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Preparation method and application of human prostate apoptosis response protein 4 and apoptin 2 ligand fusion protein

A fusion protein, fusion protein technology, applied in the field of genetic engineering to prepare the fusion protein, can solve the problems of tumor cell apoptosis and the like

Inactive Publication Date: 2012-10-31
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This region can be located not only in the nucleus of tumor cells, but also in the nucleus of normal and immortal cells, but it only selectively induces the apoptosis of tumor cells

Method used

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  • Preparation method and application of human prostate apoptosis response protein 4 and apoptin 2 ligand fusion protein
  • Preparation method and application of human prostate apoptosis response protein 4 and apoptin 2 ligand fusion protein
  • Preparation method and application of human prostate apoptosis response protein 4 and apoptin 2 ligand fusion protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Establish cDNA library and screen to obtain Par4 and Apo2L genes

[0053] 1. Preparation of total RNA from human peripheral blood mononuclear cells

[0054] Separate human peripheral blood lymphocytes with lymphocyte separation medium according to routine, use RPMI-1640 medium (GIBCO company), add 10% newborn calf serum (Hangzhou Sijiqing Biological Company), penicillin and streptomycin and culture until adherent, The mononuclear cells were obtained, and then stimulated with 10 μg / L endotoxin of Escherichia coli R595 for 2 hours to activate the mononuclear cells, and collected 10 7 Cells were extracted with a total RNA extraction kit (Qiagen Company) to obtain total RNA from human peripheral blood mononuclear cells.

[0055] 2. Establish cDNA library and screen Apo2L gene

[0056]The total RNA obtained above was purified with an Oligo-dT column (Qiagen Company) to obtain total mRNA, and the cDNA synthesis kit (Clontch Company) was used to synthesize the first and seco...

Embodiment 2

[0062] The coding sequence of Par4 was obtained by reverse transcription-polymerase chain reaction (ie RT-PCR)

[0063] 1. RT to obtain the first strand of cDNA of Par4

[0064] Using human prostate cell total RNA purchased from Clontech (USA) as a template and P1 as a primer, the first strand of Par4 cDNA was catalyzed by reverse transcriptase. Primers:

[0065] P1: 5'-CCA CAT TGA GTC TTG AAT CC-3' (SEQ ID NO.11)

[0066] 2. Polymerase chain reaction (PCR) amplification to obtain the Par4 coding sequence

[0067] Using the above-mentioned first strand of cDNA as a template, P1 and P2 as primers, Tag DNA polymerase catalyzes the synthesis and amplification of the Par4 coding sequence. After sequence analysis, the obtained DNA sequence was consistent with the Par4 coding sequence displayed in various databases, that is, the Par4 coding nucleotide sequence was obtained. Primers:

[0068] P2: 5'-TAC AAG CTC CTC CAA GC-3' (SEQ ID NO.12)

Embodiment 3

[0070] The coding sequence of Apo2L was obtained by RT-PCR

[0071] 1. Preparation of total RNA from human peripheral blood mononuclear cells

[0072] (1) Separating lymphocytes from human peripheral blood according to conventional methods, and then obtaining mononuclear cells by the adherence method;

[0073] (2) The mononuclear cells were activated with bacterial endotoxin to stimulate the cells to express more genes and increase the abundance of RNA, and the total RNA was extracted with an RNA extraction kit (Qiagen Company).

[0074] 2. Reverse transcription (RT) to obtain the first strand of cDNA of Apo2L

[0075] Using the above total RNA as a template and P3 as a primer, reverse transcriptase catalyzes the synthesis of the first strand of Apo2L cDNA. Primers:

[0076] P3: 5'-TCC AGG TCA GTT AGC CAA C-3' (SEQ ID NO.13)

[0077] 3. Polymerase chain reaction (PCR) amplification to obtain the Apo2L coding sequence

[0078] Using the above-mentioned first strand of cDNA...

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Abstract

The invention provides a human prostate apoptosis response 4(Par4) protein or a selective for apoptosis induction cancer cells domain (SAC) and apoptin 2 ligand (Apo2L) fused protein, namely Par4-Apo2L or SAC-Apo2L fused protein, a DNA (Deoxyribonucleic Acid) sequence for coding the protein, a vector containing the DNA sequence for coding, a host cell containing the vector, a method for preparing the protein by using the gene engineering, and a medicinal composition containing the protein. The Par4 or the SAC and the Apo2L have an effect of selectively inducing the apoptosis of cancer cells, so the fused protein can be used for preparing medicaments for treating diseases such as cancers.

Description

technical field [0001] The invention relates to the field of DNA recombination technology and biotechnology medicine. More specifically, the present invention relates to human prostate apoptosis response protein 4 (Par4) or its selective apoptotic cancer cell region (SAC), a protein fused with apoptin 2 ligand (Apo2L), a DNA encoding the fusion protein sequence, a vector containing the DNA sequence, a host cell containing the vector, a method for preparing the fusion protein by genetic engineering, and the application of the fusion protein in the treatment of diseases such as tumors. Background technique [0002] Par4 is an apoptosis-inducing gene that was first isolated from apoptotic prostate cancer cells by Sells et al. in 1994 by differential hybridization, so it is named: Prostate apoptosis response 4 protein (Par4) [Cell Growth Differ, 1994; 5(4): 457-466.]. The Par4 gene is located on human chromosome 12q21.2 and consists of 7 exons and 6 introns. The coding sequenc...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21C12N1/19C12N5/10C12N15/70A61K38/19A61P35/00C12R1/19
Inventor 王梁华张威吴腾云
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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