Efficient heterogeneous expression process of drosophila antitumor activity protein rd-1

An anti-tumor activity and heterologous expression technology is applied in the field of using recombinant genetically engineered bacteria to produce anti-tumor active proteins, which can solve the problems such as the difficulty of feeding fruit flies and the complicated extraction process, so as to facilitate large-scale production and promotion, and simplify the purification process. , the effect of high toxicity

Inactive Publication Date: 2010-01-13
陆建新
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Problems solved by technology

[0003] The purpose of the present invention is to provide a method for heterologous expression of rd-1 utilizing recombinant engineering bacteria, which can overcome the problems existing in the prior art such as the diffic

Method used

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Embodiment 1

[0015] Embodiment 1: The efficient heterologous expression process of Drosophila anti-tumor active protein rd-1 is characterized in that it is realized by the following steps:

[0016] (1) Pass through Sepharose CL-4B affinity chromatography column and use 0.2M D-galactose to elute to extract rd-1 protein and sequence the N-terminus and C-terminus;

[0017] (2) Design and synthesize degenerate primers Degenerate primer R and Degenerate primer F according to the sequencing results;

[0018] (3) Construction of Drosophila pupal cDNA library by SMART method;

[0019] (4) Obtain the full gene of rd-1 protein from the Drosophila pupal cDNA library by PCR method;

[0020] (5) connecting the rd-1 gene and the expression vector to construct a recombinant expression vector;

[0021] (6) Transfer the recombinant expression vector into Pichia pastoris for heterologous expression;

[0022] (7) Optimizing the fermentation conditions of the recombinant Saccharomyces japonica in order to ...

Embodiment 2

[0025] Embodiment 2: The efficient heterologous expression process of Drosophila anti-tumor active protein rd-1 is characterized in that it is realized by the following steps:

[0026] (1) Pass the Sepharose CL-4B affinity chromatography column, and use 0.2M D-galactose to elute to extract the rd-1 protein and sequence the N-terminus and C-terminus:

[0027] (2) Design and synthesize nucleic acid probe DNA probe 1 according to the sequencing results;

[0028] (3) Construction of Drosophila pupal cDNA library by SMART method;

[0029] (4) Obtain the full gene of rd-1 protein from the Drosophila pupal cDNA library by Southern blot technique;

[0030] (5) connecting the rd-1 gene and the expression vector to construct a recombinant expression vector;

[0031] (6) Transfer the recombinant expression vector into Pichia pastoris for heterologous expression;

[0032] (7) Optimizing the fermentation conditions of the recombinant Saccharomyces japonica in order to obtain a high leve...

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Abstract

The invention relates to an efficient heterogeneous expression production process of drosophila antitumor activity protein rd-1, comprising the following steps of designing and synthesizing corresponding probes, selecting gene for encoding the protein from a cDNA library constructed by adopting SMART technology according to the sequencing result of an N end and a C end of the protein, by carrying out modification of condon preference and ribosome bind site on the protein, connecting the tb1 gene and pPIC9 carrier to construct pPIC9-tbi expression carrier, switching the expression carrier into GS115 Pichia pastoris expression host strain by electroporation, constructing recombinant Pichia pastoris engineering bacteria and expressing in Pichia pastoris, determining the additive amounts by optimizing fermentation condition as follows: 4% of glycerol, 1.25% of YNB, pH 6.0 of culture medium and 1% of methanol induced concentration and obtaining the optimized fermentation time of 84h. The rd-1 protein prepared by the invention has higher antitumor bioactivity and smaller toxic and adverse effect, and can be served as main ingredients of health care products.

Description

(1) Technical field: [0001] The invention relates to a technology for producing active protein by microbial fermentation, in particular to a production process for producing anti-tumor active protein by using recombinant genetically engineered bacteria. The produced active protein can be used as the main component of anti-tumor medicine and health care product. (two) background technology: [0002] Protein is the material basis of life and consists of more than 20 amino acids. It is an important component of body cells and tissues. rd-1 protein is a protein with special biological activity selected through functional screening. After entering the human body, it mainly exerts strong pharmacological effects such as anti-tumor and anti-virus. The anti-tumor mechanism of this protein is predicted by the experiments that have been carried out. By inhibiting the growth and proliferation of tumor cells and inducing tumor cell apoptosis, it can induce the expression of a series of ...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12P21/02C07K14/435A61P35/00C12R1/84
Inventor 陆建新
Owner 陆建新
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