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Application of 2s-4b protein of peanut to induction of cell apoptosis

A 2s-4b, inducing cell technology, applied in the application of peanut 2s-4b fusion protein in inducing apoptosis, can solve the problem of less than 10% cure rate, limited early diagnosis of lung cancer, and research on the medicinal value of 2s albumin Fewer problems, etc., to achieve the effect of facilitating industrial production and efficient expression

Inactive Publication Date: 2012-07-04
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limited level of early diagnosis of lung cancer, 70% to 80% of patients are diagnosed at an advanced stage, and the overall cure rate is less than 10%.
However, there are few studies on the medicinal value of 2s albumin in peanuts

Method used

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  • Application of 2s-4b protein of peanut to induction of cell apoptosis
  • Application of 2s-4b protein of peanut to induction of cell apoptosis
  • Application of 2s-4b protein of peanut to induction of cell apoptosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 2s-4b cDNA cloning

[0039] 1. Using the conventional Trizol method in the art to extract peanut total RNA.

[0040] 2. RT-PCR detection

[0041] 3. Synthesis of First-Strand cDNA and 3'-cDNA from Peanut Total RNA

[0042] Use TaKaRa PrimeScriptTM 1st Strand cDNA Synthesis Kit for reverse transcription to synthesize the first strand of cDNA. For the reaction system and reaction conditions, please refer to the relevant kit instructions ( figure 1 ).

[0043] 4. Obtaining the 2s-4b Gene Degenerate Primer Fragment

[0044] According to the 2s protein MS-MS results obtained in our laboratory, the design 2s-4b The degenerate upstream and downstream primers are shown in SEQ ID NO:6~7. Where R=A / G, Y=C / T, M=A / C, K=G / T, S=C / G, W=A / T, H=A / C / T, B=C / G / T,=A / C / G, D=A / G / T, N=A / C / G / T. Prepare a 60 μL PCR reaction in a 200 μL Eppendorf thin-walled tube.

[0045] 5. Obtaining the 3' end of 2s-4b cDNA

[0046] According to the sequencing results obtained by deg...

Embodiment 2

[0058] Example 2 pET - 32a - 2s-4b Construction and identification of prokaryotic expression vector

[0059] 1. 2s-4b Purification of Gene PCR Products

[0060] Using the TaKaRa DNA Fragment Purification Kit for 2s-4b PCR products were purified.

[0061] 2. 2s-4b Double digestion of gene purification product and pET-32a plasmid

[0062] 3. Gel recovery and purification of enzyme digestion products

[0063] 2s-4b Purification of the digested product was carried out using TaKaRa DNA Fragment Purification Kit.

[0064] 4. 2s-4b Ligation of gene to pET-32a plasmid

[0065] Ligation was performed using T4 DNA Ligase from TaKaRa Corporation.

[0066] 5. Preparation of E. coli Competent Cells

[0067] 6. Recombinant plasmid pET-32a- 2s-4b transformation

[0068] 7. Detection of recombinant plasmids

[0069] Identification of pET-32a plasmid ligation in E. coli on resistant plates by PCR 2s-4b Full-length cDNA fragments. pET-32a- 2s-4b Double enzyme...

Embodiment 3

[0070] Example 3 AtACDO1 homologous gene OsACDO1 Antisense Subtractive Vectors and Forward Overexpression

[0071] 1. Inducible expression of 2s-4b fusion protein

[0072] (1) Transform the pET-32a-2s-4b recombinant plasmid into Escherichia coli Rosetta-gami respectively.

[0073] (2) Pick a single colony and inoculate it in 2 ml LB liquid medium containing four antibodies, and incubate at 37 °C, 200 rpm for 12 h.

[0074] (3) Take 200 μl of the initial shaking bacteria solution in 20 ml of LB liquid medium containing four antibodies, and incubate at 37 °C, 200 rpm for 4-5 h (determination of OD590 of the bacteria solution = 0.5-1.9).

[0075] (4) When the OD590 of the bacteria solution reaches 0.5~1.9, take out 1 ml of the bacteria solution, centrifuge at 10,000 × g for 1 min at room temperature, discard the culture solution, add 100 μl of sterilized PBS to resuspend the bacteria, and store at -20 °C.

[0076] (5) Add IPTG to the remaining culture medium to make the...

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Abstract

The invention discloses application of a 2s-4b protein of a peanut 2s albumin component to induction of cancer cell apoptosis. The 2s-4b protein disclosed by the invention can be used for inducing the apoptosis of lung adenocarcinoma A549 cells; an apoptosis mechanism of the 2s-4b protein is researched; and the 2s-4b protein has broad application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of peanut 2s-4b fusion protein in inducing cell apoptosis. Background technique [0002] Cancer is currently one of the "three major killers" that seriously threaten human health and life, and its incidence is increasing year by year. In 2008, there were 2,500,0000 cancer patients in the world, and an estimated 1,200,0000 new cancer cases, 700,0000 Millions of people die from cancer. It is estimated that there will be 27 million cancer patients and 17 million cancer deaths in 2030 (World cancer report 2008). More than half of all cancer cases and 60% of cancer deaths occur in moderately developed countries. Among all cancers, lung cancer has the highest morbidity and mortality. Non-small cell lung cancer (NSCLC) is the main type of lung cancer, accounting for 75% to 80% of lung cancer. Surgery is the treatment of choice. However, due to the limited level of early...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/70C12N5/09C07K14/81
Inventor 宾金华张子明植智聪龙明慧
Owner SOUTH CHINA NORMAL UNIVERSITY
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