Method for preparing allophycocyanin tripolymer fluorescent protein

A technology for allophycocyanin and fluorescent protein is applied in the field of preparing allophycocyanin trimer fluorescent protein, which can solve the problems of poor water solubility and stability of recombinant fluorescent phycocyanin, low fluorescence intensity, unfavorable development and research, etc. To achieve the effect of excellent fluorescence properties, high purity and easy cultivation

Inactive Publication Date: 2011-06-15
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

The spectral properties of the α subunit of allophycocyanin are significantly different from those of naturally occurring allophycocyanin, and the fluorescence intensity is low. The obtained recombinant fluorescent phycocyanin has poor water solubility and stability, which is not conducive to further research. development research

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  • Method for preparing allophycocyanin tripolymer fluorescent protein
  • Method for preparing allophycocyanin tripolymer fluorescent protein
  • Method for preparing allophycocyanin tripolymer fluorescent protein

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Embodiment Construction

[0018] The present invention is specifically illustrated by the following examples:

[0019] a. Gene cloning

[0020] From the National Center for Biotechnology Information (NCBI) database ( http: / / www.ncbi.nlm.nih.gov / ) to obtain the sequences of the apcA, apcB, cpcS, cpcU, ho1, pcyA genes of Synechocystis sp.PCC6803, thereby designing primers (see Table 1) for respectively constructing apcA, apcB, cpcS, cpcU, ho1 or pcyA gene fragments, and then using Synechocystis Genomic DNA of sp.PCC6803 was used as a template, and the corresponding primers designed to construct different gene fragments were used for PCR amplification, and apcA, apcB, cpcS, cpcU, ho1 or pcyAPCR products were respectively cloned. The PCR reaction system is 14.4 μl of double distilled water, containing Mg 2+ Buffer 20 μl, dNTP 4 μl, primer (forward) 1 μl, primer (reverse) 1 μl, Taq enzyme 0.2 μl, Synechocystis sp. PCC 6803 genome 1 μl.

[0021] The PCR reaction conditions adopted for the clone apcA, ap...

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Abstract

The invention belongs to the field of fluorescent protein materials in biotechnology, and specifically relates to a method for preparing an allophycocyanin tripolymer fluorescent protein. The method comprises the following steps: firstly, cloning allophycocyanin alpha sub-gene apoprotein genes and phycobilin biosynthesis enzyme genes hol and pcyA into an expression vector; and secondly, cloning allophycocyanin beta sub-gene apoprotein genes and chromophore lyase genes cpcS and cpcU into another vector, using the two expression vectors to simultaneously transform Escherichia coli, and screening out an engineering bacterium simultaneously expressing the genes, thus obtaining the allophycocyanin tripolymer fluorescent protein. By using the Escherichia coli as the raw material instead of algae to produce the recombinant photo-activated protein, the Escherichia coli is easy to culture and grows fast, thereby greatly shortening the production cycle. Compared with algae, the cell wall of the Escherichia coli can be broken easily, energy can be saved in the purification process, and a high utilization rate can be achieved.

Description

technical field [0001] The invention belongs to the field of fluorescent protein materials in biotechnology, and in particular relates to a method for preparing allophycocyanin trimer fluorescent protein. Background technique [0002] Phycobiliproteins mainly exist in prokaryotic cyanobacteria, eukaryotic red algae, cryptophyta and dinoflagellates (Pyrrophyta). According to the different absorption spectra, phycobiliproteins can be divided into four categories: phycoerythrin (PE), phycoerythrocyanin (PEC), phycocyanin (PC) and allophycocyanin ( allophycocyanin, APC). The apoprotein of phycobiliprotein contains α and β subunits, and the chromophore bound in phycobiliprotein is called phycobilin (phycobilin), and the A or D ring of phycobilin, or both rings of A and D are combined with apophycobilin at the same time. The cysteine ​​residues of the base protein are covalently linked by thioether bonds. Phycobilin covalently binds to apoproteins to form a specific conformatio...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/52C12N15/70C07K14/405
Inventor 刘少芳陈英杰路延笃陈华新李富超秦松
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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