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建立利用萤光素酶快速检测细胞活性的方法

Keywords: 萤火虫萤光素酶,蛋白表达,蛋白纯化,细胞活性

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Abstract:

运用PCR的方法,从萤火虫萤光素酶基因载体pGL4.26扩增萤火虫萤光素酶基因片段,将其插入连接于原核表达载体pET24a中,构建重组表达载体pET24a-Luc.经酶切鉴定及序列分析后,将重组载体转化到表达菌株大肠杆菌BL21(DE3)中,获得阳性重组菌BL21/pET24a-Luc.IPTG诱导蛋白高效表达并通过镍柱亲和层析纯化萤火虫萤光素酶.该目的蛋白活性用Bright-GloTM试剂进行验证并用于建立一种基于测量ATP含量的检测细胞生物活性的方法.与传统的细胞生物活性检测试剂盒MTT,CCK-8以及AlamarBlue比较,该方法具有反应迅速、活力高、灵敏度好、生产方便的优点,具有实际应用的潜力.

References

[1]  {15} LEGOCKI R P, LEGOCKI M, BALDWIN T O, et al. Bioluminescence
[2]  in soybean root nodules: Demonstration of a general approach to
[3]  assay gene expression in vivo by using bacterial luciferase[J]. Proc
[4]  Natl Acad Sci U S A, 1986, 83(23): 9080-9084.
[5]  {16} DE WET J R, WOOD K V, HELINSKI D R, et al. Cloning of
[6]  firefly luciferase cDNA and the expression of active luciferase in
[7]  Escherichia coli[J]. Proc Natl Acad Sci U S A, 1985, 82(23):
[8]  70-7873.
[9]  {17}ARAKAWA H, SHIOKAWA M, IMAMURA O, et al. Novel bioluminescent assay
[10]  of alkaline phosphatase using
[11]  adenosine-3′-phosphate-5′-phosphosulfate as substrate and the
[12]  luciferin-luciferase reaction and its application[J]. Anal Biochem,
[13]  03, 314(2): 206-211.
[14]  ISHIYAMA M, TOMINAGA H, SHIGA M, et al. A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet[J]. Biol Pharm Bull, 1996, 19(11): 1518-1520.
[15]  SCHREER A, TINSON C, SHERRY J P, et al. Application of Alamar blue/5-carboxyfluorescein diacetate acetoxymethyl ester as a noninvasive cell viability assay in primary hepatocytes from rainbow trout[J]. Anal Biochem, 2005, 344(1): 76-85.
[16]  SYKES M L, AVERY VM. Development of an Alamar Blue viability assay in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427[J]. Am J Trop Med Hyg, 2009, 81(4): 665-674.
[17]  UGAROVA N N, BROVKO L, KUTUZOVA G D.Bioluminescence and bioluminescent analysis: development of certain aspects of the problem over the last decade[J]. Biokhimiia, 1993, 58(9): 1351-1372.
[18]  PAULMURUGAN R, GAMBHIR S S. Firefly luciferase enzyme fragment complementation for imaging in cells and living animals[J]. Anal Chem, 2005, 77(5): 1295-1302.
[19]  MARQUES S M, ESTEVES D A, SILVA J C. Firefly bioluminescence: a mechanistic approach of luciferase catalyzed reactions[J]. IUBMB Life, 2009, 61(1): 6-17.
[20]  OBA Y, SATO M, OHTA Y, et al. Identification of paralogous genes of firefly luciferase in the Japanese firefly, Luciola cruciata[J]. Gene, 2006, 368: 53-60.
[21]  GREEN A A, MCELROY W D. Crystalline firefly luciferase[J]. Biochim Biophys Acta, 1956, 20(1): 170-176.
[22]  DENBURG J L, MCELROY WD. Catalytic subunit of firefly luciferase[J]. Biochemistry, 1970, 9(24): 4619-4624.
[23]  OW D W, JR DEW, HELINSKI D R, et al. Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants[J]. Science, 1986, 234(4778): 856-859.
[24]  HERBST R, GAST K, SECKLER R. Folding of firefly (Photinus pyralis) luciferase: aggregation and reactivation of unfolding intermediates[J]. Biochemistry, 1998, 37(18): 6586-6597.
[25]  MOSMANN T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytoto-xicity assays[J]. Immunol Methods, 1983, 65(1-2): 55-63.
[26]  BUTTKE T M, MCCUBREY J A, OWEN TC. Use of an aqueous soluble tetrazolium/formazan assay to measure viability and proliferation of lymphokine-dependent cell lines[J]. Immunol Methods, 1993, 157(1-2): 233-240.
[27]  ROEHM N W, RODGERS G H, HATFIELD S M, et al. An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT[J]. Immunol Methods, 1991, 142(2): 257-265.

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