二种多管水母光蛋白基因的分离、表达及生物活性初步研究

罗文新; 张军; 李少伟; 程通; 陈敏; 李少菁; 夏宁邵

二种多管水母光蛋白基因的分离、表达及生物活性初步研究

1.
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;厦门大学海洋与环境学院, 福建, 厦门, 361005
2.
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"高科技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).

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出版历程
- 收稿日期: 2001-04-16
- 修回日期: 2001-08-13

Cloning and expression of the aequorin genes from jellyfish aequorea and characterization of aequorins activities

1.
Cell Biology and Tumor Cell Engineering Laboratory of Ministry of Education Xiamen University, Xiamen 361005, China;College of Oceanography and Environmental Sciences, Xiamen University, Xiamen 361005, China
2.
Cell Biology and Tumor Cell Engineering Laboratory of Ministry of Education Xiamen University, Xiamen 361005, China

摘要

摘要: 分别从厦门东海域的大型多管水母和细小多管水母中分离到了新的光蛋白基因aeqxm和aeqxxm,并在大肠杆菌中进行了表达.aeqxm和aeqxxmDNA序列的编码区总长均为585bp,无内含子序列,推导的氨基酸序列总长均为195个氨基酸.aeqxm,aeqxxmDNA和AEVAQ440XcDNA之间的核苷酸序列同源性分别为80.7%,85.1%,aeqxm和aeqxxmDNA的核苷酸序列同源性为87.2%.原光蛋白apoaeqxm,apoaeqxxm与AEVAQ440X编码的氨基酸序列之间的同源性分别为84.7%,84.2%,apoaeqxm和apoaeqxxm的氨基酸序列之间的同源性为94.4%.分别将aeqxm和aeqxxm基因克隆至pTO-T7表达载体,apoaeqxm,apoaeqxxm在大肠杆菌中的表达量都达到40%左右.取菌体超声上清与腔肠动物荧光素f、巯基乙醇混合再生后,加入CaC_l2,用荧光全谱仪检测到470nm处的瞬时发光,表明表达的apoaeqxm,apoaeqxxm具有正常的生物学功能.
- 大型多管水母 /
- 细小多管水母 /
- 光蛋白基因 /
- 表达
Abstract: Two new aequorin genes aeqxm and aeqxxm were isolated from fellyfish Aequorea macr odactyla and A.parva respectively,which are commonly found in the warmer waters on the coastal region of East China Sea.The DNA sequences of the two genes have no introns and each one contains an ORF of 585 bp in full-length encoding a 195-aa protein.The two genes of aeqxm and aeqxxm share nucleotide homologies of 80.7% and 85.1% with AEVAQ 440X respectively,and the corresponding proteins share amino acid homologies of 84.7% and 84.2% with AEVAQ 440X.High amino acid homology was found between apoaeqxm and apoaeqx xm.The two genes were cloned into expression vector pTO-T7 respectively,and the expression yields were amounted to 40% of the total protein in E.coli BL21.The activities of the two photoproteins were reconstituted by incubating the expressed apoproteins with coelenterazine f.In the presence of Caion,both of the regenerated aeqxm and aeqxxm exhibited an emission peak at the wave length of 470 nm.
- Aequorea macrodactyla /
- Aequoreaparva /
- aequorin /
- expression

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参考文献(19)

SALA-NEWBY G B,TAYLOR K M,BADMINTON M N,et al.Imaging bioluminescent indicators show Ca²⁺ and ATP permeability thresholds in live cells attacked by complement [J].Immunology,1998,93:601-609.

HAMPTON T G,AMENDE I,TRAVERS K E,et al.Intracellular calcium dynamics in mouse model of myocardial stunning [J].Am J Physiol,1998,274:H1821-7.

CESSNA S G,CHANDRA S,LOW P S.Hypo-osmotic shock of tobacco cells stimulates Ca²⁺fluxes deriving first from external and then internal Ca²⁺ stores [J].J Biol Chem,1998,273:27 286-27 291.

LEUNG C F,WEBB S E,MILLER A L.Calcium transients accompany ooplasmic segregation in zebrafish embryos [J].Dev Growth Differ,1998,40:313-326.

CASADEI J,POWELL M J,KENTEN J H.Expression and secretion of aequorin as a chimeric antibody by means of a mammalian expression vector [J].Proc Natl Acad Sci USA,1990,87:2 047-2 051.

ZENNO S,INOUYE S.Bioluminescent immunoassay using a fusion protein of protein A and the photoprotein aequorin [J].Biochem Biophys Res Commun,1990,171:169-174.

VERHAEGEN M,CHRISTOPOULOS T K.Quantitative polymerase chain reaction based on a dual-analyte chemiluminescence hybridization assay for target DNA and internal standard [J].Anal Chem,1998,70:4 120-4 125.

ACTOR J K,KUFFNER T,DEZZUTTI C S,et al.A flash-type bioluminescent immunoasasy that is more sensitive than radioimaging:quantitative detection of cytokine cDNA in activated and resting human cells [J].J Immunol Methods,1998,211:65-77.

INOUYE S,NOGUCHI M,SAKAKI Y,et al.Cloning and sequence analysis of cDNA for the luminescent protein aequorin[J].Proc Natl Acad Sci USA,1985,82:3 154-3 158.

INOUYE S,SAKAKI Y,GOTO T,et al.Expression of apoaequorin complementary DNA in Escherichia coli [J].Biochemistry,1986,25:8 425-8 429.

CHARBONNEAU H,WALSH K A,MCCANN R O,et al.Amino acid sequence of the calcium-dependent photoprotein aequorin [J].American Chemical Society,1985,24:6 762-6 771.

PRASHER D,MCCANN R O,CORMIER M J.Cloning and expression of the cDNA cloning for aequorin:a bioluminescent calcium-binding protein [J].Biochem Biophys Res Commun,1985,126(3):1 295-1 268.

萨姆布鲁克J,弗里奇E F,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.

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

SHIMOMURA O,JOHNSON F H.Regeneration of the photoprotein aequorin [J].Nature,1975,17; 256(5514):236-238.

CORMIER M J,PRASHER D C,LONGIARU M,et al.The erzymology and molecular biology of the Ca²⁺activated photoprotein,aequorin [J].Photochem Photobiol,1989,49:59-512.

NOMURA M,INOUYE S,OHMIYA Y,et al.A C-terminal proline is required for bioluminescence of the Ca²⁺-binding photoprotein,aequorin [J].FEBS Lett,1991,295:63-66.

SHIMOMURA O,INOUYE S,MUSICKI B,et al.Recombinant aequorin and recombinant semi-synthetic aequorins:cellular Ca²⁺ ion indicators [J].Biochem J,1990,270:309-312.

KUROSE K,INOUYE S,SAKAKI Y,et al.Bioluminescence of the Ca²⁺-binding photoprotein aequorin after cysteine modification [J].Proc Natl Acad Sci USA,1989,86:80-84.

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