
以pUC19-LhcⅡ为模板LHCⅡ-F和LHCⅡ-PCA-R为引物，pCDFDuet-cpcA-cpcEF-hox1-pcyA质粒为模板LHCⅡ-PCA-F和PCA-R为引物，分别扩增LhcⅡ和cpcA基因，其目的条带理论值分别为687bp和492bp。经电泳检测如图1所示，参照Marker为D2000，发现紫外照射下条带清晰，LHCⅡ基因实际大小在Marker的第三条带750bp处稍靠下的位置与理论值基本一致，PCA基因实际大小在Marker的第四条带500bp处与理论值基本相符合，图1中，a为LhcⅡ片段；b为Marker；c为cpcA片段。
实施例2
重叠PCR
以胶回收得到的LhcⅡ、cpcA基因作为模板，进行重叠PCR，将两片段连接。LHCⅡ-PCA融合片段大小为1200bp，经电泳检测，参照Marker为D2000，发现目的条带大小在1000bp稍微偏上的位置，与理论值1200bp基本一致，证明融合片段连接成功，实验结果如图2所示，图2中a为LhcⅡ-cpcA片段；b为Marker：
实施例3
双酶切
以Marker(1kb)参照，经过BamHⅠ、SacⅠ双酶切的质粒载体pCDFDuet-cpcA-cpcEF-hox1-pcyA和pCDFDuet-cpcA-cpcEF-hox1-pebS，可看到清晰的大分子量的目的条带片段，且与理论值6kb基本一致，且在下方小分子量处还有切除的小基因片段，结果如图3所示，图3中a为含有PEB的载体；b为切除的小基因片段；c为含有PCB的载体；d为切除的小基因片段：
实施例4
重组表达载体的构建及菌液PCR验证
将融合片段LhcⅡ-cpcA及上述酶切后载体的双酶切产物在T4 DNA ligase酶的作用下
过夜连接后转化感受态细胞。
挑单克隆菌落，进行菌落PCR验证，经电泳检测如图4所示。a-j号是LHCⅡ-PCA-PCB菌液PCR的电泳结果，A-J是LHCⅡ-PCA-PEB菌液PCR的电泳结果。以Marker(D2000)为参照，可以看出在a、b、d、e、f、i、j、B、C、D、E、F、G、H、I、J都有条带，且片段大小在1000bp以上与理论值(约1200bp)基本一致，可以初步判断已经成功构建重组蛋白LHCⅡ-PCA-PCB(pCDFDuet-LhcⅡ-cpcA-cpcE/F-hox1-pcyA)表达载体和重组蛋白LHCⅡ-PCA-PEB(pCDFDuet-LhcⅡ-cpcA-cpcE/F-hox1-pebS)表达载体。取条带清晰的单克隆测序，进一步进行分析判断。
将菌液PCR中有条带的菌液提质粒送测序，测序结果均正确，所以将测序正确的质粒导入大肠杆菌感受态细胞BL21中，进行扩大培养。
实施例5
目的蛋白的SDS-PAGE电泳与锌离子检测
(1)SDS-PAGE电泳
将纯化出的蛋白进行SDS-PAGE电泳，通过判断条带的大小来确定是否是我们的目的蛋白，目的蛋白的理论值是45kDa，电泳结果显示目的蛋白条带在42kDa和51kDa之间，与理论值45kDa基本一致，可以判断纯化出的蛋白是目的蛋白。通过NanoDrop 2000spectrophotometer测定LHCⅡ-PCA-PCB蛋白浓度为0.19mg/mL，LHCⅡ-PCA-PEB蛋白浓度为1.26mg/mL。电泳结果图5所示，图中，a为Marker；b为LHCⅡ-PCA-PEB；c为LHCⅡ-PCA-PCB：
(2)锌离子检测
Zn2+能够螯合色素蛋白质上共价结合的四吡咯色素，在紫外光激发下发出红色荧光，所以可用锌电泳检测SDS-PAGE电泳条带是否为目的蛋白，如图6所示，图中，a为Marker；b为SDS-PAGE电泳；c为锌离子检测；LHCⅡ-PCA-PEB的理论分子质量为45kDa左右，我们可以看到泳道1在45KDa处有明显的条带，而泳道2显示，经过锌离子电泳检测，我们发现在同一位置有明显的荧光条带，说明藻红胆素PEB已经成功结合到融合蛋白LHCⅡ-PCA上，此处即为荧光蛋白LHCⅡ-PCA-PEB的条带。
实施例6
融合蛋白的光谱检测
重组蛋白的脱辅基蛋白上连有发光色团-藻胆素，通过检测色素蛋白的光谱，由其光谱特征可以确定色素蛋白的类型以及相关的理化特性。经过紫外分光光度计的检测显示，从大肠杆菌工程菌株中提取的LHCⅡ-PCA-PEB，最大特征吸收峰为λex＝556nm，经过荧光光谱仪的检测，LHCⅡ-PCA-PEB的最大发射峰为λem＝566nm，与文献描述一致，进一步证明融合蛋白LHCⅡ-PCA-PEB提取成功。
实施例7
融合蛋白与叶绿素的体外结合
将融合蛋白与叶绿素在体外进行结合，反复冻融三个循环(-20℃，1h；室温，30min)之后，经过脱盐柱除去未结合的游离叶绿素a，将所得样品进行光谱检测。如图7所示，除了在418nm和663nm处有两个叶绿素的特征吸收峰，而且在550nm左右还出现了藻红蛋白的特征吸收峰，初步证明LHCⅡ-PCA-PEB与叶绿素a在体外成功结合，构建了一个既包含叶绿素结合蛋白的吸收光谱又包含藻红蛋白吸收光谱的融合蛋白分子。
Claims (2)
1.一种宽吸收光谱重组光合蛋白分子，其特征在于，由叶绿素结合蛋白与全色基藻胆蛋白的融合蛋白以及叶绿素a在体外进行结合得到；
所述融合蛋白为藻蓝蛋白α亚基cpcA基因与叶绿素结合蛋白LhcⅡ基因经重叠PCR融合后所构建表达的蛋白分子。
2.一种宽吸收光谱重组光合蛋白分子的构建方法，其特征在于，包括以下步骤：
(1)分别以pUC19-LhcⅡ为模板，LHCⅡ-F和LHCⅡ-PCA-R为引物；以pCDFDuet-cpcA-cpcEF-hox1-pcyA质粒为模板，LHCⅡ-PCA-F和PCA-R为引物配制PCR体系，进行藻蓝蛋白α亚基cpcA基因与叶绿素结合蛋白LhcⅡ基因中目的片段LhcⅡ和cpcA的PCR扩增，然后切胶，进行胶回收；
(2)以胶回收得到的LhcⅡ片段和cpcA片段为模板，LHCⅡ-F、PCA-R为引物进行重叠PCR，得到融合片段LhcⅡ-cpcA，将PCR产物进行回收，得到融合基因片段，-20℃冰箱保存；
(3)将融合基因片段与T载体进行连接，连接体系由体积比为4:1:5的LhcⅡ-cpcA、pMD19-T和SolutionⅠ组成，得到pMD19-T-LhcⅡ-cpcA质粒，再取实验前期构建并保存于超低温冰箱的pCDFDuet-cpcA-cpcEF-hox1-
pcyA和pCDFDuet-cpcA-cpcEF-hox1-pebS质粒，分别使用限制性内切酶BamHⅠ、SacⅠ，37℃恒温水浴锅中分别对三种质粒进行双酶切，时长为16h，体系中DNA、BamHⅠ、SacⅠ、10×CutSmart Buffer、H2O的体积比为30:1:1:5:13，进行连接后构建得到可表达的重组质粒，重组质粒为pCDFDuet-LhcII-cpcA-cpcEF-hox1-pcyA和pCDFDuet-LhcII-cpcA-cpcEF-
hox1-pebS，并将其分别转化进大肠杆菌工程菌株BL21感受态细胞中，将重组质粒进行表达制备；
(4)将收集的菌体进行悬浮、破碎、离心、过滤、上样、咪唑梯度洗脱，纯化出的蛋白进行脱盐后保存在-80℃冰箱；
(5)将纯化成功的融合蛋白与叶绿素a在体外进行超声处理、反复冻融以及脱盐，即得宽吸收光谱重组光合蛋白分子。
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