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Recombinant TAT-HOX family protein with N-terminal labeled histamine acid and its preparation

A protein and HOX technology, applied in the field of recombinant TAT-HOX family proteins labeled with N-terminal histidine and their preparation, can solve the problems of decreased cells, defective endogenous hematopoiesis, increased resistance to anti-mitotic drugs, etc.

Pending Publication Date: 2020-07-21
TAIWAN ADVANCE BIO PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In addition, Dr. Stefan Karlsson of the University of British Columbia (University of British Columbia) and his team pointed out that both HOXB3 and HOXB4 are involved in the regulation of hematopoietic function; experimental data showed that mice lacking HOXB3 and HOXB4 had fewer hematopoietic organ cells and endogenous Hematopoiesis is defective, and hematopoietic stem cells lacking HOXB3 and HOXB4 have slowed cell cycle kinetics leading to increased resistance to antimitotic drugs

Method used

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  • Recombinant TAT-HOX family protein with N-terminal labeled histamine acid and its preparation
  • Recombinant TAT-HOX family protein with N-terminal labeled histamine acid and its preparation
  • Recombinant TAT-HOX family protein with N-terminal labeled histamine acid and its preparation

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1. Construction of HOX Family Recombinant Protein Expression Plastids

[0039] The inventors of the present application have previously constructed a plastid containing six histidine residues at the N-terminus and HIV-1 transcription activator (transactivator of transcription, TAT) fragments that can freely pass through the cell membrane through genetic engineering technology pTAT-HOXB4 (SEQ ID No.1, see figure 1 shown). Using the plastid as a template, cut it with restriction enzymes NdeI and XhoI to obtain the pTAT plastid template XhoI-plastid-6xHis-TAT-NdeI containing the cleavage sites NdeI and XhoI at both ends.

[0040] At the same time, NdeI and XhoI restriction enzyme cleavage sites were respectively designed before and after the gene sequences encoding HOX family proteins such as HOXA4, HOXC4, HOXD4, HOXB3, and HOXB6 by genetic engineering technology, and then combined with the aforementioned plastid template XhoI-plastid-6xHis-TAT- NdeI was bonded, ...

Embodiment 2

[0041] Example 2, Purification of HOX Family Recombinant Proteins

[0042] Expression and purification of His-TAT-HOXC4 and His-TAT-HOXD4 recombinant proteins

[0043] Transform pTAT-HOXC4 and pTAT-HOXD4 into E. coli strain BL21- (DE3)-RP (Agilent). The transformed Escherichia coli cells were cultured at 37° C. with 150 rpm rotation and shaking for 16 hours. Dilute this culture solution to fresh culture solution at a ratio of 1:50. The initial absorbance value OD600 at a wavelength of 600nm is about 0.05, and grow at 37°C with 200rpm rotation and shaking until the OD600 value is 0.7-0.8. Propylthio-β-D-galactose (IPTG) was used for protein induction, and isopropylthio-β-D-galactose at a final concentration of 1 mM was added, and the mixture was rotated and shaken at 37° C. at 200 rpm for 3 hours.

[0044] After the induction, the cells were collected by centrifugation and lysed back to the bacterial cells with buffer A (8M urea, 20mM HEPES, 100mM NaCl, pH 8.0). The cell s...

Embodiment 3

[0053] Example 3. Evaluation of Hematopoietic Stem Cell Proliferation Capacity of His-TAT-HOX Family Recombinant Proteins in Vitro

[0054] Erythrocytes were removed from human cord blood by lysis in 0.83 wt% ammonium chloride (pH 7.0) containing 0.1 wt% sodium bicarbonate at 4°C. After co-cultivating the collected white blood cells with the specific antibody bound to the magnetic beads and the cells, the magnetic cell separation procedure was carried out through the Stemsep column separation method to collect the cells with CD34 + cells, and the fraction representing mature human leukocytes was removed. Cells at a specific density were cultured in a six-well plate, and in the morning and evening, a specific concentration of BSA or various recombinant proteins of the His-TAT-HOX family was added for 4 consecutive days. On the 5th day, the ISHAGE method (LIN / CD34 staining plus forward and side scattered light) to count the number of stem cells and analyze.

[0055] Depend on...

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Abstract

The invention relates to a recombinant HOX family protein containing labeled histamine acid and activator of transcription (TAT) fragment at an N-terminal, the N-terminal containing at least six histamine acid residues and Tat fragments. N-terminally labeled histidine TAT-HOX family recombinant proteins (His-TAT-HOX family proteins) made according to the present invention include recombinant His-TAT-HOXA4, His-TAT-HOXC4, His-TAT-HOXD4, His-TAT-HOXB3, His-TAT-HOXB6 and the like, and after the recombinant proteins are expressed in escherichia coli host cells and purified, the recombinant proteins have the capacity of proliferating hematopoietic stem cells. The invention also discloses a preparation method and a composition of the His-TAT-HOX family recombinant proteins.

Description

technical field [0001] The invention relates to a recombinant TAT-HOX family protein whose N-terminus is marked with histidine and a preparation method thereof. More particularly, the present invention relates to a recombinant TAT-HOX family protein containing at least 6 histidine tags at the N-terminus prepared by a genetic engineering method, which includes recombinant His-TAT-HOXA4, His-TAT-HOXC4, His-TAT-HOXD4, His-TAT-HOXB3 and His-TAT-HOXB6 proteins. Background technique [0002] Hematopoietic stem cells (HSCs) are stem cells that can differentiate into all blood cells. Hematopoietic stem cells have the characteristics of "pluripotent" (pluripotent), which can differentiate into a variety of specific blood cells; "self-renewal" (self-renewal), that is, among the offspring of cells after division, at least one of the offspring The ability of cells to maintain the same characteristics as before division and to continue to divide; and the ability to move from bone marro...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/22C07K1/20C07K1/18C07K1/16C12N5/0789
CPCC07K14/4702C07K2319/10C07K2319/21C12N5/0647C12N2501/998
Inventor 黄济鸿谢淑如龚文玫陈钰霖苏昱祯
Owner TAIWAN ADVANCE BIO PHARM INC
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