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Preparation and purification of telomerase activity inhibition protein

An activity inhibitor, a technology of telomerase, which is applied in the fields of biotechnology and biochemical engineering, and can solve the problems of high production cost and inappropriate mass preparation.

Inactive Publication Date: 2007-04-18
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the high production cost of the affinity chromatography column purification method is not suitable for large-scale preparation at all

Method used

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  • Preparation and purification of telomerase activity inhibition protein
  • Preparation and purification of telomerase activity inhibition protein
  • Preparation and purification of telomerase activity inhibition protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Preparation and induced expression of genetically engineered bacteria of embodiment 1.LPGENE1 and LPGENE2

[0081] (a) Preparation of LPGENE1 and LPGENE2 genes

[0082] Using the full-length LPTS gene sequence (see Chinese patent ZL00115395.1 for the preparation method) as a template, use the following primers,

[0083] P1 (upstream primer): 5'-CATATGTCTATGCTGGCTGAACGTCGG-3' (SEQ ID NO: 7)

[0084] P2 (downstream primer): 5'-CTCGAGTTTGGAATCTTTCTTCTT-3' (SEQ ID NO: 8)

[0085] Through the PCR reaction, obtain the PCR amplification product (LPGENE1 gene) containing the full-length LPTS coding sequence with restriction sites NdeI and Xho1 at both ends, the sequence is shown in SEQ ID NO: 1; PCR reaction conditions are: in 50 μ l system To proceed, include 1 μl template, 1 μl dNTP mix (10 mM), 1 μl each of upstream and downstream primers (20 μM), 5 μl 10×PYROBEST buffer, 0.5 μl PYROBEST enzyme, and add deionized water to 50 μl. The reaction conditions were: 94°C for 3 mi...

Embodiment 2

[0100] Example 2. Separation and purification of LPGENE1 and LPGENE2

[0101] A small amount of purified LPGENE protein prepared in the laboratory can be used for affinity chromatography with a commercially available Hi-Sepharose 4B column. In order to prepare a large amount of purified LPGENE protein, separation methods such as ion exchange were used in this example to obtain high-purity LPGENE protein.

[0102] The specific separation and purification process may include the following steps:

[0103] (1) Ultrasonic disruption of bacteria

[0104] Get the thalli of centrifugation gained, add the buffer solution (30mmol / L KH 2 PO 4 -NaOH) to resuspend the cells, and then add 2‰ of 100mM PMSF (phenylmethylsulfonyl fluoride) (final concentration 0.2mM). Then under the condition of ice bath, the cell is ultrasonically broken, (ultrasonic instrument, manufactured by Ningbo Xinzhi Company), the ultrasonic conditions are as follows: working time 7 seconds, interval time 25 secon...

Embodiment 3

[0117] Example 3. Detection of biological activity of LPGENE1 and LPGENE2

[0118] LPGENE is a strong inhibitor of telomerase activity. The present invention uses the TPAP method to measure the biological activity of the LPGEGE prepared and purified in Example 2.

[0119] Telomerase comes from the lysate of conventional SMMC-7721 liver cancer cells. The specific preparation method is as follows: Inoculate SMMC-7721 liver cancer cells in a 10ml culture flask, the medium is RPMI 1640, the temperature is 37 ° C, 5% CO 2 Under the condition of adherent culture, collect the cultured cells after the bottle is full. The culture medium was aspirated dry, the cells were washed once with PBS, and then the cells were digested with trypsin, washed with PBS, sucked into a centrifuge tube, and centrifuged at 4000rpm to collect the cells. Then wash buffer (10mM Hepes-KOH (pH7.5), 1.5mM MgCl 2, 10mM KCl, 1mMDTT (dithiothreitol)) wash the cells once, and centrifuge. Use ice-cold lysis buff...

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Abstract

This invention discloses a method of using gene recombination technology to prepare telomerase activation profiling. This invention can prepare telomerase activation profilin which is high purity and high biologic activity with high efficiency and large scale.

Description

technical field [0001] The present invention relates to the fields of biotechnology and biochemical engineering. More specifically, the present invention relates to a method for preparing telomerase activity inhibitory protein by gene recombination technology, which can efficiently and large-scale prepare high-purity and high biological activity inhibitory protein for telomerase activity. Background technique [0002] Telomerase (Telomerase) is a ribonucleoprotein that synthesizes and extends the telomere of cell chromosomes. It consists of two basic components: reverse transcriptase catalytic subunit hTERT and RNA component hTR. Telomerase can use its own RNA as a template to reverse-transcribe and synthesize telomere repeats, which are added to the end of chromosomes to compensate for the loss of telomere DNA during cell division and maintain the length of telomeres. [0003] Studies have shown that telomerase activity is almost undetectable in normal human cells. Therefo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K19/00C07K1/18
Inventor 赵慕钧陈光明赵静孙成副陈国元李载平
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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