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Preparation method of tumor necrosis factor related apoptosis induction ligand fusion protein

An apoptosis-inducing ligand and tumor necrosis factor technology, applied in the biological field, can solve the problems of high cost of yeast expression system, cumbersome and complicated purification process, and low yield of soluble protein

Active Publication Date: 2014-12-17
JIANGSU SIMCERE PHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The cost of the yeast expression system is high and it is easy to cause group modification of the TRAIL protein; most of the TRAIL protein expressed in the E. coli expression system is an inclusion body, the yield of soluble protein is low, and the purification process is cumbersome and complicated

Method used

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  • Preparation method of tumor necrosis factor related apoptosis induction ligand fusion protein
  • Preparation method of tumor necrosis factor related apoptosis induction ligand fusion protein
  • Preparation method of tumor necrosis factor related apoptosis induction ligand fusion protein

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Experimental program
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Effect test

Embodiment 1

[0105] After a lot of experiments, the inventors found that the constructed fusion protein LZ-TRAIL95 can completely eliminate tumors, and the state of nude mice is significantly better than that of the positive control group given human TRAIL protein, which shows that compared with TRAIL, the fusion protein LZ-TRAIL95 can completely disappear. TRAIL95 is less toxic. In addition, the in vivo half-life of the fusion protein is 218 minutes, which is significantly higher than that of the human TRAIL protein.

[0106] The fusion protein LZ-TRAIL95 contains 3 parts: cross-linking region, LZ (leucine zipper) and TRAIL extracellular region. Among them, the cross-linking region contains two cysteines. In the trimer structure of the active form, two pairs form an intermolecular disulfide bond to strengthen the TRAIL trimer. Its sequence is as follows:

[0107] MEEDPCACESLVKFQAKVEGLLQALTRKLEAVSKRLAILENTVVGSTSEETISTVQEK QQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSN LHLRNG...

Embodiment 2

[0113] Example 2. Construction of fusion protein expression vector

[0114] Using the leucine zipper (LZ, human leucine zipper domain, codon optimized to be suitable for expression in E. coli) fragment synthesized by Shanghai Xuguan Biotechnology Development Co., Ltd. as a template, amplified by PCR The required DNA fragments, primers LZ1 and LZ2 used in the primers were synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd. Among them, LZ1 mutated the 6th and 8th cysteine ​​into serine; LZ2 not only contained the 3' end sequence of the LZ fragment, but also added a sequence encoding the GlySer linker peptide after its 3' end.

[0115] LZ1: 5'

[0116] GGAATTCCATATGGAGGAAGACCCGTCGGCCTCGGAAAGCCTGGTGAAATTTC

[0117] LZ2: 5'CGCGGATCCGACAACGGTGTTTTCCAGG

[0118] The TRAIL fragment is the 95th to 281st amino acid of the complete sequence of TRAIL, which is amplified from the cDNA library of human fetal liver bank (purchased from Clontech LaNoratories Inc.USA) by PCR ...

Embodiment 3

[0123] Embodiment 3: Preparation of TRAIL fusion protein

[0124] The engineered bacteria LZ-TRAIL95-pET25b-BL (DE3) CGMCC No.4953 was inserted into the primary culture bottle and cultivated for 8 hours, and then inoculated from the primary culture bottle into the secondary culture bottle according to 5% inoculation amount, and cultured 10 hours. After the fermenter is balanced, the secondary seed liquid is inserted to start fermentation. Cultivate at 37°C until the cell concentration OD 600nm ≈30, lower the temperature to 25°C, add IPTG to a final concentration of 0.1mmol / L, and start the induction, and the induction time is 11 hours. After the fermentation was completed, the fermented liquid was centrifuged at 14000rpm for 10 minutes, and the thalline was collected, weighed, and 125g of the wet thallus was obtained for every liter of fermented liquid, and the washed thallus was placed in a -20°C freezer for preservation.

[0125] Dissolve the fermented cells in Buffer A (...

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Abstract

The invention relates to the biological technical field, and provided s preparation method of a tumor necrosis factor related apoptosis induction ligand fusion protein. The preparation method comprises the following steps: step a, selecting an engineering bacterium LZ-TRAIL95-pET25b-BL(DE3) CGMCC No.4953 as the bacterium strain to carry out fermentation culture; step b, washing and grinding the obtained bacteria; step c, subjecting the grinded bacteria to an ammonium sulfate precipitation treatment; step d, subjecting the obtained bacteria in the ammonium sulfate precipitation treatment to a chromatographic purification treatment; step e, subjecting the purified bacteria to a desalination treatment, and finally changing the buffer solution. Compared to the prior art, the preparation method can achieved the industrial production of TRAIL fusion protein, and thus the practical application of TRAIL fusion protein will be realized soon.

Description

technical field [0001] The invention relates to the biological field, in particular to a preparation method of a tumor necrosis factor-related apoptosis-inducing ligand fusion protein. Background technique [0002] In 1995, Wiley SR and others reported that a gene encoding an anti-tumor protein was screened from a human expression tag sequence library (EST, expressed sequence tag). The protein encoded by the gene was called tumor necrosis factor-related apoptosis ligand (tumor necrosis factor-related apoptosis-inducing ligand, TRAIL), also known as apoptin 2 ligand (Apo2L), belongs to the tumor necrosis factor superfamily (Wiley SR, et al., Immunity3:673-682, 1995). The human TRAIL gene is located on chromosome 3q26, with a full length of 1769bp, encoding a total of 281 amino acids. TRAIL protein is a type II transmembrane protein with a molecular weight of 32.5KDa and a theoretical isoelectric point of 7.63. The 114-281 amino acids at the C-terminal constitute the extracel...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/22C07K1/20C07K1/18C12P21/02C12R1/19
Inventor 苏云鹏刘晓斌周宇王猛邓磊修
Owner JIANGSU SIMCERE PHARMA
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