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Ocean bacillus protein having antitumor activity and preparation and application thereof

A marine bacillus, anti-tumor activity technology, applied in the fields of genetic engineering and biomedicine, can solve the problems of difficult drug source, large-scale preparation, low content, etc., achieve good research and application value, good technical support and material basis Effect

Inactive Publication Date: 2016-08-24
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the vast majority of anti-tumor lead compounds have low content in the body, and it is difficult to prepare them on a large scale. Therefore, the source of anti-tumor lead compounds from natural sources is a big problem at present.

Method used

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  • Ocean bacillus protein having antitumor activity and preparation and application thereof
  • Ocean bacillus protein having antitumor activity and preparation and application thereof
  • Ocean bacillus protein having antitumor activity and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Construction of recombinant bacteria

[0023] 1) Use specific primers to amplify the gene encoding the protein. Design NcoI and HindIII restriction sites on the primers while amplifying, the primers are:

[0024] Forward: 5’-CATGCCATGGATGCAAGTACAGGCAGTC-3’

[0025] Reverse: 5’-CCCAAGCTTATTTTATCTGTACCACGC-3’

[0026] The PCR amplification system is: 2×ESTaqMasterMix 25μL, upstream primer 2μL, downstream primer 2μL, DNA template 2μL, add FreeWater to 50μL. The PCR amplification conditions were: pre-denaturation at 90°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 73°C for 1.0 min, 30 cycles, and finally extension at 73°C for 10 minutes.

[0027] 2) Double digestion of PET22b plasmid and amplified product with NcoI and HindIII respectively, and recover the target product after 1% electrophoresis. Then use T4DNA ligase to ligate at 16°C overnight to ligate the target gene with the vector. Finally, the ligation produ...

Embodiment 2

[0028] 3) Identify the grown-up positive clones by bacterial liquid PCR, double enzyme digestion of plasmids and sequencing. Example 2: Fermentation and culture of recombinant bacteria and separation and purification of recombinant proteins

[0029] The recombinant strain was inoculated into the sterilized initial fermentation broth, and fermented at 37°C, 150r / min. When it was cultured to OD600=0.70, add IPTG with a final concentration of 1.02mM, 30°C, 1500r / min for induction 9h. Obtain fermentation broth. The initial fermentation broth, in terms of mass fraction, includes 1.2% tryptone, 1.0% sodium chloride, 0.5% yeast extract, and the balance is water.

[0030] Centrifuge the fermentation broth at 10000rpm, 10min, 4℃, take the supernatant, and then use the 3kd, 50kd ultrafiltration membrane to intercept; then use 30%-60% ammonium sulfate precipitation, re-dissolve the obtained precipitate, dialysis and desalination ; Then use QFastFlow cation exchange chromatography to get pur...

Embodiment 3

[0032] Inhibitory effect of active protein on tumor cells: In this example, the active protein inhibits the 7 tumor cell lines tested, confirming that the active protein has a broad-spectrum anti-tumor effect.

[0033] 1. Cell culture and active protein preparation:

[0034] Inoculate tumor cells in a multi-well plate, and use DMEM medium containing 10% fetal bovine serum in 5% CO 2 Incubate in an incubator at 37°C for different time, 0.25% trypsin digestion and passage. The sample to be tested is diluted with PBS buffer into multiple concentration gradients and sterilized by filtration with a 0.22μm sterile filter membrane.

[0035] 2. MTT method detects the inhibitory effect of active protein on the growth of a variety of cancer cells

[0036] Take the tumor cells in the logarithmic growth phase, trypsinize them into a single cell suspension, collect the cell suspension, and count the cells. Inoculate 180μL per well in a 96-well cell culture plate, adjust the cell density so that t...

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Abstract

The invention relates to an ocean bacillus protein having antitumor activity and preparation and application thereof and belongs to the field of genetic engineering and biological medicines. The nucleotide sequence of the protein is as shown SEQ ID No. 1; the sequence can encode the ocean bacillus protein, and the amino acid sequence of the protein is as shown SEQ ID No. 2. The ocean bacillus protein can inhibit growth of various tumor cells and have the potential of being developed into antitumor drugs and / or healthcare products.

Description

Technical field [0001] The invention belongs to the fields of genetic engineering and biomedicine, and particularly relates to a marine bacillus protein with anti-tumor activity and its preparation and application. Background technique [0002] Malignant tumors are a major disease that seriously endangers human health. It is one of the main causes of death in our country. In 2005, about 7.5 million people died of malignant tumors worldwide. About 1.3 million people died of cancer every year in my country, and the incidence rate is increasing year by year. Upward trend. Drug therapy is an important part of comprehensive therapy for malignant tumors. Classical cytotoxic anti-tumor drugs are currently the main methods of chemotherapy and will still occupy a dominant position for a long time in the future. Therefore, it is a new trend of current anti-tumor drug research to find new structural types or new anti-tumor active ingredients with new mechanisms of action from natural organi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/32C12N15/31C12N15/70C12P21/02A61K38/16A61P35/00C12R1/19
Inventor 郑兰红杨康利朱美虹康道乐梁方方
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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