Preparation method of fusion fluorescent protein in escherichia coli

A technology of Escherichia coli and protein, applied in the direction of introducing foreign genetic material, DNA/RNA fragments, fermentation, etc. using vectors, can solve the problems of complex process and high cost, and achieve the effect of simplifying the purification process, reducing the preparation cost, and being easy to cultivate.

Active Publication Date: 2015-11-04
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, no matter whether phycobiliprotein and antibody are coupled through direct cross-linking or indirect coupling through BAS system, the process is complicated and the cost of preparation is very high.

Method used

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  • Preparation method of fusion fluorescent protein in escherichia coli
  • Preparation method of fusion fluorescent protein in escherichia coli
  • Preparation method of fusion fluorescent protein in escherichia coli

Examples

Experimental program
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Embodiment 1

[0039] 1. Cloning of genes

[0040] Synechococcus CC9311 apcA, Synechocytissp.PCC 6803 Ho1, Prochlorococcus phage P-SSM2 pebS, Synechococcus elongatus BP-1 cpcS and AFP single-chain antibody scFv gene sequences were obtained from the National Center for Biotechnology Information (NCBI) database (Accession No. AGQ46838). Accordingly, specific primers for amplifying apcA, Ho1, cpcS and scFv genes were designed respectively (Table 1). The apcA gene used CC9311 genomic DNA as a template, Ho1 used Synechocytissp.PCC 6803 genomic DNA as a template, cpcS used Synechococcus elongatus BP-1 genomic DNA as a template, and was amplified according to conventional PCR conditions. The pebS, scFv gene and linker sequences were artificially synthesized by Nanjing GenScript Biotechnology Co., Ltd. In order to facilitate the fusion PCR reaction, during the artificial synthesis of the linker sequence, partial sequences of the scFv gene and the apcA gene were added to its 5' end and 3' end, resp...

Embodiment 2

[0058] 1. Cloning of genes

[0059] Obtain Synechocytissp.PCC 6803 apcA, Synechocytissp.PCC 6803 Ho1, Prochlorococcus phage P-SSM2 pebS, Synechococcus elongatus BP-1 cpcS and AFP single-chain antibody scFv gene sequences from the National Center for Biotechnology Information (NCBI) database (Accession No. AGQ46838). Accordingly, specific primers for amplifying apcA, Ho1, cpcS and scFv genes were designed respectively (Table 1). Genomic DNA of Synechocytissp.PCC 6803 was used as template for apcA gene, genomic DNA of Synechocytissp.PCC 6803 was used as template for Ho1, and genomic DNA of Synechococcus elongatus BP-1 was used as template for cpcS gene, which were amplified according to conventional PCR conditions. The pebS, scFv gene and linker sequences were artificially synthesized by Nanjing GenScript Biotechnology Co., Ltd. In order to facilitate the fusion PCR reaction, during the artificial synthesis of the linker sequence, partial sequences of the scFv gene and the apc...

Embodiment 3

[0074] 1. Cloning of genes

[0075] Synechococcus elongatus BP-1 apcA, Synechocytissp.PCC 6803 Ho1, Prochlorococcus phage P-SSM2 pebS, Synechococcus elongatus BP-1 cpcS and AFP single-chain antibody scFv genes were obtained from the National Center for Biotechnology Information (NCBI) database sequence (Accession No. AGQ46838). Accordingly, specific primers for amplifying apcA, Ho1, cpcS and scFv genes were designed respectively (Table 1). Genomic DNA of Synechococcus elongatus BP-1 was used as template for apcA gene, genomic DNA of Synechocytissp.PCC 6803 was used as template for Ho1, and genomic DNA of Synechococcus elongatus BP-1 was used as template for cpcS. The pebS, scFv gene and linker sequences were artificially synthesized by Nanjing GenScript Biotechnology Co., Ltd. In order to facilitate the fusion PCR reaction, during the artificial synthesis of the linker sequence, partial sequences of the scFv gene and the apcA gene were added to its 5' end and 3' end, respect...

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Abstract

The invention belongs to the field of fluorescent protein and recombinant antibodies in biotechnology and particularly relates to a preparation method of fusion fluorescent protein in escherichia coli. The method is characterized in that AFP single-chain antibody genes and allophycocyanin alpha subunit genes are spliced through linker sequence to form chimeric genes, and the chimeric genes, phycobiliprotein lyase genes and phycobilin biosynthetic enzyme genes are expressed in the escherichia coli to obtain fusion fluorescent protein covalently binding phycoerythrin or fusion fluorescent protein covalently binding phycocyanobilin. The method has the advantages that biotechnology is used to produce the fusion protein, of single-chain antibodies and allophycocyanin alpha subunits, in the escherichia coli, the method is environmental friendly and low in cost, and the fusion protein is applicable to fields such as biology and biomedical detection.

Description

technical field [0001] The invention belongs to the field of fluorescent protein and recombinant antibody in biotechnology, and specifically relates to a preparation method of fusion fluorescent protein in Escherichia coli. Background technique [0002] Phycobiliprotein is an important pigment protein of algae, which has the function of light energy capture and transmission. Each molecule of phycobiliprotein contains two structurally similar polypeptide chains α and β, and the α subunit and β subunit contain about 160-180 amino acid residues, respectively, and the ratio of the two is usually 1:1. Cysteine ​​residues in subunits are covalently bonded to phycobilichromes through thioether bonds, and the type of phycobilichromes and their interactions with apoproteins determine the spectroscopic properties of phycobiliproteins. According to the different absorption spectra, phycobiliproteins can be divided into four categories: phycoerythrin PE, λ max =540nm~570nm, phycoeryth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12N15/70C12N15/62
Inventor 陈华新武静姜鹏赵瑾
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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