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Preparation method and application of a recombinant tumor necrosis factor-related apoptosis-inducing ligand protein

A technology of recombinant bacteria and protein, which is applied in the field of preparation of recombinant tumor necrosis factor-related apoptosis-inducing ligand protein, can solve the problems of poor stability of recombinant TRAIL protein, low refolding rate of inclusion body, easy precipitation, etc., to achieve convenient Purification and detection, apoptosis induction, high solubility effect

Active Publication Date: 2020-08-04
SHANDONG UNIV QILU HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, TRAIL with a monomeric structure cannot induce apoptosis. In order to achieve the purpose of TRAIL-induced apoptosis, in the prior art, recombinant TRAIL forms dimers or multimers, and then binds to death receptors to induce apoptosis. the role of
However, the preparation process of recombinant TRAIL protein reported so far has problems such as poor stability, low renaturation rate of inclusion bodies, and easy precipitation.

Method used

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  • Preparation method and application of a recombinant tumor necrosis factor-related apoptosis-inducing ligand protein
  • Preparation method and application of a recombinant tumor necrosis factor-related apoptosis-inducing ligand protein
  • Preparation method and application of a recombinant tumor necrosis factor-related apoptosis-inducing ligand protein

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Effect test

Embodiment 1

[0043] Example 1: Preparation of recombinant human HM-TRAIL protein

[0044] 1. Construction of pQE16-HM-TRAIL expression vector

[0045] will have His 6 -The amino acid sequence of the Myc tag (MRGSHHHHHHGSEQKLISEEDLNLQ) is inserted behind the 95-281 amino acid sequence of the human TRAIL protein (sequence such as SEQ ID NO.1), PCR amplifies the nucleotide sequence encoding the above-mentioned recombinant human HM-TRAIL protein ( The sequence is shown as SEQ ID NO.2), and inserted into the multiple cloning site of the pQE16 expression vector by double restriction digestion, and the sequence was verified by sequencing.

[0046] 2. Transformation of pQE16-HM-TRAIL expression vector (construction of recombinant bacteria)

[0047] (1) Take out the E.coli TOP10 competent bacteria (100ul) stored at -80°C and put them on ice to thaw.

[0048] (2) Add the ligation product (about 20ul) into the competent bacteria, flick the bottom of the EP tube, mix well, and then ice-bath for 30m...

Embodiment 2

[0100] Embodiment 2: in vitro experiment

[0101] In order to study the induction effect of recombinant human HM-TRAIL protein on tumor cell apoptosis in vitro, we used MTT method to detect the effect of various cell lines on HM-TRAIL in many breast cancer cell lines and normal breast epithelial cell line MCF-10A. sensitivity.

[0102] 1. Cell culture and culture conditions

[0103] Human breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-468 were cultured in DMEM containing 10% fetal bovine serum, 100 U / ml penicillin and 100 μg / ml streptomycin (Dulbecco's modified MEM medium ) in high glucose medium. Human breast cancer cell lines SK-BR-3 and ZR-75-1 were cultured in RPMI 1640 medium containing 10% fetal bovine serum and antibiotics. T47D breast cancer cell lines were cultured in RPMI 1640 medium containing 10 μg / ml bovine insulin and 10% fetal bovine serum. All cells were kept at 37°C and 5% CO 2 cultured under normal conditions.

[0104] 2. MTT method to detect the...

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Abstract

The invention discloses recombination human HM-TRALL protein with an amino acid sequence shown in the SEQ ID NO.1. The method comprises the steps that a human recombination HM-TRALL protein expression carrier containing a His6-Myc label is built at first, recombination bacteria for expressing human recombination HM-TRALL protein are built through the carrier, recombinant expression is carried out on the recombination human HM-TRALL protein through IPTG induction, extraction and purification are carried out, and high-purity HM-TRAIL protein is obtained. The recombination human HM-TRALL protein can selectively induce apoptosis of breast cancer cells in vitro, and will not cause normal breast cell apoptosis. The prepared recombination human HM-TRALL protein has the advantages of being good in stability and high in dissolving degree, can obviously induce apoptosis of tumor cells and is good in application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method and application of a recombinant tumor necrosis factor-related apoptosis-inducing ligand protein. Background technique [0002] Tumor has become one of the serious threats to human life and health. Although comprehensive treatment based on surgery has achieved good results, postoperative recurrence and metastasis are still the main factors affecting survival and mortality. In recent years, biotechnology has opened up new avenues for the treatment of tumors. [0003] Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, can induce apoptosis by activating extrinsic apoptotic pathways. TRAIL can bind to two receptors containing death domains (TRAILR1 / DR4 and TRAILR2 / DR5) to trigger cell death, and TRAIL can also bind to two decoy receptors (TRAILR3 / DcR1 and TRAILR4 / DcR2) to trigger cell death. to the protective eff...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21C07K1/36C07K1/34C07K1/22A61K38/17A61P35/00C12R1/19
CPCA61K38/00C07K14/70575C07K2319/21C07K2319/41C12N15/70C12N2800/101
Inventor 杨其峰张宁
Owner SHANDONG UNIV QILU HOSPITAL
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