光学活性重组别藻蓝蛋白的初步研究

IOCAS-IR > 海洋环流与波动重点实验室

	光学活性重组别藻蓝蛋白的初步研究
其他题名	Primary Study of Recombinant Allophycocyanins with Optical Activity
	杨雨
学位类型	博士
	2007-05-29
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	基因重组别藻蓝蛋白光学活性荧光
摘要	藻胆蛋白是某些原核和真核藻类中一类重要的捕光天线蛋白，由脱辅基蛋白和开环四吡咯结构的色基共价结合组成。天然藻胆蛋白具有抗肿瘤、抗病毒、抗炎抗氧化等多种生物活性，而借助基因工程技术可以高效表达构效稳定的藻胆蛋白，应用于医药、食品、生化试剂等领域。本研究在本实验室以前研制的抗肿瘤新药雷普克（rAPC）以及光学活性重组C-藻蓝蛋白α亚基的基础上，将别藻蓝蛋白α亚基基因apcA连入表达载体pCDFDuet-1中，构建了酶催化连接与自主催化连接两种新型载体，使大肠杆菌同时合成脱辅基蛋白与色基，而且二者能够在菌体内结合，表达出具有光学活性的重组别藻蓝蛋白α亚基。以重组菌BL21(DE3)作为研究对象，对酶催化连接的重组蛋白进行了5L规模的发酵，菌体密度OD600值达到23.52，并初步优化了发酵工艺，摸索了如何减少发酵过程中包涵体的生成。采用金属螯合亲和层析的方法，建立了重组蛋白快速高效的纯化工艺。对比两种重组蛋白的光谱活性，发现藻蓝蛋白裂合酶的存在影响了重组蛋白的荧光吸收特性，推测藻胆蛋白的荧光特性可能是主要由脱辅基蛋白和色基空间构象决定，而不是肽链的一级结构。这些研究结果为解释藻胆蛋白荧光活性的机制提供了线索。
其他摘要	Phycobiliproteins, important light-harvesting antenna proteins in some prokaryotic and eukaryotic algae, are made up of apoproteins and noncyclic tetrapyrrole chromophones. Since many bioactivities, such as antioxidative, antiinflammatory, antitumor, antivirus and so on, have been found from natural phycobiliproteins, it is essential to produce these proteins with stable structures and effects by gene engineering. Based on former research about recombinant allophycocyanin (rAPC) and recombinant α subunit of C-phycocyanin (optically active), the gene of α subunit of allophycocyanin, apcA, was combined with a pCDFDuet-1 vector, and two new recombinant vectors (enzymatic and self catalyzed) were constructed. The vectors could play a role in Escherichia coli to produce apolipoprotein and chromophone and connect with each other. Finally, new recombinant proteins with optical activity were produced. The recombinant strain E.coli BL21(DE3) was cultured to OD600=23.52 in a 5L fermentor. The fermentation technique was improved to reduce the production of inclusion bodies. The recombinant proteins were purified by metal chelating affinity chromatography. Compared with the two new proteins expressed by two vectors, we found that the phycocyanin lyase E and F (CpcE/F) could influence the fluorescent spectrum of recombinant proteins, which would provide a clew to find the mechanism of phycobiliproteins' fluorescent activities.
页数	80
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/353
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	杨雨. 光学活性重组别藻蓝蛋白的初步研究[D]. 海洋研究所. 中国科学院海洋研究所,2007.

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