Human insulin-like growth factor conjugated protein 3 eucaryon expression vector, construction and use

A technology for eukaryotic expression vectors and growth factors, which can be used in the field of biomedicine and can solve problems such as clinical application limitations

Inactive Publication Date: 2009-07-15
HEBEI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, considering that the prokaryotic expressed IGFBP-3 is a fusion protein, its clinical application will be limited

Method used

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  • Human insulin-like growth factor conjugated protein 3 eucaryon expression vector, construction and use
  • Human insulin-like growth factor conjugated protein 3 eucaryon expression vector, construction and use
  • Human insulin-like growth factor conjugated protein 3 eucaryon expression vector, construction and use

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Embodiment 1

[0064] Embodiment 1, the construction method of human insulin-like growth factor binding protein 3 eukaryotic expression vector

[0065] A. Design primers:

[0066] According to the cDNA sequence No.NM_000598 of 291 amino acids encoding IGFBP-3 signal peptide and mature peptide in the American gene database, the specific primers of sequence 2 were designed, the EcoRI restriction site was introduced into the upstream sequence, and the XbaI restriction site was introduced into the downstream sequence, The primer sequences are as follows:

[0067] Upstream primer: 5′-g gaattc atgcagcgggcgcgacccacgct-3′

[0068] Downstream primer: 5′-gc tctaga ttatcacttgctctgcatgctgtagcagtgcacg-3′;

[0069] The enzyme cleavage sites are underlined in the primers.

[0070] B. Amplification of the target gene:

[0071] Use the human fetal liver cDNA library as a template for PCR amplification: template 1 μl, upstream and downstream primers 10 pmol / μl each 2 μl, 2.5 mmol / L dNTP substrate 2 μl,...

Embodiment 2

[0100] The full-length sequencing of the recombinant eukaryotic expression vector was carried out, and the sequencing results showed that the size of the recombinant eukaryotic expression vector sequence 1 was 6339bp, and the IGFBP-3 sequence containing 873bp was inserted between 1101bp-1974bp, which was compared with the gene database (Genbank) The IGFBP-3 sequence is completely consistent, indicating that the recombinant eukaryotic expression vector named pCI-IGFBP-3 was successfully constructed, as shown in Sequence 1. Example 2, Research on the biological activity of pCI-IGFBP-3 recombinant eukaryotic expression vector

[0101] A. Extraction and purification of recombinant eukaryotic expression vector

[0102] Using the pCI-neo empty vector as a control, the pCI-IGFBP-3 used for transfection was extracted and purified with an endotoxin-free small plasmid extraction kit (product of OMEGA, USA). The specific operation steps were carried out according to the instructions in t...

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Abstract

The invention relates to the construction and the application of a eukaryotic expression vector of human insulin-like growth factor-binding protein-3 which is named as pCI-IGFBP-3. Sequence 1 is obtained through the recombination of human IGFBP-3 gene fragment and eukaryotic expression vector pCI-neo. The sequence 1 is summed up to 6399bp, wherein, IGFBP-3 sequence 3 containing 873bp is inserted between 1101bp and 1974bp. Primers are designed by the invention according to the cDNA sequence of human IGFBP-3 in Genbank. Human fetal liver cDNA library is used as a template to obtain an IGFBP-3cDNA sequence containing single peptide sequence by a pCR method in a cloning way. Target genes are directionally inserted into the eukaryotic expression vector pCI-neo by the double digestion of EcoR I and Xba I. The secretory IGFBP-3 eukaryotic expression vector is constructed successfully. Experimental results show that the eukaryotic expression vector of the human insulin-like growth factor-binding protein-3 can secrete and express IGFBP-3 with biological activities in eukaryotic cells. The secreted and expressed IGFBP-3 directly acts on host cells to play a role of promoting apoptosis. The construction and the application of the eukaryotic expression vector of the human insulin-like growth factor-binding protein-3 are clinically applied to the preparation of antitumor drugs with the IGFBP-3 eukaryotic expression vector as the effective component and lay the foundation for the development of naked plasmid anti-oncogene therapeutic drugs.

Description

technical field [0001] The invention relates to a human insulin-like growth factor binding protein 3 (IGFBP-3) eukaryotic expression vector, a construction method and application, belonging to the field of biomedicine. Background technique [0002] IGFBP-3 belongs to the insulin-like growth factor binding protein family, its gene is located at 7p12-14, its mRNA length is 2.4kb, its polypeptide chain consists of 264 amino acids (including 18 cys), its molecular weight is about 29kD, and its primary structure contains 3 An N-type glycosylation site and a heparin binding site can be phosphorylated at the Ser site, and there is also a nuclear localization sequence (NLS) in its amino acid sequence. A variety of tissues can secrete IGFBP-3, and IGFBP-3 plays an important role in regulating cell growth and apoptosis through autocrine and paracrine. More and more evidence shows that IGFBP-3 can directly promote apoptosis and inhibit proliferation of various cancer cells, and has be...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N15/66A61K48/00A61P35/00
Inventor 吴琛单耀军宋文倩王嘉丽王艳芳
Owner HEBEI UNIVERSITY
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