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Expression vector and fused protein of human differential initiator 3 and preparation thereof

A technology of differentiation inhibitors and fusion proteins, which is applied in the fields of genetic engineering and medical biology, can solve the problems that recombinant hId3 protein anti-hId3 antibodies have not been reported, and achieve high-efficiency expression

Inactive Publication Date: 2008-10-01
NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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AI Technical Summary

Problems solved by technology

The prokaryotic expression and purification of recombinant hId3 protein and the preparation of anti-hId3 antibody have not been reported at home and abroad

Method used

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  • Expression vector and fused protein of human differential initiator 3 and preparation thereof
  • Expression vector and fused protein of human differential initiator 3 and preparation thereof
  • Expression vector and fused protein of human differential initiator 3 and preparation thereof

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Embodiment 1

[0054] 1. According to the hId3 cDNA coding sequence provided in GeneBank, design a pair of specific primers, introduce an EcoR I restriction site at the 5' end of the upstream primer; add a Sal I restriction site at the 5' end of the downstream primer, which also includes a stop codon Child TAA.

[0055] Upstream primer: 5'-CG GAATTC AAGGCGCTGAGCCCGGTG-3' (SEQ ID No. 2)

[0056] (The underline is the EcoR I restriction site)

[0057] Downstream primer: 5'-ACGC GTC GAC TTAGTGGCAAAAGCTCCTTTTG-3' (SEQ ID No. 3)

[0058] (The underlined part is the Sal I restriction site).

[0059] With pGEM-T / Id3 vector (see Li Xiaojun et al., construction of Id3-EGFP fusion gene expression vector and its expression in human lung cancer cell A549, "Chinese Journal of Laboratory Medicine", Volume 29, No. 12, December 2006) as Template (the gene fragment containing hId3 that can also be obtained by conventional methods is template), carries out PCR amplification with above-mentioned primer ...

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Abstract

The present invention belongs to genetic engineering and medical biotechnology field, providing a human differentiation inhibitor 3 expression vector, fusion protein and their preparing method. After PCR amplification, the expression vector makes hId3 gene clone between sites EcoR I and Sal I of fusion expression vector pET32a, recombinant expression vector hId3 / pET32a is produced. The invention makes hId3 encoding gene directly clone in prokaryotic expression vector, hId3 fusion protein expression vector is produced, realizing a high level expression of hId3 in escherichia coli, the expression accounts for 27% of bacterial protein, a whole set engineering bacteria induced expression and purification process are provided at the same time, rabbit anti-hId3 polyclonal antibody is produced by using hId3 fusion protein as an immunogen, laying a good foundation for researching biological function of hId3 protein and its application in clinical diagnose.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and medical biology, and relates to a human differentiation inhibitor 3 (hId3) expression vector, hId3 fusion protein, anti-hId3 polyclonal antibody and a preparation method thereof. Background technique [0002] Inhibitors of differentiation / DNAbinding (Id), a member of the helix-loop-helix (HLH) transcription factor family, negatively regulates the activity of basic helix-loop-helix (bHLH) transcription factors, thereby inhibiting Cell Differentiation [1-4] . Mammalian cells contain four Id factors (Id1-Id4). Id3 as a serum-induced immediate early gene identified for the first time in a mouse fibroblast cell line [5] . It is involved in a variety of cell biological processes, including T cell and B cell development, skeletal muscle differentiation, vascular smooth muscle proliferation, embryonic neurogenesis, osteogenesis, and tumor-induced angiogenesis, etc. [5-12] . Aberrant expressio...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/12C07K19/00C07K16/18
Inventor 李晓军贾丽仲爱芳虞伟
Owner NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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