大型多管水母的绿色荧光蛋白

罗文新; 张军; 杨海杰; 谢小燕; 覃映雪; 逄淑强; 李少菁; 夏宁邵

大型多管水母的绿色荧光蛋白

厦门大学, 细胞生物学与肿瘤细胞工程教育部重点实验室, 福建, 厦门, 361005

基金项目: 国家海洋“863”计划领域青年基金资助项目(819-Q-06);国家自然科学基金资助项目(C01040101);福建省自然科学基金资助项目(C0010001);国家海洋局海洋生物工程重点实验室开放基金资助项目(HY9703)

计量
- 文章访问数: 855
- HTML全文浏览量: 6
- PDF下载量: 1580
- 被引次数: 0
出版历程
- 收稿日期: 2001-02-26
- 修回日期: 2001-04-26

Green fluorescent protein of the jellyfish Aequorea macrodactyla

Ministry of Education, Cell Biology and Tumor Cell Engineering Laboratory, Xiamen University, Xiamen 361005, China

摘要

摘要: 采用PCR扩增和southern杂交筛选相结合的方法,从厦门水域的大型多管水母中分离到了新的绿色荧光蛋白基因gfpxm,并在大肠杆菌中进行了表达.gfpxmDNA序列编码区长为1042bp,包含3个外显子和两个内含子,cDNA编码区全长为717bp.gfpxmcDNA与已知野生型Aeqgfp 10cDNA长度一致,核苷酸同源性为81.9%,推导的氨基酸序列全长为238个氨基酸,与AeqGFP10的氨基酸同源性为83.6%.将gfpxm克隆至pTO-T7表达载体,GFPxm在大肠杆菌BL21中的表达量达菌体总蛋白的50%左右.荧光性质和强度分析结果表明,GFPxm蛋白的激发峰为476nm,发射峰为496nm,荧光量子产率为1. GFPxm蛋白的荧光很稳定,对热、碱性、变性剂和盐等有较强抗性.
- 大型多管水母 /
- 绿色荧光蛋白 /
- 克隆表达 /
- 荧光性质
Abstract: A new green fluor escent protein gene-gfpxm was isolated from jellyfish Aequorea macrodactyla in the coastal region of East China Sea by the method of combination of PCR amplification and southern hybridization analysis.The gfpxm gene contains three extrons and two introns spread over 1042 bp of genomic DNA,the entire coding region of cDNAis 717 bp,being identical to the wild-type gfp-Aeqgfp 10 cDNA in length.The amino acid sequence of GFPxm deduced from the nucleotide sequence is 238-aa-residue,sharing homology of 83.6% with that of AeqGFP10.The entire coding sequence was cloned into the pTO-T7 expression vector and expressed in Ecoli. The expression yield of GFPxm was amounted to 50% of the total protein.Compared with GFP of Avictoria,the expressed GFPxm exhibited an excitation peak at a higher wave length of 476nm and an emission peak at a lower wave length 496 nm with a higher quantum yield of 10.The fluorescence of GFPxm is significant stable,showing strong resistant to heat,alkaline,denaturants and salts.
- Aequorea macrodactyla /
- green fluorescent protein gene /
- cloning and expression /
- fluorescent property

HTML全文

参考文献(21)

WARD W W, CODY C W, HART R C, et al. Sepectrophoton etric identity of the energy transfer chromophores in Renilla and Aequorea green fluorescent proteins[J]. Photobiochem Photobio, 1980, 31:611-615.

KAIN S R, ADAMS M, KONDEPUDI A, et al. Green fluorescent protein as a reporter of gene expression and protein localization[J]. Biotechniques, 1995, 19: 650-655.

WANG S, HAZELRIGG T. Implications for bcd mRNA localization from spatial distribution of in drosophila oogendsis[J].Nature, 1994, 369: 400-403.

BARTHMAIER P, FYRBERY E. Rapid communication monitoring development and pathology of drosophila indirect flight muscles using green fluorescent protein[J]. Dev Biol, 1995, 169: 770-774.

SCALES S J, PEPPERKOK R, KREIS T E. Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action for COPII and COPI[J]. Cell, 1997, 90:1 137-1 148.

ULLMAN E F, MARSHALL W F, SEDAT J W, et al. Mitosis in living budding yeast:anaphase A but no metaphase plate[J]. Science, 1999, 277: 574-578.

HU W, CHENG C L. Expression of Aequorea green fluorescent protein in plant cells[J]. FEBS Letters, 1995, 369: 331-334.

MOSS J B, ROSENTHAL N. Analysis of gene expression patterns in the embryonic mouse myotome with the green fluorescent protein[J]. J Cell Biochem, 1994, 180: 489-495.

许振祖,张金标.奥东一闽南近海的浮游水螅水母类,管水母类和钵水母类[J]厦门大学学报(自然科学版),1978,17(4):32

萨姆布鲁克J,弗里奇EF,曼尼阿蒂斯T.分子克隆操作指南(第二版)[M].金冬雁,黎孟枫等译北京:科学出版社,1992.

陈国珍,黄贤智,许金钩,等.荧光分析法(第二版)[M]北京:科学出版社,1990.16-17

PRASHER D C, ECKENRODE V K, WARD W W, et al. Primary structure of the Aequorea victoria green-fluorescent protein[J]. Gene, 1992, 111: 229-233.

罗文新,张军,夏宁邵,等一种带增强子的原核表达载体的构建及初步应用[J].生物工程学报,2000,16(5):578-581.

MORISE J G, SHIMOMURA O, JOHNSON F H, et al. Intermolecular energy transfer in the bioluminescent system of Aequorea[J]. Biochemistry, 13(194): 2 656～2 662.

WARD W W. Properties of the coelenterate green-fluorescent proteins[A]. Deluca M, Mcelroy W D. Bioluminescence and chemiluminescence:basic chemistry and analytical application[M]. New York: Academic Press Inc, 1981. 235-242.

WARD W W, BOKMAN S H. Reversible denaturation of aequorea green fluorescent protein: physical separation and characterization of the renatured protein[J]. Biochemistry, 1982, 21:4 535-4 540.

CHALFIE M, TU Y, EUSKIRCHEN G, et al. Green fluorescent protein as a marker for gene expression[J]. Science,1994, 263: 802-808.

HEIM R, CUBITT A, TSIEN R Y. Improved green fluorescence[J]. Nature, 1995, 373: 663-664.

HEIM R, PRASHER D C, TSIEN R Y. Wavelength mutations and posttrans-lational autoxidation of green fluorescent protein[J]. Proc Natl Acad Sci USA, 1994, 91:12 501-12 504.

HEIM R, TSIEN R Y. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer[J]. Curr Biol, 1996, 6: 178-182.

MIRO R D, SILVA C M, YOUVAN D C. Fluorescence resonance energy transfer between blueemitting and red-shifted excitation derivatives of the green fluorescent protein[J]. Gene, 1996, 173: 13-17.

施引文献

资源附件(0)

访问统计