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Gene-recombined TNF-alpha derivative RMP16 and preparation method and application thereof

A technology of RMP16 and gene recombination, applied in the field of genetic engineering, can solve the problems of shock death, limited practical clinical application, adverse reactions, etc., and achieve the effects of high biological activity, low cost and long half-life

Active Publication Date: 2014-12-03
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If you want to achieve the curative effect, you must frequently inject large doses of TNF-α, which will lead to serious adverse reactions and even shock death
The above defects of TNF-α seriously limit its practical clinical application,

Method used

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  • Gene-recombined TNF-alpha derivative RMP16 and preparation method and application thereof
  • Gene-recombined TNF-alpha derivative RMP16 and preparation method and application thereof
  • Gene-recombined TNF-alpha derivative RMP16 and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Construction and Expression of Expression Engineering Bacteria pTXB1-RMP16 / ER2566

[0056] Specific steps are as follows:

[0057] (1) Obtaining the coding sequence of RMP16:

[0058] Design the cDNA encoding RMP16 according to the codon preference of Escherichia coli, design 3 primers, and adopt the two-step method to obtain the sequence (such as figure 1 shown):

[0059] Primer F1:

[0060] 5'-GGTGGT CATATG TGGCAGCGCCCGAGCAGCTGGATTGAAGGTCGCCTG-3';

[0061] Primer F2:

[0062] 5'-GCTCACCGCAATGCGGCTAATGGTATGGGTCAGCAGGCGACCTTC-3';

[0063] Primer F3:

[0064] 5'-CCACCAT GCTCTTCCGCA CAGCAGATTCACTTTGGTCTGATAGCTCACCGCAAT-3';

[0065] in, CATATG is the Nde Ⅰ restriction site, GCTCTTCCGCA It is the restriction site of Sap Ⅰ; GGTGGT and CCACCAT are the protection bases.

[0066] ① Chain extension reaction:

[0067] The reaction system is: 2 μL of primer F1 (250 μM / L), 2 μL of primer F2 (250 μM / L), 10×TaKaRa Buffer (containing 4 mmol / L MgCl 2 ) 2 μL, dNTP 2 μL,...

Embodiment 2

[0083] Purification, Preparation and Identification of Recombinant TNF-α Derivative RMP16

[0084] Get 20mL chitin beads (NEB company product #S6651L) to fill a 1.5×20cm chromatographic column, wash the column with 10 times the column volume of Buffer A solution; Note: Use 10 times the column volume of Buffer A solution to pass through the column at 2mL / min to wash away impurities or non-affinity-bound fusion proteins, and use 60mL Buffer B solution (20mM Tris-HCl, 0.5M NaCl, 1mM EDTA, 50mM β-mercaptoethanol, pH 8.0) to pass through the column naturally, so that β-mercaptoethanol is evenly distributed and soaked in the column packing, and then the inlet and outlet ends are sealed. The above reaction system is reacted at 23°C for 24h to induce the N-terminus of intein Cleave and release the target polypeptide RMP16. Elute with Buffer A solution and collect the target polypeptide solution with a clean beaker, and then purify by high performance liquid chromatography (HPLC) to p...

Embodiment 3

[0086]Inhibition of the prepared recombinant polypeptide RMP16 on the growth of various tumor cells

[0087] Prostate cancer Du145, lymphoma Raji, and leukemia K562 cell lines (all purchased from American Type Culture Collection (ATCC)) were cultured in RPMI-1640+10% FBS medium (product of Gibco, USA). Cervical cancer HeLa and breast cancer MCF-7 cell lines (both purchased from American Type Culture Collection (ATCC)) were cultured in DMEM+10% FBS medium (product of Gibco, USA). All cell lines were cultured at 37°C, saturated humidity and 5% CO 2 In the incubator, the medium was changed or subcultured every 2-3 days, and the cells in the logarithmic growth phase were collected and divided into 6×10 3 Inoculate each well in a 96-well plate, 100 μL per well, set in CO 2 After culturing in an incubator for 24 hours to adhere to the wall, treat with a certain concentration of drugs for 48 hours, set 6 replicate wells for each concentration, set up the experimental group, control...

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Abstract

The invention discloses a gene-recombined TNF-alpha derivative RMP16 and a preparation method and application thereof. The RMP16 is obtained through gene recombination, has higher stability and physiological activity in blood compared with those of the natural TNF-alpha, can remarkably inhibit the growth of cells of tumors, such as prostatic carcinoma Du145, lymphoma Raji, leukemia K562, cervical carcinoma HeLa, breast cancer MCF-7 and the like, has an especially outstanding inhibition effect on DU145, can inhibit the growth of remarkable Du145 cell transplantable tumor, and has pharmacodynamic effects similar to those of estramustine; proved by results of tests on nude mice with Du145 cell transplantable tumor, the recombinant polypeptide RMP16 has toxic and side effects, which are obviously lower than those of estramustine, and is free from obvious toxic and side effect performance. The gene-recombined TNF-alpha derivative RMP16 can be applied to the preparation of drugs with the functions of treating the tumors, such as prostatic carcinoma, lymphoma, chronic myelogenous leukemia, cervical carcinoma, breast cancer and the like.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a gene recombined TNF-α derivative RMP16 and its preparation method and application. Background technique [0002] TNF-α is one of the members of tumor necrosis factor family discovered earlier and studied most deeply. It has strong anti-tumor effect and participates in the regulation of various physiological and pathological processes. Human TNF-α is a single-copy gene located on chromosome 6 (6P 12-13 ), tightly linked with the MHC gene group; the cDNA size of TNF-α is about 2.76kb, consisting of 4 exons and 3 introns. The human TNF-α precursor consists of 233 amino acid residues, including a signal peptide of 76 amino acid residues encoded by exons 1 and 2; after the signal peptide is excised, a non-glycosylated mature human TNF-α is formed , is 157 amino acid residues, and the relative molecular weight is 17kDa. Among them, the 69th and 101st positions are two ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/525C12N15/66C12N15/70C12N1/21C07K1/16A61K38/19A61P35/00
CPCA61K38/00C07K14/525C12N15/70
Inventor 马义洪岸姜石松
Owner JINAN UNIVERSITY
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