Gene recombinant tnf-α derivative rmp16 and its preparation method and application
A technology of RMP16 and gene recombination, applied in the field of genetic engineering, can solve problems such as adverse reactions, limitation of actual clinical application, shock death, etc., and achieve high biological activity, low cost, and broad application prospects
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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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